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18 November 2021 A look at how viagra for sale online some of our members marked National Pathology Week from 1-7 November, 2021. Swansea Bay University Health Board Pathology staff at Morriston Hospital, Singleton Hospital, Neath Porth Talbot Hospital and Princess of Wales Hospital promoted pathology services and biomedical science at their respected hospitals throughout the week with a series of engagement events – all under the week’s theme, #AllTogetherNow. #NationalPathologyWeek @princess of Wales Hospital viagra for sale online.

@IBMScience @RCPath @SBUPathology pic.twitter.com/YzTugwZ3Pw — Kim Lewis (@KimChrisLewis) November 2, 2021 Display stands were put up in front of pathology services, where staff were able to answer any questions from other Hospital staff as well as patients. The stands were visited by lots of different groups, including- medical students studying at Cardiff University, parents who were curious about career journeys in pathology on behalf of their children, and visitors wanting to understand what we do with their bloods after collection. Day viagra for sale online 4 of #nationalpathologyweek2021.

Some of our Pathology staff came out to support Harvey's Gang and Blood bike Wales @Princess of Wales Hospital. @IBMScience @GangHarveys @SBUPathology @RCPath pic.twitter.com/qst5T9VTgz — Kim Lewis (@KimChrisLewis) November 4, 2021 today our staff went on a walk round our hospital sites to raise funds for @Laird_Admiral and @BloodBikesWales pic.twitter.com/5NlZLi2f1r — Swansea Bay UHB Pathology (@SBUPathology) November 4, 2021 Pathology staff also held a Harvey’s Gang tour, where a young boy was taken around the laboratory and shown his blood films and other laboratory tests. After the tour, Pathology staff across all viagra for sale online sites wore plastic aprons and marched around the Hospital in support of Harvey’s Gang and Blood Bike.

With this hospital march, the department was able to raise money to support and promote Harvey’s Gang and Blood Bike Wales. “After the event there was a huge boost of morale in each department. Staff particularly liked the walk around the hospital and the departmental Kahoot! viagra for sale online.

quiz. In effort to promote staff well-being, the management have agreed to routine departmental engagement such as these to further support staff well-being. Overall, this was a successful event with positive outcomes.” Kimberly Lewis, Specialist Biomedical Scientist in Clinical Biochemistry at Princess of Wales Hospital To finish off the week, Swansea Bay UHB viagra for sale online held a departmental quiz.

Staff formed teams within their department (i.e. Biochemistry, haematology, microbiology and cellular pathology). Biochemistry took viagra for sale online the win!.

And the winners are …. Biochemistry at viagra for sale online POW!. Trophy and prizes to be delivered next week.

Thank you to all who have supported us this week #NationalPathologyWeek2021 #AllTogetherNow @RCPath @IBMScience @Rutharoo15 @RhodDavies1 @ChrissieMoz @maggsheidi pic.twitter.com/GnxXxntGVL — Swansea Bay UHB Pathology (@SBUPathology) November 5, 2021 Christie Pathology Partnership To mark National Pathology Week, IBMS Council Member Tahmina Hussain organised a week of lunchtime pathology featuring staff at The Christie Pathology Partners (Manchester). Each day they delivered a lunchtime session on a different discipline in Pathology - covering Blood Sciences Specimen viagra for sale online Reception, a Histology lab tour, Cytogenetics, Mortuary and Bereavement Suite and the Blood Transfusion laboratory. “These sessions gave a really interesting insight into Pathology and the roles each and every one of us plays in the patient care pathway.

Often, we are so busy working in our own departments, we are not aware of what the role of our team members are in different departments so this was a really good way of getting ‘All together now’, meeting other team members and learning something new!. Due to the success of these sessions, many of viagra for sale online the staff members who were not able to attend have requested a repeat!. € Tahmina Hussain Specialist Biomedical Scientist in Haematology &.

Blood Transfusion at The Christie Pathology Partnership Support from IBMS Chief Executive David Wells As IBMS Chief Executive and former Head of Pathology at NHS England, David Wells shared a message of support for National Pathology Week on social media. Whatever you have done to celebrate #nationalpathologyweek viagra for sale online thank you!. @IBMScience @RCPath pic.twitter.com/uxGEQCfl7e — David Wells (@DavidRWells) November 5, 2021 Thank you to everyone across the profession who came together to raise awareness and celebrate National Pathology Week 2021!.

11 November 2021 The Association of British HealthTech Industries (ABHI) and the British In Vitro Diagnostics Association (BIVDA) write to Dr Jenny Harries OBE, Chief Executive Officer for UK Health Security Agency The letter raises concerns over validation processes and the quality of some erectile dysfunction treatment tests available on the market. They argue that implementation of the erectile dysfunction Test Device Approvals (CTDA) process has viagra for sale online damaged the UK diagnostics industry and led to the needless withdrawal of erectile dysfunction treatment tests with no identified deficiency which has weakened supply resilience in the UK and could disrupt testing capacity and capability. The letter was shared with David Wells, IBMS Chief Executive and Professor Allan Wilson, IBMS President, as well as politicians and representatives from the diagnostics industry.

David Wells, IBMS Chief Executive said. "The IBMS welcomes legislation that supports the supply of high-quality diagnostic tests for our patients and the general viagra for sale online public. In enacting this legislation, the Government must ensure that existing capacity and capability is maintained to support the NHS over the coming months.

Therefore, industry concerns, together with those of laboratory experts should be taken into consideration, to ensure the supply chain and resilience of the availability of tests is maintained to meet the needs of the country.” Read the letter in full>>.

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With low natural viagra foods incomes (e.g., 31% for those with income under $10,000). And Black and Hispanic beneficiaries (25% and 22%, respectively).The new analysis also provides an overview of coverage of dental, hearing, and vision services in Medicare Advantage plans. While most plans offer coverage for these services, the extent of coverage varies and has limits.Nearly all Medicare Advantage enrollees with access to dental coverage have preventive care benefits, and most have access to more extensive dental natural viagra foods benefits. Cost sharing for more extensive dental services is typically 50 percent for in-network care, and typically is subject to an annual dollar cap on plan payments.Similarly, almost all Medicare Advantage enrollees have access to hearing exams and hearing aid coverage. The coverage generally is subject to either a maximum annual dollar cap and/or frequency limits on how often natural viagra foods plans cover the service.Virtually all Medicare Advantage enrollees have access to vision exams and eyewear coverage, typically subject to maximum annual limits averaging about $160 per year.The findings come as policymakers in Congress are considering adding dental, hearing, and vision benefits to Medicare as part of the budget reconciliation bill, one of several competing spending priorities in the debate.

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About 16 percent of all Medicare beneficiaries reported in viagra for sale online 2019 that there was a time in the last year that they could not get dental, hearing, or vision care, but this was reported by a greater percentage of beneficiaries under age 65 with long-term disabilities (35%). Those enrolled in both Medicare and Medicaid (35%). With low incomes (e.g., 31% for those with income under viagra for sale online $10,000).

And Black and Hispanic beneficiaries (25% and 22%, respectively).The new analysis also viagra online usa provides an overview of coverage of dental, hearing, and vision services in Medicare Advantage plans. While most plans offer coverage for these viagra for sale online services, the extent of coverage varies and has limits.Nearly all Medicare Advantage enrollees with access to dental coverage have preventive care benefits, and most have access to more extensive dental benefits. Cost sharing for more extensive dental services is typically 50 percent for in-network care, and typically is subject to an annual dollar cap on plan payments.Similarly, almost all Medicare Advantage enrollees have access to hearing exams and hearing aid coverage.

The coverage generally is subject viagra for sale online to either a maximum annual dollar cap and/or frequency limits on how often plans cover the service.Virtually all Medicare Advantage enrollees have access to vision exams and eyewear coverage, typically subject to maximum annual limits averaging about $160 per year.The findings come as policymakers in Congress are considering adding dental, hearing, and vision benefits to Medicare as part of the budget reconciliation bill, one of several competing spending priorities in the debate. It would be the largest expansion of Medicare benefits since the Part D drug benefit was launched in 2006. (A similar viagra for sale online 2019 proposal would have increased Medicare spending by more than $300 billion over 10 years according to the Congressional Budget Office.)For the full analysis and other KFF data and analyses about Medicare, including the recent Medicare and Dental Coverage.

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Last summer, two truck drivers employed by an Oklahoma City roofing company shared a serious safety concern with grapefruit juice and viagra their manager. They reported that the tires on the company truck they were driving were damaged, and that they were concerned not only about their own safety, but also the safety of others on the road. Until the issue was grapefruit juice and viagra addressed, they weren't comfortable driving the vehicle. The employer’s response?.

Firing both drivers. The whistleblower division of the grapefruit juice and viagra U.S. Department of Labor’s Occupational Safety and Health Administration investigated and found that the employer violated the Surface Transportation Assistance Act, one of more than 20 whistleblower statutes we enforce. We ordered the company to grapefruit juice and viagra reinstate both drivers, and to pay them more than $23,000 in back wages and $70,000 in punitive damages.

The company is also required to train managers and employees on workers’ rights under the STAA to help prevent any future illegal retaliation against employees. Under federal law, workers have the right to raise concerns if they believe their rights are being violated in the workplace. Similarly, the statute enforced by the department’s Mine Safety and Health Administration provides grapefruit juice and viagra strong protections for miners. Under the Federal Mine Safety and Health Act of 1977, miners have the right to report hazardous conditions and can refuse to work in unsafe conditions.

Miners may not be fired, demoted, harassed, intimidated, transferred, refused employment, discriminated against or suffer any loss of wages for exercising their rights under the Mine Act. These are grapefruit juice and viagra critical protections that allow miners to actively participate in the safety of their workplace, and MSHA takes enforcement of hazard and discrimination complaints seriously. And similar rights apply to pay concerns, too – If employers don’t pay workers all the wages they’ve earned, they can speak up without fear of retaliation. The department’s Wage and Hour Division enforces laws on the federal minimum wage, overtime pay, recordkeeping and child grapefruit juice and viagra labor requirements, as well as laws about working conditions and payment for agricultural and migrant workers.

The division protects workers subject to wage garnishment, too. Any worker protected by any of the laws that the division enforces is protected by their anti-retaliation provisions. View our online resources to determine grapefruit juice and viagra whether a particular law applies. We enforce the law regardless of a worker’s immigration status, and can communicate with workers in more than 200 languages.

National Whistleblower Appreciation Day As we recognize National Whistleblower Appreciation Day on July 30, we celebrate all of the brave workers, like these drivers, who speak out against unsafe or unfair working conditions, and thank them for their contributions to ensuring a safer, more just workforce for themselves and their co-workers. Because of their grapefruit juice and viagra actions, lives and livelihoods have been saved. The U.S. Department of Labor is committed to protecting every worker’s right to speak up without retaliation if they are mistreated, denied their rights or grapefruit juice and viagra have concerns about safety.

Unfortunately, we know that many workers are not protected by their employers when they speak up, and that they need a place to turn if they’re punished, lose their job or are simply afraid to say something directly to their employer. Employers who retaliate against workers are not only breaking the law, they’re creating an unsafe, unfair work environment for all of their employees. Acts of retaliation can include reducing pay or work hours, firing grapefruit juice and viagra or suspending workers, reassignment, discipline or demotion, threats, harassment or intimidation, and blacklisting from future hiring. While we’re committed to strong enforcement, we’re also dedicated to education and outreach so that employers know the rules and have access to resources to ensure workers have a platform to speak up.

An open grapefruit juice and viagra line of communication goes a long way in the workplace, and we encourage employers to create an anti-retaliation program to help improve employee engagement. Any risk to workers’ safety is unacceptable, and the erectile dysfunction treatment viagra has presented unprecedented challenges for employers when it comes to keeping workers safe on the job. While many businesses are acting in good faith, we’ve seen many that have failed to implement basic steps to protect workers. In 2020, OSHA issued nearly $4 million in citations from 300 inspections for violations related to the erectile dysfunction at businesses that failed to provide grapefruit juice and viagra the necessary personal protective equipment or other resources to help keep workers safe during the viagra.

Many of these cases were reported to us by concerned workers who feared for their lives, as well as the lives of their family and co-workers, before the availability of the treatment. Report a concern Learn more about our whistleblower protection programs online, including how to file a complaint, or contact OSHA at 1-800-321-OSHA (6742) with workplace safety or health concerns. Report a mining accident or a hazardous condition at a grapefruit juice and viagra mine, impoundment or abandoned mine online or by calling 1-800-745-1553. You can learn more about your wage and hour rights or file a complaint by contacting the Wage and Hour Division online or by calling at 1-866-4-US-WAGE (1-866-487-9243).

Jeannette Galanis is the deputy assistant secretary for the Mine Safety and Health Administration, Jim Frederick is the acting assistant secretary for the Occupational Safety and Health Administration and Jessica Looman grapefruit juice and viagra is the acting administrator for the Wage and Hour Division. Follow @MSHA_DOL, @OSHA_DOL and @WHD_DOL on Twitter.President William Howard Taft signed the act creating the U.S. Department of Labor in his final hours as president on March 4, 1913. In the decades following, the department has supported the interests of workers, jobseekers and retirees across grapefruit juice and viagra the country.

We’ve reduced workplace fatalities and recovered billions of dollars in stolen wages and benefits. Here are a few things you might not know about the Labor Department.1. We’re younger than Labor grapefruit juice and viagra Day. Americans first celebrated Labor Day in 1882, and it became a federal holiday in 1894 – nearly 20 years before the creation of the Labor Department.2.

We put our own spin on the idea of “ladies first.” The Labor Department grapefruit juice and viagra was the first Cabinet agency led by a woman. Frances Perkins. Six women have held the title since then, giving us the record not just for the first, but for the most women secretaries as well. Many of the women who followed in Perkins’ footsteps have grapefruit juice and viagra blazed their own trails.

Elaine Chao and Hilda Solis were the first Asian American and Hispanic women in the Cabinet, respectively. Alexis Herman was the first African American to serve as secretary of labor. And Elizabeth Dole was the first woman to lead two different departments for grapefruit juice and viagra two different presidents (Labor and Transportation).3. Clam chowder could be a contentious dish at a Labor Secretary reunion.

Our 29 secretaries come from over a dozen states and three countries, but about one-fifth of them have called New York or Massachusetts home grapefruit juice and viagra. Current Secretary Marty Walsh isn’t the first to hail from Boston. In fact, he’s not even the first Mayor of Boston to serve as Secretary of Labor. It’s an grapefruit juice and viagra honor he shares with Maurice J.

Tobin. 4. We didn’t always wear pants. The women, at least.

In fact, when the department’s female employees started wearing pants to work, it was notable enough to merit a mention in the newsletter. €œFashion forward” isn’t a phrase typically used to describe the department, but in the 1970s, we were ahead of other federal agencies in officially allowing women to work in pants, thanks to Elizabeth Duncan Koontz, the director of the Women's Bureau and an advocate for women's rights. Pants proved to be very practical, and the rest is history.5. Our secretaries’ side hustles are next level.

Being a Cabinet member would be a pretty impressive accomplishment for most people, but for the 29 men and women who have served as secretary of labor, it’s just the beginning. Our first secretary, William Wilson, wrote poetry. Arthur Goldberg served as both a Supreme Court justice and ambassador to the United Nations. George Shultz also served as the director of the Office of Management and Budget, Secretary of the Treasury and Secretary of State.

And Frances Perkins is a saint in the Episcopal church.6. Our assistant secretaries are pretty impressive too. If you ever start to feel smug about your personal accomplishments, take a look at J. Ernest Wilkins’ resume.

As assistant secretary of labor for international affairs, Wilkins was the first African American to attend a Cabinet meeting. He earned a PhD from the University of Chicago before his 20th birthday (the third of five science degrees he would earn in his lifetime). He was also a mathematician, mechanical engineer and nuclear scientist, who worked as a physicist for the Manhattan Project.7. Our PSAs have seen stars.

RJ Mitte, Montel Williams, R. Lee Ermey, Carroll O’Connor, Colin Powell, the Flintstones, Johnny Cash, Mary Tyler Moore, Milton Berle, Ricardo Montalban, Vincent Price, Adam West, and Batgirl portrayer Yvonne Craig are among the celebrities who have helped educate the public about the department’s resources and the laws we enforce.8. We are the original weekend warriors. In that we enforce the law that created the 40-hour workweek.

Early twentieth century labor activists had long advocated for clearly defined work hours and a minimum wage – two causes championed by Secretary Frances Perkins. The 40-hour workweek and the minimum wage became law with the passage of the Fair Labor Standards Act in 1938, and we’ve been enforcing it ever since. Can’t get enough Labor Department history?. Learn more about our department here.

Laura McGinnis is a public affairs specialist in the U.S. Department of Labor.MDEL Bulletin, June 24 2021, from the Medical Devices Compliance Program On this page Fees for Medical Device Establishment Licences (MDELs) We issue Medical Device Establishment Licences (MDELs) to. class I manufacturers importers or distributors of all device classes for human use in Canada The MDEL fee is a flat fee, regardless of when we receive your initial application. The same fee applies to applications for.

a new MDEL the reinstatement of a suspended MDEL the annual licence review (ALR) of an MDEL If you submit any of these applications, you must pay the MDEL fee when you receive an invoice. See Part 3, Division 2 of the Fees in Respect of Drugs and Medical Devices Order. Normally, we collect the MDEL fee before we review an application. However, to help meet the demand for medical devices during the erectile dysfunction treatment viagra, we have been reviewing and processing MDEL applications before collecting the fees.

As a result, some MDEL holders still haven't paid the fees for their 2020 initial MDEL application, despite multiple reminders. Authority to withhold services in case of non-payment As stated in the Food and Drug Act, Health Canada has the authority to withhold services, approvals, rights and/or privileges, if the fee for an MDEL application is not paid. Non-payment of fees 30.64. The Minister may withdraw or withhold a service, the use of a facility, a regulatory process or approval or a product, right or privilege under this Act from any person who fails to pay the fee fixed for it under subsection 30.61(1).

For more information, please refer to. Cancellation of existing MDELs We will cancel MDELs for existing MDEL holders with outstanding fees for. initial applications or annual licence review applications If your establishment licence is cancelled, you are no longer authorized to conduct licensable activities (such as manufacturing, distributing or importing medical devices). You must stop licensable activities as soon as you receive your cancellation notice.

Resuming activities after MDEL cancellation To resume licensable activities, you must re-apply for a new establishment licence and pay the MDEL fee. See section 45 of the Medical Device Regulations. To find out how to re-apply for a MDEL, please refer to our Guidance on medical device establishment licensing (GUI-0016). In line with the Compliance and Enforcement Policy (POL-0001), Health Canada monitors activities for compliance.

If your MDEL has been cancelled, you may be subject to compliance and enforcement actions if you conduct non-compliant activities. If you have questions about a MDEL or the application process, please contact the Medical Device Establishment Licensing Unit at [email protected]. If you have questions about invoicing and fees for an MDEL application, please contact the Cost Recovery Invoicing Unit at [email protected]. Related links.

Last summer, two truck drivers employed by an Oklahoma City roofing company shared a serious safety concern blog with viagra for sale online their manager. They reported that the tires on the company truck they were driving were damaged, and that they were concerned not only about their own safety, but also the safety of others on the road. Until the issue was addressed, they weren't comfortable driving viagra for sale online the vehicle. The employer’s response?. Firing both drivers.

The viagra for sale online whistleblower division of the U.S. Department of Labor’s Occupational Safety and Health Administration investigated and found that the employer violated the Surface Transportation Assistance Act, one of more than 20 whistleblower statutes we enforce. We ordered the company viagra for sale online to reinstate both drivers, and to pay them more than $23,000 in back wages and $70,000 in punitive damages. The company is also required to train managers and employees on workers’ rights under the STAA to help prevent any future illegal retaliation against employees. Under federal law, workers have the right to raise concerns if they believe their rights are being violated in the workplace.

Similarly, the statute enforced by viagra for sale online the department’s Mine Safety and Health Administration provides strong protections for miners. Under the Federal Mine Safety and Health Act of 1977, miners have the right to report hazardous conditions and can refuse to work in unsafe conditions. Miners may not be fired, demoted, harassed, intimidated, transferred, refused employment, discriminated against or suffer any loss of wages for exercising their rights under the Mine Act. These are critical protections viagra for sale online that allow miners to actively participate in the safety of their workplace, and MSHA takes enforcement of hazard and discrimination complaints seriously. And similar rights apply to pay concerns, too – If employers don’t pay workers all the wages they’ve earned, they can speak up without fear of retaliation.

The department’s Wage and Hour Division enforces laws on the federal minimum wage, overtime pay, recordkeeping and child labor requirements, as well as laws about viagra for sale online working conditions and payment for agricultural and migrant workers. The division protects workers subject to wage garnishment, too. Any worker protected by any of the laws that the division enforces is protected by their anti-retaliation provisions. View our online resources to determine whether viagra for sale online a particular law applies. We enforce the law regardless of a worker’s immigration status, and can communicate with workers in more than 200 languages.

National Whistleblower Appreciation Day As we recognize National Whistleblower Appreciation Day on July 30, we celebrate all of the brave workers, like these drivers, who speak out against unsafe or unfair working conditions, and thank them for their contributions to ensuring a safer, more just workforce for themselves and their co-workers. Because of their actions, lives and livelihoods have been saved viagra for sale online. The U.S. Department of Labor is committed to protecting every worker’s right to speak up without retaliation if they are mistreated, denied their rights or have viagra for sale online concerns about safety. Unfortunately, we know that many workers are not protected by their employers when they speak up, and that they need a place to turn if they’re punished, lose their job or are simply afraid to say something directly to their employer.

Employers who retaliate against workers are not only breaking the law, they’re creating an unsafe, unfair work environment for all of their employees. Acts of viagra for sale online retaliation can include reducing pay or work hours, firing or suspending workers, reassignment, discipline or demotion, threats, harassment or intimidation, and blacklisting from future hiring. While we’re committed to strong enforcement, we’re also dedicated to education and outreach so that employers know the rules and have access to resources to ensure workers have a platform to speak up. An open line of communication goes a long way in the workplace, and we encourage employers to create viagra for sale online an anti-retaliation program to help improve employee engagement. Any risk to workers’ safety is unacceptable, and the erectile dysfunction treatment viagra has presented unprecedented challenges for employers when it comes to keeping workers safe on the job.

While many businesses are acting in good faith, we’ve seen many that have failed to implement basic steps to protect workers. In 2020, OSHA issued nearly $4 million in citations from 300 inspections for violations related to the erectile dysfunction at businesses that failed to provide the necessary viagra for sale online personal protective equipment or other resources to help keep workers safe during the viagra. Many of these cases were reported to us by concerned workers who feared for their lives, as well as the lives of their family and co-workers, before the availability of the treatment. Report a concern Learn more about our whistleblower protection programs online, including how to file a complaint, or contact OSHA at 1-800-321-OSHA (6742) with workplace safety or health concerns. Report a mining accident or a hazardous condition at a mine, impoundment or abandoned viagra for sale online mine online or by calling 1-800-745-1553.

You can learn more about your wage and hour rights or file a complaint by contacting the Wage and Hour Division online or by calling at 1-866-4-US-WAGE (1-866-487-9243). Jeannette Galanis is the deputy assistant secretary for the Mine Safety and Health Administration, Jim Frederick is the acting assistant secretary for the Occupational Safety and viagra for sale online Health Administration and Jessica Looman is the acting administrator for the Wage and Hour Division. Follow @MSHA_DOL, @OSHA_DOL and @WHD_DOL on Twitter.President William Howard Taft signed the act creating the U.S. Department of Labor in his final hours as president on March 4, 1913. In the decades following, the department has supported the interests viagra for sale online of workers, jobseekers and retirees across the country.

We’ve reduced workplace fatalities and recovered billions of dollars in stolen wages and benefits. Here are a few things you might not know about the Labor Department.1. We’re younger than viagra for sale online Labor Day. Americans first celebrated Labor Day in 1882, and it became a federal holiday in 1894 – nearly 20 years before the creation of the Labor Department.2. We put our own spin on the idea of “ladies first.” The Labor Department was the first Cabinet agency led viagra for sale online by a woman.

Frances Perkins. Six women have held the title since then, giving us the record not just for the first, but for the most women secretaries as well. Many of the women who viagra for sale online followed in Perkins’ footsteps have blazed their own trails. Elaine Chao and Hilda Solis were the first Asian American and Hispanic women in the Cabinet, respectively. Alexis Herman was the first African American to serve as secretary of labor.

And Elizabeth viagra for sale online Dole was the first woman to lead two different departments for two different presidents (Labor and Transportation).3. Clam chowder could be a contentious dish at a Labor Secretary reunion. Our 29 viagra for sale online secretaries come from over a dozen states and three countries, but about one-fifth of them have called New York or Massachusetts home. Current Secretary Marty Walsh isn’t the first to hail from Boston. In fact, he’s not even the first Mayor of Boston to serve as Secretary of Labor.

It’s an honor he shares viagra for sale online with Maurice J. Tobin. 4. We didn’t always wear pants. The women, at least.

In fact, when the department’s female employees started wearing pants to work, it was notable enough to merit a mention in the newsletter. €œFashion forward” isn’t a phrase typically used to describe the department, but in the 1970s, we were ahead of other federal agencies in officially allowing women to work in pants, thanks to Elizabeth Duncan Koontz, the director of the Women's Bureau and an advocate for women's rights. Pants proved to be very practical, and the rest is history.5. Our secretaries’ side hustles are next level. Being a Cabinet member would be a pretty impressive accomplishment for most people, but for the 29 men and women who have served as secretary of labor, it’s just the beginning.

Our first secretary, William Wilson, wrote poetry. Arthur Goldberg served as both a Supreme Court justice and ambassador to the United Nations. George Shultz also served as the director of the Office of Management and Budget, Secretary of the Treasury and Secretary of State. And Frances Perkins is a saint in the Episcopal church.6. Our assistant secretaries are pretty impressive too.

If you ever start to feel smug about your personal accomplishments, take a look at J. Ernest Wilkins’ resume. As assistant secretary of labor for international affairs, Wilkins was the first African American to attend a Cabinet meeting. He earned a PhD from the University of Chicago before his 20th birthday (the third of five science degrees he would earn in his lifetime). He was also a mathematician, mechanical engineer and nuclear scientist, who worked as a physicist for the Manhattan Project.7.

Our PSAs have seen stars. RJ Mitte, Montel Williams, R. Lee Ermey, Carroll O’Connor, Colin Powell, the Flintstones, Johnny Cash, Mary Tyler Moore, Milton Berle, Ricardo Montalban, Vincent Price, Adam West, and Batgirl portrayer Yvonne Craig are among the celebrities who have helped educate the public about the department’s resources and the laws we enforce.8. We are the original weekend warriors. In that we enforce the law that created the 40-hour workweek.

Early twentieth century labor activists had long advocated for clearly defined work hours and a minimum wage – two causes championed by Secretary Frances Perkins. The 40-hour workweek and the minimum wage became law with the passage of the Fair Labor Standards Act in 1938, and we’ve been enforcing it ever since. Can’t get enough Labor Department history?. Learn more about our department here. Laura McGinnis is a public affairs specialist in the U.S.

Department of Labor.MDEL Bulletin, June 24 2021, from the Medical Devices Compliance Program On this page Fees for Medical Device Establishment Licences (MDELs) We issue Medical Device Establishment Licences (MDELs) to. class I manufacturers importers or distributors of all device classes for human use in Canada The MDEL fee is a flat fee, regardless of when we receive your initial application. The same fee applies to applications for. a new MDEL the reinstatement of a suspended MDEL the annual licence review (ALR) of an MDEL If you submit any of these applications, you must pay the MDEL fee when you receive an invoice. See Part 3, Division 2 of the Fees in Respect of Drugs and Medical Devices Order.

Normally, we collect the MDEL fee before we review an application. However, to help meet the demand for medical devices during the erectile dysfunction treatment viagra, we have been reviewing and processing MDEL applications before collecting the fees. As a result, some MDEL holders still haven't paid the fees for their 2020 initial MDEL application, despite multiple reminders. Authority to withhold services in case of non-payment As stated in the Food and Drug Act, Health Canada has the authority to withhold services, approvals, rights and/or privileges, if the fee for an MDEL application is not paid. Non-payment of fees 30.64.

The Minister may withdraw or withhold a service, the use of a facility, a regulatory process or approval or a product, right or privilege under this Act from any person who fails to pay the fee fixed for it under subsection 30.61(1). For more information, please refer to. Cancellation of existing MDELs We will cancel MDELs for existing MDEL holders with outstanding fees for. initial applications or annual licence review applications If your establishment licence is cancelled, you are no longer authorized to conduct licensable activities (such as manufacturing, distributing or importing medical devices). You must stop licensable activities as soon as you receive your cancellation notice.

Resuming activities after MDEL cancellation To resume licensable activities, you must re-apply for a new establishment licence and pay the MDEL fee. See section 45 of the Medical Device Regulations. To find out how to re-apply for a MDEL, please refer to our Guidance on medical device establishment licensing (GUI-0016). In line with the Compliance and Enforcement Policy (POL-0001), Health Canada monitors activities for compliance. If your MDEL has been cancelled, you may be subject to compliance and enforcement actions if you conduct non-compliant activities.

If you have questions about a MDEL or the application process, please contact the Medical Device Establishment Licensing Unit at [email protected]. If you have questions about invoicing and fees for an MDEL application, please contact the Cost Recovery Invoicing Unit at [email protected]. Related links.

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Start Preamble over the counter viagra for women Announcement Type. New. Funding Announcement over the counter viagra for women Number. HHS-2021-IHS-TPI-0001. Assistance Listing (Catalog of Federal Domestic Assistance or CFDA) Number.

93.382. Key Dates Application Deadline Date. September 1, 2021. Earliest Anticipated Start Date. September 30, 2021.

I. Funding Opportunity Description Statutory Authority The Indian Health Service (IHS) is accepting applications for grants for the Community Health Aide Program (CHAP) Tribal Planning and Implementation (TPI) program. The CHAP is authorized under the Snyder Act, 25 U.S.C. 13. The Transfer Act, 42 U.S.C.

2001(a). And the Indian Health Care Improvement Act, 25 U.S.C. 16161. This grant program is described in the Assistance Listings located at https://beta.sam.gov (formerly known as Catalog of Federal Domestic Assistance) under 93.382. Background The national CHAP will provide a network of health aides trained to support licensed health professionals while providing direct health care, Start Printed Page 41045health promotion, and disease prevention services.

These providers will work within a referral relationship under the supervision of licensed clinical providers that includes clinics, service units, and hospitals. The program will increase access to direct health services, including inpatient and outpatient visits. The Alaska CHAP has become a model for efficient and high quality health care delivery in rural Alaska, providing approximately 300,000 patient encounters per year and responding to emergencies 24 hours a day, seven days a week. Specialized providers in dental and behavioral health were later introduced to respond to the needs of patients and address the health disparities in oral health and mental health among American Indians and Alaska Natives. The national CHAP is a workforce model that includes three different provider types that act as extenders of their licensed clinical supervisor.

The national CHAP currently includes a behavioral health aide, community health aide, and dental health aide. Each of the health aide categories operate in a tiered level practice system. The national CHAP model provides an opportunity for increased access to care through the extension of primary care, dental, and behavioral health clinicians. In 2010, under the permanent reauthorization of the Indian Health Care Improvement Act (IHCIA), Congress provided the Secretary of the U.S. Department of Health and Human Services, acting through the IHS, the authority to expand the Alaska CHAP program.

In 2016, the IHS initiated Tribal Consultation on expanding the CHAP to the contiguous 48 states. In 2018, the IHS formed the CHAP Tribal Advisory Group (TAG) and began developing the program. In 2020, the IHS announced the national CHAP policy, which formally created the national CHAP. Purpose The purpose of the TPI program is to support the planning and implementation for Tribes and Tribal Organizations (T/TO) positioned to begin operating a CHAP or support a growing CHAP in the contiguous 48 states. The grant program is designed to support the regional flexibility required for T/TO to implement a CHAP unique to the needs of their individual communities across the country through the identification of feasibility factors.

The focus of the program is to. 1. Develop clinical supervisor support for primary care, behavioral health, and dental health clinicians providing both direct and indirect supervision of prospective health aides. 2. Identify area and community-specific health care needs of patients that can be addressed by the health aides.

3. Identify and develop a technology infrastructure plan for the mobility and success of health aides in anticipation of providing services. 4. Develop a training plan to include partners across the T/TO's geographic region to enhance the training opportunities available to prospective health aides to include continuing education and clinical practice. 5.

Identify best practices for integrating a CHAP workforce into an existing Tribal health system. 6. Address social determinants of health that impact the recruitment and retention of prospective health aides. And 7. Identify the total cost of full implementation of a CHAP within an existing Tribal health system.

II. Award Information Funding Instrument—Grant Estimated Funds Available The total funding identified for fiscal year (FY) 2021 is approximately $1,500,000. Individual award amounts are anticipated to be between $450,000 and $500,000. The funding available for competing awards issued under this announcement is subject to the availability of appropriations and budgetary priorities of the Agency. The IHS is under no obligation to make awards that are selected for funding under this announcement.

Anticipated Number of Awards Approximately three awards will be issued under this program announcement. The IHS intends to award no more than one grant per IHS area. Period of Performance The period of performance is two years. III. Eligibility Information 1.

Eligibility To be eligible for this new FY 2021 funding opportunity, an applicant must be one of the following, as defined under 25 U.S.C. 1603. A federally recognized Indian Tribe as defined by 25 U.S.C. 1603(14). The term “Indian Tribe” means any Indian Tribe, band, nation, or other organized group or community, including any Alaska Native village or group, or regional or village corporation, as defined in or established pursuant to the Alaska Native Claims Settlement Act (85 Stat.

688) [43 U.S.C. 1601 et seq.], which is recognized as eligible for the special programs and services provided by the United States to Indians because of their status as Indians. A Tribal organization as defined by 25 U.S.C. 1603(26). The term “Tribal organization” has the meaning given the term in section 4 of the Indian Self-Determination and Education Assistance Act (25 U.S.C.

5304). €œTribal organization” means the recognized governing body of any Indian Tribe. Any legally established organization of Indians which is controlled, sanctioned, or chartered by such governing body or which is democratically elected by the adult members of the Indian community to be served by such organization and which includes the maximum participation of Indians in all phases of its activities. Provided that, in any case where a contract is let or grant made to an organization to perform services benefiting more than one Indian Tribe, the approval of each such Indian Tribe shall be a prerequisite to the letting or making of such contract or grant. Applicant shall submit letters of support and/or Tribal Resolutions from the Tribes to be served.

An applicant may not apply to both this opportunity, TPI, and the CHAP Tribal Assessment and Planning (TAP) opportunity (number HHS-2021-IHS-TAP-0001). An organization currently carrying out a CHAP in the United States, in accordance with 25 U.S.C. 1616l through an Indian Self-Determination and Education Assistance Act (ISDEAA) agreement, is eligible to apply, but may not utilize the funds to carry out a CHAP. The Program Office will notify any applicants deemed ineligible. Note.

Please refer to Section IV.2 (Application and Submission Information/Subsection 2, Content and Form of Application Submission) for additional proof of applicant status documents required, such as Tribal Resolutions, proof of nonprofit status, etc. 2. Cost Sharing or Matching The IHS does not require matching funds or cost sharing for grants or cooperative agreements. 3. Other Requirements Applications with budget requests that exceed the highest dollar amount outlined under Section II Award Information, Estimated Funds Available, or exceed the Period of Performance outlined under Section II Award Information, Period of Performance, will Start Printed Page 41046be considered not responsive and will not be reviewed.

The Division of Grants Management (DGM) will notify the applicant. Additional Required Documentation Tribal Resolution The DGM must receive an official, signed Tribal Resolution prior to issuing a Notice of Award (NoA) to any applicant selected for funding. An Indian Tribe or Tribal organization that is proposing a project affecting another Indian Tribe must include resolutions from all affected Tribes to be served. However, if an official, signed Tribal Resolution cannot be submitted with the application prior to the application deadline date, a draft Tribal Resolution must be submitted with the application by the deadline date in order for the application to be considered complete and eligible for review. The draft Tribal Resolution is not in lieu of the required signed resolution but is acceptable until a signed resolution is received.

If an application without a signed Tribal Resolution is selected for funding, the applicant will be contacted by the Grants Management Specialist (GMS) listed in this funding announcement and given 90 days to submit an official, signed Tribal Resolution to the GMS. If the signed Tribal Resolution is not received within 90 days, the award will be forfeited. Tribes organized with a governing structure other than a Tribal council may submit an equivalent document commensurate with their governing organization. Proof of Nonprofit Status Organizations claiming nonprofit status must submit a current copy of the 501(c)(3) Certificate with the application. IV.

Application and Submission Information 1. Obtaining Application Materials The application package and detailed instructions for this announcement are hosted on https://www.Grants.gov. Please direct questions regarding the application process to Mr. Paul Gettys at (301) 443-2114 or (301) 443-5204. 2.

Content and Form Application Submission Mandatory documents for all applicants include. Abstract (one page) summarizing the project. Application forms. 1. SF-424, Application for Federal Assistance.

2. SF-424A, Budget Information—Non-Construction Programs. 3. SF-424B, Assurances—Non-Construction Programs. Project Narrative (not to exceed 15 pages).

See Section IV.2.A Project Narrative for instructions. 1. Background information on the organization. 2. Proposed scope of work, objectives, and activities that provide a description of what the applicant plans to accomplish.

Budget Justification and Narrative (not to exceed 5 pages). See Section IV.2.B Budget Narrative for instructions. One-page Timeframe Chart. Tribal Resolution(s). Letters of Support from organization's Board of Directors (if applicable).

501(c)(3) Certificate. Biographical sketches for all Key Personnel. Contractor/Consultant resumes or qualifications and scope of work. Disclosure of Lobbying Activities (SF-LLL), if applicant conducts reportable lobbying. Certification Regarding Lobbying (GG-Lobbying Form).

Copy of current Negotiated Indirect Cost rate (IDC) agreement (required in order to receive IDC). Organizational Chart (optional). Documentation of current Office of Management and Budget (OMB) Financial Audit (if applicable). Acceptable forms of documentation include. 1.

Email confirmation from Federal Audit Clearinghouse (FAC) that audits were submitted. Or 2. Face sheets from audit reports. Applicants can find these on the FAC website at https://harvester.census.gov/​facdissem/​Main.aspx. Public Policy Requirements All Federal public policies apply to IHS grants and cooperative agreements.

Pursuant to 45 CFR 80.3(d), an individual shall not be deemed subjected to discrimination by reason of their exclusion from benefits limited by Federal law to individuals eligible for benefits and services from the IHS. See https://www.hhs.gov/​grants/​grants/​grants-policies-regulations/​index.html. Requirements for Project and Budget Narratives A. Project Narrative This narrative should be a separate document that is no more than 15 pages and must. (1) Have consecutively numbered pages.

(2) use black font 12 points or larger. (3) be single-spaced. And (4) be formatted to fit standard letter paper (81/2 x 11 inches). Be sure to succinctly answer all questions listed under the evaluation criteria (refer to Section V.1, Evaluation Criteria) and place all responses and required information in the correct section noted below or they will not be considered or scored. If the narrative exceeds the page limit, the application will be considered not responsive and will not be reviewed.

The 15-page limit for the narrative does not include the work plan, standard forms, Tribal Resolutions, budget, budget justifications, narratives, and/or other items. There are three parts to the narrative. Part 1—Program Information. Part 2—Program Plan. And Part 3—Program Evaluation.

See below for additional details about what must be included in the narrative. The page limits below are for each narrative and budget submitted. Part 1. Program Information (Limit—4 pages) Section 1. Community Profile Describe the demographics of the community including, but not limited to, geography, languages, age, and socioeconomic status.

The community profile should include data specific to the community that would benefit from the implementation of CHAP. Section 2. Health &. Infrastructure Needs Describe the community's current health disparities related to primary, behavioral, and oral health care. The needs section should provide facts and evidence related to infrastructure barriers (e.g., recruitment, retention, and access to facilities).

Section 3. Organizational Capacity Describe the T/TO's current health program activities, how long it has been operating, and what programs or services are currently being provided. Describe in full the organization's infrastructure and its ability to assess the feasibility of implementing a CHAP and identifying significant barriers that could prohibit the implementation. Part 2. Program Plan (Limit—6 pages) Section 1.

Program Plan Describe in full the direction the T/TO plans to take in the CHAP TPI. The program plan should identify the plan to address Tribal infrastructure needs specific to. Clinical supervisor support and clinical operations. Enhanced scope of work to address community and region specific needs. Training infrastructure (including continuing education).Start Printed Page 41047 Technology infrastructure.

System integration. Support to prospective health aides that address social determinants of health. Section 2. Program Activities Describe in full how the applicant will develop a robust clinical support system for the clinical supervision of providers. The activities should also include how the applicant will correlate the community health needs to additional requirements to be included into the scope of work of health aides, a detailed plan of how to adjust the clinical operations to incorporate a CHAP, and the training plan to include continuing education for prospective health aides.

Describe the resources the applicant will provide for health aides once the CHAP is operating, including technology investments to aide in mobility of providers and auxiliary supports to address critical social determinants of health. The program plan activities should also include how the applicant plans to calculate the full implementation. Section 3. Staffing Plan Describe key staff tasked with carrying out the program activities in Section 2. Applicants are highly encouraged to partner with other key stakeholders within the T/TO's region for a robust understanding of the needs and implications of implementing a CHAP into their respective communities.

Section 4. Timeline Describe a timeline not to exceed two years for the completion of the program plan, activities, and evaluation plan. Provide a timeline chart depicting a realistic timeline that details all major activities, milestones, and applicable staffing plans. The timeline should include the projected progress report due at the midpoint of the project period. The timeline chart should not exceed one page.

Part 3. Program Evaluation (Limit—5 pages) Section 1. Evaluation Plan Please identify and describe significant program activities and achievements associated with the delivery of quality health services. Provide a plan to provide a comparison of the actual accomplishments to the goals established for the project period, or if applicable, provide justification for the lack of progress. The evaluation plan should address major categories related to (See Sample Logic Model in Related Documents in Grants.gov).

Clinical supervision support. Enhanced scope of practice. Training infrastructure (including continuing education). Technology needs. Integration best practices.

Auxiliary supports for prospective health aides working within the system. Calculating total implementation cost. B. Budget Narrative (Limit—5 pages) Provide a budget narrative that explains the amounts requested for each line item of the budget from the SF-424A (Budget Information for Non-Construction Programs). The budget narrative should specifically describe how each item will support the achievement of proposed objectives.

Be very careful about showing how each item in the “Other” category is justified. For subsequent budget years (see Multi-Year Project Requirements in Section V.1. Application Review Information, Evaluation Criteria), the narrative should highlight the changes from year 1 or clearly indicate that there are no substantive budget changes during the period of performance. Do NOT use the budget narrative to expand the project narrative. 3.

Submission Dates and Times Applications must be submitted through Grants.gov by 11:59 p.m. Eastern Time on the Application Deadline Date. Any application received after the application deadline will not be accepted for review. Grants.gov will notify the applicant via email if the application is rejected. If technical challenges arise and assistance is required with the application process, contact Grants.gov Customer Support (see contact information at https://www.Grants.gov).

If problems persist, contact Mr. Paul Gettys ([email protected]), Acting Director, DGM, by telephone at (301) 443-2114 or (301) 443-5204. Please be sure to contact Mr. Gettys at least ten days prior to the application deadline. Please do not contact the DGM until you have received a Grants.gov tracking number.

In the event you are not able to obtain a tracking number, call the DGM as soon as possible. The IHS will not acknowledge receipt of applications. 4. Intergovernmental Review Executive Order 12372 requiring intergovernmental review is not applicable to this program. 5.

Funding Restrictions Pre-award costs are allowable up to 90 days before the start date of the award provided the costs are otherwise allowable if awarded. Pre-award costs are incurred at the risk of the applicant. The available funds are inclusive of direct and indirect costs. Only one grant may be awarded per applicant. 6.

Electronic Submission Requirements All applications must be submitted via Grants.gov. Please use the https://www.Grants.gov website to submit an application. Find the application by selecting the “Search Grants” link on the homepage. Follow the instructions for submitting an application under the Package tab. No other method of application submission is acceptable.

If the applicant cannot submit an application through Grants.gov, a waiver must be requested. Prior approval must be requested and obtained from Mr. Paul Gettys, Acting Director, DGM. A written waiver request must be sent to [email protected] with a copy to [email protected]. The waiver request must be documented in writing (emails are acceptable) before submitting an application by some other method, and include clear justification for the need to deviate from the required application submission process.

Once the waiver request has been approved, the applicant will receive a confirmation of approval email containing submission instructions. A copy of the written approval must be included with the application that is submitted to the DGM. Applications that are submitted without a copy of the signed waiver from the Acting Director of the DGM will not be reviewed. The Grants Management Officer of the DGM will notify the applicant via email of this decision. Applications submitted under waiver must be received by the DGM no later than 5:00 p.m., Eastern Time, on the Application Deadline Date.

Late applications will not be accepted for processing. Applicants that do not register for both the System for Award Management (SAM) and Grants.gov and/or fail to request timely assistance with technical issues will not be considered for a waiver to submit an application via alternative method. Please be aware of the following. Please search for the application package in https://www.Grants.gov by entering the Assistance Listing (CFDA) number or the Funding Opportunity Number. Both numbers are located in the header of this announcement.

If you experience technical challenges while submitting your application, please contact Grants.gov Customer Support (see contact information at https://www.Grants.gov).Start Printed Page 41048 Upon contacting Grants.gov, obtain a tracking number as proof of contact. The tracking number is helpful if there are technical issues that cannot be resolved and a waiver from the agency must be obtained. Applicants are strongly encouraged not to wait until the deadline date to begin the application process through Grants.gov as the registration process for SAM and Grants.gov could take up to 20 working days. Please follow the instructions on Grants.gov to include additional documentation that may be requested by this funding announcement. Applicants must comply with any page limits described in this funding announcement.

After submitting the application, the applicant will receive an automatic acknowledgment from Grants.gov that contains a Grants.gov tracking number. The IHS will not notify the applicant that the application has been received. Dun and Bradstreet (D&B) Data Universal Numbering System (DUNS) Applicants and grantee organizations are required to obtain a DUNS number and maintain an active registration in the SAM database. The DUNS number is a unique 9-digit identification number provided by D&B that uniquely identifies each entity. The DUNS number is site specific.

Therefore, each distinct performance site may be assigned a DUNS number. Obtaining a DUNS number is easy, and there is no charge. To obtain a DUNS number, please access the request service through https://fedgov.dnb.com/​webform or call (866) 705-5711. The Federal Funding Accountability and Transparency Act of 2006, as amended (“Transparency Act”), requires all HHS recipients to report information on sub-awards. Accordingly, all IHS grantees must notify potential first-tier sub-recipients that no entity may receive a first-tier sub-award unless the entity has provided its DUNS number to the prime grantee organization.

This requirement ensures the use of a universal identifier to enhance the quality of information available to the public pursuant to the Transparency Act. System for Award Management (SAM) Organizations that are not registered with SAM must have a DUNS number first, then access the SAM online registration through the SAM home page at https://sam.gov (U.S. Organizations will also need to provide an Employer Identification Number from the Internal Revenue Service that may take an additional 2-5 weeks to become active). Please see SAM.gov for details on the registration process and timeline. Registration with the SAM is free of charge but can take several weeks to process.

Applicants may register online at https://sam.gov. Additional information on implementing the Transparency Act, including the specific requirements for DUNS and SAM, are available on the DGM Grants Management, Policy Topics web page. Https://www.ihs.gov/​dgm/​policytopics/​. V. Application Review Information Possible points assigned to each section are noted in parentheses.

The 15-page project narrative should include only the first year of activities. Information for multi-year projects should be included as a separate document. See “Multi-year Project Requirements” at the end of this section for more information. The narrative section should be written in a manner that is clear to outside reviewers unfamiliar with prior related activities of the applicant. It should be well organized, succinct, and contain all information necessary for reviewers to fully understand the project.

Points will be assigned to each evaluation criteria adding up to a total of 100 possible points. Points are assigned as follows. 1. Evaluation Criteria A. Introduction and Need for Assistance (10 points) Identify the proposed project and plans to fully implement a CHAP within their community.

The needs should clearly identify the existing health system and how the CHAP will be integrated to meet the health needs of the community in the fields of behavioral, oral, and primary health care. B. Project Objective(s), Work Plan, and Approach (30 points) The work plan should be comprised of two key parts. Program Information and Program Plan. Provide information related to three key sections.

Community profile. Health and infrastructure. And organizational capacity. The Program Information part should demonstrate a robust community profile that highlights the existing health system, demographic data of community members and user population, and a detailed description of the T/TO carrying out the proposed activity. An acceptable Program Plan expecting to receive full points should include details of the applicants plan to address the program objective.

The Program Plan should address, at a minimum, key activities related to clinical supervisor support, scope of work, technology infrastructure, training infrastructure, integration best practices, and auxiliary support to health aides that address social determinants. C. Program Evaluation (30 points) The program evaluation should be comprised of two key sections. Evaluation plan and outcome report. The evaluation plan should address major categories related to.

Clinical supervisor support. Enhanced scope of work. Technology infrastructure. Training infrastructure. Integration best practices.

Auxiliary support. And full implementation costs (See Sample Logic Model in Related Documents in Grants.gov). The evaluation plan should identify how the T/TO plans to fully integrate CHAP. The evaluation should include total implementation costs based on the implementation plan and program plan identified, including any significant implementation barriers. List measurable and attainable goals with explicit timelines that detail expectation of findings.

The Outcome Report should describe, in full, the findings of the program plan, evaluation, and determination on stage of readiness for implementation. The outcome report should organize the findings into at least five of the seven categories. 1. Clinical Supervisor Support. 2.

Scope of Work. 3. Technology Infrastructure. 4. Training Infrastructure.

5. Integration Planning. 6. Auxiliary Support. 7.

Implementation Cost. Applicants are encouraged to identify additional categories above the seven aforementioned and may choose to develop subcategories that best fit the program plan. D. Organizational Capabilities, Key Personnel, and Qualifications (10 points) Provide a detailed biographical sketch of each member of key personnel assigned to carry out the objectives of the program plan. The sketches should detail the qualifications and expertise of identified staff.

E. Categorical Budget and Budget Justification (20 points) Provide a detailed budget of each expenditure directly related to the identified program activities. Multi-Year Project Requirements Applications must include a brief project narrative and budget (one Start Printed Page 41049additional page per year) addressing the developmental plans for each additional year of the project. This attachment will not count as part of the project narrative or the budget narrative. Additional documents can be uploaded as Other Attachments in Grants.gov Work plan, logic model, and/or timeline for proposed objectives.

Position descriptions for key staff. Resumes of key staff that reflect current duties. Consultant or contractor proposed scope of work and letter of commitment (if applicable). Current Indirect Cost Rate Agreement. Organizational chart.

Map of area identifying project location(s). Additional documents to support narrative (i.e., data tables, key news articles, etc.). 2. Review and Selection Each application will be prescreened for eligibility and completeness, as outlined in the funding announcement. Applications that meet the eligibility criteria shall be reviewed for merit by the Objective Review Committee (ORC) based on evaluation criteria.

Incomplete applications and applications that are not responsive to the administrative thresholds (budget limit, project period limit) will not be referred to the ORC and will not be funded. The applicant will be notified of this determination. Applicants must address all program requirements and provide all required documentation. 3. Notifications of Disposition All applicants will receive an Executive Summary Statement from the IHS Office of Clinical and Preventive Services within 30 days of the conclusion of the ORC outlining the strengths and weaknesses of their application.

The summary statement will be sent to the Authorizing Official identified on the face page (SF-424) of the application. A. Award Notices for Funded Applications The NoA is the authorizing document for which funds are dispersed to the approved entities and reflects the amount of Federal funds awarded, the purpose of the grant, the terms and conditions of the award, the effective date of the award, and the budget/project period. Each entity approved for funding must have a user account in GrantSolutions in order to retrieve the NoA. Please see the Agency Contacts list in Section VII for the systems contact information.

B. Approved But Unfunded Applications Approved applications not funded due to lack of available funds will be held for one year. If funding becomes available during the course of the year, the application may be reconsidered. Note. Any correspondence other than the official NoA executed by an IHS grants management official announcing to the project director that an award has been made to their organization is not an authorization to implement their program on behalf of the IHS.

VI. Award Administration Information 1. Administrative Requirements Awards issued under this announcement are subject to, and are administered in accordance with, the following regulations and policies. A. The Criteria as Outlined in This Program Announcement B.

Administrative Regulations for Grants C. Grants Policy D. Cost Principles Uniform Administrative Requirements for HHS Awards, “Cost Principles,” at 45 CFR part 75, subpart E. E. Audit Requirements Uniform Administrative Requirements for HHS Awards, “Audit Requirements,” at 45 CFR part 75, subpart F.

F. As of August 13, 2020, 2 CFR 200 has been updated to include a prohibition on certain telecommunications and video surveillance services or equipment. This prohibition is described in 2 CFR 200.216. This will also be described in the terms and conditions of every IHS grant and cooperative agreement awarded on or after August 13, 2020. 2.

Indirect Costs This section applies to all recipients that request reimbursement of indirect costs (IDC) in their application budget. In accordance with HHS Grants Policy Statement, Part II-27, IHS requires applicants to obtain a current IDC rate agreement and submit it to the DGM prior to the DGM issuing an award. The rate agreement must be prepared in accordance with the applicable cost principles and guidance as provided by the cognizant agency or office. A current rate covers the applicable grant activities under the current award's budget period. If the current rate agreement is not on file with the DGM at the time of award, the IDC portion of the budget will be restricted.

The restrictions remain in place until the current rate agreement is provided to the DGM. Per 45 CFR 75.414(f) Indirect (F&A) costs, “any non-Federal entity [i.e., applicant] that has never received a negotiated indirect cost rate,. . . May elect to charge a de minimis rate of 10 percent of modified total direct costs (MTDC) which may be used indefinitely.

As described in Section 75.403, costs must be consistently charged as either indirect or direct costs, but may not be double charged or inconsistently charged as both. If chosen, this methodology once elected must be used consistently for all Federal awards until such time as the non-Federal entity chooses to negotiate for a rate, which the non-Federal entity may apply to do at any time.” Electing to charge a de minimis rate of 10 percent only applies to applicants that have never received an approved negotiated indirect cost rate from HHS or another cognizant federal agency. Applicants awaiting approval of their indirect cost proposal may request the 10 percent de minimis rate. When the applicant chooses this method, costs included in the indirect cost pool must not be charged as direct costs to the grant. Available funds are inclusive of direct and appropriate indirect costs.

Approved indirect funds are awarded as part of the award amount, and no additional funds will be provided. Generally, IDC rates for IHS grantees are negotiated with the Division of Cost Allocation (DCA) at https://rates.psc.gov/​ or the Department of the Interior (Interior Business Center) at Start Printed Page 41050 https://ibc.doi.gov/​ICS/​tribal. For questions regarding the indirect cost policy, please call the Grants Management Specialist listed under “Agency Contacts” or the main DGM office at (301) 443-5204. 3. Reporting Requirements The grantee must submit required reports consistent with the applicable deadlines.

Failure to submit required reports within the time allowed may result in suspension or termination of an active grant, withholding of additional awards for the project, or other enforcement actions such as withholding of payments or converting to the reimbursement method of payment. Continued failure to submit required reports may result in the imposition of special award provisions and/or the non-funding or non-award of other eligible projects or activities. This requirement applies whether the delinquency is attributable to the failure of the awardee organization or the individual responsible for preparation of the reports. Per DGM policy, all reports must be submitted electronically by attaching them as a “Grant Note” in GrantSolutions. Personnel responsible for submitting reports will be required to obtain a login and password for GrantSolutions.

Please see the Agency Contacts list in Section VII for the systems contact information. The reporting requirements for this program are noted below. A. Progress Reports Program progress reports are required semi-annually. The progress reports are due within 30 days after the budget period ends (specific dates will be listed in the NoA Terms and Conditions).

These reports must include a brief comparison of actual accomplishments to the goals established for the period, a summary of progress to date or, if applicable, provide sound justification for the lack of progress, and other pertinent information as required. A final report must be submitted within 90 days of expiration of the period of performance. B. Financial Reports Federal Cash Transaction Reports are due 30 days after the close of every calendar quarter to the Payment Management Services at https://pms.psc.gov. Failure to submit timely reports may result in adverse award actions blocking access to funds.

Federal Financial Reports are due 30 days after the end of each budget period, and a final report is due 90 days after the end of the Period of Performance. Grantees are responsible and accountable for accurate information being reported on all required reports. The Progress Reports, the Federal Cash Transaction Report, and the Federal Financial Report. C. Data Collection and Reporting At the conclusion of the program period, the outcome report should detail how the T/TO plans to completely integrate CHAP into their Tribal health system and list major barriers that could potentially impact full integration.

The Outcome Report should describe, in full, the findings of the program plan and evaluation, and plans for implementation. The outcome report should organize the findings of the key categories. 1. Clinical Supervisor Support. 2.

Scope of Practice. 3. Technology Infrastructure. 4. Training Plan.

5. System Integration. 6. Auxiliary Support to Address Social Determinants. Based on the findings and measurable outcomes of the categories, the applicant should explicitly identify the implementation plan and projected cost associated with full implementation.

D. Federal Sub-Award Reporting System (FSRS) This award may be subject to the Transparency Act sub-award and executive compensation reporting requirements of 2 CFR part 170. The Transparency Act requires the OMB to establish a single searchable database, accessible to the public, with information on financial assistance awards made by Federal agencies. The Transparency Act also includes a requirement for recipients of Federal grants to report information about first-tier sub-awards and executive compensation under Federal assistance awards. IHS has implemented a Term of Award into all IHS Standard Terms and Conditions, NoAs, and funding announcements regarding the FSRS reporting requirement.

This IHS Term of Award is applicable to all IHS grant and cooperative agreements issued on or after October 1, 2010, with a $25,000 sub-award obligation threshold met for any specific reporting period. For the full IHS award term implementing this requirement and additional award applicability information, visit the DGM Grants Management website at https://www.ihs.gov/​dgm/​policytopics/​. E. Compliance With Executive Order 13166 Implementation of Services Accessibility Provisions for All Grant Application Packages and Funding Opportunity Announcements Recipients of Federal financial assistance (FFA) from HHS must administer their programs in compliance with Federal civil rights laws that prohibit discrimination on the basis of race, color, national origin, disability, age, and, in some circumstances, religion, conscience, and sex. This includes ensuring programs are accessible to persons with limited English proficiency.

The HHS Office for Civil Rights provides guidance on complying with civil rights laws enforced by HHS. Please see https://www.hhs.gov/​civil-rights/​for-providers/​provider-obligations/​index.html and http://www.hhs.gov/​ocr/​civilrights/​understanding/​section1557/​index.html. Please contact the HHS Office for Civil Rights for more information about obligations and prohibitions under Federal civil rights laws at https://www.hhs.gov/​ocr/​about-us/​contact-us/​index.html or call 1-800-368-1019 or TDD 1-800-537-7697. F. Federal Awardee Performance and Integrity Information System (FAPIIS) The IHS is required to review and consider any information about the applicant that is in the Federal Awardee Performance and Integrity Information System (FAPIIS) at https://www.fapiis.gov before making any award in excess of the simplified acquisition threshold (currently $250,000) over the period of performance.

An applicant may review and comment on any information about itself that a Federal awarding agency previously entered. The IHS will consider any comments by the applicant, in addition to other information in FAPIIS, in making a judgment about the applicant's integrity, business ethics, and record of performance under Federal awards when completing the review of risk posed by applicants as described in 45 CFR 75.205. As required by 45 CFR part 75, appendix XII, of the Uniform Guidance, non-Federal entities (NFEs) are required to disclose in FAPIIS any information about criminal, civil, and administrative proceedings, and/or affirm that there is no new information to provide. This applies to NFEs that receive Federal awards (currently active grants, cooperative agreements, and procurement contracts) greater than $10,000,000 for any period of time during the period of performance of an award/project. Mandatory Disclosure Requirements As required by 2 CFR part 200 of the Uniform Guidance and the HHS implementing regulations at 45 CFR part 75, the IHS must require an NFE or an applicant for a Federal award to disclose, in a timely manner, in writing to the IHS or pass-through entity all violations of Federal criminal law involving fraud, bribery, or gratuity violations potentially affecting the Federal award.

Submission is required for all applicants and recipients, in writing, to the IHS and to the HHS Office of Inspector General, all information related to violations of Federal criminal law involving fraud, bribery, or gratuity violations potentially affecting the Federal award. 45 CFR 75.113. Disclosures must be sent in writing to. U.S. Department of Health and Human Services, Indian Health Service, Division of Grants Management, ATTN.

Paul Gettys, Acting Director, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857, (Include “Mandatory Grant Disclosures” in subject line), Office. (301) 443-5204, Fax. (301) 594-0899, Email. [email protected].

And U.S. Department of Health and Human Services, Office of Inspector General, ATTN. Mandatory Grant Disclosures, Intake Coordinator, 330 Independence Avenue SW, Cohen Building, Room 5527, Washington, DC 20201, URL. Https://oig.hhs.gov/​fraud/​report-fraud/​, (Include “Mandatory Grant Disclosures” in subject line), Fax. (202) 205-0604 (Include “Mandatory Grant Disclosures” in subject line) or, Email.

[email protected]. Failure to make required disclosures can result in any of the remedies described in 45 CFR 75.371 Remedies for noncompliance, including suspension or debarment (see 2 CFR parts 180 &. 376). VII. Agency Contacts 1.

Questions on the programmatic issues may be directed to. Minette C. Galindo, Public Health Advisor, Indian Health Service, Office of Clinical and Preventive Services, 5600 Fishers Lane, Mail Stop. 08N34A, Rockville, MD 20857, Phone. (301) 443-4644, Fax.

(301) 594-6213, Email. [email protected]. 2. Questions on grants management and fiscal matters may be directed to. Donald Gooding, Grants Management Specialist, Indian Health Service, Division of Grants Management, 5600 Fishers Lane, Mail Stop.

09E70, Rockville, MD 20857, Phone. (301) 443-2298, Email. [email protected]. 3. Questions on systems matters may be directed to.

Paul Gettys, Acting Director, Indian Health Service, Division of Grants Management, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857, Phone. (301) 443-2114. Or the DGM main line (301) 443-5204, email. [email protected].

VIII. Other Information The Public Health Service strongly encourages all grant, cooperative agreement, and contract recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of the facility) in which regular or routine education, library, day care, health care, or early childhood development services are provided to children. This is consistent with the HHS mission to protect and advance the physical and mental health of the American people. Start Signature Elizabeth A.

Fowler, Acting Director, Indian Health Service. End Signature End Preamble [FR Doc. 2021-16283 Filed 7-29-21. 8:45 am]BILLING CODE 4165-16-PStart Preamble Announcement Type. New.

Funding Announcement Number. HHS-2021-IHS-TAP-0001. Assistance Listing (Catalog of Federal Domestic Assistance or CFDA) Number. 93.382. Key Dates Application Deadline Date.

September 6, 2021. Earliest Anticipated Start Date. September 30, 2021. I. Funding Opportunity Description Statutory Authority The Indian Health Service (IHS) is accepting applications for grants for the Community Health Aide Program (CHAP) Tribal Assessment and Planning (TAP) program.

The CHAP is authorized under the Snyder Act, 25 U.S.C. 13. The Transfer Act, 42 U.S.C. 2001(a). And the Indian Health Care Improvement Act, 25 U.S.C.

1616l. This grant program is described in the Assistance Listings located at https://sam.gov/​content/​home (formerly known as Catalog of Federal Domestic Assistance) under 93.382. Background The national CHAP will provide a network of health aides trained to support licensed health professionals while providing direct health care, health promotion, and disease prevention services. These providers will work within a referral relationship under the supervision of licensed clinical providers that include clinics, service units, and hospitals. The CHAP aides will increase access to direct health services, including inpatient and outpatient visits.Start Printed Page 41052 The Alaska CHAP has become a model for efficient and high quality health care delivery in rural Alaska, providing approximately 300,000 patient encounters per year and responding to emergencies 24 hours a day, seven days a week.

Specialized providers in dental and behavioral health were later introduced to respond to the needs of patients and address the health disparities in oral health and mental health among American Indian and Alaska Natives. The national CHAP is a workforce model that includes three different provider types that act as extenders of their licensed clinical supervisor. The national CHAP currently includes a behavioral health aide, community health aide, and dental health aide. Each of the health aide categories operate in a tiered level practice system. The national CHAP model provides an opportunity for increased access to care through the extension of primary care, dental, and behavioral health clinicians.

In 2010, under the permanent reauthorization of the Indian Health Care Improvement Act (IHCIA), Congress provided the Secretary of the U.S. Department of Health and Human Services, acting through the IHS, the authority to expand the CHAP nationally. In 2016, the IHS initiated Tribal Consultation on expanding the CHAP to the contiguous 48 states. In 2018, the IHS formed the CHAP Tribal Advisory Group (TAG) and began developing the program. In 2020, the IHS announced the national CHAP policy, which formally created the national CHAP.

Purpose The purpose of the TAP program is to support the assessment and planning of Tribes and Tribal Organizations (T/TO) in determining the feasibility of implementing CHAP in their respective communities. The program is designed to support the regional flexibility required for T/TO to design a program unique to the needs of their individual communities across the country through the identification of feasibility factors. The focus of the program is to. 1. Assess whether the T/TO can integrate CHAP into the Tribal health system, including the health care workforce.

2. Identify systemic barriers that prohibit the complete integration of CHAP into an existing health care system. The barriers should be related to. Clinical infrastructure. Workforce barriers.

Certification of providers. Training of providers. Inclusion of culture in the services provided by a CHAP provider. 3. Plan partnerships across the T/TO geographic region to address the barriers, including reimbursement, training, education, clinical infrastructure, implementation cost, and determination of system integration.

II. Award Information Funding Instrument—Grant Estimated Funds Available The total funding identified for fiscal year (FY) 2021 is approximately $2,340,000. Individual award amounts for the first budget year are anticipated to be between $250,000 and $260,000. The funding available for competing and subsequent continuation awards issued under this announcement is subject to the availability of appropriations and budgetary priorities of the Agency. The IHS is under no obligation to make awards that are selected for funding under this announcement.

Anticipated Number of Awards Approximately nine awards will be issued under this program announcement. The IHS intends to award no more than one grant per IHS area. Period of Performance The period of performance is two years. III. Eligibility Information 1.

Eligibility To be eligible for this new FY 2021 funding opportunity, an applicant must be one of the following, as defined under 25 U.S.C. 1603. A federally recognized Indian Tribe as defined by 25 U.S.C. 1603(14). The term “Indian Tribe” means any Indian Tribe, band, nation, or other organized group or community, including any Alaska Native village or group, or regional or village corporation, as defined in or established pursuant to the Alaska Native Claims Settlement Act (85 Stat.

688) [43 U.S.C. 1601 et seq.], which is recognized as eligible for the special programs and services provided by the United States to Indians because of their status as Indians. A Tribal organization as defined by 25 U.S.C. 1603(26). The term “Tribal organization” has the meaning given the term in section 4 of the Indian Self-Determination and Education Assistance Act (25 U.S.C.

5304). €œTribal organization” means the recognized governing body of any Indian Tribe. Any legally established organization of Indians which is controlled, sanctioned, or chartered by such governing body or which is democratically elected by the adult members of the Indian community to be served by such organization and which includes the maximum participation of Indians in all phases of its activities. Provided that, in any case where a contract is let or grant made to an organization to perform services benefiting more than one Indian Tribe, the approval of each such Indian Tribe shall be a prerequisite to the letting or making of such contract or grant. Applicant shall submit letters of support and/or Tribal Resolutions from the Tribes to be served.

An applicant may not apply to both this opportunity, TAP, and the CHAP Tribal Planning and Implementation (TPI) opportunity (number HHS-2021-IHS-TPI-0001). An organization currently carrying out a CHAP in the United States, in accordance with 25 U.S.C. 1616l through an Indian Self-Determination and Education Assistance Act (ISDEAA) agreement, is also not eligible to apply. The Program office will notify any applicants deemed ineligible. Note.

Please refer to Section IV.2 (Application and Submission Information/Subsection 2, Content and Form of Application Submission) for additional proof of applicant status documents required, such as Tribal Resolutions, proof of nonprofit status, etc. 2. Cost Sharing or Matching The IHS does not require matching funds or cost sharing for grants or cooperative agreements. 3. Other Requirements Applications with budget requests that exceed the highest dollar amount outlined under Section II Award Information, Estimated Funds Available, or exceed the Period of Performance outlined under Section II Award Information, Period of Performance, will be considered not responsive and will not be reviewed.

The Division of Grants Management (DGM) will notify the applicant. Additional Required Documentation Tribal Resolution The DGM must receive an official, signed Tribal Resolution prior to issuing a Notice of Award (NoA) to any applicant selected for funding. An Indian Tribe or Tribal organization that is proposing a project affecting another Indian Tribe must include resolutions from all affected Tribes to be served. However, if an official, signed Tribal Resolution cannot be submitted with the application prior to the application Start Printed Page 41053deadline date, a draft Tribal Resolution must be submitted with the application by the deadline date in order for the application to be considered complete and eligible for review. The draft Tribal Resolution is not in lieu of the required signed resolution but is acceptable until a signed resolution is received.

If an application without a signed Tribal Resolution is selected for funding, the applicant will be contacted by the Grants Management Specialist (GMS) listed in this funding announcement and given 90 days to submit an official, signed Tribal Resolution to the GMS. If the signed Tribal Resolution is not received within 90 days, the award will be forfeited. Tribes organized with a governing structure other than a Tribal council may submit an equivalent document commensurate with their governing organization. Proof of Nonprofit Status Organizations claiming nonprofit status must submit a current copy of the 501(c)(3) Certificate with the application. IV.

Application and Submission Information 1. Obtaining Application Materials The application package and detailed instructions for this announcement are hosted on https://www.Grants.gov. Please direct questions regarding the application process to Mr. Paul Gettys at (301) 443-2114 or (301) 443-5204. 2.

Content and Form Application Submission Mandatory documents for all applicants include. Abstract (one page) summarizing the project. Application forms. 1. SF-424, Application for Federal Assistance.

2. SF-424A, Budget Information—Non-Construction Programs. 3. SF-424B, Assurances—Non-Construction Programs. Project Narrative (not to exceed 15 pages).

See Section IV.2.A Project Narrative for instructions. 1. Background information on the organization. 2. Proposed scope of work, objectives, and activities that provide a description of what the applicant plans to accomplish.

Budget Justification and Narrative (not to exceed 5 pages). See Section IV.2.B Budget Narrative for instructions. One-page Timeframe Chart. Tribal Resolution(s). Letters of Support from organization's Board of Directors (if applicable).

501(c)(3) Certificate. Biographical sketches for all Key Personnel. Contractor/Consultant resumes or qualifications and scope of work. Disclosure of Lobbying Activities (SF-LLL), if applicant conducts reportable lobbying. Certification Regarding Lobbying (GG-Lobbying Form).

Copy of current Negotiated Indirect Cost rate (IDC) agreement (required in order to receive IDC). Organizational Chart (optional). Documentation of current Office of Management and Budget (OMB) Financial Audit (if applicable). Acceptable forms of documentation include. 1.

Email confirmation from Federal Audit Clearinghouse (FAC) that audits were submitted. Or 2. Face sheets from audit reports. Applicants can find these on the FAC website at https://harvester.census.gov/​facdissem/​Main.aspx. Public Policy Requirements All Federal public policies apply to IHS grants and cooperative agreements.

Pursuant to 45 CFR 80.3(d), an individual shall not be deemed subjected to discrimination by reason of their exclusion from benefits limited by Federal law to individuals eligible for benefits and services from the IHS. See https://www.hhs.gov/​grants/​grants/​grants-policies-regulations/​index.html. Requirements for Project and Budget Narratives A. Project Narrative This narrative should be a separate document that is no more than 15 pages and must. (1) Have consecutively numbered pages.

(2) use black font 12 points or larger. (3) be single-spaced. And (4) be formatted to fit standard letter paper (81/2 x 11 inches). Be sure to succinctly answer all questions listed under the evaluation criteria (refer to Section V.1, Evaluation Criteria) and place all responses and required information in the correct section noted below or they will not be considered or scored. If the narrative exceeds the page limit, the application will be considered not responsive and not be reviewed.

The 15-page limit for the narrative does not include the work plan, standard forms, Tribal Resolutions, budget, budget justifications, narratives, and/or other items. There are three parts to the narrative. Part 1—Program Information. Part 2—Program Plan. And Part 3—Program Evaluation and Outcome Report.

See below for additional details about what must be included in the narrative. The page limits below are for each narrative and budget submitted. Part 1. Program Information (Limit—4 Pages) Section 1. Community Profile Describe the demographics of the community including, but not limited to, geography, languages, age, and socioeconomic status.

The community profile should include data specific to the community that would benefit from the implementation of CHAP. Section 2. Health &. Infrastructure Needs Describe the community's current health disparities related to primary, behavioral, and oral health care. Section 3.

Organizational Capacity Describe the T/TO's current health program activities, how long it has been operating, and what programs or services are currently being provided. Describe in full the organization's infrastructure and its ability to assess the barriers that could impact the integration of CHAP and identify significant barriers that could prohibit the implementation. Part 2. Program Plan (Limit—6 Pages) Section 1. Program Plan Describe in full the direction the T/TO plans to take in the CHAP TAP.

The program plan should first clearly identify the problems within the community related to behavioral, primary, and oral health. The program plan should then include the plan to assess the problem(s). This should include a timeline for the assessment. The program plan should identify a timeline to determine whether CHAP can address the barriers identified. Section 2.

Program Activities Describe in full the activities to identify problems creating barriers within the community related to behavioral, primary, and oral health. These activities should be categorized (at a minimum) within key factors related to clinical infrastructure, workforce barriers, training infrastructure, and cultural inclusion. Describe in full how the applicant plans to assess the problems identified. Finally, describe in detail the activities and associated timeline to determine whether CHAP is feasible and activities to quantify the cost associated with CHAP. The program activities should detail which partners will aid in Start Printed Page 41054identifying and assessing barriers related to clinical infrastructure, workforce barriers, training infrastructure, and cultural inclusion.

Section 3. Staffing Plan Describe key staff tasked with carrying out the program activities in Section 2. Applicants should account for potential stakeholder partnerships following the assessment of barriers in the staffing plan. Section 4. Timeline Describe a timeline not to exceed two years for the completion of the program plan, activities, and evaluation plan.

Provide a timeline chart depicting a realistic timeline that details all major activities, milestones, and applicable staffing plans. The timeline should include the projected progress report due at the midpoint of the project period. The timeline chart should not exceed one page. Part 3. Program Evaluation &.

Outcome Report (Limit—5 Pages) Section 1. Evaluation Plan The evaluation plan should identify and describe significant program activities and achievements associated with the assessment and planning of whether CHAP can address identified barriers within the existing Tribal health system. Provide a comparison of the actual accomplishments to the goals established for the project period, or if applicable, provide justification for the lack of progress. The evaluation plan should organize all identified problems that lead to barriers into major categories related to clinical infrastructure, workforce barriers, training infrastructure, and cultural inclusion specific to the scope of practice of prospective CHAP providers. The evaluation plan should detail how these barriers can be quantified.

The evaluation plan should detail how the applicant will measure the assessment of whether CHAP can address the issues identified including number of partnerships for each major category of barriers, other factors that may impact feasibility, and sustainability. Finally, the evaluation plan should detail how the applicant plans to calculate the total cost associated with integrating CHAP as part of the planning process. Section 2. Outcome Report At the conclusion of the program period, using the findings from the evaluation, the T/TO should determine the feasibility of implementing a CHAP within their own community. The Outcome Report should describe in full the findings of the program plan, evaluation, and determination on stage of readiness for implementation.

The outcome report should organize the findings into at least five categories. 1. Clinical Infrastructure. 2. Workforce Barriers.

3. Training Infrastructure. 4. Cultural Inclusion. 5.

Implementation Cost. Based on the findings and measurable outcomes of the categories, the applicant should explicitly identify whether CHAP is feasible for implementation into their respective community. Applicants should develop an organized report that highlights the categories succinctly and includes data (quantitative or qualitative) from the evaluation plan. The outcome report should explicitly detail the cost associated with integrating CHAP if it is found that CHAP can address the barriers identified in the assessment phase. B.

Budget Narrative (Limit—5 Pages) Provide a budget narrative that explains the amounts requested for each line item of the budget from the SF-424A (Budget Information for Non-Construction Programs). The budget narrative should specifically describe how each item will support the achievement of proposed objectives. Be very careful about showing how each item in the “Other” category is justified. For subsequent budget years (see Multi-Year Project Requirements in Section V.1. Application Review Information, Evaluation Criteria), the narrative should highlight the changes from year 1 or clearly indicate that there are no substantive budget changes during the period of performance.

Do NOT use the budget narrative to expand the project narrative. 3. Submission Dates and Times Applications must be submitted through Grants.gov by 11:59 p.m. Eastern Time on the Application Deadline Date. Any application received after the application deadline will not be accepted for review.

Grants.gov will notify the applicant via email if the application is rejected. If technical challenges arise and assistance is required with the application process, contact Grants.gov Customer Support (see contact information at https://www.Grants.gov). If problems persist, contact Mr. Paul Gettys ([email protected]), Acting Director, DGM, by telephone at (301) 443-2114 or (301) 443-5204. Please be sure to contact Mr.

Gettys at least ten days prior to the application deadline. Please do not contact the DGM until you have received a Grants.gov tracking number. In the event you are not able to obtain a tracking number, call the DGM as soon as possible. The IHS will not acknowledge receipt of applications. 4.

Intergovernmental Review Executive Order 12372 requiring intergovernmental review is not applicable to this program. 5. Funding Restrictions Pre-award costs are allowable up to 90 days before the start date of the award provided the costs are otherwise allowable if awarded. Pre-award costs are incurred at the risk of the applicant. The available funds are inclusive of direct and indirect costs.

Only one grant may be awarded per applicant. 6. Electronic Submission Requirements All applications must be submitted via Grants.gov. Please use the https://www.Grants.gov website to submit an application. Find the application by selecting the “Search Grants” link on the homepage.

Follow the instructions for submitting an application under the Package tab. No other method of application submission is acceptable. If the applicant cannot submit an application through Grants.gov, a waiver must be requested. Prior approval must be requested and obtained from Mr. Paul Gettys, Acting Director, DGM.

A written waiver request must be sent to [email protected] with a copy to [email protected]. The waiver request must. (1) Be documented in writing (emails are acceptable) before submitting an application by some other method, and (2) include clear justification for the need to deviate from the required application submission process. Once the waiver request has been approved, the applicant will receive a confirmation of approval email containing submission instructions. A copy of the written approval must be included with the application that is submitted to the DGM.

Applications that are submitted without a copy of the signed waiver from the Acting Director of the DGM will not be reviewed. The Grants Management Officer of the DGM will notify the applicant via email of this decision. Applications submitted under waiver must be received by the DGM no later than 5:00 p.m., Eastern Time, on the Application Deadline Date. Late applications will not be accepted for processing. Applicants that do not register for both the System for Award Management (SAM) and Grants.gov and/or fail to request timely assistance with technical issues will not be Start Printed Page 41055considered for a waiver to submit an application via alternative method.

Please be aware of the following. Please search for the application package in https://www.Grants.gov by entering the Assistance Listing (CFDA) number or the Funding Opportunity Number. Both numbers are located in the header of this announcement. If you experience technical challenges while submitting your application, please contact Grants.gov Customer Support (see contact information at https://www.Grants.gov). Upon contacting Grants.gov, obtain a tracking number as proof of contact.

The tracking number is helpful if there are technical issues that cannot be resolved and a waiver from the agency must be obtained. Applicants are strongly encouraged not to wait until the deadline date to begin the application process through Grants.gov as the registration process for SAM and Grants.gov could take up to 20 working days. Please follow the instructions on Grants.gov to include additional documentation that may be requested by this funding announcement. Applicants must comply with any page limits described in this funding announcement. After submitting the application, the applicant will receive an automatic acknowledgment from Grants.gov that contains a Grants.gov tracking number.

The IHS will not notify the applicant that the application has been received. Dun and Bradstreet (D&B) Data Universal Numbering System (DUNS) Applicants and grantee organizations are required to obtain a DUNS number and maintain an active registration in the SAM database. The DUNS number is a unique 9-digit identification number provided by D&B that uniquely identifies each entity. The DUNS number is site specific. Therefore, each distinct performance site may be assigned a DUNS number.

Obtaining a DUNS number is easy, and there is no charge. To obtain a DUNS number, please access the request service through https://fedgov.dnb.com/​webform or call (866) 705-5711. The Federal Funding Accountability and Transparency Act of 2006, as amended (“Transparency Act”), requires all HHS recipients to report information on sub-awards. Accordingly, all IHS grantees must notify potential first-tier sub-recipients that no entity may receive a first-tier sub-award unless the entity has provided its DUNS number to the prime grantee organization. This requirement ensures the use of a universal identifier to enhance the quality of information available to the public pursuant to the Transparency Act.

System for Award Management (SAM) Organizations that are not registered with SAM must have a DUNS number first, then access the SAM online registration through the SAM home page at https://sam.gov (U.S. Organizations will also need to provide an Employer Identification Number from the Internal Revenue Service that may take an additional 2-5 weeks to become active). Please see SAM.gov for details on the registration process and timeline. Registration with the SAM is free of charge but can take several weeks to process. Applicants may register online at https://sam.gov.

Additional information on implementing the Transparency Act, including the specific requirements for DUNS and SAM, are available on the DGM Grants Management, Policy Topics web page. Https://www.ihs.gov/​dgm/​policytopics/​. V. Application Review Information Possible points assigned to each section are noted in parentheses. The 15-page project narrative should include only the first year of activities.

Information for multi-year projects should be included as a separate document. See “Multi-year Project Requirements” at the end of this section for more information. The narrative section should be written in a manner that is clear to outside reviewers unfamiliar with prior related activities of the applicant. It should be well organized, succinct, and contain all information necessary for reviewers to fully understand the project. Attachments requested in the criteria do not count toward the 15-page limit for the project narrative.

Points will be assigned to each evaluation criteria adding up to a total of 100 possible points. Points are assigned as follows. 1. Evaluation Criteria A. Introduction and Need for Assistance (10 Points) Identify the proposed project and plans to identify the feasibility of implementing a CHAP within their community.

The needs should clearly identify the existing health system and how the CHAP may be a viable workforce model for the community needs. B. Project Objective(s), Work Plan, and Approach (30 Points) The work plan should be comprised of two key parts. Program Information and Program Plan. Acceptable Program Information should provide information related to three (3) key sections.

Community profile. Health and infrastructure. And organizational capacity. The Program Information part should demonstrate a robust community profile that highlights the existing health system, demographic data of community members and user population, and a detailed description of the T/TO carrying out the proposed activity. An acceptable Program Plan should include details of the applicant's plan to address the program objective.

The Program Plan should address, at a minimum, key activities related to clinical infrastructure, workforce barriers, and training infrastructure. C. Program Evaluation (30 Points) The program evaluation should address how the applicant intends to measure major categories related to clinical infrastructure. Workforce barriers. Training infrastructure.

Cultural inclusion (See Sample Logic Model in Related Documents in Grants.gov) specific to the scope of practice of prospective CHAP providers. And implementation costs. The evaluation plan should identify. how the applicant plans to determine the feasibility of CHAP integration into the Tribal system. Measurement of significant systematic barriers.

Implementation cost associated with CHAP. And planning for the scope of work. The applicant may choose to develop a readiness assessment to measure the feasibility. List measurable and attainable goals with explicit timelines that detail expectation of findings. D.

Organizational Capabilities, Key Personnel, and Qualifications (10 Points) Provide a detailed biographical sketch of each member of key personnel assigned to carry out the objectives of the program plan. The sketches should detail the qualifications and expertise of identified staff. E. Categorical Budget and Budget Justification (20 Points) Provide a detailed budget of each expenditure directly related to the identified program activities. Multi-Year Project Requirements Applications must include a brief project narrative and budget (one additional page per year) addressing the developmental plans for each additional year of the project.

This attachment will Start Printed Page 41056not count as part of the project narrative or the budget narrative. Additional documents can be uploaded as Other Attachments in Grants.gov. Work plan, logic model, and/or timeline for proposed objectives. Position descriptions for key staff. Resumes of key staff that reflect current duties.

Consultant or contractor proposed scope of work and letter of commitment (if applicable). Current Indirect Cost Rate Agreement. Organizational chart. Map of area identifying project location(s). Additional documents to support narrative (i.e., data tables, key news articles, etc.).

2. Review and Selection Each application will be prescreened for eligibility and completeness, as outlined in the funding announcement. Applications that meet the eligibility criteria shall be reviewed for merit by the Objective Review Committee (ORC) based on evaluation criteria. Incomplete applications and applications that are not responsive to the administrative thresholds (budget limit, project period limit) will not be referred to the ORC and will not be funded. The applicant will be notified of this determination.

Applicants must address all program requirements and provide all required documentation. 3. Notifications of Disposition All applicants will receive an Executive Summary Statement from the IHS Office of Clinical and Preventive Services within 30 days of the conclusion of the ORC outlining the strengths and weaknesses of their application. The summary statement will be sent to the Authorizing Official identified on the face page (SF-424) of the application. A.

Award Notices for Funded Applications The NoA is the authorizing document for which funds are dispersed to the approved entities and reflects the amount of Federal funds awarded, the purpose of the grant, the terms and conditions of the award, the effective date of the award, and the budget/project period. Each entity approved for funding must have a user account in GrantSolutions in order to retrieve the NoA. Please see the Agency Contacts list in Section VII for the systems contact information. B. Approved but Unfunded Applications Approved applications not funded due to lack of available funds will be held for one year.

If funding becomes available during the course of the year, the application may be reconsidered. Note. Any correspondence other than the official NoA executed by an IHS grants management official announcing to the project director that an award has been made to their organization is not an authorization to implement their program on behalf of the IHS. VI. Award Administration Information 1.

Administrative Requirements Awards issued under this announcement are subject to, and are administered in accordance with, the following regulations and policies. A. The Criteria as Outlined in This Program Announcement B. Administrative Regulations for Grants C. Grants Policy D.

Cost Principles Uniform Administrative Requirements for HHS Awards, “Cost Principles,” at 45 CFR part 75, subpart E. E. Audit Requirements Uniform Administrative Requirements for HHS Awards, “Audit Requirements,” at 45 CFR part 75, subpart F. F. As of August 13, 2020, 2 CFR 200 was updated to include a prohibition on certain telecommunications and video surveillance services or equipment.

This prohibition is described in 2 CFR 200.216. This will also be described in the terms and conditions of every IHS grant and cooperative agreement awarded on or after August 13, 2020. 2. Indirect Costs This section applies to all recipients that request reimbursement of indirect costs (IDC) in their application budget. In accordance with HHS Grants Policy Statement, Part II-27, IHS requires applicants to obtain a current IDC rate agreement and submit it to the DGM prior to the DGM issuing an award.

The rate agreement must be prepared in accordance with the applicable cost principles and guidance as provided by the cognizant agency or office. A current rate covers the applicable grant activities under the current award's budget period. If the current rate agreement is not on file with the DGM at the time of award, the IDC portion of the budget will be restricted. The restrictions remain in place until the current rate agreement is provided to the DGM. Per 45 CFR 75.414(f) Indirect (F&A) costs, “any non-Federal entity [i.e., applicant] that has never received a negotiated indirect cost rate,.

. . May elect to charge a de minimis rate of 10 percent of modified total direct costs (MTDC) which may be used indefinitely. As described in Section 75.403, costs must be consistently charged as either indirect or direct costs, but may not be double charged or inconsistently charged as both. If chosen, this methodology once elected must be used consistently for all Federal awards until such time as the non-Federal entity chooses to negotiate for a rate, which the non-Federal entity may apply to do at any time.” Electing to charge a de minimis rate of 10 percent only applies to applicants that have never received an approved negotiated indirect cost rate from HHS or another cognizant federal agency.

Applicants awaiting approval of their indirect cost proposal may request the 10 percent de minimis rate. When the applicant chooses this method, costs included in the indirect cost pool must not be charged as direct costs to the grant. Available funds are inclusive of direct and appropriate indirect costs. Approved indirect funds are awarded as part of the award amount, and no additional funds will be provided. Generally, IDC rates for IHS grantees are negotiated with the Division of Cost Allocation (DCA) at https://rates.psc.gov/​ or the Department of the Interior (Interior Business Center) at https://ibc.doi.gov/​ICS/​tribal.

For Start Printed Page 41057questions regarding the indirect cost policy, please call the Grants Management Specialist listed under “Agency Contacts” or the main DGM office at (301) 443-5204. 3. Reporting Requirements The grantee must submit required reports consistent with the applicable deadlines. Failure to submit required reports within the time allowed may result in suspension or termination of an active grant, withholding of additional awards for the project, or other enforcement actions such as withholding of payments or converting to the reimbursement method of payment. Continued failure to submit required reports may result in the imposition of special award provisions and/or the non-funding or non-award of other eligible projects or activities.

This requirement applies whether the delinquency is attributable to the failure of the awardee organization or the individual responsible for preparation of the reports. Per DGM policy, all reports must be submitted electronically by attaching them as a “Grant Note” in GrantSolutions. Personnel responsible for submitting reports will be required to obtain a login and password for GrantSolutions. Please see the Agency Contacts list in Section VII for the systems contact information. The reporting requirements for this program are noted below.

A. Progress Reports Program progress reports are required semi-annually. The progress reports are due within 30 days after the budget period ends (specific dates will be listed in the NoA Terms and Conditions). These reports must include a brief comparison of actual accomplishments to the goals established for the period, a summary of progress to date or, if applicable, provide sound justification for the lack of progress, and other pertinent information as required. A final report must be submitted within 90 days of expiration of the period of performance.

B. Financial Reports Federal Cash Transaction Reports are due 30 days after the close of every calendar quarter to the Payment Management Services at https://pms.psc.gov. Failure to submit timely reports may result in adverse award actions blocking access to funds. Federal Financial Reports are due 30 days after the end of each budget period, and a final report is due 90 days after the end of the Period of Performance. Grantees are responsible and accountable for accurate information being reported on all required reports.

The Progress Reports and Federal Financial Report. C. Data Collection and Reporting To satisfy the reporting requirements, the applicant is expected to develop an outcome report. The outcome report should explicitly state whether CHAP implementation and integration into the existing health care system is viable or not. The Outcome Report should describe, in full, the findings of the program plan, evaluation, and determination on stage of readiness for implementation.

The outcome report should organize the findings into at least five categories. 1. Clinical Infrastructure. 2. Workforce Barriers.

3. Training Infrastructure. 4. Cultural Inclusion. 5.

Implementation Cost. Applicants are encouraged to identify additional categories above the five aforementioned and may choose to develop subcategories that best fit the program plan. D. Federal Sub-Award Reporting System (FSRS) This award may be subject to the Transparency Act sub-award and executive compensation reporting requirements of 2 CFR part 170. The Transparency Act requires the OMB to establish a single searchable database, accessible to the public, with information on financial assistance awards made by Federal agencies.

The Transparency Act also includes a requirement for recipients of Federal grants to report information about first-tier sub-awards and executive compensation under Federal assistance awards. IHS has implemented a Term of Award into all IHS Standard Terms and Conditions, NoAs, and funding announcements regarding the FSRS reporting requirement. This IHS Term of Award is applicable to all IHS grant and cooperative agreements issued on or after October 1, 2010, with a $25,000 sub-award obligation threshold met for any specific reporting period. For the full IHS award term implementing this requirement and additional award applicability information, visit the DGM Grants Management website at https://www.ihs.gov/​dgm/​policytopics/​. E.

Compliance With Executive Order 13166 Implementation of Services Accessibility Provisions for All Grant Application Packages and Funding Opportunity Announcements Recipients of Federal financial assistance (FFA) from HHS must administer their programs in compliance with Federal civil rights laws that prohibit discrimination on the basis of race, color, national origin, disability, age, and, in some circumstances, religion, conscience, and sex. This includes ensuring programs are accessible to persons with limited English proficiency. The HHS Office for Civil Rights provides guidance on complying with civil rights laws enforced by HHS. Please see https://www.hhs.gov/​civil-rights/​for-providers/​provider-obligations/​index.html and http://www.hhs.gov/​ocr/​civilrights/​understanding/​section1557/​index.html. Please contact the HHS Office for Civil Rights for more information about obligations and prohibitions under Federal civil rights laws at https://www.hhs.gov/​ocr/​about-us/​contact-us/​index.html or call 1-800-368-1019 or TDD 1-800-537-7697.

F. Federal Awardee Performance and Integrity Information System (FAPIIS) The IHS is required to review and consider any information about the applicant that is in the Federal Awardee Performance and Integrity Information System (FAPIIS), at https://www.fapiis.gov, before making any award in excess of the simplified acquisition threshold (currently $250,000) over the period of performance. An applicant may review and comment on any information about itself that a Federal awarding agency previously entered. IHS will consider any comments by the applicant, in addition to other information in FAPIIS, in making a judgment about the applicant's integrity, business ethics, and record of performance under Federal awards when completing the review of risk posed by applicants as described in 45 CFR 75.205. As required by 45 CFR part 75, appendix XII, of the Uniform Guidance, non-Federal entities (NFEs) are required to disclose in FAPIIS any information about criminal, civil, and administrative proceedings, and/or affirm that there is no new information to provide.

This applies to NFEs that receive Federal awards (currently active grants, cooperative agreements, and procurement contracts) greater than $10,000,000 for any period of time during the period of performance of an award/project. Mandatory Disclosure Requirements As required by 2 CFR part 200 of the Uniform Guidance and the HHS implementing regulations at 45 CFR part 75, the IHS must require an NFE or an applicant for a Federal award to disclose, in a timely manner, in writing to the IHS or pass-through entity all violations of Federal criminal law involving fraud, bribery, or gratuity violations potentially affecting the Federal award. Submission is required for all applicants and recipients, in writing, to the IHS and to the HHS Office of Inspector General of all information related to violations of Federal criminal law involving fraud, bribery, or gratuity violations potentially affecting the Federal award. 45 CFR 75.113. Disclosures must be sent in writing to.

U.S. Department of Health and Human Services, Indian Health Service, Division of Grants Management, ATTN. Paul Gettys, Acting Director, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857 (Include “Mandatory Grant Disclosures” in subject line), Office. (301) 443-5204, Fax.

(301) 594-0899, Email. [email protected]. And U.S. Department of Health and Human Services, Office of Inspector General, ATTN. Mandatory Grant Disclosures, Intake Coordinator, 330 Independence Avenue SW, Cohen Building, Room 5527, Washington, DC 20201, URL.

Https://oig.hhs.gov/​fraud/​report-fraud/​ (Include “Mandatory Grant Disclosures” in subject line), Fax. (202) 205-0604 (Include “Mandatory Grant Disclosures” in subject line) or Email. [email protected]. Failure to make required disclosures can result in any of the remedies described in 45 CFR 75.371 Remedies for noncompliance, including suspension or debarment (see 2 CFR parts 180 &. 376).

VII. Agency Contacts 1. Questions on the programmatic issues may be directed to. Minette C. Galindo, Public Health Advisor, Indian Health Service, Office of Clinical and Preventive Services, 5600 Fishers Lane, Mail Stop.

08N34A, Rockville, MD 20857, Phone. (301) 443-4644, Email. [email protected]. 2. Questions on grants management and fiscal matters may be directed to.

Donald Gooding, Grants Management Specialist, Indian Health Service, Division of Grants Management, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857, Phone. (301) 443-2298, Email. [email protected]. 3.

Questions on systems matters may be directed to. Paul Gettys, Acting Director, Indian Health Service, Division of Grants Management, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857, Phone. (301) 443-2114. Or the DGM main line (301) 443-5204, Email.

[email protected]. VIII. Other Information The Public Health Service strongly encourages all grant, cooperative agreement and contract recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of the facility) in which regular or routine education, library, day care, health care, or early childhood development services are provided to children. This is consistent with the HHS mission to protect and advance the physical and mental health of the American people.

Start Signature Elizabeth A. Fowler, Acting Director, Indian Health Service. End Signature End Preamble [FR Doc. 2021-16280 Filed 7-29-21. 8:45 am]BILLING CODE 4165-16-P.

Start Preamble viagra for sale online Announcement Type. New. Funding Announcement viagra for sale online Number. HHS-2021-IHS-TPI-0001. Assistance Listing (Catalog of Federal Domestic Assistance or CFDA) Number.

93.382. Key Dates Application Deadline Date. September 1, 2021. Earliest Anticipated Start Date. September 30, 2021.

I. Funding Opportunity Description Statutory Authority The Indian Health Service (IHS) is accepting applications for grants for the Community Health Aide Program (CHAP) Tribal Planning and Implementation (TPI) program. The CHAP is authorized under the Snyder Act, 25 U.S.C. 13. The Transfer Act, 42 U.S.C.

2001(a). And the Indian Health Care Improvement Act, 25 U.S.C. 16161. This grant program is described in the Assistance Listings located at https://beta.sam.gov (formerly known as Catalog of Federal Domestic Assistance) under 93.382. Background The national CHAP will provide a network of health aides trained to support licensed health professionals while providing direct health care, Start Printed Page 41045health promotion, and disease prevention services.

These providers will work within a referral relationship under the supervision of licensed clinical providers that includes clinics, service units, and hospitals. The program will increase access to direct health services, including inpatient and outpatient visits. The Alaska CHAP has become a model for efficient and high quality health care delivery in rural Alaska, providing approximately 300,000 patient encounters per year and responding to emergencies 24 hours a day, seven days a week. Specialized providers in dental and behavioral health were later introduced to respond to the needs of patients and address the health disparities in oral health and mental health among American Indians and Alaska Natives. The national CHAP is a workforce model that includes three different provider types that act as extenders of their licensed clinical supervisor.

The national CHAP currently includes a behavioral health aide, community health aide, and dental health aide. Each of the health aide categories operate in a tiered level practice system. The national CHAP model provides an opportunity for increased access to care through the extension of primary care, dental, and behavioral health clinicians. In 2010, under the permanent reauthorization of the Indian Health Care Improvement Act (IHCIA), Congress provided the Secretary of the U.S. Department of Health and Human Services, acting through the IHS, the authority to expand the Alaska CHAP program.

In 2016, the IHS initiated Tribal Consultation on expanding the CHAP to the contiguous 48 states. In 2018, the IHS formed the CHAP Tribal Advisory Group (TAG) and began developing the program. In 2020, the IHS announced the national CHAP policy, which formally created the national CHAP. Purpose The purpose of the TPI program is to support the planning and implementation for Tribes and Tribal Organizations (T/TO) positioned to begin operating a CHAP or support a growing CHAP in the contiguous 48 states. The grant program is designed to support the regional flexibility required for T/TO to implement a CHAP unique to the needs of their individual communities across the country through the identification of feasibility factors.

The focus of the program is to. 1. Develop clinical supervisor support for primary care, behavioral health, and dental health clinicians providing both direct and indirect supervision of prospective health aides. 2. Identify area and community-specific health care needs of patients that can be addressed by the health aides.

3. Identify and develop a technology infrastructure plan for the mobility and success of health aides in anticipation of providing services. 4. Develop a training plan to include partners across the T/TO's geographic region to enhance the training opportunities available to prospective health aides to include continuing education and clinical practice. 5.

Identify best practices for integrating a CHAP workforce into an existing Tribal health system. 6. Address social determinants of health that impact the recruitment and retention of prospective health aides. And 7. Identify the total cost of full implementation of a CHAP within an existing Tribal health system.

II. Award Information Funding Instrument—Grant Estimated Funds Available The total funding identified for fiscal year (FY) 2021 is approximately $1,500,000. Individual award amounts are anticipated to be between $450,000 and $500,000. The funding available for competing awards issued under this announcement is subject to the availability of appropriations and budgetary priorities of the Agency. The IHS is under no obligation to make awards that are selected for funding under this announcement.

Anticipated Number of Awards Approximately three awards will be issued under this program announcement. The IHS intends to award no more than one grant per IHS area. Period of Performance The period of performance is two years. III. Eligibility Information 1.

Eligibility To be eligible for this new FY 2021 funding opportunity, an applicant must be one of the following, as defined under 25 U.S.C. 1603. A federally recognized Indian Tribe as defined by 25 U.S.C. 1603(14). The term “Indian Tribe” means any Indian Tribe, band, nation, or other organized group or community, including any Alaska Native village or group, or regional or village corporation, as defined in or established pursuant to the Alaska Native Claims Settlement Act (85 Stat.

688) [43 U.S.C. 1601 et seq.], which is recognized as eligible for the special programs and services provided by the United States to Indians because of their status as Indians. A Tribal organization as defined by 25 U.S.C. 1603(26). The term “Tribal organization” has the meaning given the term in section 4 of the Indian Self-Determination and Education Assistance Act (25 U.S.C.

5304). €œTribal organization” means the recognized governing body of any Indian Tribe. Any legally established organization of Indians which is controlled, sanctioned, or chartered by such governing body or which is democratically elected by the adult members of the Indian community to be served by such organization and which includes the maximum participation of Indians in all phases of its activities. Provided that, in any case where a contract is let or grant made to an organization to perform services benefiting more than one Indian Tribe, the approval of each such Indian Tribe shall be a prerequisite to the letting or making of such contract or grant. Applicant shall submit letters of support and/or Tribal Resolutions from the Tribes to be served.

An applicant may not apply to both this opportunity, TPI, and the CHAP Tribal Assessment and Planning (TAP) opportunity (number HHS-2021-IHS-TAP-0001). An organization currently carrying out a CHAP in the United States, in accordance with 25 U.S.C. 1616l through an Indian Self-Determination and Education Assistance Act (ISDEAA) agreement, is eligible to apply, but may not utilize the funds to carry out a CHAP. The Program Office will notify any applicants deemed ineligible. Note.

Please refer to Section IV.2 (Application and Submission Information/Subsection 2, Content and Form of Application Submission) for additional proof of applicant status documents required, such as Tribal Resolutions, proof of nonprofit status, etc. 2. Cost Sharing or Matching The IHS does not require matching funds or cost sharing for grants or cooperative agreements. 3. Other Requirements Applications with budget requests that exceed the highest dollar amount outlined under Section II Award Information, Estimated Funds Available, or exceed the Period of Performance outlined under Section II Award Information, Period of Performance, will Start Printed Page 41046be considered not responsive and will not be reviewed.

The Division of Grants Management (DGM) will notify the applicant. Additional Required Documentation Tribal Resolution The DGM must receive an official, signed Tribal Resolution prior to issuing a Notice of Award (NoA) to any applicant selected for funding. An Indian Tribe or Tribal organization that is proposing a project affecting another Indian Tribe must include resolutions from all affected Tribes to be served. However, if an official, signed Tribal Resolution cannot be submitted with the application prior to the application deadline date, a draft Tribal Resolution must be submitted with the application by the deadline date in order for the application to be considered complete and eligible for review. The draft Tribal Resolution is not in lieu of the required signed resolution but is acceptable until a signed resolution is received.

If an application without a signed Tribal Resolution is selected for funding, the applicant will be contacted by the Grants Management Specialist (GMS) listed in this funding announcement and given 90 days to submit an official, signed Tribal Resolution to the GMS. If the signed Tribal Resolution is not received within 90 days, the award will be forfeited. Tribes organized with a governing structure other than a Tribal council may submit an equivalent document commensurate with their governing organization. Proof of Nonprofit Status Organizations claiming nonprofit status must submit a current copy of the 501(c)(3) Certificate with the application. IV.

Application and Submission Information 1. Obtaining Application Materials The application package and detailed instructions for this announcement are hosted on https://www.Grants.gov. Please direct questions regarding the application process to Mr. Paul Gettys at (301) 443-2114 or (301) 443-5204. 2.

Content and Form Application Submission Mandatory documents for all applicants include. Abstract (one page) summarizing the project. Application forms. 1. SF-424, Application for Federal Assistance.

2. SF-424A, Budget Information—Non-Construction Programs. 3. SF-424B, Assurances—Non-Construction Programs. Project Narrative (not to exceed 15 pages).

See Section IV.2.A Project Narrative for instructions. 1. Background information on the organization. 2. Proposed scope of work, objectives, and activities that provide a description of what the applicant plans to accomplish.

Budget Justification and Narrative (not to exceed 5 pages). See Section IV.2.B Budget Narrative for instructions. One-page Timeframe Chart. Tribal Resolution(s). Letters of Support from organization's Board of Directors (if applicable).

501(c)(3) Certificate. Biographical sketches for all Key Personnel. Contractor/Consultant resumes or qualifications and scope of work. Disclosure of Lobbying Activities (SF-LLL), if applicant conducts reportable lobbying. Certification Regarding Lobbying (GG-Lobbying Form).

Copy of current Negotiated Indirect Cost rate (IDC) agreement (required in order to receive IDC). Organizational Chart (optional). Documentation of current Office of Management and Budget (OMB) Financial Audit (if applicable). Acceptable forms of documentation include. 1.

Email confirmation from Federal Audit Clearinghouse (FAC) that audits were submitted. Or 2. Face sheets from audit reports. Applicants can find these on the FAC website at https://harvester.census.gov/​facdissem/​Main.aspx. Public Policy Requirements All Federal public policies apply to IHS grants and cooperative agreements.

Pursuant to 45 CFR 80.3(d), an individual shall not be deemed subjected to discrimination by reason of their exclusion from benefits limited by Federal law to individuals eligible for benefits and services from the IHS. See https://www.hhs.gov/​grants/​grants/​grants-policies-regulations/​index.html. Requirements for Project and Budget Narratives A. Project Narrative This narrative should be a separate document that is no more than 15 pages and must. (1) Have consecutively numbered pages.

(2) use black font 12 points or larger. (3) be single-spaced. And (4) be formatted to fit standard letter paper (81/2 x 11 inches). Be sure to succinctly answer all questions listed under the evaluation criteria (refer to Section V.1, Evaluation Criteria) and place all responses and required information in the correct section noted below or they will not be considered or scored. If the narrative exceeds the page limit, the application will be considered not responsive and will not be reviewed.

The 15-page limit for the narrative does not include the work plan, standard forms, Tribal Resolutions, budget, budget justifications, narratives, and/or other items. There are three parts to the narrative. Part 1—Program Information. Part 2—Program Plan. And Part 3—Program Evaluation.

See below for additional details about what must be included in the narrative. The page limits below are for each narrative and budget submitted. Part 1. Program Information (Limit—4 pages) Section 1. Community Profile Describe the demographics of the community including, but not limited to, geography, languages, age, and socioeconomic status.

The community profile should include data specific to the community that would benefit from the implementation of CHAP. Section 2. Health &. Infrastructure Needs Describe the community's current health disparities related to primary, behavioral, and oral health care. The needs section should provide facts and evidence related to infrastructure barriers (e.g., recruitment, retention, and access to facilities).

Section 3. Organizational Capacity Describe the T/TO's current health program activities, how long it has been operating, and what programs or services are currently being provided. Describe in full the organization's infrastructure and its ability to assess the feasibility of implementing a CHAP and identifying significant barriers that could prohibit the implementation. Part 2. Program Plan (Limit—6 pages) Section 1.

Program Plan Describe in full the direction the T/TO plans to take in the CHAP TPI. The program plan should identify the plan to address Tribal infrastructure needs specific to. Clinical supervisor support and clinical operations. Enhanced scope of work to address community and region specific needs. Training infrastructure (including continuing education).Start Printed Page 41047 Technology infrastructure.

System integration. Support to prospective health aides that address social determinants of health. Section 2. Program Activities Describe in full how the applicant will develop a robust clinical support system for the clinical supervision of providers. The activities should also include how the applicant will correlate the community health needs to additional requirements to be included into the scope of work of health aides, a detailed plan of how to adjust the clinical operations to incorporate a CHAP, and the training plan to include continuing education for prospective health aides.

Describe the resources the applicant will provide for health aides once the CHAP is operating, including technology investments to aide in mobility of providers and auxiliary supports to address critical social determinants of health. The program plan activities should also include how the applicant plans to calculate the full implementation. Section 3. Staffing Plan Describe key staff tasked with carrying out the program activities in Section 2. Applicants are highly encouraged to partner with other key stakeholders within the T/TO's region for a robust understanding of the needs and implications of implementing a CHAP into their respective communities.

Section 4. Timeline Describe a timeline not to exceed two years for the completion of the program plan, activities, and evaluation plan. Provide a timeline chart depicting a realistic timeline that details all major activities, milestones, and applicable staffing plans. The timeline should include the projected progress report due at the midpoint of the project period. The timeline chart should not exceed one page.

Part 3. Program Evaluation (Limit—5 pages) Section 1. Evaluation Plan Please identify and describe significant program activities and achievements associated with the delivery of quality health services. Provide a plan to provide a comparison of the actual accomplishments to the goals established for the project period, or if applicable, provide justification for the lack of progress. The evaluation plan should address major categories related to (See Sample Logic Model in Related Documents in Grants.gov).

Clinical supervision support. Enhanced scope of practice. Training infrastructure (including continuing education). Technology needs. Integration best practices.

Auxiliary supports for prospective health aides working within the system. Calculating total implementation cost. B. Budget Narrative (Limit—5 pages) Provide a budget narrative that explains the amounts requested for each line item of the budget from the SF-424A (Budget Information for Non-Construction Programs). The budget narrative should specifically describe how each item will support the achievement of proposed objectives.

Be very careful about showing how each item in the “Other” category is justified. For subsequent budget years (see Multi-Year Project Requirements in Section V.1. Application Review Information, Evaluation Criteria), the narrative should highlight the changes from year 1 or clearly indicate that there are no substantive budget changes during the period of performance. Do NOT use the budget narrative to expand the project narrative. 3.

Submission Dates and Times Applications must be submitted through Grants.gov by 11:59 p.m. Eastern Time on the Application Deadline Date. Any application received after the application deadline will not be accepted for review. Grants.gov will notify the applicant via email if the application is rejected. If technical challenges arise and assistance is required with the application process, contact Grants.gov Customer Support (see contact information at https://www.Grants.gov).

If problems persist, contact Mr. Paul Gettys ([email protected]), Acting Director, DGM, by telephone at (301) 443-2114 or (301) 443-5204. Please be sure to contact Mr. Gettys at least ten days prior to the application deadline. Please do not contact the DGM until you have received a Grants.gov tracking number.

In the event you are not able to obtain a tracking number, call the DGM as soon as possible. The IHS will not acknowledge receipt of applications. 4. Intergovernmental Review Executive Order 12372 requiring intergovernmental review is not applicable to this program. 5.

Funding Restrictions Pre-award costs are allowable up to 90 days before the start date of the award provided the costs are otherwise allowable if awarded. Pre-award costs are incurred at the risk of the applicant. The available funds are inclusive of direct and indirect costs. Only one grant may be awarded per applicant. 6.

Electronic Submission Requirements All applications must be submitted via Grants.gov. Please use the https://www.Grants.gov website to submit an application. Find the application by selecting the “Search Grants” link on the homepage. Follow the instructions for submitting an application under the Package tab. No other method of application submission is acceptable.

If the applicant cannot submit an application through Grants.gov, a waiver must be requested. Prior approval must be requested and obtained from Mr. Paul Gettys, Acting Director, DGM. A written waiver request must be sent to [email protected] with a copy to [email protected]. The waiver request must be documented in writing (emails are acceptable) before submitting an application by some other method, and include clear justification for the need to deviate from the required application submission process.

Once the waiver request has been approved, the applicant will receive a confirmation of approval email containing submission instructions. A copy of the written approval must be included with the application that is submitted to the DGM. Applications that are submitted without a copy of the signed waiver from the Acting Director of the DGM will not be reviewed. The Grants Management Officer of the DGM will notify the applicant via email of this decision. Applications submitted under waiver must be received by the DGM no later than 5:00 p.m., Eastern Time, on the Application Deadline Date.

Late applications will not be accepted for processing. Applicants that do not register for both the System for Award Management (SAM) and Grants.gov and/or fail to request timely assistance with technical issues will not be considered for a waiver to submit an application via alternative method. Please be aware of the following. Please search for the application package in https://www.Grants.gov by entering the Assistance Listing (CFDA) number or the Funding Opportunity Number. Both numbers are located in the header of this announcement.

If you experience technical challenges while submitting your application, please contact Grants.gov Customer Support (see contact information at https://www.Grants.gov).Start Printed Page 41048 Upon contacting Grants.gov, obtain a tracking number as proof of contact. The tracking number is helpful if there are technical issues that cannot be resolved and a waiver from the agency must be obtained. Applicants are strongly encouraged not to wait until the deadline date to begin the application process through Grants.gov as the registration process for SAM and Grants.gov could take up to 20 working days. Please follow the instructions on Grants.gov to include additional documentation that may be requested by this funding announcement. Applicants must comply with any page limits described in this funding announcement.

After submitting the application, the applicant will receive an automatic acknowledgment from Grants.gov that contains a Grants.gov tracking number. The IHS will not notify the applicant that the application has been received. Dun and Bradstreet (D&B) Data Universal Numbering System (DUNS) Applicants and grantee organizations are required to obtain a DUNS number and maintain an active registration in the SAM database. The DUNS number is a unique 9-digit identification number provided by D&B that uniquely identifies each entity. The DUNS number is site specific.

Therefore, each distinct performance site may be assigned a DUNS number. Obtaining a DUNS number is easy, and there is no charge. To obtain a DUNS number, please access the request service through https://fedgov.dnb.com/​webform or call (866) 705-5711. The Federal Funding Accountability and Transparency Act of 2006, as amended (“Transparency Act”), requires all HHS recipients to report information on sub-awards. Accordingly, all IHS grantees must notify potential first-tier sub-recipients that no entity may receive a first-tier sub-award unless the entity has provided its DUNS number to the prime grantee organization.

This requirement ensures the use of a universal identifier to enhance the quality of information available to the public pursuant to the Transparency Act. System for Award Management (SAM) Organizations that are not registered with SAM must have a DUNS number first, then access the SAM online registration through the SAM home page at https://sam.gov (U.S. Organizations will also need to provide an Employer Identification Number from the Internal Revenue Service that may take an additional 2-5 weeks to become active). Please see SAM.gov for details on the registration process and timeline. Registration with the SAM is free of charge but can take several weeks to process.

Applicants may register online at https://sam.gov. Additional information on implementing the Transparency Act, including the specific requirements for DUNS and SAM, are available on the DGM Grants Management, Policy Topics web page. Https://www.ihs.gov/​dgm/​policytopics/​. V. Application Review Information Possible points assigned to each section are noted in parentheses.

The 15-page project narrative should include only the first year of activities. Information for multi-year projects should be included as a separate document. See “Multi-year Project Requirements” at the end of this section for more information. The narrative section should be written in a manner that is clear to outside reviewers unfamiliar with prior related activities of the applicant. It should be well organized, succinct, and contain all information necessary for reviewers to fully understand the project.

Points will be assigned to each evaluation criteria adding up to a total of 100 possible points. Points are assigned as follows. 1. Evaluation Criteria A. Introduction and Need for Assistance (10 points) Identify the proposed project and plans to fully implement a CHAP within their community.

The needs should clearly identify the existing health system and how the CHAP will be integrated to meet the health needs of the community in the fields of behavioral, oral, and primary health care. B. Project Objective(s), Work Plan, and Approach (30 points) The work plan should be comprised of two key parts. Program Information and Program Plan. Provide information related to three key sections.

Community profile. Health and infrastructure. And organizational capacity. The Program Information part should demonstrate a robust community profile that highlights the existing health system, demographic data of community members and user population, and a detailed description of the T/TO carrying out the proposed activity. An acceptable Program Plan expecting to receive full points should include details of the applicants plan to address the program objective.

The Program Plan should address, at a minimum, key activities related to clinical supervisor support, scope of work, technology infrastructure, training infrastructure, integration best practices, and auxiliary support to health aides that address social determinants. C. Program Evaluation (30 points) The program evaluation should be comprised of two key sections. Evaluation plan and outcome report. The evaluation plan should address major categories related to.

Clinical supervisor support. Enhanced scope of work. Technology infrastructure. Training infrastructure. Integration best practices.

Auxiliary support. And full implementation costs (See Sample Logic Model in Related Documents in Grants.gov). The evaluation plan should identify how the T/TO plans to fully integrate CHAP. The evaluation should include total implementation costs based on the implementation plan and program plan identified, including any significant implementation barriers. List measurable and attainable goals with explicit timelines that detail expectation of findings.

The Outcome Report should describe, in full, the findings of the program plan, evaluation, and determination on stage of readiness for implementation. The outcome report should organize the findings into at least five of the seven categories. 1. Clinical Supervisor Support. 2.

Scope of Work. 3. Technology Infrastructure. 4. Training Infrastructure.

5. Integration Planning. 6. Auxiliary Support. 7.

Implementation Cost. Applicants are encouraged to identify additional categories above the seven aforementioned and may choose to develop subcategories that best fit the program plan. D. Organizational Capabilities, Key Personnel, and Qualifications (10 points) Provide a detailed biographical sketch of each member of key personnel assigned to carry out the objectives of the program plan. The sketches should detail the qualifications and expertise of identified staff.

E. Categorical Budget and Budget Justification (20 points) Provide a detailed budget of each expenditure directly related to the identified program activities. Multi-Year Project Requirements Applications must include a brief project narrative and budget (one Start Printed Page 41049additional page per year) addressing the developmental plans for each additional year of the project. This attachment will not count as part of the project narrative or the budget narrative. Additional documents can be uploaded as Other Attachments in Grants.gov Work plan, logic model, and/or timeline for proposed objectives.

Position descriptions for key staff. Resumes of key staff that reflect current duties. Consultant or contractor proposed scope of work and letter of commitment (if applicable). Current Indirect Cost Rate Agreement. Organizational chart.

Map of area identifying project location(s). Additional documents to support narrative (i.e., data tables, key news articles, etc.). 2. Review and Selection Each application will be prescreened for eligibility and completeness, as outlined in the funding announcement. Applications that meet the eligibility criteria shall be reviewed for merit by the Objective Review Committee (ORC) based on evaluation criteria.

Incomplete applications and applications that are not responsive to the administrative thresholds (budget limit, project period limit) will not be referred to the ORC and will not be funded. The applicant will be notified of this determination. Applicants must address all program requirements and provide all required documentation. 3. Notifications of Disposition All applicants will receive an Executive Summary Statement from the IHS Office of Clinical and Preventive Services within 30 days of the conclusion of the ORC outlining the strengths and weaknesses of their application.

The summary statement will be sent to the Authorizing Official identified on the face page (SF-424) of the application. A. Award Notices for Funded Applications The NoA is the authorizing document for which funds are dispersed to the approved entities and reflects the amount of Federal funds awarded, the purpose of the grant, the terms and conditions of the award, the effective date of the award, and the budget/project period. Each entity approved for funding must have a user account in GrantSolutions in order to retrieve the NoA. Please see the Agency Contacts list in Section VII for the systems contact information.

B. Approved But Unfunded Applications Approved applications not funded due to lack of available funds will be held for one year. If funding becomes available during the course of the year, the application may be reconsidered. Note. Any correspondence other than the official NoA executed by an IHS grants management official announcing to the project director that an award has been made to their organization is not an authorization to implement their program on behalf of the IHS.

VI. Award Administration Information 1. Administrative Requirements Awards issued under this announcement are subject to, and are administered in accordance with, the following regulations and policies. A. The Criteria as Outlined in This Program Announcement B.

Administrative Regulations for Grants C. Grants Policy D. Cost Principles Uniform Administrative Requirements for HHS Awards, “Cost Principles,” at 45 CFR part 75, subpart E. E. Audit Requirements Uniform Administrative Requirements for HHS Awards, “Audit Requirements,” at 45 CFR part 75, subpart F.

F. As of August 13, 2020, 2 CFR 200 has been updated to include a prohibition on certain telecommunications and video surveillance services or equipment. This prohibition is described in 2 CFR 200.216. This will also be described in the terms and conditions of every IHS grant and cooperative agreement awarded on or after August 13, 2020. 2.

Indirect Costs This section applies to all recipients that request reimbursement of indirect costs (IDC) in their application budget. In accordance with HHS Grants Policy Statement, Part II-27, IHS requires applicants to obtain a current IDC rate agreement and submit it to the DGM prior to the DGM issuing an award. The rate agreement must be prepared in accordance with the applicable cost principles and guidance as provided by the cognizant agency or office. A current rate covers the applicable grant activities under the current award's budget period. If the current rate agreement is not on file with the DGM at the time of award, the IDC portion of the budget will be restricted.

The restrictions remain in place until the current rate agreement is provided to the DGM. Per 45 CFR 75.414(f) Indirect (F&A) costs, “any non-Federal entity [i.e., applicant] that has never received a negotiated indirect cost rate,. . . May elect to charge a de minimis rate of 10 percent of modified total direct costs (MTDC) which may be used indefinitely.

As described in Section 75.403, costs must be consistently charged as either indirect or direct costs, but may not be double charged or inconsistently charged as both. If chosen, this methodology once elected must be used consistently for all Federal awards until such time as the non-Federal entity chooses to negotiate for a rate, which the non-Federal entity may apply to do at any time.” Electing to charge a de minimis rate of 10 percent only applies to applicants that have never received an approved negotiated indirect cost rate from HHS or another cognizant federal agency. Applicants awaiting approval of their indirect cost proposal may request the 10 percent de minimis rate. When the applicant chooses this method, costs included in the indirect cost pool must not be charged as direct costs to the grant. Available funds are inclusive of direct and appropriate indirect costs.

Approved indirect funds are awarded as part of the award amount, and no additional funds will be provided. Generally, IDC rates for IHS grantees are negotiated with the Division of Cost Allocation (DCA) at https://rates.psc.gov/​ or the Department of the Interior (Interior Business Center) at Start Printed Page 41050 https://ibc.doi.gov/​ICS/​tribal. For questions regarding the indirect cost policy, please call the Grants Management Specialist listed under “Agency Contacts” or the main DGM office at (301) 443-5204. 3. Reporting Requirements The grantee must submit required reports consistent with the applicable deadlines.

Failure to submit required reports within the time allowed may result in suspension or termination of an active grant, withholding of additional awards for the project, or other enforcement actions such as withholding of payments or converting to the reimbursement method of payment. Continued failure to submit required reports may result in the imposition of special award provisions and/or the non-funding or non-award of other eligible projects or activities. This requirement applies whether the delinquency is attributable to the failure of the awardee organization or the individual responsible for preparation of the reports. Per DGM policy, all reports must be submitted electronically by attaching them as a “Grant Note” in GrantSolutions. Personnel responsible for submitting reports will be required to obtain a login and password for GrantSolutions.

Please see the Agency Contacts list in Section VII for the systems contact information. The reporting requirements for this program are noted below. A. Progress Reports Program progress reports are required semi-annually. The progress reports are due within 30 days after the budget period ends (specific dates will be listed in the NoA Terms and Conditions).

These reports must include a brief comparison of actual accomplishments to the goals established for the period, a summary of progress to date or, if applicable, provide sound justification for the lack of progress, and other pertinent information as required. A final report must be submitted within 90 days of expiration of the period of performance. B. Financial Reports Federal Cash Transaction Reports are due 30 days after the close of every calendar quarter to the Payment Management Services at https://pms.psc.gov. Failure to submit timely reports may result in adverse award actions blocking access to funds.

Federal Financial Reports are due 30 days after the end of each budget period, and a final report is due 90 days after the end of the Period of Performance. Grantees are responsible and accountable for accurate information being reported on all required reports. The Progress Reports, the Federal Cash Transaction Report, and the Federal Financial Report. C. Data Collection and Reporting At the conclusion of the program period, the outcome report should detail how the T/TO plans to completely integrate CHAP into their Tribal health system and list major barriers that could potentially impact full integration.

The Outcome Report should describe, in full, the findings of the program plan and evaluation, and plans for implementation. The outcome report should organize the findings of the key categories. 1. Clinical Supervisor Support. 2.

Scope of Practice. 3. Technology Infrastructure. 4. Training Plan.

5. System Integration. 6. Auxiliary Support to Address Social Determinants. Based on the findings and measurable outcomes of the categories, the applicant should explicitly identify the implementation plan and projected cost associated with full implementation.

D. Federal Sub-Award Reporting System (FSRS) This award may be subject to the Transparency Act sub-award and executive compensation reporting requirements of 2 CFR part 170. The Transparency Act requires the OMB to establish a single searchable database, accessible to the public, with information on financial assistance awards made by Federal agencies. The Transparency Act also includes a requirement for recipients of Federal grants to report information about first-tier sub-awards and executive compensation under Federal assistance awards. IHS has implemented a Term of Award into all IHS Standard Terms and Conditions, NoAs, and funding announcements regarding the FSRS reporting requirement.

This IHS Term of Award is applicable to all IHS grant and cooperative agreements issued on or after October 1, 2010, with a $25,000 sub-award obligation threshold met for any specific reporting period. For the full IHS award term implementing this requirement and additional award applicability information, visit the DGM Grants Management website at https://www.ihs.gov/​dgm/​policytopics/​. E. Compliance With Executive Order 13166 Implementation of Services Accessibility Provisions for All Grant Application Packages and Funding Opportunity Announcements Recipients of Federal financial assistance (FFA) from HHS must administer their programs in compliance with Federal civil rights laws that prohibit discrimination on the basis of race, color, national origin, disability, age, and, in some circumstances, religion, conscience, and sex. This includes ensuring programs are accessible to persons with limited English proficiency.

The HHS Office for Civil Rights provides guidance on complying with civil rights laws enforced by HHS. Please see https://www.hhs.gov/​civil-rights/​for-providers/​provider-obligations/​index.html and http://www.hhs.gov/​ocr/​civilrights/​understanding/​section1557/​index.html. Please contact the HHS Office for Civil Rights for more information about obligations and prohibitions under Federal civil rights laws at https://www.hhs.gov/​ocr/​about-us/​contact-us/​index.html or call 1-800-368-1019 or TDD 1-800-537-7697. F. Federal Awardee Performance and Integrity Information System (FAPIIS) The IHS is required to review and consider any information about the applicant that is in the Federal Awardee Performance and Integrity Information System (FAPIIS) at https://www.fapiis.gov before making any award in excess of the simplified acquisition threshold (currently $250,000) over the period of performance.

An applicant may review and comment on any information about itself that a Federal awarding agency previously entered. The IHS will consider any comments by the applicant, in addition to other information in FAPIIS, in making a judgment about the applicant's integrity, business ethics, and record of performance under Federal awards when completing the review of risk posed by applicants as described in 45 CFR 75.205. As required by 45 CFR part 75, appendix XII, of the Uniform Guidance, non-Federal entities (NFEs) are required to disclose in FAPIIS any information about criminal, civil, and administrative proceedings, and/or affirm that there is no new information to provide. This applies to NFEs that receive Federal awards (currently active grants, cooperative agreements, and procurement contracts) greater than $10,000,000 for any period of time during the period of performance of an award/project. Mandatory Disclosure Requirements As required by 2 CFR part 200 of the Uniform Guidance and the HHS implementing regulations at 45 CFR part 75, the IHS must require an NFE or an applicant for a Federal award to disclose, in a timely manner, in writing to the IHS or pass-through entity all violations of Federal criminal law involving fraud, bribery, or gratuity violations potentially affecting the Federal award.

Submission is required for all applicants and recipients, in writing, to the IHS and to the HHS Office of Inspector General, all information related to violations of Federal criminal law involving fraud, bribery, or gratuity violations potentially affecting the Federal award. 45 CFR 75.113. Disclosures must be sent in writing to. U.S. Department of Health and Human Services, Indian Health Service, Division of Grants Management, ATTN.

Paul Gettys, Acting Director, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857, (Include “Mandatory Grant Disclosures” in subject line), Office. (301) 443-5204, Fax. (301) 594-0899, Email. [email protected].

And U.S. Department of Health and Human Services, Office of Inspector General, ATTN. Mandatory Grant Disclosures, Intake Coordinator, 330 Independence Avenue SW, Cohen Building, Room 5527, Washington, DC 20201, URL. Https://oig.hhs.gov/​fraud/​report-fraud/​, (Include “Mandatory Grant Disclosures” in subject line), Fax. (202) 205-0604 (Include “Mandatory Grant Disclosures” in subject line) or, Email.

[email protected]. Failure to make required disclosures can result in any of the remedies described in 45 CFR 75.371 Remedies for noncompliance, including suspension or debarment (see 2 CFR parts 180 &. 376). VII. Agency Contacts 1.

Questions on the programmatic issues may be directed to. Minette C. Galindo, Public Health Advisor, Indian Health Service, Office of Clinical and Preventive Services, 5600 Fishers Lane, Mail Stop. 08N34A, Rockville, MD 20857, Phone. (301) 443-4644, Fax.

(301) 594-6213, Email. [email protected]. 2. Questions on grants management and fiscal matters may be directed to. Donald Gooding, Grants Management Specialist, Indian Health Service, Division of Grants Management, 5600 Fishers Lane, Mail Stop.

09E70, Rockville, MD 20857, Phone. (301) 443-2298, Email. [email protected]. 3. Questions on systems matters may be directed to.

Paul Gettys, Acting Director, Indian Health Service, Division of Grants Management, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857, Phone. (301) 443-2114. Or the DGM main line (301) 443-5204, email. [email protected].

VIII. Other Information The Public Health Service strongly encourages all grant, cooperative agreement, and contract recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of the facility) in which regular or routine education, library, day care, health care, or early childhood development services are provided to children. This is consistent with the HHS mission to protect and advance the physical and mental health of the American people. Start Signature Elizabeth A.

Fowler, Acting Director, Indian Health Service. End Signature End Preamble [FR Doc. 2021-16283 Filed 7-29-21. 8:45 am]BILLING CODE 4165-16-PStart Preamble Announcement Type. New.

Funding Announcement Number. HHS-2021-IHS-TAP-0001. Assistance Listing (Catalog of Federal Domestic Assistance or CFDA) Number. 93.382. Key Dates Application Deadline Date.

September 6, 2021. Earliest Anticipated Start Date. September 30, 2021. I. Funding Opportunity Description Statutory Authority The Indian Health Service (IHS) is accepting applications for grants for the Community Health Aide Program (CHAP) Tribal Assessment and Planning (TAP) program.

The CHAP is authorized under the Snyder Act, 25 U.S.C. 13. The Transfer Act, 42 U.S.C. 2001(a). And the Indian Health Care Improvement Act, 25 U.S.C.

1616l. This grant program is described in the Assistance Listings located at https://sam.gov/​content/​home (formerly known as Catalog of Federal Domestic Assistance) under 93.382. Background The national CHAP will provide a network of health aides trained to support licensed health professionals while providing direct health care, health promotion, and disease prevention services. These providers will work within a referral relationship under the supervision of licensed clinical providers that include clinics, service units, and hospitals. The CHAP aides will increase access to direct health services, including inpatient and outpatient visits.Start Printed Page 41052 The Alaska CHAP has become a model for efficient and high quality health care delivery in rural Alaska, providing approximately 300,000 patient encounters per year and responding to emergencies 24 hours a day, seven days a week.

Specialized providers in dental and behavioral health were later introduced to respond to the needs of patients and address the health disparities in oral health and mental health among American Indian and Alaska Natives. The national CHAP is a workforce model that includes three different provider types that act as extenders of their licensed clinical supervisor. The national CHAP currently includes a behavioral health aide, community health aide, and dental health aide. Each of the health aide categories operate in a tiered level practice system. The national CHAP model provides an opportunity for increased access to care through the extension of primary care, dental, and behavioral health clinicians.

In 2010, under the permanent reauthorization of the Indian Health Care Improvement Act (IHCIA), Congress provided the Secretary of the U.S. Department of Health and Human Services, acting through the IHS, the authority to expand the CHAP nationally. In 2016, the IHS initiated Tribal Consultation on expanding the CHAP to the contiguous 48 states. In 2018, the IHS formed the CHAP Tribal Advisory Group (TAG) and began developing the program. In 2020, the IHS announced the national CHAP policy, which formally created the national CHAP.

Purpose The purpose of the TAP program is to support the assessment and planning of Tribes and Tribal Organizations (T/TO) in determining the feasibility of implementing CHAP in their respective communities. The program is designed to support the regional flexibility required for T/TO to design a program unique to the needs of their individual communities across the country through the identification of feasibility factors. The focus of the program is to. 1. Assess whether the T/TO can integrate CHAP into the Tribal health system, including the health care workforce.

2. Identify systemic barriers that prohibit the complete integration of CHAP into an existing health care system. The barriers should be related to. Clinical infrastructure. Workforce barriers.

Certification of providers. Training of providers. Inclusion of culture in the services provided by a CHAP provider. 3. Plan partnerships across the T/TO geographic region to address the barriers, including reimbursement, training, education, clinical infrastructure, implementation cost, and determination of system integration.

II. Award Information Funding Instrument—Grant Estimated Funds Available The total funding identified for fiscal year (FY) 2021 is approximately $2,340,000. Individual award amounts for the first budget year are anticipated to be between $250,000 and $260,000. The funding available for competing and subsequent continuation awards issued under this announcement is subject to the availability of appropriations and budgetary priorities of the Agency. The IHS is under no obligation to make awards that are selected for funding under this announcement.

Anticipated Number of Awards Approximately nine awards will be issued under this program announcement. The IHS intends to award no more than one grant per IHS area. Period of Performance The period of performance is two years. III. Eligibility Information 1.

Eligibility To be eligible for this new FY 2021 funding opportunity, an applicant must be one of the following, as defined under 25 U.S.C. 1603. A federally recognized Indian Tribe as defined by 25 U.S.C. 1603(14). The term “Indian Tribe” means any Indian Tribe, band, nation, or other organized group or community, including any Alaska Native village or group, or regional or village corporation, as defined in or established pursuant to the Alaska Native Claims Settlement Act (85 Stat.

688) [43 U.S.C. 1601 et seq.], which is recognized as eligible for the special programs and services provided by the United States to Indians because of their status as Indians. A Tribal organization as defined by 25 U.S.C. 1603(26). The term “Tribal organization” has the meaning given the term in section 4 of the Indian Self-Determination and Education Assistance Act (25 U.S.C.

5304). €œTribal organization” means the recognized governing body of any Indian Tribe. Any legally established organization of Indians which is controlled, sanctioned, or chartered by such governing body or which is democratically elected by the adult members of the Indian community to be served by such organization and which includes the maximum participation of Indians in all phases of its activities. Provided that, in any case where a contract is let or grant made to an organization to perform services benefiting more than one Indian Tribe, the approval of each such Indian Tribe shall be a prerequisite to the letting or making of such contract or grant. Applicant shall submit letters of support and/or Tribal Resolutions from the Tribes to be served.

An applicant may not apply to both this opportunity, TAP, and the CHAP Tribal Planning and Implementation (TPI) opportunity (number HHS-2021-IHS-TPI-0001). An organization currently carrying out a CHAP in the United States, in accordance with 25 U.S.C. 1616l through an Indian Self-Determination and Education Assistance Act (ISDEAA) agreement, is also not eligible to apply. The Program office will notify any applicants deemed ineligible. Note.

Please refer to Section IV.2 (Application and Submission Information/Subsection 2, Content and Form of Application Submission) for additional proof of applicant status documents required, such as Tribal Resolutions, proof of nonprofit status, etc. 2. Cost Sharing or Matching The IHS does not require matching funds or cost sharing for grants or cooperative agreements. 3. Other Requirements Applications with budget requests that exceed the highest dollar amount outlined under Section II Award Information, Estimated Funds Available, or exceed the Period of Performance outlined under Section II Award Information, Period of Performance, will be considered not responsive and will not be reviewed.

The Division of Grants Management (DGM) will notify the applicant. Additional Required Documentation Tribal Resolution The DGM must receive an official, signed Tribal Resolution prior to issuing a Notice of Award (NoA) to any applicant selected for funding. An Indian Tribe or Tribal organization that is proposing a project affecting another Indian Tribe must include resolutions from all affected Tribes to be served. However, if an official, signed Tribal Resolution cannot be submitted with the application prior to the application Start Printed Page 41053deadline date, a draft Tribal Resolution must be submitted with the application by the deadline date in order for the application to be considered complete and eligible for review. The draft Tribal Resolution is not in lieu of the required signed resolution but is acceptable until a signed resolution is received.

If an application without a signed Tribal Resolution is selected for funding, the applicant will be contacted by the Grants Management Specialist (GMS) listed in this funding announcement and given 90 days to submit an official, signed Tribal Resolution to the GMS. If the signed Tribal Resolution is not received within 90 days, the award will be forfeited. Tribes organized with a governing structure other than a Tribal council may submit an equivalent document commensurate with their governing organization. Proof of Nonprofit Status Organizations claiming nonprofit status must submit a current copy of the 501(c)(3) Certificate with the application. IV.

Application and Submission Information 1. Obtaining Application Materials The application package and detailed instructions for this announcement are hosted on https://www.Grants.gov. Please direct questions regarding the application process to Mr. Paul Gettys at (301) 443-2114 or (301) 443-5204. 2.

Content and Form Application Submission Mandatory documents for all applicants include. Abstract (one page) summarizing the project. Application forms. 1. SF-424, Application for Federal Assistance.

2. SF-424A, Budget Information—Non-Construction Programs. 3. SF-424B, Assurances—Non-Construction Programs. Project Narrative (not to exceed 15 pages).

See Section IV.2.A Project Narrative for instructions. 1. Background information on the organization. 2. Proposed scope of work, objectives, and activities that provide a description of what the applicant plans to accomplish.

Budget Justification and Narrative (not to exceed 5 pages). See Section IV.2.B Budget Narrative for instructions. One-page Timeframe Chart. Tribal Resolution(s). Letters of Support from organization's Board of Directors (if applicable).

501(c)(3) Certificate. Biographical sketches for all Key Personnel. Contractor/Consultant resumes or qualifications and scope of work. Disclosure of Lobbying Activities (SF-LLL), if applicant conducts reportable lobbying. Certification Regarding Lobbying (GG-Lobbying Form).

Copy of current Negotiated Indirect Cost rate (IDC) agreement (required in order to receive IDC). Organizational Chart (optional). Documentation of current Office of Management and Budget (OMB) Financial Audit (if applicable). Acceptable forms of documentation include. 1.

Email confirmation from Federal Audit Clearinghouse (FAC) that audits were submitted. Or 2. Face sheets from audit reports. Applicants can find these on the FAC website at https://harvester.census.gov/​facdissem/​Main.aspx. Public Policy Requirements All Federal public policies apply to IHS grants and cooperative agreements.

Pursuant to 45 CFR 80.3(d), an individual shall not be deemed subjected to discrimination by reason of their exclusion from benefits limited by Federal law to individuals eligible for benefits and services from the IHS. See https://www.hhs.gov/​grants/​grants/​grants-policies-regulations/​index.html. Requirements for Project and Budget Narratives A. Project Narrative This narrative should be a separate document that is no more than 15 pages and must. (1) Have consecutively numbered pages.

(2) use black font 12 points or larger. (3) be single-spaced. And (4) be formatted to fit standard letter paper (81/2 x 11 inches). Be sure to succinctly answer all questions listed under the evaluation criteria (refer to Section V.1, Evaluation Criteria) and place all responses and required information in the correct section noted below or they will not be considered or scored. If the narrative exceeds the page limit, the application will be considered not responsive and not be reviewed.

The 15-page limit for the narrative does not include the work plan, standard forms, Tribal Resolutions, budget, budget justifications, narratives, and/or other items. There are three parts to the narrative. Part 1—Program Information. Part 2—Program Plan. And Part 3—Program Evaluation and Outcome Report.

See below for additional details about what must be included in the narrative. The page limits below are for each narrative and budget submitted. Part 1. Program Information (Limit—4 Pages) Section 1. Community Profile Describe the demographics of the community including, but not limited to, geography, languages, age, and socioeconomic status.

The community profile should include data specific to the community that would benefit from the implementation of CHAP. Section 2. Health &. Infrastructure Needs Describe the community's current health disparities related to primary, behavioral, and oral health care. Section 3.

Organizational Capacity Describe the T/TO's current health program activities, how long it has been operating, and what programs or services are currently being provided. Describe in full the organization's infrastructure and its ability to assess the barriers that could impact the integration of CHAP and identify significant barriers that could prohibit the implementation. Part 2. Program Plan (Limit—6 Pages) Section 1. Program Plan Describe in full the direction the T/TO plans to take in the CHAP TAP.

The program plan should first clearly identify the problems within the community related to behavioral, primary, and oral health. The program plan should then include the plan to assess the problem(s). This should include a timeline for the assessment. The program plan should identify a timeline to determine whether CHAP can address the barriers identified. Section 2.

Program Activities Describe in full the activities to identify problems creating barriers within the community related to behavioral, primary, and oral health. These activities should be categorized (at a minimum) within key factors related to clinical infrastructure, workforce barriers, training infrastructure, and cultural inclusion. Describe in full how the applicant plans to assess the problems identified. Finally, describe in detail the activities and associated timeline to determine whether CHAP is feasible and activities to quantify the cost associated with CHAP. The program activities should detail which partners will aid in Start Printed Page 41054identifying and assessing barriers related to clinical infrastructure, workforce barriers, training infrastructure, and cultural inclusion.

Section 3. Staffing Plan Describe key staff tasked with carrying out the program activities in Section 2. Applicants should account for potential stakeholder partnerships following the assessment of barriers in the staffing plan. Section 4. Timeline Describe a timeline not to exceed two years for the completion of the program plan, activities, and evaluation plan.

Provide a timeline chart depicting a realistic timeline that details all major activities, milestones, and applicable staffing plans. The timeline should include the projected progress report due at the midpoint of the project period. The timeline chart should not exceed one page. Part 3. Program Evaluation &.

Outcome Report (Limit—5 Pages) Section 1. Evaluation Plan The evaluation plan should identify and describe significant program activities and achievements associated with the assessment and planning of whether CHAP can address identified barriers within the existing Tribal health system. Provide a comparison of the actual accomplishments to the goals established for the project period, or if applicable, provide justification for the lack of progress. The evaluation plan should organize all identified problems that lead to barriers into major categories related to clinical infrastructure, workforce barriers, training infrastructure, and cultural inclusion specific to the scope of practice of prospective CHAP providers. The evaluation plan should detail how these barriers can be quantified.

The evaluation plan should detail how the applicant will measure the assessment of whether CHAP can address the issues identified including number of partnerships for each major category of barriers, other factors that may impact feasibility, and sustainability. Finally, the evaluation plan should detail how the applicant plans to calculate the total cost associated with integrating CHAP as part of the planning process. Section 2. Outcome Report At the conclusion of the program period, using the findings from the evaluation, the T/TO should determine the feasibility of implementing a CHAP within their own community. The Outcome Report should describe in full the findings of the program plan, evaluation, and determination on stage of readiness for implementation.

The outcome report should organize the findings into at least five categories. 1. Clinical Infrastructure. 2. Workforce Barriers.

3. Training Infrastructure. 4. Cultural Inclusion. 5.

Implementation Cost. Based on the findings and measurable outcomes of the categories, the applicant should explicitly identify whether CHAP is feasible for implementation into their respective community. Applicants should develop an organized report that highlights the categories succinctly and includes data (quantitative or qualitative) from the evaluation plan. The outcome report should explicitly detail the cost associated with integrating CHAP if it is found that CHAP can address the barriers identified in the assessment phase. B.

Budget Narrative (Limit—5 Pages) Provide a budget narrative that explains the amounts requested for each line item of the budget from the SF-424A (Budget Information for Non-Construction Programs). The budget narrative should specifically describe how each item will support the achievement of proposed objectives. Be very careful about showing how each item in the “Other” category is justified. For subsequent budget years (see Multi-Year Project Requirements in Section V.1. Application Review Information, Evaluation Criteria), the narrative should highlight the changes from year 1 or clearly indicate that there are no substantive budget changes during the period of performance.

Do NOT use the budget narrative to expand the project narrative. 3. Submission Dates and Times Applications must be submitted through Grants.gov by 11:59 p.m. Eastern Time on the Application Deadline Date. Any application received after the application deadline will not be accepted for review.

Grants.gov will notify the applicant via email if the application is rejected. If technical challenges arise and assistance is required with the application process, contact Grants.gov Customer Support (see contact information at https://www.Grants.gov). If problems persist, contact Mr. Paul Gettys ([email protected]), Acting Director, DGM, by telephone at (301) 443-2114 or (301) 443-5204. Please be sure to contact Mr.

Gettys at least ten days prior to the application deadline. Please do not contact the DGM until you have received a Grants.gov tracking number. In the event you are not able to obtain a tracking number, call the DGM as soon as possible. The IHS will not acknowledge receipt of applications. 4.

Intergovernmental Review Executive Order 12372 requiring intergovernmental review is not applicable to this program. 5. Funding Restrictions Pre-award costs are allowable up to 90 days before the start date of the award provided the costs are otherwise allowable if awarded. Pre-award costs are incurred at the risk of the applicant. The available funds are inclusive of direct and indirect costs.

Only one grant may be awarded per applicant. 6. Electronic Submission Requirements All applications must be submitted via Grants.gov. Please use the https://www.Grants.gov website to submit an application. Find the application by selecting the “Search Grants” link on the homepage.

Follow the instructions for submitting an application under the Package tab. No other method of application submission is acceptable. If the applicant cannot submit an application through Grants.gov, a waiver must be requested. Prior approval must be requested and obtained from Mr. Paul Gettys, Acting Director, DGM.

A written waiver request must be sent to [email protected] with a copy to [email protected]. The waiver request must. (1) Be documented in writing (emails are acceptable) before submitting an application by some other method, and (2) include clear justification for the need to deviate from the required application submission process. Once the waiver request has been approved, the applicant will receive a confirmation of approval email containing submission instructions. A copy of the written approval must be included with the application that is submitted to the DGM.

Applications that are submitted without a copy of the signed waiver from the Acting Director of the DGM will not be reviewed. The Grants Management Officer of the DGM will notify the applicant via email of this decision. Applications submitted under waiver must be received by the DGM no later than 5:00 p.m., Eastern Time, on the Application Deadline Date. Late applications will not be accepted for processing. Applicants that do not register for both the System for Award Management (SAM) and Grants.gov and/or fail to request timely assistance with technical issues will not be Start Printed Page 41055considered for a waiver to submit an application via alternative method.

Please be aware of the following. Please search for the application package in https://www.Grants.gov by entering the Assistance Listing (CFDA) number or the Funding Opportunity Number. Both numbers are located in the header of this announcement. If you experience technical challenges while submitting your application, please contact Grants.gov Customer Support (see contact information at https://www.Grants.gov). Upon contacting Grants.gov, obtain a tracking number as proof of contact.

The tracking number is helpful if there are technical issues that cannot be resolved and a waiver from the agency must be obtained. Applicants are strongly encouraged not to wait until the deadline date to begin the application process through Grants.gov as the registration process for SAM and Grants.gov could take up to 20 working days. Please follow the instructions on Grants.gov to include additional documentation that may be requested by this funding announcement. Applicants must comply with any page limits described in this funding announcement. After submitting the application, the applicant will receive an automatic acknowledgment from Grants.gov that contains a Grants.gov tracking number.

The IHS will not notify the applicant that the application has been received. Dun and Bradstreet (D&B) Data Universal Numbering System (DUNS) Applicants and grantee organizations are required to obtain a DUNS number and maintain an active registration in the SAM database. The DUNS number is a unique 9-digit identification number provided by D&B that uniquely identifies each entity. The DUNS number is site specific. Therefore, each distinct performance site may be assigned a DUNS number.

Obtaining a DUNS number is easy, and there is no charge. To obtain a DUNS number, please access the request service through https://fedgov.dnb.com/​webform or call (866) 705-5711. The Federal Funding Accountability and Transparency Act of 2006, as amended (“Transparency Act”), requires all HHS recipients to report information on sub-awards. Accordingly, all IHS grantees must notify potential first-tier sub-recipients that no entity may receive a first-tier sub-award unless the entity has provided its DUNS number to the prime grantee organization. This requirement ensures the use of a universal identifier to enhance the quality of information available to the public pursuant to the Transparency Act.

System for Award Management (SAM) Organizations that are not registered with SAM must have a DUNS number first, then access the SAM online registration through the SAM home page at https://sam.gov (U.S. Organizations will also need to provide an Employer Identification Number from the Internal Revenue Service that may take an additional 2-5 weeks to become active). Please see SAM.gov for details on the registration process and timeline. Registration with the SAM is free of charge but can take several weeks to process. Applicants may register online at https://sam.gov.

Additional information on implementing the Transparency Act, including the specific requirements for DUNS and SAM, are available on the DGM Grants Management, Policy Topics web page. Https://www.ihs.gov/​dgm/​policytopics/​. V. Application Review Information Possible points assigned to each section are noted in parentheses. The 15-page project narrative should include only the first year of activities.

Information for multi-year projects should be included as a separate document. See “Multi-year Project Requirements” at the end of this section for more information. The narrative section should be written in a manner that is clear to outside reviewers unfamiliar with prior related activities of the applicant. It should be well organized, succinct, and contain all information necessary for reviewers to fully understand the project. Attachments requested in the criteria do not count toward the 15-page limit for the project narrative.

Points will be assigned to each evaluation criteria adding up to a total of 100 possible points. Points are assigned as follows. 1. Evaluation Criteria A. Introduction and Need for Assistance (10 Points) Identify the proposed project and plans to identify the feasibility of implementing a CHAP within their community.

The needs should clearly identify the existing health system and how the CHAP may be a viable workforce model for the community needs. B. Project Objective(s), Work Plan, and Approach (30 Points) The work plan should be comprised of two key parts. Program Information and Program Plan. Acceptable Program Information should provide information related to three (3) key sections.

Community profile. Health and infrastructure. And organizational capacity. The Program Information part should demonstrate a robust community profile that highlights the existing health system, demographic data of community members and user population, and a detailed description of the T/TO carrying out the proposed activity. An acceptable Program Plan should include details of the applicant's plan to address the program objective.

The Program Plan should address, at a minimum, key activities related to clinical infrastructure, workforce barriers, and training infrastructure. C. Program Evaluation (30 Points) The program evaluation should address how the applicant intends to measure major categories related to clinical infrastructure. Workforce barriers. Training infrastructure.

Cultural inclusion (See Sample Logic Model in Related Documents in Grants.gov) specific to the scope of practice of prospective CHAP providers. And implementation costs. The evaluation plan should identify. how the applicant plans to determine the feasibility of CHAP integration into the Tribal system. Measurement of significant systematic barriers.

Implementation cost associated with CHAP. And planning for the scope of work. The applicant may choose to develop a readiness assessment to measure the feasibility. List measurable and attainable goals with explicit timelines that detail expectation of findings. D.

Organizational Capabilities, Key Personnel, and Qualifications (10 Points) Provide a detailed biographical sketch of each member of key personnel assigned to carry out the objectives of the program plan. The sketches should detail the qualifications and expertise of identified staff. E. Categorical Budget and Budget Justification (20 Points) Provide a detailed budget of each expenditure directly related to the identified program activities. Multi-Year Project Requirements Applications must include a brief project narrative and budget (one additional page per year) addressing the developmental plans for each additional year of the project.

This attachment will Start Printed Page 41056not count as part of the project narrative or the budget narrative. Additional documents can be uploaded as Other Attachments in Grants.gov. Work plan, logic model, and/or timeline for proposed objectives. Position descriptions for key staff. Resumes of key staff that reflect current duties.

Consultant or contractor proposed scope of work and letter of commitment (if applicable). Current Indirect Cost Rate Agreement. Organizational chart. Map of area identifying project location(s). Additional documents to support narrative (i.e., data tables, key news articles, etc.).

2. Review and Selection Each application will be prescreened for eligibility and completeness, as outlined in the funding announcement. Applications that meet the eligibility criteria shall be reviewed for merit by the Objective Review Committee (ORC) based on evaluation criteria. Incomplete applications and applications that are not responsive to the administrative thresholds (budget limit, project period limit) will not be referred to the ORC and will not be funded. The applicant will be notified of this determination.

Applicants must address all program requirements and provide all required documentation. 3. Notifications of Disposition All applicants will receive an Executive Summary Statement from the IHS Office of Clinical and Preventive Services within 30 days of the conclusion of the ORC outlining the strengths and weaknesses of their application. The summary statement will be sent to the Authorizing Official identified on the face page (SF-424) of the application. A.

Award Notices for Funded Applications The NoA is the authorizing document for which funds are dispersed to the approved entities and reflects the amount of Federal funds awarded, the purpose of the grant, the terms and conditions of the award, the effective date of the award, and the budget/project period. Each entity approved for funding must have a user account in GrantSolutions in order to retrieve the NoA. Please see the Agency Contacts list in Section VII for the systems contact information. B. Approved but Unfunded Applications Approved applications not funded due to lack of available funds will be held for one year.

If funding becomes available during the course of the year, the application may be reconsidered. Note. Any correspondence other than the official NoA executed by an IHS grants management official announcing to the project director that an award has been made to their organization is not an authorization to implement their program on behalf of the IHS. VI. Award Administration Information 1.

Administrative Requirements Awards issued under this announcement are subject to, and are administered in accordance with, the following regulations and policies. A. The Criteria as Outlined in This Program Announcement B. Administrative Regulations for Grants C. Grants Policy D.

Cost Principles Uniform Administrative Requirements for HHS Awards, “Cost Principles,” at 45 CFR part 75, subpart E. E. Audit Requirements Uniform Administrative Requirements for HHS Awards, “Audit Requirements,” at 45 CFR part 75, subpart F. F. As of August 13, 2020, 2 CFR 200 was updated to include a prohibition on certain telecommunications and video surveillance services or equipment.

This prohibition is described in 2 CFR 200.216. This will also be described in the terms and conditions of every IHS grant and cooperative agreement awarded on or after August 13, 2020. 2. Indirect Costs This section applies to all recipients that request reimbursement of indirect costs (IDC) in their application budget. In accordance with HHS Grants Policy Statement, Part II-27, IHS requires applicants to obtain a current IDC rate agreement and submit it to the DGM prior to the DGM issuing an award.

The rate agreement must be prepared in accordance with the applicable cost principles and guidance as provided by the cognizant agency or office. A current rate covers the applicable grant activities under the current award's budget period. If the current rate agreement is not on file with the DGM at the time of award, the IDC portion of the budget will be restricted. The restrictions remain in place until the current rate agreement is provided to the DGM. Per 45 CFR 75.414(f) Indirect (F&A) costs, “any non-Federal entity [i.e., applicant] that has never received a negotiated indirect cost rate,.

. . May elect to charge a de minimis rate of 10 percent of modified total direct costs (MTDC) which may be used indefinitely. As described in Section 75.403, costs must be consistently charged as either indirect or direct costs, but may not be double charged or inconsistently charged as both. If chosen, this methodology once elected must be used consistently for all Federal awards until such time as the non-Federal entity chooses to negotiate for a rate, which the non-Federal entity may apply to do at any time.” Electing to charge a de minimis rate of 10 percent only applies to applicants that have never received an approved negotiated indirect cost rate from HHS or another cognizant federal agency.

Applicants awaiting approval of their indirect cost proposal may request the 10 percent de minimis rate. When the applicant chooses this method, costs included in the indirect cost pool must not be charged as direct costs to the grant. Available funds are inclusive of direct and appropriate indirect costs. Approved indirect funds are awarded as part of the award amount, and no additional funds will be provided. Generally, IDC rates for IHS grantees are negotiated with the Division of Cost Allocation (DCA) at https://rates.psc.gov/​ or the Department of the Interior (Interior Business Center) at https://ibc.doi.gov/​ICS/​tribal.

For Start Printed Page 41057questions regarding the indirect cost policy, please call the Grants Management Specialist listed under “Agency Contacts” or the main DGM office at (301) 443-5204. 3. Reporting Requirements The grantee must submit required reports consistent with the applicable deadlines. Failure to submit required reports within the time allowed may result in suspension or termination of an active grant, withholding of additional awards for the project, or other enforcement actions such as withholding of payments or converting to the reimbursement method of payment. Continued failure to submit required reports may result in the imposition of special award provisions and/or the non-funding or non-award of other eligible projects or activities.

This requirement applies whether the delinquency is attributable to the failure of the awardee organization or the individual responsible for preparation of the reports. Per DGM policy, all reports must be submitted electronically by attaching them as a “Grant Note” in GrantSolutions. Personnel responsible for submitting reports will be required to obtain a login and password for GrantSolutions. Please see the Agency Contacts list in Section VII for the systems contact information. The reporting requirements for this program are noted below.

A. Progress Reports Program progress reports are required semi-annually. The progress reports are due within 30 days after the budget period ends (specific dates will be listed in the NoA Terms and Conditions). These reports must include a brief comparison of actual accomplishments to the goals established for the period, a summary of progress to date or, if applicable, provide sound justification for the lack of progress, and other pertinent information as required. A final report must be submitted within 90 days of expiration of the period of performance.

B. Financial Reports Federal Cash Transaction Reports are due 30 days after the close of every calendar quarter to the Payment Management Services at https://pms.psc.gov. Failure to submit timely reports may result in adverse award actions blocking access to funds. Federal Financial Reports are due 30 days after the end of each budget period, and a final report is due 90 days after the end of the Period of Performance. Grantees are responsible and accountable for accurate information being reported on all required reports.

The Progress Reports and Federal Financial Report. C. Data Collection and Reporting To satisfy the reporting requirements, the applicant is expected to develop an outcome report. The outcome report should explicitly state whether CHAP implementation and integration into the existing health care system is viable or not. The Outcome Report should describe, in full, the findings of the program plan, evaluation, and determination on stage of readiness for implementation.

The outcome report should organize the findings into at least five categories. 1. Clinical Infrastructure. 2. Workforce Barriers.

3. Training Infrastructure. 4. Cultural Inclusion. 5.

Implementation Cost. Applicants are encouraged to identify additional categories above the five aforementioned and may choose to develop subcategories that best fit the program plan. D. Federal Sub-Award Reporting System (FSRS) This award may be subject to the Transparency Act sub-award and executive compensation reporting requirements of 2 CFR part 170. The Transparency Act requires the OMB to establish a single searchable database, accessible to the public, with information on financial assistance awards made by Federal agencies.

The Transparency Act also includes a requirement for recipients of Federal grants to report information about first-tier sub-awards and executive compensation under Federal assistance awards. IHS has implemented a Term of Award into all IHS Standard Terms and Conditions, NoAs, and funding announcements regarding the FSRS reporting requirement. This IHS Term of Award is applicable to all IHS grant and cooperative agreements issued on or after October 1, 2010, with a $25,000 sub-award obligation threshold met for any specific reporting period. For the full IHS award term implementing this requirement and additional award applicability information, visit the DGM Grants Management website at https://www.ihs.gov/​dgm/​policytopics/​. E.

Compliance With Executive Order 13166 Implementation of Services Accessibility Provisions for All Grant Application Packages and Funding Opportunity Announcements Recipients of Federal financial assistance (FFA) from HHS must administer their programs in compliance with Federal civil rights laws that prohibit discrimination on the basis of race, color, national origin, disability, age, and, in some circumstances, religion, conscience, and sex. This includes ensuring programs are accessible to persons with limited English proficiency. The HHS Office for Civil Rights provides guidance on complying with civil rights laws enforced by HHS. Please see https://www.hhs.gov/​civil-rights/​for-providers/​provider-obligations/​index.html and http://www.hhs.gov/​ocr/​civilrights/​understanding/​section1557/​index.html. Please contact the HHS Office for Civil Rights for more information about obligations and prohibitions under Federal civil rights laws at https://www.hhs.gov/​ocr/​about-us/​contact-us/​index.html or call 1-800-368-1019 or TDD 1-800-537-7697.

F. Federal Awardee Performance and Integrity Information System (FAPIIS) The IHS is required to review and consider any information about the applicant that is in the Federal Awardee Performance and Integrity Information System (FAPIIS), at https://www.fapiis.gov, before making any award in excess of the simplified acquisition threshold (currently $250,000) over the period of performance. An applicant may review and comment on any information about itself that a Federal awarding agency previously entered. IHS will consider any comments by the applicant, in addition to other information in FAPIIS, in making a judgment about the applicant's integrity, business ethics, and record of performance under Federal awards when completing the review of risk posed by applicants as described in 45 CFR 75.205. As required by 45 CFR part 75, appendix XII, of the Uniform Guidance, non-Federal entities (NFEs) are required to disclose in FAPIIS any information about criminal, civil, and administrative proceedings, and/or affirm that there is no new information to provide.

This applies to NFEs that receive Federal awards (currently active grants, cooperative agreements, and procurement contracts) greater than $10,000,000 for any period of time during the period of performance of an award/project. Mandatory Disclosure Requirements As required by 2 CFR part 200 of the Uniform Guidance and the HHS implementing regulations at 45 CFR part 75, the IHS must require an NFE or an applicant for a Federal award to disclose, in a timely manner, in writing to the IHS or pass-through entity all violations of Federal criminal law involving fraud, bribery, or gratuity violations potentially affecting the Federal award. Submission is required for all applicants and recipients, in writing, to the IHS and to the HHS Office of Inspector General of all information related to violations of Federal criminal law involving fraud, bribery, or gratuity violations potentially affecting the Federal award. 45 CFR 75.113. Disclosures must be sent in writing to.

U.S. Department of Health and Human Services, Indian Health Service, Division of Grants Management, ATTN. Paul Gettys, Acting Director, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857 (Include “Mandatory Grant Disclosures” in subject line), Office. (301) 443-5204, Fax.

(301) 594-0899, Email. [email protected]. And U.S. Department of Health and Human Services, Office of Inspector General, ATTN. Mandatory Grant Disclosures, Intake Coordinator, 330 Independence Avenue SW, Cohen Building, Room 5527, Washington, DC 20201, URL.

Https://oig.hhs.gov/​fraud/​report-fraud/​ (Include “Mandatory Grant Disclosures” in subject line), Fax. (202) 205-0604 (Include “Mandatory Grant Disclosures” in subject line) or Email. [email protected]. Failure to make required disclosures can result in any of the remedies described in 45 CFR 75.371 Remedies for noncompliance, including suspension or debarment (see 2 CFR parts 180 &. 376).

VII. Agency Contacts 1. Questions on the programmatic issues may be directed to. Minette C. Galindo, Public Health Advisor, Indian Health Service, Office of Clinical and Preventive Services, 5600 Fishers Lane, Mail Stop.

08N34A, Rockville, MD 20857, Phone. (301) 443-4644, Email. [email protected]. 2. Questions on grants management and fiscal matters may be directed to.

Donald Gooding, Grants Management Specialist, Indian Health Service, Division of Grants Management, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857, Phone. (301) 443-2298, Email. [email protected]. 3.

Questions on systems matters may be directed to. Paul Gettys, Acting Director, Indian Health Service, Division of Grants Management, 5600 Fishers Lane, Mail Stop. 09E70, Rockville, MD 20857, Phone. (301) 443-2114. Or the DGM main line (301) 443-5204, Email.

[email protected]. VIII. Other Information The Public Health Service strongly encourages all grant, cooperative agreement and contract recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of the facility) in which regular or routine education, library, day care, health care, or early childhood development services are provided to children. This is consistent with the HHS mission to protect and advance the physical and mental health of the American people.

Start Signature Elizabeth A. Fowler, Acting Director, Indian Health Service. End Signature End Preamble [FR Doc. 2021-16280 Filed 7-29-21. 8:45 am]BILLING CODE 4165-16-P.

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Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment can you get viagra without a prescription (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of can you get viagra without a prescription a predominantly central memory T cell (TCM cell) phenotype.

We present a bicistronic retroviral vector encoding both a tumor vasculature–targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward can you get viagra without a prescription the acceleration of effective therapies reaching the clinic.

The adoptive cell transfer (ACT) of ex vivo–expanded T lymphocytes has yielded robust and durable clinical responses against several cancer-types, such as tumor-infiltrating lymphocyte therapy of advanced melanoma (Mardiana et al., 2019). Another approach to ACT involves the redirection of peripheral blood T cells to tumor antigens by engineering them to express a chimeric antigen receptor (CAR) that triggers cellular activation upon tumor antigen can you get viagra without a prescription binding. CAR-T cell therapy against hematologic malignancies, by targeting the B cell lineage antigens CD19 or the B cell maturation antigen, has proven efficacious in the clinic, and there is optimism that similar success will be achieved for some solid tumors (Geyer and Brentjens, 2016.

Irving et al., 2017). A range can you get viagra without a prescription of physical (Lanitis et al., 2015) and immunometabolic barriers that can prevent T cell homing, transendothelial migration across tumor blood vessels, engraftment/persistence, and effector function limit the potency of CAR-T cell therapy against solid tumors (Brown et al., 2016. Louis et al., 2011).

Moreover, chronic antigen exposure and a lack of sufficient costimulation in the tumor microenvironment (TME) can cause CAR-T cell exhaustion (Irving et al., 2017). Coengineering of can you get viagra without a prescription CAR-T cells may help to overcome some of these obstacles (Lanitis et al., 2020). Genetic modifications, for example, can be made to enable better homing and tumor penetration or render CAR-T cells resistant to suppressive mechanisms in the TME (Stromnes et al., 2010).

In addition, CAR-T cells can be armed with secretory molecules can you get viagra without a prescription or additional receptors to support CAR-T cell activity and/or harness endogenous immunity (Adachi et al., 2018. Pegram et al., 2012). Preclinical evaluation of CAR-T cells has, for the most part, been performed with xenograft tumor models in immunodeficient mice (Lee et al., 2011.

Mardiros et can you get viagra without a prescription al., 2013. Lanitis et al., 2012). Although this approach can be used to evaluate human CAR-T cell persistence, homing, tumor control, and survival following ACT, critical parameters, including potential toxicity against normal tissues (Tran et al., 2013), and the impact of endogenous immunity on both tumor control and escape are not addressed in such models (Spear et al., 2012.

Avanzi et al., can you get viagra without a prescription 2018). As varying obstacles must be overcome to enhance CAR-T cell responses against different solid tumor types, comprehensive studies in immunocompetent syngeneic tumor models would enable more accurate screening of T cell engineering strategies and provide important insights into improving coengineering and combinatorial treatment approaches (Lanitis et al., 2020). A key limitation of CAR evaluation in syngeneic models stems from inadequate methodologies can you get viagra without a prescription for efficient murine T cell transduction and expansion.

Indeed, unless T cells derived from multiple donor spleens are transduced or the engineered T cells are restimulated for further expansion, which among other drawbacks are costly and can promote exhaustion and apoptosis (Bucks et al., 2009), respectively, current protocols yield insufficient numbers of CAR-T cells for ACT studies (Lee et al., 2009). The efficiency of cell-surface expression of second-generation (2G) CARs, comprising the endodomain (ED) of CD3ζ and one costimulatory ED (e.g., CD28 or 4-1BB), generally reaches 40–60% (Kochenderfer et al., 2010. Davila et can you get viagra without a prescription al., 2013.

Wang et al., 2014. Fu et al., 2013). Although retroviral transduction rates as high as 70–80% for murine T cells have been reported, this was assessed at 2 to 3 d after transduction can you get viagra without a prescription (Tran et al., 2013.

Kuhn et al., 2019. Kusabuka et al., 2016) and thus may include false positives can you get viagra without a prescription due to transient expression from nonintegrated vector DNA (i.e., pseudo-transduction. Case et al., 1999, Costello et al., 2000).

Moreover, short-term transduction efficiency is often based on reporter genes like GFP, which may overestimate CAR expression levels (Kusabuka et al., 2016. Kuhn et can you get viagra without a prescription al., 2019. Davila et al., 2013).

Finally, while can you get viagra without a prescription stable retroviral packaging and producer cell lines may enable transduction efficiencies for 2G and third-generation (3G. I.e., a CAR having two or more costimulatory EDs) CARs of >60% (Fu et al., 2013), this is a laborious approach if multiple CAR designs are to be compared (Chinnasamy et al., 2010). Here, we report the development of an efficient and highly reproducible protocol for primary murine T cell retroviral transduction and expansion, yielding functional murine 2G-CAR-T cells, as well as fourth-generation (4G)-CAR-T cells coengineered to express murine IL-15 (mIL-15) for enhanced in vitro and in vivo function and TME reprogramming.

Overall, our work provides important tools for enabling the systematic evaluation of 4G-CAR-T cells in immunocompetent, syngeneic tumor-bearing can you get viagra without a prescription mice, which we believe is critical for effective therapies reaching the clinic. We sought to optimize murine T cell activation, transduction, and expansion methods for preclinical CAR therapy evaluation in immunocompetent, syngeneic tumor-bearing mice. The final protocol we developed is summarized in Fig.

1 and is can you get viagra without a prescription described in detail in Materials and methods. We used a 2G-CAR targeting vascular endothelial cell growth factor receptor 2 (VEGFR-2), comprising the well-characterized single-chain variable fragment (scFv) DC101 (Chinnasamy et al., 2010), a CD8α hinge and transmembrane domain, and the murine EDs of CD28 and CD3ζ. The anti-VEGFR-2 can you get viagra without a prescription CAR retroviral vector is abbreviated as DC101-28z (Fig.

2 A). Because retroviagraes infect proliferating cells (Kusabuka et al., 2016. Chinnasamy et al., can you get viagra without a prescription 2010.

Hu et al., 2017), we first compared three commonly used methods for inducing T cell activation. (i) magnetic beads coated with anti-(α) CD3 antibody (Ab) and αCD28 Ab (αCD3/CD28 beads) plus recombinant human IL-2 (hIL-2), (ii) plate-immobilized αCD3 Ab along with soluble αCD28 Ab (αCD3-plate/CD28) plus hIL-2, and (iii) Concanavalin A plus hIL-2 and hIL-7. Stimulation with αCD3/CD28 can you get viagra without a prescription beads consistently resulted in the highest frequency of CD44+ CD62L− (recently activated, memory), CD25+ or CD69+ (activated), and Ki67+ (proliferating) CD3+ T cells (Fig.

2 B and Fig. S1 A) can you get viagra without a prescription. We next found that concentration of viral particles through ultracentrifugation yielded higher viral titers (>3 × 107 transducing units/ml.

Fig. 2 C) and enabled significantly higher transduction of primary activated primary murine T cells as can you get viagra without a prescription compared with unconcentrated retroviagra (Fig. 2 D), reaching a plateau at a multiplicity of (MOI) of 5 (∼80% CAR expression.

Fig. 2 E). A single transduction at 24 h after activation versus transduction at both 24 and 48 h did not affect the efficiency in terms of either percentage of cells transduced or CAR expression level per cell (i.e., mean fluorescence intensity [MFI].

Fig. 2, E and F). We observed, however, that the transduction efficiency at 48 h after activation was inferior to that obtained at 24 h after activation (Fig.

2, E and F). A schema of the T cell activation and transduction approaches compared are depicted in Fig. 2 G.

Finally, we observed highest CAR transduction efficiency in CD3+ lymphocytes activated with αCD3/CD28 beads in the presence of hIL-2 as compared with the other aforementioned activation methods (Fig. 2, H and I). Similar results were observed for CD8+ T cells, while for CD4+ T cells, the percentage CAR expression was the same for both αCD3/CD28-bead and αCD3-plate/CD28 activation (Fig.

S1 B). Thus, αCD3/CD28-bead activation was used for all further experiments. Notably, we also investigated concentrated lentiviral transduction of αCD3/CD28-bead–activated murine T cells using the same anti-VEGFR-2 CAR, and consistent with another study (Kerkar et al., 2011), we obtained very low transduction efficiency (∼10%, data not shown).

While long-term T cell culture in IL-2 drives terminal differentiation, the common γ-chain cytokines IL-7 and IL-15 have been reported to promote a central memory T cell (TCM cell) phenotype enabling superior persistence and in vivo tumor control upon ACT (Klebanoff et al., 2005). Thus, we next compared the expansion and functional properties of transduced murine CAR-T cells cultured in hIL-2 alone versus hIL-2 for the first 3 days, followed by hIL-7/IL-15 for the remainder of the culture period (Fig. 3 A).

Both hIL-7 and hIL-15 have been previously demonstrated to act on murine T cells to promote homeostatic proliferation and survival (Eisenman et al., 2002. Nanjappa et al., 2008). As for hIL-2–expanded CAR-T cells (Fig.

2 G), we observed that a single transduction of T cells at 24 h and subsequent expansion in hIL-7/IL-15 was sufficient to achieve a robust and stable transduction efficiency at a MOI as low as 5 (Fig. 3 B). Both culture conditions (hIL-2 alone versus hIL-2 followed by hIL-7/IL-15) enabled high CAR expression on day 7 (Fig.

3 C). On day 9, however, we observed a 26-fold expansion of CAR-T cells exposed to hIL-7/IL-15 as compared with a 9-fold expansion in the presence of hIL-2 alone at a standard concentration of 50 IU/ml (Fig. 3 D).

Moreover, CAR-T cells cultured with hIL-7/IL-15 continued to expand for at least 14 d, while T cells cultured in hIL-2 alone reached a plateau after 1 wk (Fig. 3 D) and exhibited significantly higher levels of cell death starting early in the culture (Fig. 3 E).

We also observed a significantly higher frequency of CD8+ T cells in the hIL-7/IL-15 culture (Fig. 3 F). Finally, transduced T cells expanded with hIL-7/IL-15 had a significantly higher proportion of TCM cells based on cell-surface expression of the hyaluronic acid receptor CD44 and the L-selectin CD62L from day 5 after cytokine addition (Fig.

3, G and H). We sought to evaluate the in vitro reactivity of hIL-2 only versus hIL-7/IL-15 expanded CAR-T cells against target antigen. On day 7 after transduction, we co-cultured CAR-T cells with bEnd3 murine endothelial cells expressing VEGFR-2, as well as with control VEGFR-2− H5V murine endothelial cells (Fig.

3 I). HIL-7/IL-15 expanded CAR-T cells secreted significantly higher levels of IFN-γ, granzyme B, and IL-2 (Fig. 3 J) after bEnd3 target cell recognition in vitro.

Because CAR-T cell expansion with hIL-7/IL-15 results in a higher frequency of CD8+ T cells as compared with hIL-2 only, we next sorted CD8+ T cells on day 7 after transduction and performed a co-culture with bEnd3 and H5V cells. Higher levels of granzyme B, IL-2, and IFN-γ were secreted by hIL-7/IL-15–expanded CD8+ CAR-T cells than hIL-2–expanded ones (Fig. S2).

Moreover, hIL-7/IL-15–expanded CAR-T cells exhibited significantly higher persistence (Fig. 3 K), division rates (Fig. 3 L), and numbers of proliferating CD8+ T cells after 4 d of co-culture (Fig.

3 M). Thus, as compared with hIL-2 alone, CAR-T cell expansion with hIL-7/IL-15 promotes higher viability and favors a TCM cell phenotype, more robust expansion, and superior secretion of cytokines and long-term proliferative capacity upon challenge with target cells. The high transduction efficiency achieved with our optimized method encouraged us to evaluate the coexpression of transgenes and test the impact of additional cargo on CAR-T cell performance.

Given the enhanced functional properties of CAR-T cells exposed to hIL-7/IL-15 at 48 h after transduction as opposed to hIL-2 alone, we focused on coengineering T cells to constitutively produce mIL-15. Notably, hIL-15 has been previously demonstrated to significantly improve the antitumor activity of human CAR-T cells targeting glioblastoma (Krenciute et al., 2017). A bicistronic retroviral vector encoding mIL-15 and the DC101 CAR, both driven by the 5′ LTR of the retroviagra (de Felipe et al., 1999) and separated by a self-cleaving 2A peptide sequence (T2A.

Liu et al., 2017), was built to express this 4G-CAR construct (Fig. 4 A). With a single round of transduction at a MOI as low as 5, we achieved a similarly high expression of the 4G- as the 2G-CAR (Fig.

4, B and C), as well as high intracellular expression of mIL-15 (Fig. 4 D). Significant mIL-15 was also detected by ELISA upon lysis of 4G-CAR-T cells (Fig.

4 E), but very low levels of mIL-15 were found in the culture supernatant (data not shown), presumably due to sequestration of the cytokine by cell-surface IL-15 receptor-α (IL-15-Rα), as has been previously observed for human T cells engineered to secrete hIL-15 (Markley and Sadelain, 2010). Our hypothesis was supported by the fact that we detected high levels of soluble mIL-15 in the supernatants of transfected human Phoenix Eco cells (i.e., the retroviagra producer cell line. Fig.

4 F). Moreover, 4G-CAR–transduced C1498 leukemia cells (which do not express IL-15-Rα. Fig.

S3 A) secreted high levels of mIL-15 (Fig. 4, G and H). Finally, we activated both 2G- and 4G-CAR-T cells with cognate antigen and found significant secretion of mIL-15 by the 4G-CAR-T cells (Fig.

4 I), as has similarly been reported in the context of engineered human T cells (Krenciute et al., 2017). We next sought to investigate the impact of mIL-15 coexpression on CAR-T cell signaling and phenotype. In the absence of exogenous cytokine in the culture supernatant, we observed elevated pSTAT5 in the 4G- versus 2G-CAR-T cells both in terms of frequency and level per cell (Fig.

4, J and K). We further evaluated IL-15-Rα expression and detected lower levels on 4G-CAR-T cells (Fig. 4, L and M), presumably due to receptor internalization (Dubois et al., 2002) and/or mIL-15 occupancy blocking the Ab binding site.

Subsequently, we assessed expression of the antiapoptotic protein Bcl-2, previously reported to enhance 2G- versus first-generation (1G)–CAR-T cell persistence (Song et al., 2012), and found higher expression levels on days 2 and 5 after transduction for 4G- as compared with 2G-CAR-T cells in the absence of exogenous cytokines (Fig. S3, B and C). In addition, we observed significantly higher frequencies of Ki67+ Bcl-2+ 4G-CAR-T cells on days 2 and 5 after transduction (Fig.

5, A and B). Thus, mIL-15 coexpression appears to augment both CAR-T cell survival and proliferation. We further assessed the phenotype of CAR-T cells in the absence of exogenous cytokines in the culture medium and found that on day 2 following transduction, 2G- and 4G-CAR-T cells displayed no differences in the proportion of naive (CD62Lhigh CD44low), central memory (CM.

CD62Lhigh CD44high) and effector memory (EM. CD62Llow CD44high) T cell phenotype populations. However, by day 5 after transduction, 4G-CAR-T cells had a higher proportion of naive and CM cells and fewer EM cells, as compared with 2G-CAR-T cells (Fig.

5, C and D). Notably, there were significantly lower levels of the inhibitory receptor programmed cell death 1 (PD-1. Both percentage and MFI) on 4G- compared with 2G-CAR-T cells (Fig.

5, E and F). Consistent with the above findings, we observed that in the absence of exogenous cytokine the 4G-CAR-T cells exhibited increased expansion during the first 2 d after transduction as compared with the 2G-CAR-T cells (Fig. 5 G).

Both 2G- and 4G-CAR-T cells began to contract at a similar rate from day 2 after transduction, but there were significantly more 4G- than 2G-CAR-T cells on days 5 and 7 (Fig. 5 G). Finally, we observed higher viability of 4G-CAR-T cells over time (Fig.

5 H). Thus, with our optimized protocol, we achieved a high rate of T cell transduction with retroviagra coexpressing a CAR and mIL-15, and in the absence of exogenous cytokines, these 4G-CAR-T cells exhibit a less differentiated and inhibitory phenotype as well as enhanced expansion and viability in vitro. We next sought to evaluate the expansion of 4G- versus 2G-CAR-T cells in the presence of exogenous hIL-7/IL-15.

We observed continuous expansion of 4G- and 2G-CAR-T cells for 2 wk but at a significantly higher rate for the 4G-CAR-T cells (Fig. 6 A). Viability was similarly high for both over a 10-d period (Fig.

6 B). Notably, 4G-CAR-T cells cultured in hIL-2 demonstrated enhanced expansion at days 5 and 9 as compared with similarly cultured 2G-CAR-T cells (Fig. 6 C).

We subsequently sought to determine if increasing hIL-15 levels in the medium could augment 2G-CAR-T cell expansion. We demonstrated that 2G-CAR-T cells cultured in the presence of increasing concentrations of hIL-15 (while maintaining hIL-7 at 10 ng/ml) achieved significant increases in fold expansion, reaching or surpassing that of 4G-CAR-T cells (cultured in standard 10 ng/ml hIL-15) at day 9 after transduction in the presence of 50 ng/ml or 100 ng/ml hIL-15, respectively (Fig. 6 D and Fig.

S3 D). Notably, increasing the concentration of hIL-15 in the culture medium from 10 to 50 or 100 ng/ml significantly increased the expansion of 4G-CAR-T cells (Fig. 6 E), and the fold expansion of 4G-CAR-T cells was nearly double compared to that of 2G-CAR-T cells (cultured in equivalent increased hIL-15 concentrations) on day 9 after transduction (Fig.

6 E and Fig. S3 D). We next tested the effector capacity of 4G- as compared with 2G-CAR-T cells against target cells.

Significantly higher levels of IL-2 were produced by 4G- than 2G-CAR-T cells upon co-culture with VEGFR-2+ bEnd3 cells at 1 wk after transduction, while neither reacted against VEGFR-2− H5V cells (Fig. 6 F). We further observed mIL-15 secretion by 4G-CAR-T cells only upon co-culture with bEnd3 cells and not H5V cells (Fig.

6 G). In addition, there was significantly higher expansion of 4G- than 2G-CAR-T cells at day 4 after co-culture with bEnd3 cells, and neither expanded upon co-culture with H5V cells (Fig. 6, H and I).

The 4G-CAR-T cells also exhibited significantly higher proliferation (Fig. 6 J) and numbers of dividing CD8+ T cells compared with 2G-CAR- or control T cells at day 4 of the co-culture (Fig. 6, K and L).

The ability of 4G- and 2G-CAR-T cells to induce apoptosis of target cells was equivalent (Fig. 6 M, and N), in accordance with previous evaluation of hIL-15-CAR-T cells (Krenciute et al., 2017). We further tested the 4G- and 2G-CAR-T cells in vivo against subcutaneous B16 melanoma tumors.

Briefly, on day 8 after tumor cell injection, with tumors approaching 20–40 mm3 in volume, CD45.2+ C57BL/6 mice were lymphodepleted by sublethal total body irradiation and subsequently received two intravenous T cell injections (8–9 × 106 CD45.1+ cells at each injection. Fig. 7 A).

In mice treated with control T cells, the tumors grew rapidly, while modest tumor control was observed in mice that received 2G-CAR-T cells, similar to previous reports for this tumor vasculature targeting CAR (Chinnasamy et al., 2010, 2012). Mice treated with 4G-CAR-T cells, however, had significantly attenuated tumor growth (Fig. 7 B).

Ex vivo analysis of transferred CD45.1+ T cells in the blood, spleen, and tumor on day 11 after ACT revealed significantly higher engraftment of 4G- than 2G-CAR-T cells and control T cells (Fig. 7, C–E). In addition, CAR expression levels were higher for 4G- than 2G-CAR-T cells in blood, spleen, and tumor (Fig.

7, C, D, and F). Notably, we observed sustained presence of the mIL-15 transgene in the spleens and tumors of mice treated with 4G-CAR-T cells (Fig. 7, D and F).

Finally, in agreement with our in vitro data, 4G-CAR-T cells expressed significantly higher levels of the antiapoptotic protein Bcl-2 in vivo (Fig. 7 G. Flow cytometry gating strategy shown in Fig.

S4). Thus, mIL-15 coexpression by CAR-T cells enhances not only expansion and in vitro effector functions but also in vivo persistence and tumor control. Finally, we sought to comprehensively evaluate the effect of mIL-15 coexpression on CAR-T cells in vivo and to determine if endogenous immune cells are also impacted.

Following the same ACT strategy as demonstrated above (Fig. 8 A), we observed that 4G-CAR-T cells in the spleen (Fig. 8, B and C) and tumor-draining lymph nodes (Fig.

S5, A and B) exhibited a higher frequency of Ki67 (cellular marker for proliferation) than 2G-CAR-T cells. In the tumor, despite that Ki67 expression levels were similar for both 4G- and 2G-CAR-T cells (Fig. 8, D and E), the 4G-CAR-T cells displayed significantly lower levels of PD-1 (Fig.

8, F and G). Analysis of endogenous immune infiltrate revealed significantly higher coexpression of CD69 and Ki67 by natural killer (NK) cells in 4G- as compared with 2G-CAR-T cell–treated tumors (Fig. 8, H and I).

In addition, in 4G-CAR-T cell–treated mice there were lower levels of tumor-residing M2 (F4/80+ CD206+) macrophages, which are often associated with immunosuppression in the TME (Fig. 8 J, K). Both the activation of NK cells and lower levels of M2 macrophages may contribute to tumor control in the context of 4G-CAR-T cell transfer.

Tumor-residing B cells (CD19+ MHC II+) were not detected (Fig. S5, C and D), and there were no differences in splenic B cell frequency in any of the treated mice (Fig. S5, E and F).

Finally, similar frequencies of tumor-residing dendritic cells (DCs. CD11b− CD11c+) were observed among the control and CAR-T cell–treated mice (Fig. S5, G and H).

The flow cytometry gating strategy for the ex vivo characterization of the different immune cell populations is shown in Fig. S4. Thus, 4G-CAR-T cells coexpressing mIL-15, in addition to conferring enhanced tumor control as compared with 2G-CAR-T cells, also reprogram the TME in favor of protective endogenous immunity.

CAR-T cell therapy has yielded unprecedented clinical responses against some hematological malignancies, but not against epithelial-derived solid tumors (Irving et al., 2017). Rational combinatorial treatments and innovative CAR-T cell coengineering strategies (Lanitis et al., 2020) offer solutions for overcoming obstacles in the solid TME, but these are best evaluated in immunocompetent mice to enable the interplay of the endogenous immune system. In this study, we have presented optimized conditions for murine T cell activation, retroviral transduction, and expansion that allowed us to achieve consistently high and stable transgene expression levels, as well as robust expansion of both 2G- and 4G-CAR-T cells having a predominantly TCM cell phenotype, which is favored for ACT (Melchionda et al., 2005.

Gattinoni et al., 2005. Zhou et al., 2005). We have also elucidated the beneficial impact of mIL-15 coexpression by murine CAR-T cells both in vitro and in vivo.

Retroviral vectors, most commonly derived from the murine stem cell viagra (MSCV), a derivative of the Moloney murine leukemia viagra, have proven to be the most effective approach for stably introducing genes into murine T cells (Kerkar et al., 2011). Lentiviagra, however, has demonstrated poor gene transfer in murine T cells, likely due to impaired completion of reverse transcription and of nuclear import of the viral preintegration complex (Baumann et al., 2004. Tsurutani et al., 2007).

Most examples of efficient murine T cell retroviral transduction are for small and easily expressed reporter genes like GFP (Kurachi et al., 2017. Zhang et al., 2003) or 1G-CARs comprising the CD3ζ endodomain only (Lee et al., 2009). Retroviagra-mediated expression of 2G-CARs has proven less robust both in terms of percentage transduction and expression level per T cell (Kochenderfer et al., 2010.

Davila et al., 2013. Fu et al., 2013). Moreover, the long-term stability of CAR expression by murine T cells has not previously been thoroughly evaluated (Kusabuka et al., 2016.

Kochenderfer et al., 2010). Despite that it is common procedure to concentrate lentiviagra via ultracentrifugation, this is usually not performed for CAR-encoding retroviagraes. In this study, we demonstrated that retroviagra can be efficiently concentrated, leading to significantly improved CAR transduction efficiencies.

We further observed a correlation between CD8+ T cell activation levels (the highest level was achieved by αCD3/CD28 bead stimulation) and transduction efficiency. Previous studies have presented CAR expression early after transduction (2–3 d. Tran et al., 2013.

Kusabuka et al., 2016. Kochenderfer et al., 2010) and thus cannot distinguish from pseudo-transduction (Case et al., 1999. Costello et al., 2000).

In addition, some studies have applied antibiotic selection for enrichment of CAR-T cells (Kusabuka et al., 2016) or have measured GFP (or other markers) that can overestimate transduction efficiency. Here, we have demonstrated robust, long-term CAR expression in murine T cells by staining with recombinant target antigen and in the absence of any selection/enrichment method. In this study, we have also shown the utility of the common γ-chain cytokines hIL-7/IL-15 for enhanced CAR-T cell expansion and survival, as well as for promoting a TCM cell phenotype and ameliorating effector function.

Others have reported superior tumor control by IL-7/IL-15 than IL-2–expanded T cells (Cha et al., 2010. Gattinoni et al., 2005. Mueller et al., 2008).

It has also been previously demonstrated that exposure of murine T cells to IL-2 can potentiate apoptosis by suppressing the inhibitor of Fas signaling, FLIP (FLICE-inhibitory protein), and enhancing the expression of the proapoptotic molecule Fas ligand (Lenardo, 1991. Refaeli et al., 1998). In contrast, IL-7 and IL-15 inhibit activation-induced cell death, support the proliferation and survival of T cells (Waldmann, 2015.

Jiang et al., 2004. Cha et al., 2010), promote a TCM cell phenotype characterized by longer telomeres, and elevate T cell persistence and antitumor efficacy (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005. Klebanoff et al., 2004. Le et al., 2009).

Similarly, it has been shown that IL-7 and IL-15 enable enhanced human CAR-T cell effector function upon antigen recognition (Xu et al., 2014. Zhou et al., 2019) and that exogenous IL-15 can expand anti-CD19 CAR-T cells in treated patients by up to 180-fold (Ramanayake et al., 2015). Contradictory reports of lower murine T cell function in vitro following culture in IL-7/IL-15 versus IL-2 alone are presumably due to the method of T cell stimulation used, differences in the concentration of IL-2 used, and the duration of expansion (Cha et al., 2010.

Gattinoni et al., 2005. Mueller et al., 2008). We further showed that our methodologies enable the efficient coexpression of mIL-15 and a CAR (encoded by a bicistronic vector) in murine T cells.

Human CAR-T cells coexpressing hIL-15 as a fusion protein tethered to the cell surface, or in a secreted form, have previously demonstrated enhanced expansion and persistence upon antigen stimulation (both in vitro and in vivo), as well as increased tumor control (Hoyos et al., 2010. Markley and Sadelain, 2010). As such, there are high expectations for clinical efficacy of IL-15–CAR-T cells.

In nonactivated murine 4G-CAR-T cells, we observed very low levels of mIL-15 in the culture supernatant, but upon antigenic stimulation, significantly higher amounts were detected, in line with reports for hIL-15 CAR-T cells (Krenciute et al., 2017. Hoyos et al., 2010). Elevated levels of pSTAT5 in the 4G- versus 2G-CAR-T cells indicated active signaling by cytokine/receptor engagement.

The functional integrity of the coexpressed mIL-15 was further supported by enhanced 4G-CAR-T cell proliferation and survival, possibly due to up-regulation of the antiapoptotic molecule Bcl-2 (Wu et al., 2002. Shenoy et al., 2014). In addition, mIL-15 coexpression promoted a TCM cell phenotype, limited PD-1 up-regulation, and conferred superior effector function upon antigenic challenge.

The culture methods presented herein comprising hIL-7/hIL-15 in the medium permitted efficient murine CAR-T cell expansion, which was significantly reinforced upon mIL-15 coexpression by CAR-T cells. This enabled us to further investigate the efficacy of 4G-CAR-T cells in vivo against B16 melanoma tumors. We observed higher tumor control and persistence of 4G- as compared with the 2G-CAR-T cells and sustained expression of the mIL-15 transgene.

Moreover 4G-CAR-T cells exhibited higher Bcl-2 levels, in line with our in vitro data, suggesting that mIL-15 can render CAR-T cells more resistant to apoptosis in vivo. The coexpression of mIL-15 was also associated with significantly lower up-regulation of PD-1, an inhibitory receptor that can impair T cell function in the TME (Ahmadzadeh et al., 2009). Finally, evaluation of endogenous tumor immune infiltrate revealed a significantly higher frequency of activated (CD69+ Ki67+) NK cells and fewer M2 (F4/80+ CD206+) macrophages upon 4G- versus 2G-CAR-T cell transfer.

As NK cells are associated with delayed melanoma tumor growth (Nath et al., 2019), and M2 macrophages have been shown to contribute to tumor progression and metastasis (Poh and Ernst, 2018), the observed TME remodeling upon 4G-CAR-T cell transfer is favorable for tumor control. Our findings are consistent with prior studies. For example, coadministration of IL-15 with tumor-directed monoclonal antibodies enhanced Ab-dependent cellular cytotoxicity by augmenting both NK cell and macrophage activation (Zhang et al., 2018).

In another study, it was shown that the absence of IL-15 in immunocompetent mice promotes the formation of M2 macrophages (Gillgrass et al., 2014). In summary, we have presented comprehensive and highly reproducible methods for efficient retroviral transduction and robust expansion of murine CAR-T cells endowed with favorable properties for ACT studies in immunocompetent mice. We further demonstrated that coexpression of mIL-15 directly promotes CAR-T cell fitness and function and remodels the TME to favor tumor control.

As it is becoming apparent that endogenous immunity can play a critical role in either suppressing or supporting CAR-T cell function in the TME (Kuhn et al., 2019), comprehensive studies in immunocompetent mice are critical for accelerating the translation of effective CAR therapies to the clinic. The murine brain endothelioma cell line bEnd3, the murine immortalized heart endothelial cell line H5V, and the murine leukemia cell line C1498 were cultured in DMEM-GlutaMAX comprising 4,500 mg/liter glucose and 110 mg/liter sodium pyruvate and supplemented with 10% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. The melanoma cell line B16-F10 was grown as a monolayer in DMEM-GlutaMAX supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin sulfate.

Cells were passaged twice weekly to maintain them under exponential growth conditions and were routinely tested for mycoplasma contamination. The Phoenix Eco retroviral ecotropic packaging cell line, derived from immortalized normal human embryonic kidney cells, was maintained in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Primary murine T cells were cultured in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, and 10 mM nonessential amino acids (referred to as murine T cell culture medium).

Murine T cell culture medium was further supplemented with human cytokines as described in the method for T cell expansion. The retroviral vector pMSGV (murine stem cell viagra [MSCV]–based splice-gag vector) comprising the MSCV LTR was used as the backbone for all CAR constructs. A 2G-CAR consisting of the anti-VEGFR-2 scFv, DC101, the CD8α hinge (H), and TM region, followed by the EDs of CD28 and CD3ζ (DC101-28-z), was kindly provided by Dr.

Steven A. Rosenberg (National Cancer Institute, Bethesda, MD. Chinnasamy et al., 2010).

The DC101-28-z CAR was built by PCR amplification of a 362-bp fragment from the 2G construct with the primers. 5′-ACG​CGC​GGC​CGC​AAC​TAC​TAC​CAA​GC-3′ and 5′-ACG​CGT​CGA​CGG​GGC​GGT​ACG​CTG​CAA​AGT​CTC-3′ followed by NotI and SalI digestion of both the PCR product and the parental 2G vector, gel purification, and ligation. To generate the 4G-CAR construct encoding both mIL-15 and the VEGFR-2–directed CAR (mIL-15-T2A-DC101-28-z), a gene-string encoding the murine Igκ leader sequence followed by codon-optimized mIL-15 and T2A, flanked by XhoI and EcoRI restriction sites at the 5′ and 3′ ends, respectively, was synthesized.

The DC101-28-z construct and fragment were then digested (XhoI and EcoRI), gel purified, and ligated together. All genes strings were synthesized by GeneArt AG, and all constructs were fully sequenced by Microsynth AG. High-titer, replication-defective retroviagra was produced and concentrated as depicted in Fig.

1. Briefly, Phoenix Eco cells were seeded at 107 per T-150 tissue culture flask in 35 ml culture medium (Fig. 1 A, 1) 24 h before transfection with 14.4 µg pCL-Eco Retroviagra Packaging Vector and 21.4 µg pMSGV transfer plasmid using Turbofect (Thermo Fisher Scientific.

Fig. 1 A, 2). All plasmids were purified using HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Thermo Fisher Scientific).

For the transfection mixture, a 3:1 ratio of Turbofect/plasmid was prepared in 2 ml Opti-MEM and incubated for 30 min at room temperature (RT. Fig. 1 A, 2).

Medium was then removed from T-150 flasks bearing 80–90% confluent Phoenix Eco cells and the transfection mixture was applied and incubated for 1 min, followed by addition of 31 ml fresh medium (Fig. 1 A, 2). The viral supernatant was discarded 20–24 h after transfection and replaced with 33 ml fresh medium (Fig.

1 A, 5) after transfection, the supernatant was harvested, and viral particles were concentrated by ultracentrifugation for 2 h at 24,000 g at 4°C with a Beckman JS-24 rotor (Beckman Coulter) and resuspended in 0.4 ml murine T cell medium. The retroviagra was then used immediately, or aliquoted, frozen on dry ice, and stored at −80°C. As depicted in Fig.

1 B, murine T cells were isolated from single-cell suspensions of dissociated spleens from CD45.1+ congenic C57BL/6 mice bred in-house at the animal facility of the University of Lausanne (UNIL. Epalinges, Switzerland) using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies. Fig.

1 B, 1.1). T cells were plated at 106/ml in 24- or 48-well plates in T cell medium (described above) and stimulated with αCD3/CD28 Ab-coated beads (Invitrogen) at a bead to cell ratio of 2:1 and 50 IU/ml hIL-2 (Glaxo. Fig.

1 B, 1.1). Non–treated cell-culture grade 48- or 24-well plates (Corning Falcon) were precoated with 0.25 ml or 0.5 ml, respectively, of recombinant RetroNectin (Takara Bio) at a final concentration of 20 μg/ml, overnight (O/N) at 4°C (Fig. 1 B, 1.2).

1 d after T cell activation, the retronectin-precoated plates were washed with PBS, blocked with 2% BSA in PBS for 30 min at RT (Fig. 1 B, 2.1). Subsequently, plates were washed once, retroviagra was added at the MOI indicated in the figures, and plates were then spun at 2,000 g for 1.5 h at 32°C (Fig.

1 B, 2.2). The supernatants were then aspirated, and 0.5 to 106 of 24 h activated T cells were transferred to each coated well (48- or 24-well plates. Fig.

1 B, 2.3). The plates were centrifuged for 10 min at 300 g and incubated O/N (Fig. 1 B, 2.3).

In some experiments the transduction procedure was performed at 48 h, or at both 24 and 48 h after activation. The cultures were maintained at a cell density of 0.5 to 106 cells/ml and replenished with fresh T cell medium (supplemented with hIL-2 alone or hIL-2 followed by hIL-7/IL-15 on day 2 after transduction) every other day (Fig. 1 B, 3).

At day 7, CAR surface expression was assessed by flow cytometric analysis (as described below), and the rested engineered T cells were adjusted for equal expression before functional in vitro and in vivo assays (Fig. 1 B, 4). Murine C1498 leukemia cells were transduced as described above for primary murine T cells, except that they were not activated and were maintained afterwards in DMEM-GlutaMAX complete medium at a cell density of 3 × 105 viable cells/ml.

For flow cytometric analysis, cells were surface stained using antibodies against CD3ε (145-2C11), CD4 (GK1.5, RM4-5), CD8α (53–6.7), CD25 (PC61), CD44 (IM7), CD45.1 (A20), CD45 (30F/11), CD62L (MEL-14), CD69 (H1-2F3), IL-15-Rα (6B4C88), PD-1 (29F.1A12), Ly-6G (1A8), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD206 (C068C2), NK-1.1 (PK136), CD19 (6D5), and MHC class II (M5/114.15.2). Abs were purchased from eBioscience and BioLegend or produced in-house from hybridomas by the flow cytometry platform. DC101-CAR expression by retrovirally transduced T cells was detected by incubation with soluble mouse VEGFR-2–hIgG-Fc fusion protein (R&D Systems) followed by staining with labeled goat anti-hIgG Fc (clone HP6017.

Biolegend). Thy1.1-T cells were stained in parallel as a negative control. VEGFR-2 expression by mouse endothelial cell lines was evaluated by cell-surface staining with rat anti-VEGFR-2 Ab (clone Avas12.

BioLegend) and matched isotype control (Rat IgG2a κ isotype. Clone RTK2758. BioLegend).

For detection of phosphorylated STAT5, cells were fixed with BD Cytofix Fixation Buffer at 4°C for 15 min and permeabilized with BD Phosflow Perm Buffer III for 30 min at 4°C. Intracellular phospho-staining was performed for 1 h at RT in the dark with Ab against phospho-STAT5 (Tyr694. D47E7 XP Rabbit mAb 4322.

Cell Signaling). For intracellular staining of mIL-15 (clone AIO.3. EBioscience), Bcl-2 (clone 10C4.

EBioscience), and Ki67 (clone SolA15. EBioscience), T cells were fixed and then permeabilized using the FoxP3 transcription factor staining buffer set (eBioscience) according to the manufacturer’s recommendations. For the detection of mIL-15, the cells were further washed and incubated for 30 min with anti-rat IgG2a.

To discriminate dead cells, 7-AAD (BioLegend) staining was performed. Live/dead fixable Aqua Dead cell staining was used to exclude dead cells in the ex vivo analysis of immune cells derived from the spleens, tumors, and tumor-draining lymph nodes according to the manufacturer’s instructions (Molecular Probes, Life Technologies). Data were acquired with a BD flow cytometer and analyzed using FlowJo software (Tree Star).

Cells extracted from dissociated tumors were lysed using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). Total RNA was isolated using the RNeasy Mini Kit (Qiagen). After treatment with RNase-free DNase I (Qiagen), 400 ng of total RNA was reverse transcribed using PrimeScript First Strand cDNA Synthesis Kit (Takara Bio), as indicated by the manufacturer.

Quantitative real-time PCR was performed according to the commercial protocol using SYBR Green Fast PCR Master Mix (Thermo Fisher Scientific) and the 7500 Fast Real-Time PCR System (Applied Biosystems). Primers to specifically amplify regions of the DC101 scFv of the CAR cassette, or the mIL-15 transgene, were designed using the GenScript website and are as follows. DC101 forward, 5′-GCA​ACC​CAA​ACT​CCT​CAT​CT-3′.

DC101 reverse, 5′-TAT​CAT​CAG​CCT​CCA​CAG​GA-3′. IL-15 forward, 5′-CCA​GGA​TCT​ACA​GGC​GAC​AA-3′. IL-15 reverse, 5′-ATG​CTC​TGG​ATC​AGG​CTC​TC-3′.

PCR amplification of the housekeeping gene GAPDH was performed as a control, and to allow normalization of samples. The following primers were used for GAPDH. GAPDH forward, 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′.

GAPDH reverse, 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′. Each sample was run in triplicate, and each experiment included three nontemplate control wells. The relative mRNA levels (fold change) of each transgene among the different samples were quantified using the comparative 2−ΔΔCt method.

We wish to thank members of the Flow Cytometry Platform and the Animal Care Facility of UNIL for their excellent support. We also kindly thank Dr. Steven A.

Rosenberg (National Cancer Institute, Bethesda, MD) for sharing a second generation anti-VEGFR-2 CAR construct comprising the scFv DC101. This work was generously supported by Ludwig Cancer Research, the European Research Council (advanced grant 1400206AdG-322875 to G. Coukos), and the Biltema Foundation.

P. Romero is supported in part by Oncosuisse (grant KFS-4404-02-2018). Author contributions.

Lanitis conceived, designed, developed, and supervised the study and wrote the manuscript. E. Lanitis, G.

Spill conducted experiments and acquired and analyzed data. A. Spill supported the in vivo and ex vivo studies.

Romero and D. Dangaj reviewed the data and manuscript and provided suggestions. All authors read and approved the manuscript.Christopher Mapperley Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review &.

Editing 1Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK2Laboratory of Haematopoietic Stem Cell and Leukaemia Biology, Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for other works by this author on:.

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering viagra for sale online strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we viagra for sale online describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature–targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages.

The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in viagra for sale online immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic. The adoptive cell transfer (ACT) of ex vivo–expanded T lymphocytes has yielded robust and durable clinical responses against several cancer-types, such as tumor-infiltrating lymphocyte therapy of advanced melanoma (Mardiana et al., 2019). Another approach to ACT involves the redirection of peripheral blood T cells to tumor antigens by engineering them to express a chimeric antigen receptor (CAR) that triggers cellular activation upon tumor antigen binding viagra for sale online.

CAR-T cell therapy against hematologic malignancies, by targeting the B cell lineage antigens CD19 or the B cell maturation antigen, has proven efficacious in the clinic, and there is optimism that similar success will be achieved for some solid tumors (Geyer and Brentjens, 2016. Irving et al., 2017). A range of physical (Lanitis et al., viagra for sale online 2015) and immunometabolic barriers that can prevent T cell homing, transendothelial migration across tumor blood vessels, engraftment/persistence, and effector function limit the potency of CAR-T cell therapy against solid tumors (Brown et al., 2016. Louis et al., 2011).

Moreover, chronic antigen exposure and a lack of sufficient costimulation in the tumor microenvironment (TME) can cause CAR-T cell exhaustion (Irving et al., 2017). Coengineering of CAR-T cells may help to overcome viagra for sale online some of these obstacles (Lanitis et al., 2020). Genetic modifications, for example, can be made to enable better homing and tumor penetration or render CAR-T cells resistant to suppressive mechanisms in the TME (Stromnes et al., 2010). In addition, CAR-T cells can be armed with secretory molecules or additional receptors to support CAR-T cell activity viagra for sale online and/or harness endogenous immunity (Adachi et al., 2018.

Pegram et al., 2012). Preclinical evaluation of CAR-T cells has, for the most part, been performed with xenograft tumor models in immunodeficient mice (Lee et al., 2011. Mardiros et al., viagra for sale online 2013. Lanitis et al., 2012).

Although this approach can be used to evaluate human CAR-T cell persistence, homing, tumor control, and survival following ACT, critical parameters, including potential toxicity against normal tissues (Tran et al., 2013), and the impact of endogenous immunity on both tumor control and escape are not addressed in such models (Spear et al., 2012. Avanzi et al., 2018) viagra for sale online. As varying obstacles must be overcome to enhance CAR-T cell responses against different solid tumor types, comprehensive studies in immunocompetent syngeneic tumor models would enable more accurate screening of T cell engineering strategies and provide important insights into improving coengineering and combinatorial treatment approaches (Lanitis et al., 2020). A key limitation of CAR evaluation in syngeneic models stems from inadequate methodologies viagra for sale online for efficient murine T cell transduction and expansion.

Indeed, unless T cells derived from multiple donor spleens are transduced or the engineered T cells are restimulated for further expansion, which among other drawbacks are costly and can promote exhaustion and apoptosis (Bucks et al., 2009), respectively, current protocols yield insufficient numbers of CAR-T cells for ACT studies (Lee et al., 2009). The efficiency of cell-surface expression of second-generation (2G) CARs, comprising the endodomain (ED) of CD3ζ and one costimulatory ED (e.g., CD28 or 4-1BB), generally reaches 40–60% (Kochenderfer et al., 2010. Davila et al., 2013 viagra for sale online. Wang et al., 2014.

Fu et al., 2013). Although retroviral transduction rates as high as 70–80% for murine T cells have been reported, this was assessed at 2 to viagra for sale online 3 d after transduction (Tran et al., 2013. Kuhn et al., 2019. Kusabuka et al., 2016) and thus may include false positives due to transient expression from viagra for sale online nonintegrated vector DNA (i.e., pseudo-transduction.

Case et al., 1999, Costello et al., 2000). Moreover, short-term transduction efficiency is often based on reporter genes like GFP, which may overestimate CAR expression levels (Kusabuka et al., 2016. Kuhn et al., 2019 viagra for sale online. Davila et al., 2013).

Finally, while stable retroviral packaging and viagra for sale online producer cell lines may enable transduction efficiencies for 2G and third-generation (3G. I.e., a CAR having two or more costimulatory EDs) CARs of >60% (Fu et al., 2013), this is a laborious approach if multiple CAR designs are to be compared (Chinnasamy et al., 2010). Here, we report the development of an efficient and highly reproducible protocol for primary murine T cell retroviral transduction and expansion, yielding functional murine 2G-CAR-T cells, as well as fourth-generation (4G)-CAR-T cells coengineered to express murine IL-15 (mIL-15) for enhanced in vitro and in vivo function and TME reprogramming. Overall, our viagra for sale online work provides important tools for enabling the systematic evaluation of 4G-CAR-T cells in immunocompetent, syngeneic tumor-bearing mice, which we believe is critical for effective therapies reaching the clinic.

We sought to optimize murine T cell activation, transduction, and expansion methods for preclinical CAR therapy evaluation in immunocompetent, syngeneic tumor-bearing mice. The final protocol we developed is summarized in Fig. 1 and viagra for sale online is described in detail in Materials and methods. We used a 2G-CAR targeting vascular endothelial cell growth factor receptor 2 (VEGFR-2), comprising the well-characterized single-chain variable fragment (scFv) DC101 (Chinnasamy et al., 2010), a CD8α hinge and transmembrane domain, and the murine EDs of CD28 and CD3ζ.

The anti-VEGFR-2 CAR retroviral vector viagra for sale online is abbreviated as DC101-28z (Fig. 2 A). Because retroviagraes infect proliferating cells (Kusabuka et al., 2016. Chinnasamy et al., viagra for sale online 2010.

Hu et al., 2017), we first compared three commonly used methods for inducing T cell activation. (i) magnetic beads coated with anti-(α) CD3 antibody (Ab) and αCD28 Ab (αCD3/CD28 beads) plus recombinant human IL-2 (hIL-2), (ii) plate-immobilized αCD3 Ab along with soluble αCD28 Ab (αCD3-plate/CD28) plus hIL-2, and (iii) Concanavalin A plus hIL-2 and hIL-7. Stimulation with αCD3/CD28 beads viagra for sale online consistently resulted in the highest frequency of CD44+ CD62L− (recently activated, memory), CD25+ or CD69+ (activated), and Ki67+ (proliferating) CD3+ T cells (Fig. 2 B and Fig.

S1 A) viagra for sale online. We next found that concentration of viral particles through ultracentrifugation yielded higher viral titers (>3 × 107 transducing units/ml. Fig. 2 C) and enabled significantly viagra for sale online higher transduction of primary activated primary murine T cells as compared with unconcentrated retroviagra (Fig.

2 D), reaching a plateau at a multiplicity of (MOI) of 5 (∼80% CAR expression. Fig. 2 E). A single transduction at 24 h after activation versus transduction at both 24 and 48 h did not affect the efficiency in terms of either percentage of cells transduced or CAR expression level per cell (i.e., mean fluorescence intensity [MFI].

Fig. 2, E and F). We observed, however, that the transduction efficiency at 48 h after activation was inferior to that obtained at 24 h after activation (Fig. 2, E and F).

A schema of the T cell activation and transduction approaches compared are depicted in Fig. 2 G. Finally, we observed highest CAR transduction efficiency in CD3+ lymphocytes activated with αCD3/CD28 beads in the presence of hIL-2 as compared with the other aforementioned activation methods (Fig. 2, H and I).

Similar results were observed for CD8+ T cells, while for CD4+ T cells, the percentage CAR expression was the same for both αCD3/CD28-bead and αCD3-plate/CD28 activation (Fig. S1 B). Thus, αCD3/CD28-bead activation was used for all further experiments. Notably, we also investigated concentrated lentiviral transduction of αCD3/CD28-bead–activated murine T cells using the same anti-VEGFR-2 CAR, and consistent with another study (Kerkar et al., 2011), we obtained very low transduction efficiency (∼10%, data not shown).

While long-term T cell culture in IL-2 drives terminal differentiation, the common γ-chain cytokines IL-7 and IL-15 have been reported to promote a central memory T cell (TCM cell) phenotype enabling superior persistence and in vivo tumor control upon ACT (Klebanoff et al., 2005). Thus, we next compared the expansion and functional properties of transduced murine CAR-T cells cultured in hIL-2 alone versus hIL-2 for the first 3 days, followed by hIL-7/IL-15 for the remainder of the culture period (Fig. 3 A). Both hIL-7 and hIL-15 have been previously demonstrated to act on murine T cells to promote homeostatic proliferation and survival (Eisenman et al., 2002.

Nanjappa et al., 2008). As for hIL-2–expanded CAR-T cells (Fig. 2 G), we observed that a single transduction of T cells at 24 h and subsequent expansion in hIL-7/IL-15 was sufficient to achieve a robust and stable transduction efficiency at a MOI as low as 5 (Fig. 3 B).

Both culture conditions (hIL-2 alone versus hIL-2 followed by hIL-7/IL-15) enabled high CAR expression on day 7 (Fig. 3 C). On day 9, however, we observed a 26-fold expansion of CAR-T cells exposed to hIL-7/IL-15 as compared with a 9-fold expansion in the presence of hIL-2 alone at a standard concentration of 50 IU/ml (Fig. 3 D).

Moreover, CAR-T cells cultured with hIL-7/IL-15 continued to expand for at least 14 d, while T cells cultured in hIL-2 alone reached a plateau after 1 wk (Fig. 3 D) and exhibited significantly higher levels of cell death starting early in the culture (Fig. 3 E). We also observed a significantly higher frequency of CD8+ T cells in the hIL-7/IL-15 culture (Fig.

3 F). Finally, transduced T cells expanded with hIL-7/IL-15 had a significantly higher proportion of TCM cells based on cell-surface expression of the hyaluronic acid receptor CD44 and the L-selectin CD62L from day 5 after cytokine addition (Fig. 3, G and H). We sought to evaluate the in vitro reactivity of hIL-2 only versus hIL-7/IL-15 expanded CAR-T cells against target antigen.

On day 7 after transduction, we co-cultured CAR-T cells with bEnd3 murine endothelial cells expressing VEGFR-2, as well as with control VEGFR-2− H5V murine endothelial cells (Fig. 3 I). HIL-7/IL-15 expanded CAR-T cells secreted significantly higher levels of IFN-γ, granzyme B, and IL-2 (Fig. 3 J) after bEnd3 target cell recognition in vitro.

Because CAR-T cell expansion with hIL-7/IL-15 results in a higher frequency of CD8+ T cells as compared with hIL-2 only, we next sorted CD8+ T cells on day 7 after transduction and performed a co-culture with bEnd3 and H5V cells. Higher levels of granzyme B, IL-2, and IFN-γ were secreted by hIL-7/IL-15–expanded CD8+ CAR-T cells than hIL-2–expanded ones (Fig. S2). Moreover, hIL-7/IL-15–expanded CAR-T cells exhibited significantly higher persistence (Fig.

3 K), division rates (Fig. 3 L), and numbers of proliferating CD8+ T cells after 4 d of co-culture (Fig. 3 M). Thus, as compared with hIL-2 alone, CAR-T cell expansion with hIL-7/IL-15 promotes higher viability and favors a TCM cell phenotype, more robust expansion, and superior secretion of cytokines and long-term proliferative capacity upon challenge with target cells.

The high transduction efficiency achieved with our optimized method encouraged us to evaluate the coexpression of transgenes and test the impact of additional cargo on CAR-T cell performance. Given the enhanced functional properties of CAR-T cells exposed to hIL-7/IL-15 at 48 h after transduction as opposed to hIL-2 alone, we focused on coengineering T cells to constitutively produce mIL-15. Notably, hIL-15 has been previously demonstrated to significantly improve the antitumor activity of human CAR-T cells targeting glioblastoma (Krenciute et al., 2017). A bicistronic retroviral vector encoding mIL-15 and the DC101 CAR, both driven by the 5′ LTR of the retroviagra (de Felipe et al., 1999) and separated by a self-cleaving 2A peptide sequence (T2A.

Liu et al., 2017), was built to express this 4G-CAR construct (Fig. 4 A). With a single round of transduction at a MOI as low as 5, we achieved a similarly high expression of the 4G- as the 2G-CAR (Fig. 4, B and C), as well as high intracellular expression of mIL-15 (Fig.

4 D). Significant mIL-15 was also detected by ELISA upon lysis of 4G-CAR-T cells (Fig. 4 E), but very low levels of mIL-15 were found in the culture supernatant (data not shown), presumably due to sequestration of the cytokine by cell-surface IL-15 receptor-α (IL-15-Rα), as has been previously observed for human T cells engineered to secrete hIL-15 (Markley and Sadelain, 2010). Our hypothesis was supported by the fact that we detected high levels of soluble mIL-15 in the supernatants of transfected human Phoenix Eco cells (i.e., the retroviagra producer cell line.

Fig. 4 F). Moreover, 4G-CAR–transduced C1498 leukemia cells (which do not express IL-15-Rα. Fig.

S3 A) secreted high levels of mIL-15 (Fig. 4, G and H). Finally, we activated both 2G- and 4G-CAR-T cells with cognate antigen and found significant secretion of mIL-15 by the 4G-CAR-T cells (Fig. 4 I), as has similarly been reported in the context of engineered human T cells (Krenciute et al., 2017).

We next sought to investigate the impact of mIL-15 coexpression on CAR-T cell signaling and phenotype. In the absence of exogenous cytokine in the culture supernatant, we observed elevated pSTAT5 in the 4G- versus 2G-CAR-T cells both in terms of frequency and level per cell (Fig. 4, J and K). We further evaluated IL-15-Rα expression and detected lower levels on 4G-CAR-T cells (Fig.

4, L and M), presumably due to receptor internalization (Dubois et al., 2002) and/or mIL-15 occupancy blocking the Ab binding site. Subsequently, we assessed expression of the antiapoptotic protein Bcl-2, previously reported to enhance 2G- versus first-generation (1G)–CAR-T cell persistence (Song et al., 2012), and found higher expression levels on days 2 and 5 after transduction for 4G- as compared with 2G-CAR-T cells in the absence of exogenous cytokines (Fig. S3, B and C). In addition, we observed significantly higher frequencies of Ki67+ Bcl-2+ 4G-CAR-T cells on days 2 and 5 after transduction (Fig.

5, A and B). Thus, mIL-15 coexpression appears to augment both CAR-T cell survival and proliferation. We further assessed the phenotype of CAR-T cells in the absence of exogenous cytokines in the culture medium and found that on day 2 following transduction, 2G- and 4G-CAR-T cells displayed no differences in the proportion of naive (CD62Lhigh CD44low), central memory (CM. CD62Lhigh CD44high) and effector memory (EM.

CD62Llow CD44high) T cell phenotype populations. However, by day 5 after transduction, 4G-CAR-T cells had a higher proportion of naive and CM cells and fewer EM cells, as compared with 2G-CAR-T cells (Fig. 5, C and D). Notably, there were significantly lower levels of the inhibitory receptor programmed cell death 1 (PD-1.

Both percentage and MFI) on 4G- compared with 2G-CAR-T cells (Fig. 5, E and F). Consistent with the above findings, we observed that in the absence of exogenous cytokine the 4G-CAR-T cells exhibited increased expansion during the first 2 d after transduction as compared with the 2G-CAR-T cells (Fig. 5 G).

Both 2G- and 4G-CAR-T cells began to contract at a similar rate from day 2 after transduction, but there were significantly more 4G- than 2G-CAR-T cells on days 5 and 7 (Fig. 5 G). Finally, we observed higher viability of 4G-CAR-T cells over time (Fig. 5 H).

Thus, with our optimized protocol, we achieved a high rate of T cell transduction with retroviagra coexpressing a CAR and mIL-15, and in the absence of exogenous cytokines, these 4G-CAR-T cells exhibit a less differentiated and inhibitory phenotype as well as enhanced expansion and viability in vitro. We next sought to evaluate the expansion of 4G- versus 2G-CAR-T cells in the presence of exogenous hIL-7/IL-15. We observed continuous expansion of 4G- and 2G-CAR-T cells for 2 wk but at a significantly higher rate for the 4G-CAR-T cells (Fig. 6 A).

Viability was similarly high for both over a 10-d period (Fig. 6 B). Notably, 4G-CAR-T cells cultured in hIL-2 demonstrated enhanced expansion at days 5 and 9 as compared with similarly cultured 2G-CAR-T cells (Fig. 6 C).

We subsequently sought to determine if increasing hIL-15 levels in the medium could augment 2G-CAR-T cell expansion. We demonstrated that 2G-CAR-T cells cultured in the presence of increasing concentrations of hIL-15 (while maintaining hIL-7 at 10 ng/ml) achieved significant increases in fold expansion, reaching or surpassing that of 4G-CAR-T cells (cultured in standard 10 ng/ml hIL-15) at day 9 after transduction in the presence of 50 ng/ml or 100 ng/ml hIL-15, respectively (Fig. 6 D and Fig. S3 D).

Notably, increasing the concentration of hIL-15 in the culture medium from 10 to 50 or 100 ng/ml significantly increased the expansion of 4G-CAR-T cells (Fig. 6 E), and the fold expansion of 4G-CAR-T cells was nearly double compared to that of 2G-CAR-T cells (cultured in equivalent increased hIL-15 concentrations) on day 9 after transduction (Fig. 6 E and Fig. S3 D).

We next tested the effector capacity of 4G- as compared with 2G-CAR-T cells against target cells. Significantly higher levels of IL-2 were produced by 4G- than 2G-CAR-T cells upon co-culture with VEGFR-2+ bEnd3 cells at 1 wk after transduction, while neither reacted against VEGFR-2− H5V cells (Fig. 6 F). We further observed mIL-15 secretion by 4G-CAR-T cells only upon co-culture with bEnd3 cells and not H5V cells (Fig.

6 G). In addition, there was significantly higher expansion of 4G- than 2G-CAR-T cells at day 4 after co-culture with bEnd3 cells, and neither expanded upon co-culture with H5V cells (Fig. 6, H and I). The 4G-CAR-T cells also exhibited significantly higher proliferation (Fig.

6 J) and numbers of dividing CD8+ T cells compared with 2G-CAR- or control T cells at day 4 of the co-culture (Fig. 6, K and L). The ability of 4G- and 2G-CAR-T cells to induce apoptosis of target cells was equivalent (Fig. 6 M, and N), in accordance with previous evaluation of hIL-15-CAR-T cells (Krenciute et al., 2017).

We further tested the 4G- and 2G-CAR-T cells in vivo against subcutaneous B16 melanoma tumors. Briefly, on day 8 after tumor cell injection, with tumors approaching 20–40 mm3 in volume, CD45.2+ C57BL/6 mice were lymphodepleted by sublethal total body irradiation and subsequently received two intravenous T cell injections (8–9 × 106 CD45.1+ cells at each injection. Fig. 7 A).

In mice treated with control T cells, the tumors grew rapidly, while modest tumor control was observed in mice that received 2G-CAR-T cells, similar to previous reports for this tumor vasculature targeting CAR (Chinnasamy et al., 2010, 2012). Mice treated with 4G-CAR-T cells, however, had significantly attenuated tumor growth (Fig. 7 B). Ex vivo analysis of transferred CD45.1+ T cells in the blood, spleen, and tumor on day 11 after ACT revealed significantly higher engraftment of 4G- than 2G-CAR-T cells and control T cells (Fig.

7, C–E). In addition, CAR expression levels were higher for 4G- than 2G-CAR-T cells in blood, spleen, and tumor (Fig. 7, C, D, and F). Notably, we observed sustained presence of the mIL-15 transgene in the spleens and tumors of mice treated with 4G-CAR-T cells (Fig.

7, D and F). Finally, in agreement with our in vitro data, 4G-CAR-T cells expressed significantly higher levels of the antiapoptotic protein Bcl-2 in vivo (Fig. 7 G. Flow cytometry gating strategy shown in Fig.

S4). Thus, mIL-15 coexpression by CAR-T cells enhances not only expansion and in vitro effector functions but also in vivo persistence and tumor control. Finally, we sought to comprehensively evaluate the effect of mIL-15 coexpression on CAR-T cells in vivo and to determine if endogenous immune cells are also impacted. Following the same ACT strategy as demonstrated above (Fig.

8 A), we observed that 4G-CAR-T cells in the spleen (Fig. 8, B and C) and tumor-draining lymph nodes (Fig. S5, A and B) exhibited a higher frequency of Ki67 (cellular marker for proliferation) than 2G-CAR-T cells. In the tumor, despite that Ki67 expression levels were similar for both 4G- and 2G-CAR-T cells (Fig.

8, D and E), the 4G-CAR-T cells displayed significantly lower levels of PD-1 (Fig. 8, F and G). Analysis of endogenous immune infiltrate revealed significantly higher coexpression of CD69 and Ki67 by natural killer (NK) cells in 4G- as compared with 2G-CAR-T cell–treated tumors (Fig. 8, H and I).

In addition, in 4G-CAR-T cell–treated mice there were lower levels of tumor-residing M2 (F4/80+ CD206+) macrophages, which are often associated with immunosuppression in the TME (Fig. 8 J, K). Both the activation of NK cells and lower levels of M2 macrophages may contribute to tumor control in the context of 4G-CAR-T cell transfer. Tumor-residing B cells (CD19+ MHC II+) were not detected (Fig.

S5, C and D), and there were no differences in splenic B cell frequency in any of the treated mice (Fig. S5, E and F). Finally, similar frequencies of tumor-residing dendritic cells (DCs. CD11b− CD11c+) were observed among the control and CAR-T cell–treated mice (Fig.

S5, G and H). The flow cytometry gating strategy for the ex vivo characterization of the different immune cell populations is shown in Fig. S4. Thus, 4G-CAR-T cells coexpressing mIL-15, in addition to conferring enhanced tumor control as compared with 2G-CAR-T cells, also reprogram the TME in favor of protective endogenous immunity.

CAR-T cell therapy has yielded unprecedented clinical responses against some hematological malignancies, but not against epithelial-derived solid tumors (Irving et al., 2017). Rational combinatorial treatments and innovative CAR-T cell coengineering strategies (Lanitis et al., 2020) offer solutions for overcoming obstacles in the solid TME, but these are best evaluated in immunocompetent mice to enable the interplay of the endogenous immune system. In this study, we have presented optimized conditions for murine T cell activation, retroviral transduction, and expansion that allowed us to achieve consistently high and stable transgene expression levels, as well as robust expansion of both 2G- and 4G-CAR-T cells having a predominantly TCM cell phenotype, which is favored for ACT (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005). We have also elucidated the beneficial impact of mIL-15 coexpression by murine CAR-T cells both in vitro and in vivo. Retroviral vectors, most commonly derived from the murine stem cell viagra (MSCV), a derivative of the Moloney murine leukemia viagra, have proven to be the most effective approach for stably introducing genes into murine T cells (Kerkar et al., 2011). Lentiviagra, however, has demonstrated poor gene transfer in murine T cells, likely due to impaired completion of reverse transcription and of nuclear import of the viral preintegration complex (Baumann et al., 2004.

Tsurutani et al., 2007). Most examples of efficient murine T cell retroviral transduction are for small and easily expressed reporter genes like GFP (Kurachi et al., 2017. Zhang et al., 2003) or 1G-CARs comprising the CD3ζ endodomain only (Lee et al., 2009). Retroviagra-mediated expression of 2G-CARs has proven less robust both in terms of percentage transduction and expression level per T cell (Kochenderfer et al., 2010.

Davila et al., 2013. Fu et al., 2013). Moreover, the long-term stability of CAR expression by murine T cells has not previously been thoroughly evaluated (Kusabuka et al., 2016. Kochenderfer et al., 2010).

Despite that it is common procedure to concentrate lentiviagra via ultracentrifugation, this is usually not performed for CAR-encoding retroviagraes. In this study, we demonstrated that retroviagra can be efficiently concentrated, leading to significantly improved CAR transduction efficiencies. We further observed a correlation between CD8+ T cell activation levels (the highest level was achieved by αCD3/CD28 bead stimulation) and transduction efficiency. Previous studies have presented CAR expression early after transduction (2–3 d.

Tran et al., 2013. Kusabuka et al., 2016. Kochenderfer et al., 2010) and thus cannot distinguish from pseudo-transduction (Case et al., 1999. Costello et al., 2000).

In addition, some studies have applied antibiotic selection for enrichment of CAR-T cells (Kusabuka et al., 2016) or have measured GFP (or other markers) that can overestimate transduction efficiency. Here, we have demonstrated robust, long-term CAR expression in murine T cells by staining with recombinant target antigen and in the absence of any selection/enrichment method. In this study, we have also shown the utility of the common γ-chain cytokines hIL-7/IL-15 for enhanced CAR-T cell expansion and survival, as well as for promoting a TCM cell phenotype and ameliorating effector function. Others have reported superior tumor control by IL-7/IL-15 than IL-2–expanded T cells (Cha et al., 2010.

Gattinoni et al., 2005. Mueller et al., 2008). It has also been previously demonstrated that exposure of murine T cells to IL-2 can potentiate apoptosis by suppressing the inhibitor of Fas signaling, FLIP (FLICE-inhibitory protein), and enhancing the expression of the proapoptotic molecule Fas ligand (Lenardo, 1991. Refaeli et al., 1998).

In contrast, IL-7 and IL-15 inhibit activation-induced cell death, support the proliferation and survival of T cells (Waldmann, 2015. Jiang et al., 2004. Cha et al., 2010), promote a TCM cell phenotype characterized by longer telomeres, and elevate T cell persistence and antitumor efficacy (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005. Klebanoff et al., 2004. Le et al., 2009). Similarly, it has been shown that IL-7 and IL-15 enable enhanced human CAR-T cell effector function upon antigen recognition (Xu et al., 2014.

Zhou et al., 2019) and that exogenous IL-15 can expand anti-CD19 CAR-T cells in treated patients by up to 180-fold (Ramanayake et al., 2015). Contradictory reports of lower murine T cell function in vitro following culture in IL-7/IL-15 versus IL-2 alone are presumably due to the method of T cell stimulation used, differences in the concentration of IL-2 used, and the duration of expansion (Cha et al., 2010. Gattinoni et al., 2005. Mueller et al., 2008).

We further showed that our methodologies enable the efficient coexpression of mIL-15 and a CAR (encoded by a bicistronic vector) in murine T cells. Human CAR-T cells coexpressing hIL-15 as a fusion protein tethered to the cell surface, or in a secreted form, have previously demonstrated enhanced expansion and persistence upon antigen stimulation (both in vitro and in vivo), as well as increased tumor control (Hoyos et al., 2010. Markley and Sadelain, 2010). As such, there are high expectations for clinical efficacy of IL-15–CAR-T cells.

In nonactivated murine 4G-CAR-T cells, we observed very low levels of mIL-15 in the culture supernatant, but upon antigenic stimulation, significantly higher amounts were detected, in line with reports for hIL-15 CAR-T cells (Krenciute et al., 2017. Hoyos et al., 2010). Elevated levels of pSTAT5 in the 4G- versus 2G-CAR-T cells indicated active signaling by cytokine/receptor engagement. The functional integrity of the coexpressed mIL-15 was further supported by enhanced 4G-CAR-T cell proliferation and survival, possibly due to up-regulation of the antiapoptotic molecule Bcl-2 (Wu et al., 2002.

Shenoy et al., 2014). In addition, mIL-15 coexpression promoted a TCM cell phenotype, limited PD-1 up-regulation, and conferred superior effector function upon antigenic challenge. The culture methods presented herein comprising hIL-7/hIL-15 in the medium permitted efficient murine CAR-T cell expansion, which was significantly reinforced upon mIL-15 coexpression by CAR-T cells. This enabled us to further investigate the efficacy of 4G-CAR-T cells in vivo against B16 melanoma tumors.

We observed higher tumor control and persistence of 4G- as compared with the 2G-CAR-T cells and sustained expression of the mIL-15 transgene. Moreover 4G-CAR-T cells exhibited higher Bcl-2 levels, in line with our in vitro data, suggesting that mIL-15 can render CAR-T cells more resistant to apoptosis in vivo. The coexpression of mIL-15 was also associated with significantly lower up-regulation of PD-1, an inhibitory receptor that can impair T cell function in the TME (Ahmadzadeh et al., 2009). Finally, evaluation of endogenous tumor immune infiltrate revealed a significantly higher frequency of activated (CD69+ Ki67+) NK cells and fewer M2 (F4/80+ CD206+) macrophages upon 4G- versus 2G-CAR-T cell transfer.

As NK cells are associated with delayed melanoma tumor growth (Nath et al., 2019), and M2 macrophages have been shown to contribute to tumor progression and metastasis (Poh and Ernst, 2018), the observed TME remodeling upon 4G-CAR-T cell transfer is favorable for tumor control. Our findings are consistent with prior studies. For example, coadministration of IL-15 with tumor-directed monoclonal antibodies enhanced Ab-dependent cellular cytotoxicity by augmenting both NK cell and macrophage activation (Zhang et al., 2018). In another study, it was shown that the absence of IL-15 in immunocompetent mice promotes the formation of M2 macrophages (Gillgrass et al., 2014).

In summary, we have presented comprehensive and highly reproducible methods for efficient retroviral transduction and robust expansion of murine CAR-T cells endowed with favorable properties for ACT studies in immunocompetent mice. We further demonstrated that coexpression of mIL-15 directly promotes CAR-T cell fitness and function and remodels the TME to favor tumor control. As it is becoming apparent that endogenous immunity can play a critical role in either suppressing or supporting CAR-T cell function in the TME (Kuhn et al., 2019), comprehensive studies in immunocompetent mice are critical for accelerating the translation of effective CAR therapies to the clinic. The murine brain endothelioma cell line bEnd3, the murine immortalized heart endothelial cell line H5V, and the murine leukemia cell line C1498 were cultured in DMEM-GlutaMAX comprising 4,500 mg/liter glucose and 110 mg/liter sodium pyruvate and supplemented with 10% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate.

The melanoma cell line B16-F10 was grown as a monolayer in DMEM-GlutaMAX supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin sulfate. Cells were passaged twice weekly to maintain them under exponential growth conditions and were routinely tested for mycoplasma contamination. The Phoenix Eco retroviral ecotropic packaging cell line, derived from immortalized normal human embryonic kidney cells, was maintained in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Primary murine T cells were cultured in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, and 10 mM nonessential amino acids (referred to as murine T cell culture medium).

Murine T cell culture medium was further supplemented with human cytokines as described in the method for T cell expansion. The retroviral vector pMSGV (murine stem cell viagra [MSCV]–based splice-gag vector) comprising the MSCV LTR was used as the backbone for all CAR constructs. A 2G-CAR consisting of the anti-VEGFR-2 scFv, DC101, the CD8α hinge (H), and TM region, followed by the EDs of CD28 and CD3ζ (DC101-28-z), was kindly provided by Dr. Steven A.

Rosenberg (National Cancer Institute, Bethesda, MD. Chinnasamy et al., 2010). The DC101-28-z CAR was built by PCR amplification of a 362-bp fragment from the 2G construct with the primers. 5′-ACG​CGC​GGC​CGC​AAC​TAC​TAC​CAA​GC-3′ and 5′-ACG​CGT​CGA​CGG​GGC​GGT​ACG​CTG​CAA​AGT​CTC-3′ followed by NotI and SalI digestion of both the PCR product and the parental 2G vector, gel purification, and ligation.

To generate the 4G-CAR construct encoding both mIL-15 and the VEGFR-2–directed CAR (mIL-15-T2A-DC101-28-z), a gene-string encoding the murine Igκ leader sequence followed by codon-optimized mIL-15 and T2A, flanked by XhoI and EcoRI restriction sites at the 5′ and 3′ ends, respectively, was synthesized. The DC101-28-z construct and fragment were then digested (XhoI and EcoRI), gel purified, and ligated together. All genes strings were synthesized by GeneArt AG, and all constructs were fully sequenced by Microsynth AG. High-titer, replication-defective retroviagra was produced and concentrated as depicted in Fig.

1. Briefly, Phoenix Eco cells were seeded at 107 per T-150 tissue culture flask in 35 ml culture medium (Fig. 1 A, 1) 24 h before transfection with 14.4 µg pCL-Eco Retroviagra Packaging Vector and 21.4 µg pMSGV transfer plasmid using Turbofect (Thermo Fisher Scientific. Fig.

1 A, 2). All plasmids were purified using HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Thermo Fisher Scientific). For the transfection mixture, a 3:1 ratio of Turbofect/plasmid was prepared in 2 ml Opti-MEM and incubated for 30 min at room temperature (RT. Fig.

1 A, 2). Medium was then removed from T-150 flasks bearing 80–90% confluent Phoenix Eco cells and the transfection mixture was applied and incubated for 1 min, followed by addition of 31 ml fresh medium (Fig. 1 A, 2). The viral supernatant was discarded 20–24 h after transfection and replaced with 33 ml fresh medium (Fig.

1 A, 3). At 48 (Fig. 1 A, 4) and 72 h (Fig. 1 A, 5) after transfection, the supernatant was harvested, and viral particles were concentrated by ultracentrifugation for 2 h at 24,000 g at 4°C with a Beckman JS-24 rotor (Beckman Coulter) and resuspended in 0.4 ml murine T cell medium.

The retroviagra was then used immediately, or aliquoted, frozen on dry ice, and stored at −80°C. As depicted in Fig. 1 B, murine T cells were isolated from single-cell suspensions of dissociated spleens from CD45.1+ congenic C57BL/6 mice bred in-house at the animal facility of the University of Lausanne (UNIL. Epalinges, Switzerland) using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies.

Fig. 1 B, 1.1). T cells were plated at 106/ml in 24- or 48-well plates in T cell medium (described above) and stimulated with αCD3/CD28 Ab-coated beads (Invitrogen) at a bead to cell ratio of 2:1 and 50 IU/ml hIL-2 (Glaxo. Fig.

1 B, 1.1). Non–treated cell-culture grade 48- or 24-well plates (Corning Falcon) were precoated with 0.25 ml or 0.5 ml, respectively, of recombinant RetroNectin (Takara Bio) at a final concentration of 20 μg/ml, overnight (O/N) at 4°C (Fig. 1 B, 1.2). 1 d after T cell activation, the retronectin-precoated plates were washed with PBS, blocked with 2% BSA in PBS for 30 min at RT (Fig.

1 B, 2.1). Subsequently, plates were washed once, retroviagra was added at the MOI indicated in the figures, and plates were then spun at 2,000 g for 1.5 h at 32°C (Fig. 1 B, 2.2). The supernatants were then aspirated, and 0.5 to 106 of 24 h activated T cells were transferred to each coated well (48- or 24-well plates.

Fig. 1 B, 2.3). The plates were centrifuged for 10 min at 300 g and incubated O/N (Fig. 1 B, 2.3).

In some experiments the transduction procedure was performed at 48 h, or at both 24 and 48 h after activation. The cultures were maintained at a cell density of 0.5 to 106 cells/ml and replenished with fresh T cell medium (supplemented with hIL-2 alone or hIL-2 followed by hIL-7/IL-15 on day 2 after transduction) every other day (Fig. 1 B, 3). At day 7, CAR surface expression was assessed by flow cytometric analysis (as described below), and the rested engineered T cells were adjusted for equal expression before functional in vitro and in vivo assays (Fig.

1 B, 4). Murine C1498 leukemia cells were transduced as described above for primary murine T cells, except that they were not activated and were maintained afterwards in DMEM-GlutaMAX complete medium at a cell density of 3 × 105 viable cells/ml. For flow cytometric analysis, cells were surface stained using antibodies against CD3ε (145-2C11), CD4 (GK1.5, RM4-5), CD8α (53–6.7), CD25 (PC61), CD44 (IM7), CD45.1 (A20), CD45 (30F/11), CD62L (MEL-14), CD69 (H1-2F3), IL-15-Rα (6B4C88), PD-1 (29F.1A12), Ly-6G (1A8), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD206 (C068C2), NK-1.1 (PK136), CD19 (6D5), and MHC class II (M5/114.15.2). Abs were purchased from eBioscience and BioLegend or produced in-house from hybridomas by the flow cytometry platform.

DC101-CAR expression by retrovirally transduced T cells was detected by incubation with soluble mouse VEGFR-2–hIgG-Fc fusion protein (R&D Systems) followed by staining with labeled goat anti-hIgG Fc (clone HP6017. Biolegend). Thy1.1-T cells were stained in parallel as a negative control. VEGFR-2 expression by mouse endothelial cell lines was evaluated by cell-surface staining with rat anti-VEGFR-2 Ab (clone Avas12.

BioLegend) and matched isotype control (Rat IgG2a κ isotype. Clone RTK2758. BioLegend). For detection of phosphorylated STAT5, cells were fixed with BD Cytofix Fixation Buffer at 4°C for 15 min and permeabilized with BD Phosflow Perm Buffer III for 30 min at 4°C.

Intracellular phospho-staining was performed for 1 h at RT in the dark with Ab against phospho-STAT5 (Tyr694. D47E7 XP Rabbit mAb 4322. Cell Signaling). For intracellular staining of mIL-15 (clone AIO.3.

EBioscience), Bcl-2 (clone 10C4. EBioscience), and Ki67 (clone SolA15. EBioscience), T cells were fixed and then permeabilized using the FoxP3 transcription factor staining buffer set (eBioscience) according to the manufacturer’s recommendations. For the detection of mIL-15, the cells were further washed and incubated for 30 min with anti-rat IgG2a.

To discriminate dead cells, 7-AAD (BioLegend) staining was performed. Live/dead fixable Aqua Dead cell staining was used to exclude dead cells in the ex vivo analysis of immune cells derived from the spleens, tumors, and tumor-draining lymph nodes according to the manufacturer’s instructions (Molecular Probes, Life Technologies). Data were acquired with a BD flow cytometer and analyzed using FlowJo software (Tree Star). Cells extracted from dissociated tumors were lysed using TRIzol reagent (Invitrogen, Thermo Fisher Scientific).

Total RNA was isolated using the RNeasy Mini Kit (Qiagen). After treatment with RNase-free DNase I (Qiagen), 400 ng of total RNA was reverse transcribed using PrimeScript First Strand cDNA Synthesis Kit (Takara Bio), as indicated by the manufacturer. Quantitative real-time PCR was performed according to the commercial protocol using SYBR Green Fast PCR Master Mix (Thermo Fisher Scientific) and the 7500 Fast Real-Time PCR System (Applied Biosystems). Primers to specifically amplify regions of the DC101 scFv of the CAR cassette, or the mIL-15 transgene, were designed using the GenScript website and are as follows.

DC101 forward, 5′-GCA​ACC​CAA​ACT​CCT​CAT​CT-3′. DC101 reverse, 5′-TAT​CAT​CAG​CCT​CCA​CAG​GA-3′. IL-15 forward, 5′-CCA​GGA​TCT​ACA​GGC​GAC​AA-3′. IL-15 reverse, 5′-ATG​CTC​TGG​ATC​AGG​CTC​TC-3′.

PCR amplification of the housekeeping gene GAPDH was performed as a control, and to allow normalization of samples. The following primers were used for GAPDH. GAPDH forward, 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′. GAPDH reverse, 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′.

Each sample was run in triplicate, and each experiment included three nontemplate control wells. The relative mRNA levels (fold change) of each transgene among the different samples were quantified using the comparative 2−ΔΔCt method. We wish to thank members of the Flow Cytometry Platform and the Animal Care Facility of UNIL for their excellent support. We also kindly thank Dr.

Steven A. Rosenberg (National Cancer Institute, Bethesda, MD) for sharing a second generation anti-VEGFR-2 CAR construct comprising the scFv DC101. This work was generously supported by Ludwig Cancer Research, the European Research Council (advanced grant 1400206AdG-322875 to G. Coukos), and the Biltema Foundation.

P. Romero is supported in part by Oncosuisse (grant KFS-4404-02-2018). Author contributions. M.

Irving, G. Coukos, and E. Lanitis conceived, designed, developed, and supervised the study and wrote the manuscript. E.

Lanitis, G. Rota, P. Kosti, C. Ronet, and A.

Spill conducted experiments and acquired and analyzed data. A. Spill supported the in vivo and ex vivo studies. B.

Seijo built essential constructs. P. Romero and D. Dangaj reviewed the data and manuscript and provided suggestions.

All authors read and approved the manuscript.Christopher Mapperley Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review &. Editing 1Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK2Laboratory of Haematopoietic Stem Cell and Leukaemia Biology, Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for other works by this author on:.