In California, the number of HIV infected individuals continues to increase. As anti-retroviral drugs are not curative, these individuals still have to deal with the emotional, financial, and medical consequences. Our HIV stem cell gene therapy approach comprises the transplantation of a purified population of HIV-resistant blood forming stem cells which would generate an HIV-resistant immune system in a patient’s body. This would be significantly compelling to the state of California.
As the largest provider of bone marrow cell transplants in California, and the second largest in the nation, our institution has great expertise and an excellent record of safety in the delivery of stem cell treatments. We now propose to create the Alpha Clinic for Cell Therapy and Innovation (ACT-I) in which new, state-of-the-art, stem cell treatments for cancer and devastating blood-related diseases will be conducted and evaluated. As these experimental therapies prove to be effective, and become routine practice, our ACT-I Program will serve as the clinical center for delivery of these treatments. ACT-I will be an integral part of our Hematologic Malignancy and Stem Cell Transplantation Institute, placing it in the center of our institutional strengths, expertise, infrastructure and investment over the next decade. To move quickly once the CIRM award is made, ACT-I can be launched within our institution’s Day Hospital, a brand new, outpatient blood stem cell transplantation center opened in late 2013 with California Department of Health approval for 24 hour a day operation. This will ensure that ACT-I will have all the clinical and regulatory expertise, trained personnel, state-of-the-art facilities and other infrastructure in place to conduct first-in-human clinical trials and to deliver future, stem cell-based therapies for cancer and blood-related diseases, including AIDS. When our new Ambulatory Treatment Center is complete in 2018, it will double our capacity for patient visits and allow for expansion of the ACT-I pipeline of new stem cell products in a state-of-the-art facility.
Beyond our campus, we operate satellite clinics covering an area that includes urban, suburban and rural sites. More than 17.7 million people live in this area, and represent some of the greatest racial and ethnic diversity seen in any part of the country. Our ACT-I is prepared to serve a significant, diverse and underserved portion of the population of California.
CLINICAL TRIALS. Our proposal has two lead clinical trials that will be the first to be tested in ACT-I. One will deliver transplants of blood stem cells that have been modified to treat patients suffering from AIDS and lymphoma. The second will use neural stem cells to deliver drugs directly to cancer cells hiding in the brain. These studies represent some of the new and exciting biomedical technologies being developed at our institution. In addition to the two lead trials, we have several additional clinical studies poised to use and be tested in this special facility for clinical trials. In summary, ACT-I is well prepared to accommodate the long list of clinical trials and begin to fulfill the promise of providing new stem cell therapies for the citizens of California.
California’s citizens voted for the California Stem Cell Research and Cures Act to support the development of stem cell-based therapies that treat incurable diseases and relieve human suffering. To achieve this goal, we propose to establish an Alpha Clinic for Cellular Therapies and Innovation (ACT-I) as an integral part of our Hematological Malignancies and Stem Cell Transplantation Institute, and serve as the clinical center for the testing and delivery of new, cutting-edge, cellular treatments for cancer and other blood-related diseases. Our institution is uniquely well-suited to serve as a national leader in the study and delivery of stem cell therapeutics because we are the largest provider of stem cell transplants in California, and the second largest in the country. According to national benchmarking data, our Hematopoietic Cell Transplantation program is the only program in the nation to have achieved survival outcomes above expectation for each of the past nine years. This program currently offers financially sustainable, research-driven clinical care for patients with cancer, HIV and other life-threatening diseases. CIRM funding will allow the ACT-I clinic to ramp up quickly, drawing upon institutionally established protocols, personnel and infrastructure to conduct first-in-human clinical trials for assessment of efficacy. As CIRM funding winds down, ACT-I will have institutional support to offer proven cellular therapeutics to patients. The lead studies at the forefront of the ACT-I pipeline of clinical trials focus on treatments for HIV-1 infection and brain tumors, two devastating and incurable conditions. These first trials are closely followed by a robust queue of other stem cell therapeutics for leukemia, lymphoma, prostate cancer, brain cancers and thalassemia.
Our long list of proposed treatments addresses diseases that have a major impact on the lives of Californians. Thalassemia is found in up to 1 in 2,200 children born in California; prostate cancer affects 211,300 men, and HIV-1 infection occurs in 111,000 of our citizens. From 2008 to 2010, 6,705 Californians were diagnosed with brain cancers, 4,580 of whom died. In considering hematological malignancies during this same period, 2,800 patients were diagnosed with Hodgkin lymphoma (416 died), 20,351 with non-Hodgkin lymphoma (6,241 died), 13,358 with leukemia (6,961 died), 3,900 with acute myelogenous leukemia (2,972 died), 2,129 with acute lymphoblastic leukemia (648 died) and 4,198 with chronic lymphocytic leukemia (1,271 died). Standard of care fails in many cases; mortality rates for patients with hematological malignancies range from 25% to 76%. Successful stem cell therapeutics hold the promise to reduce disease-related mortality while improving disease-related survival and quality of life for the citizens of California, and for those affected by these diseases worldwide.
- The CIRM Alpha Stem Cell Clinic (ASCC) was opened at City of Hope on March 1, 2015. The award is intended to enable researchers to pursue important work that aims to bring the potential of stem cell treatments to fruition. Two clinical trials were identified to launch this center, and in the first year, we have added 8 additional studies. The current and future clinical studies include:
- • Transplants of blood stem cells that have been genetically modified to treat patients with either AIDS or with AIDS-related lymphoma
- • Use of neural stem cells to deliver drugs directly to cancers hiding in the brain and elsewhere
- • T cell immunotherapy trials to treat patients with cancer
- • Correction of hemophilia B by genetic editing of liver stem cells
- To accomplish this, the City of Hope developed a novel approach to evaluation of these new therapies. Instead of using the existing Clinical Research Unit here, a dedicated outpatient clinic was established in the City of Hope Day Hospital and staffed with clinic nurses. The reason for this is reflected in the two-fold nature of the CIRM network of Alpha Stem Cell Clinics’ goals: the first is to accelerate the development of new stem cells therapies and the second is the achievement of a fiscally sustainable clinical activity. As the largest stem cell transplantation center in California, the City of Hope plan takes advantage of our clinical nursing expertise of the Day Hospital and of the business expertise that makes this out-patient transplant center sustainable.
- Thus, the COH Alpha Clinic is an experiment in itself, testing whether this hybrid research unit is the best approach to introduction of new treatments to the clinic. CIRM funding has made it possible to bring the clinical staff together with the research staff in this way to accelerate development of stem cell research.
The overall goal of this proposal is to develop new methods and technologies to improve our ability to engineer hematopoietic stem cells. These are the adult stem cells found in the bone marrow that give rise to all of the components of the blood and immune systems. Being able to engineer these cells provides potential treatments for diseases of the blood including genetic diseases, such as sickle cell disease or severe immune deficiencies, as well as serious infections such as HIV/AIDS. We work with a new class of genetic engineering tools called targeted nucleases that have the potential to make genetic engineering of stem cells much more precise and therefore safer. In addition, we are exploring methods to deliver these reagents directly to the stem cells in the body, without the currently necessary steps of first removing the cells and performing the genetic engineering in a lab. Such capabilities would greatly improve the safety of human gene therapy, as well as facilitate its practical implementation. HIV/AIDS is our disease of focus, and we will use these techniques to develop new treatments that go beyond the current use of targeted nucleases in patients, where HIV’s co-receptor gene, called CCR5, is being disrupted. Our goal is to develop a next-generation of anti-HIV therapies and we expect that the techniques we develop will be broadly applicable to other disease of the blood and immune systems where stem cell therapies could be of benefit.
HIV/AIDS is a major social, economic and health burden to California and its citizens. The numbers are sobering: California has 14% of all US cases of HIV, second only to New York, with 220,543 cases reported through June 2014, including 98,161 deaths. With the advent of improved antiretroviral drugs, mortality has significantly decreased, but so has the length of time people need to take the drugs, and the economic burden to the state is revealed by the cost of drugs representing 85% of all AIDS-related costs. Both federal and state laws require that the AIDS Drug Assistance Program be the payer of last resort for these medications, and its budget is underwritten by the General Fund. Beyond the fiscal concerns, patients live with the potential for developing side effects to the drugs or drug-resistant virus, and accessing these life-long drug regimens is a daily struggle for many. Consequently, the development of stem cell based therapies for HIV brings the potential of one-shot and long-lasting treatments that could arm a patient’s own immune system with the capability to suppress HIV in the absence of drugs. Such an outcome would provide economic returns over the long-run by reducing spending on drugs, as well as improving the quality of life for individuals with HIV/AIDS. Beyond HIV, the development of technologies to improve the efficiency, safety and implementation of hematopoietic stem cell therapies will benefit other diseases where such cells could be curative.
The HIV-1 virus enters cells by binding to a protein called CCR5 on the cell surface. A naturally occurring mutation in CCR5, CCR5d32, has been shown to provide protection from HIV-1 infection and AIDS. All individuals carry two copies of the CCR5 gene, and those with both copies of CCR5 mutated are highly resistant to infection with HIV-1. Those carrying one mutated copy can get infected by HIV-1, but have a delayed progression to AIDS. Media and scientific magazines have discussed widely the case of the “Berlin patient,” who was cured of his HIV-1 infection after receiving therapy for his leukemia with a blood stem cell transplant from a donor whose cells had both copies of CCR5 mutated. Other HIV-1 infected patients have not been treated in this way since donors must be a near-perfect tissue match with the patient and also have the double CCR5 mutation, two rare events which almost never happen together.
To mimic the effect of the CCR5 double mutation, we propose to create blood stem cells that have a double mutation in the CCR5 gene and then test this gene therapy method in patients. The patients’ own blood stem cells will be treated in a process that can mutate the CCR5 gene, and then the stem cells will be transplanted back into the individual. These cells will carry the disrupted CCR5 gene and provide a renewable, long-lasting source of HIV-1 resistant immune cells. This novel strategy gets around the need to find a stem cell donor who carries the CCR5 double mutation and, since the stem cells come from the patient, there will be an ideal “perfect match” with no chance for rejection in the patient.
Results from mice transplanted with blood stem cells treated in this way have shown that the modified cells are functional and produce CCR5 mutant (HIV-1 resistant) progeny cells. When infected with HIV-1, these mice have reduced viral loads, and, importantly, the CCR5-disrupted CD4 cells have strong survival advantage. In the proposed clinical trial, HIV-1 infected patients with low levels of CD4 cells and no detectable HIV-1 while on antiviral medications will have their own blood stem cells modified at the CCR5 gene. Modified cells will then be re-infused into the patient after treatment with a chemotherapy agent, busulfan, which makes room for the stem cells to “take hold” in the marrow and generate an immune system with CCR5-mutant, HIV-1 resistant cells.
The applicant institution has worked with collaborating partners to develop this treatment method (a project funded by CIRM). They successfully developed this gene modification method up to the clinical testing phase. With combined expertise in stem cells, gene therapy, transplantation, treatment of HIV-related disease, this Strategic Partnership has the knowledge and skill to achieve all project goals.
California has the second highest number of persons living with HIV-1/AIDS in the United States. By the end of 2008, there were 100,366 adults and adolescents reported to be living with HIV-1 or AIDS in California (Cf. Scheer S et al, The Open AIDS Journal, 2012, 6(Suppl 1):188). This incidence translates into a medical and fiscal burden larger than any other state, except New York. A study from 2011 reported that public funding accounted for approximately $1.92 billion in HIV-1/AIDS services in California in the fiscal year 2008, of which the majority (90.4%) supported treatment (Cf. Leibowitz AA et al, J Acquir Immune Defic Syndr 2011;58:e11). In the fiscal year 2007-08, the California AIDS Drug Assistance Program (ADAP) served 32,842 clients and filled over 953,000 prescriptions for these clients. The Governor's current spending plan (2013-14 Budget Act) called for $448.4M to support this program, with sources coming from federal and California state funds.
With these huge costs and HIV-1/AIDS being a life-long infection that requires compliant daily treatment medication for a lifetime, the need for a cure that has the potential to reduce the duration of antiviral therapy is substantial. Most importantly, such a therapy would significantly impact the quality of life of persons with HIV-1/AIDS. If successful, a stem cell-based therapy, though expensive as a single treatment, would significantly reduce the cost burden on the state and federal treatment programs and save money over the lifetime of a patient. The estimated cost of lifelong cART therapy is estimated to be $420,000 -$755,000 USD, with 73% of the cost going to cART drugs (Cf. Sloan CE et al. AIDS 2012, 26, 45). Conversion to generic first line combination cART drugs is estimated to only reduce costs by ~ $42,000/patient (Cf. Walensky R.P et al. Ann Intern Med 2013, 158, 84). Furthermore, not all patients with HIV-1 infection fully respond to the therapy; in fact, about one fifth of patients have an inadequate immune response despite keeping the virus at undetectable levels. These patients are at increased risk of infection and chronic diseases, which also impact the health spending of California.
There is no treatment for such patients and, if successful, the cellular therapy derived from genetically modified blood stem cells proposed here would have a major impact on patient management and outcome. Success of this therapy would establish the safety of a possible future cure for HIV-1/AIDS. Added benefits are: 1) this stem cell-based gene therapy for HIV-1/AIDS will have a positive impact on the overall field of stem cell research with application to other diseases; 2) this demonstrates the progression of new technology supported by CIRM to the clinic, and 3) the technology was derived from California-based industry and success should have a positive economic impact in the state.
- The HIV-1 virus enters cells by binding to a protein called CCR5 on the cell surface. A naturally occurring mutation in CCR5, CCR532, has been shown to provide protection from HIV-1 infection and AIDS. Media and scientific magazines have discussed widely the case of the “Berlin patient,” who was cured of his HIV-1 infection after receiving therapy for his leukemia with a blood stem cell transplant from a donor whose cells had both copies of CCR5 mutated. This strategy is unlikely to be successful for the AIDS patient in general since donors must be a near-perfect tissue match with the patient and also have the double CCR5 mutation, two rare events which almost never happen together.
- To mimic the effect of the CCR5 double mutation, this project creates blood stem cells that have a double mutation in the CCR5 gene and then test this gene therapy method in HIV-infected research participants. The participants’ own blood stem cells are treated in a process that can mutate the CCR5 gene, and then the stem cells are infused back into the individual. These cells carry the disrupted CCR5 gene. We administer a low dose of chemotherapy called busulfan to the recipient prior to the transplant to provide space for the new stem cells. We anticipate that, upon stem cell infusion, the gene-modified cells will provide a renewable, long-lasting source of HIV-1 resistant immune cells. This novel strategy gets around the need to find a stem cell donor who carries the CCR5 double mutation and, since the stem cells come from the patient, there will be an ideal “perfect match” with no chance for rejection in the patient.
- The applicant institution has worked with collaborating partners to start the study in July 2015. Three participating clinics screen and enroll the eligible participants. So far, we have enrolled the first research participant and have successfully mobilized and collected the blood stem cells and have manufactured the gene-modified blood stem cells. We are now in the process of testing of this investigational product. Screening of potential participants continues at the clinics currently open to recruitment. It is planned to add more clinics to the study in the current funding year.
- This is a partnership among City of Hope, Sangamo Biosciences Inc, and CIRM. With combined expertise in stem cells, gene therapy, transplantation, treatment of HIV-related disease, this Strategic Partnership has the knowledge, skill and resources to achieve all project goals.
Stem cell research offers the promise of replacing missing or damaged tissues in the treatment of disease. Stem-cell-derived transplants still face problems with rejection as in traditional organ transplants. Several drugs can prevent rejection but also suppress the immune system, leaving patients vulnerable to infections and cancer. To avoid rejection without using drugs requires re-educating the immune system to “tolerate” the transplant and not see it as foreign. Because of its role in educating developing immune cells, the thymus is a critical organ in establishing what the immune system recognizes as “self” and not foreign, in a process known as immune tolerance. By growing a new thymus from stem cells matched to transplanted tissues, we can condition the immune system to be tolerant to the transplant and avoid chronic immunosuppression. We have developed a method to grow stem cells into thymic cells that become normal thymus tissue when grafted into mouse models. Notably, the new thymus can promote normal development of immune cells, indicating the potential for generating new, tolerant immune cells. We propose to induce immune tolerance to other stem-cell derived tissues using stem-cell-derived thymus tissue to engineer tolerance. We will optimize our methods of growing thymus tissue, which will be used to condition mice to accept stem-cell-derived pancreas grafts, testing their ability both to prevent rejection and to cure diabetes in a transplant model.
The proposed work aims to improve the effectiveness of stem cell treatments by preventing immunological rejection of transplanted tissue derived from stem cells. An important barrier to the clinical use of stem-cell-derived organs and tissues is the potential of the immune system to reject or damage this regenerated tissue. Improved approaches to address immune rejection are needed since stem cell therapies are underway in treating diseases that have a wide impact on the health of Californians, including diabetes, Parkinson’s disease, Alzheimer’s disease, retinal eye diseases, and musculoskeletal diseases.
The proposed studies will improve treatment for these diseases by providing a novel method to halt immunologic rejection or destruction of tissues that are derived from stem cells. We have successfully developed methods to grow thymus tissue, which controls the ability of the immune system to be “tolerant” of transplanted tissue. Here we will improve methods to generate thymus from stem cells and show that it can promote survival of transplanted tissue derived from the same cells. By using the thymus to condition the immune system towards tolerance, we hope to avoid immune rejection without the use of immunosuppressive drugs. Induction of a tolerant immune system in this way would represent a significant advance in improving stem cell therapies. Thus, this work could have a broad impact on a large number of the disease treatments that involve stem cells.
- We continue to make progress with our efforts to generate functional thymic epithelial cells that are derived from stem cell sources. Over the last year we have been able to improve our ability to differentiate thymic epithelial progenitors by using a 3-Dimenstional culture system. This system has improved our efficiency and we are currently further refining it for use in our differentiation method. In future years, this will help accelerate our progress in TEP generation. A second area that we have made progress in is the generation of a reporter stem cell line for a key transcription factor called FOXN1. These cells express a key marker that help tell us how well our protocol is working. Through the use of this new cell line we again have made substantial progress in our differentiation efficiency. In looking forward to the next years of funding, we are well positioned for our more elaborate experiments for looking at immune tolerance that is induced by our cells.
- Currently, HIV infection is studied in mouse models where human stem cells are transplanted into mice, serving as targets for HIV-1 infection. These models are used extensively for investigation of the impact of gene modification of stem cells with anti-HIV gene therapeutics. One limitation of these models is that the transplant with anti-HIV gene-modified human stem cells is established first then followed by HIV-1 infection. This model is the reverse order of a therapeutic transplant in humans where individuals are transplanted with anti-HIV gene-modified stem cells after HIV-1 infection. We propose to develop a more representative model where we first transplant stem cells to establish a human immune system in mice, then infect with HIV-1, and then conduct a second transplant with gene-modified human stem cells. This model would more closely mimic the human clinical situation. We have successfully identified conditions for two transplants in mice and are now evaluating whether this new humanized mouse model can be used to study protection from HIV-1 by anti-HIV gene modification of stem cells.
- The most common current models for anti-HIV-1 gene therapy is through the use of humanized mice, immunodeficient animals into which human cells are engrafted. Evaluation of anti-HIV-1 gene therapy in animal models has been performed by HIV-1 challenge AFTER transplant with gene modified HSPC. This experimental design allows us to gain important data in a relatively simple setting. However, the clinical application of gene therapy will be in patients already infected with HIV-1. Thus, it is important to test the impact of HIV-1 infection on the success of gene modified HSPC engraftment and differentiation and through control of HIV-1 in previously infected humanized mice. We tested a new mouse model whereby the first transplant of CD34+ cells allows human hematopoietic cell reconstitution and provides human cells for HIV-1. After HIV-1 is established, a second transplant is conducted using anti-HIV-1 gene modified CD34+ cells and thymus tissue. We find that the timing of the second transplant is important in order to maintain the integrity of the thymic organoid. In this system, repopulation of human CD45+ cells, CD3+, CD4+, CD8+ cells occurs efficiently. In HIV-1 infected animals, CD4+ cells are decreased relative to uninfected control animals. Under these conditions, we observe protection from HIV-1 by the transplant of genetically modified CD34+ cells. These studies provide the basis for a new model which should be useful for understanding and evaluating the efficacy of anti-HIV-1 gene modification of hematopoietic stem cells.
The AIDS virus infects and destroys cells of the immune system such that the bodies of infected individuals cannot fight infections or some cancers. If untreated HIV infection leads to death. Current therapies to stop virus replication in the body are expensive and can have side effects. They also do not eliminate the virus from the body. Our overall goal is to use a gene therapy approach to improve a patient’s own immune response against HIV, thus rendering them able to fight their own viral infection. We will test an approach designed to engineer a patient’s immune system so that it can directly kill cells infected by HIV, thereby preventing spread of the virus throughout the body. This would decrease virus replication, and perhaps eliminate HIV from the body. This should prevent the HIV-induced destruction of the immune system, and restore the body’s ability to mount immune responses against a variety of infectious agents and cancers, and may eliminate the need for the patient to take antiretroviral drugs. Successful completion of this project will yield an immunotherapeutic that is ready for preclinical development as a treatment for HIV-1 infection and AIDS.
California ranks second in the nation in cases of HIV/AIDS, with over 155,000 persons living with HIV infection currently. This is projected to increase steadily to over 172,000 persons in the next 5 years. Aside from the personal, social, and work productivity losses due to HIV infection and its treatment, the direct healthcare cost to California is thought to approach $1.8 billion annually (CDC).
A curative treatment is therefore a high priority, given the high costs of chronic treatment and the increasingly apparent long-term toxicities of the antiretroviral drugs. Stem cell therapy offers promise for this goal, by addressing two major immune mechanisms of failure to control HIV infection: 1) loss of both anti-HIV CD8+ T-cells and the CD4+ T-cells required for their maintenance and function, and 2) CD8+ T-cell targeting that is subject to HIV evasion through mutation and down-modulation of the class I Human Leukocyte Antigens that present HIV protein sequences to CD8+ T-cells.
In this project, we propose to develop a strategy to program stem cells to provide a self-renewing population of both CD8+ and CD4+ HIV-targeted T-cells that are resistant to direct HIV infection, and which bypass the mechanisms by which HIV usually evades the immune response. If successful, this approach would allow development of a one-time stem cell gene therapy treatment that yields long-term immune control of HIV infection.
- The overall goal of this project is to use a gene therapy approach to engineer immune system stem cells with molecules that will direct mature immune cells developed from the stem cells to attack and kill cells infected with the AIDS virus (HIV). The approach chosen, if successful, will develop a way to stably engineer the anti-viral immune response, which should provide an inexhaustible source of HIV-specific immune cells that can fight the infection in the body. We proposed to design and test this strategy in the laboratory and in a humanized mouse system that closely mimics conditions in the human body, for efficacy against HIV-1 infection. To date we have met our scheduled milestones associated with construction of anti-viral molecules and with the testing of various stem cell preparations in the laboratory, and are beginning the studies in humanized mice. The group is interacting effectively and we anticipate continue progress towards our goals.
- The overall goal of this project is to use a gene therapy approach to engineer immune system stem cells with molecules that will direct mature immune cells developed from the stem cells to attack and kill cells infected with the AIDS virus (HIV). The approach chosen, if successful, will develop a way to stably engineer the anti-viral immune response, which should provide an inexhaustible source of HIV-specific immune cells that can fight the infection in the body. We proposed to design and test this strategy in the laboratory and in a humanized mouse system that closely mimics conditions in the human body, for efficacy against HIV-1 infection. To date we have largely met our scheduled milestones associated with construction of anti-viral molecules and with the testing of various stem cell preparations in the laboratory, and are beginning the studies in humanized mice. The group is interacting effectively and we anticipate continue progress towards our goals.
The Human Immunodeficiency Virus (HIV) is still a major health problem. In both developed and underdeveloped nations, millions of people are infected with this virus. HIV infects cells of the immune system, becomes part of the cell’s genetic information, stays there for the rest of the life of these cells, and uses these cells as a factory to make more HIV. In this process, the immune cells get destroyed. Soon a condition called AIDS, the Acquired Immunodeficiency Syndrome sets in where the immune system cannot fight common infections. If left untreated, death from severe infections occurs within 8 to 10 years. Although advances in treatment using small molecule drugs have extended the life span of HIV infected individuals, neither a cure for HIV infection nor a well working vaccine could be developed. Drug treatment is currently the only option to keep HIV infected individuals alive. Patients have to take a combination of drugs daily and reliably for the rest of their lives. If not taken regularly, HIV becomes resistant to the drugs and continues to destroy immune cells. What makes this situation even more complicated is the fact that many patients cannot take these drugs due to severe side effects.
Stem cell gene therapy for HIV may offer an alternative treatment. Blood forming stem cells, also called bone marrow stem cells make all blood cells of the body, including immune system cells such as T cells and macrophages that HIV destroys. If “anti-HIV genes” were inserted into the genetic information of bone marrow stem cells, these genes would be passed on to all new immune cells and make them resistant to HIV. Anti-HIV gene containing immune cells can now multiply in the presence of HIV and fight the virus. In previous and current stem cell gene therapy clinical trials for HIV, only one anti-HIV gene has been used. Our approach, however, will use a combination of three anti-HIV genes which are much more potent. They will not only prevent HIV from entering an immune cell but will also prevent HIV from mutating, since it would have to escape the anti-HIV effect of three genes, similar to triple combination anti-HIV drug therapy. To demonstrate safety and effectiveness of our treatment, we will perform a clinical trial in HIV lymphoma patients. In such patients, the destruction of the immune system by HIV led to the development of a cancer of the lymph nodes called B cell lymphoma. High dose chemotherapy together with the transplantation of the patient’s own bone marrow stem cells cures B cell lymphoma. We will insert anti-HIV genes in the patient’s bone marrow stem cells and then transplant these gene containing cells into the HIV infected lymphoma patient. The gene containing bone marrow stem cells will produce a new immune system and newly arising immune cells will be resistant to HIV. In this case, we have not only cured the patient's cancer but have also given the patient an HIV resistant immune system which will be able to fight HIV.
As of September 30, 2010, over 198,883 cumulative HIV/AIDS cases were reported in California. Another 40,000 un-named cases of HIV were also reported before 2006 although some of them may be duplicates of the named HIV cases. Patients living with HIV/AIDS totaled 108,986 at the end of September 2010. These numbers continue to grow since new cases of HIV and AIDS are being reported on a daily basis and patients now live much longer. In fact, after New York, California has the second highest number of HIV cases in the nation. Although the current and improved anti-retroviral small molecule drugs have prolonged the life of these patients, they still have to deal with the emotional, financial, and medical consequences of this disease. The fear of side effects and the potential generation of drug resistant strains of HIV is a constant struggle that these patients have to live with for the rest of their lives. Furthermore, not every patient with HIV responds to treatment and not every complication of HIV dissipates upon starting a drug regimen. In fact, the risk of some AIDS-related cancers still remains high despite the ongoing drug therapy. Additionally, in the current economic crisis, the financial burden of the long term treatment of these patients on California taxpayers is even more obvious. In 2006, the lifetime cost of taking care of an HIV patient was calculated to be about $618,900. Most of this was related to the medication cost. With the introduction of new HIV medications that have a substantially higher price and with the increase in the survival of HIV/AIDS patients, the cost of taking care of these patients can be estimated to be very high.
The proposed budget cuts and projected shortfall in the California AIDS assistant programs such as ADAP will make the situation worse and could result in catastrophic consequences for patients who desperately need this of kind of support. Consequently, improved therapeutic approaches and the focus on developing a cure for HIV infected patients are issues of great importance to the people of California.
Our proposed anti-HIV stem cell gene therapy strategy comprises the modification of autologous hematopoietic blood forming stem cells with a triple combination of potent anti-HIV genes delivered by a single lentiviral vector construct. This approach would engineer a patient’s immune cells in a way to make them completely resistant to HIV infection. By transplanting these anti-HIV gene expressing stem cells back into an HIV infected patient, the ability of HIV to further replicate and ravage the patient’s immune system would be diminished. The prospect of such a stem cell based therapy which may require only a single treatment to cure an HIV infected patient and which would last for the life of the individual would be especially compelling to the HIV community and the people of California.
- HIV is still a major health problem. In both developed and underdeveloped nations, millions of people are infected with this virus. If left untreated, death from severe infections occurs within 8 to 10 years. Although advances in treatment using small molecule drugs have extended the life span of HIV infected individuals, neither a cure for HIV infection nor a well working vaccine could be developed. Drug treatment is currently the only option to keep HIV infected individuals alive. Patients have to take a combination of drugs daily and reliably for the rest of their lives. If not taken regularly, HIV becomes active again and may even become resistant to the drugs and continues to destroy immune cells. What makes this situation even more complicated is the fact that many patients cannot take these drugs due to severe side effects. Stem cell gene therapy for HIV may offer an alternative treatment. If “anti-HIV genes” were inserted into the genetic information of bone marrow stem cells, these genes would be passed on to all new immune cells and make them resistant to HIV. Anti-HIV gene containing immune cells can now multiply in the presence of HIV and fight the virus. In our approach, we are planning to use a combination of three anti-HIV genes which are much more potent. They will not only prevent HIV from entering an immune cell but will also prevent HIV from mutating, since it would have to escape the anti-HIV effect of three genes, similar to triple combination anti-HIV drug therapy. To demonstrate safety and effectiveness of our treatment, we have proposed a clinical trial in HIV lymphoma patients with stem cell gene therapy incorporated into their routine treatment with high dose chemotherapy together with the transplantation. The fund provided by CIRM (California Institute for Regenerative Medicine) gave us the opportunity to put together a panel of experts within the University of California at Davis and another panel of international experts in the area of gene therapy (an external advisory board). Intense discussion in multiple meeting with members of these two panels as well as many other meetings with individual researches within our institution resulted in the design of a clinical trial for treating patients with HIV disease using our gene therapy approach. It further helped us to identify the necessary means needed to support such a regulatory intensive gene therapy trial. To be able to recruit enough patients for such a trial, we used the funds from this planning grant for several presentations to our colleagues in other institutions for a multi-institutional clinical trial approach. The funds provided to us through this grant helped to calculate the budget required to 1) finish our application with Federal Drug Administration (FDA) to obtain the appropriate license for starting such a trial and 2) to manufacture the target drug and 3) to run the actual clinical trial. Finally, with the help of this grant, we have put together a CIRM disease grant proposal and have applied for necessary funds based on the above calculation.
- The original progress report was submitted to the CIRM on March 1st 2012. The no cost extension was requested to perform the necessary work related to further development of our clinical trial before submission to RAC. During this period, in multiple meetings we rewrote our clinical trial based on the comments of our external advisory board and other consultants. We submitted our clinical trial protocol and Appendix M to RAC committee and after receiving their preliminary comments, we formulated our response. As the last step, we presented our clinical trial to the members of RAC committee and received a unanimous approval to move forward with the IND application to FDA.
Despite significant advances in treatment and prevention programs, HIV infection with progression to Acquired Immunodeficiency Syndrome (AIDS) is still prevalent in California. The CDC Estimates >56,000 new cases of HIV infection each year in the US with over 148,000 cumulative cases reported in California alone (as of 2009). Multi-drug therapy has been helpful in reducing the severity of disease and prolonging lifespan but sixteen of every one hundred HIV patients will eventually fail to control the virus after attempting at least 2 drug treatment regimens. The Centers for Disease Control (CDC) recently estimated the lifetime cost of medical care for AIDS to be in excess of $600,000 per patient, over 85% of which is attributable to prescription drug costs. Additionally, medication non-compliance, intolerance of drugs due to side effects and the development of resistant strains of virus are all complicating factors in obtaining consistent clinical benefit with lifelong drug therapy. Therefore, there is a need to provide a longer lasting, cost effective therapy for this disease. Our project builds on prior work from our laboratories in which genetically engineered blood stem cells were transplanted into HIV patients and shown to give rise to gene-marked peripheral blood cells that last for up to 2 years. These cells may protect HIV patients from progression to AIDS if they are present in sufficient numbers. Our therapeutic candidate is a gene modified human blood stem cell carrying multiple anti-HIV molecules that prevent virus infection, replication and spread and a gene that allows us to chemically “enrich” the number of disease resistant cells present in a patient’s blood. The anti-HIV molecules are made of ribonucleic acid (RNA) and were developed and tested in our laboratories. We have already conducted a first generation stem cell therapy clinical trial to test these molecules with promising results. We now propose to refine and further develop this treatment with second generation RNA molecules and gene transfer procedures that will improve the number of disease resistant cells in the blood of HIV patients. We will develop an animal model system to test newer, more efficient anti-HIV molecules and a drug treatment method to enhance the number of HIV resistant stem cells circulating in the blood of patients that receive gene modified blood stem cells. At the end of the proposed experiments, we expect to have selected the most efficient combination of RNA molecules and drug selection strategy to provide a sufficient number of disease resistant cells in the peripheral blood to prevent progression to clinical immunodeficiency (AIDS).
California has ~14% of all cases of AIDS in the U.S., and this translates into a medical and fiscal burden larger than any other state except NY. Antiviral chemotherapy accounts for approximately 85% of AIDS-related medical costs, and federal and state law requires that in California the AIDS Drug Assistance Program (ADAP) be the payer of last resort for these medications. Antiretroviral drugs currently cost about $12,000 per year and account for about $350 million of the California AIDS Drug Assistance Plan's budget. The Governor's spending plan (2009-09 Budget Act) called for $418M to support this program, with funds from several sources including federal (Ryan White Care Act), from an ADAP Rebate Fund, and from the California State General Fund. The ADAP Rebate Fund consists of monies paid to the state by the manufacturers of the drugs provided to the HIV/AIDS clients under the program. The ADAP budget has grown by ~15% yearly for several years, and based on an Legislative Analyst's Office (LAO) review, the problem faced is that, as the case load is increasing, support from the Rebate Fund is decreasing. It is projected by LAO that from a level of $80.3 million at end 2008, the Fund will decrease to $24M by the end of 2010. The General Fund currently provides $96.3M to the ADAP budget, and it is projected that as the ADAP Rebate Fund shrinks, the shortfall will have to be met by increases from the General Fund by 2012. The alternative, as noted by LOA, is to implement cost-cutting measures that would likely increase the barriers to receiving care for some patients, impacting the health of some HIV/AIDS patients and increasing the associated public health risks.
- Our goal in the first year of the grant has been to establish a mouse model for HIV infection using hematopoietic stem and progenitor cells (HSPC) from healthy adult donors and to create a series of novel RNA-based vectors that will provide resistance to HIV infection in the progeny of these cells. Dr. DiGiusto's group has successfully demonstrated the ability to reliably engrafted mice with human immune system cells derived from adult HSPC. Among the engrafted cells are CD4+ T-cells and monocytes, the target cells for HIV infection. We have also demonstrated that we can expand the number of HSPC from each donor and maintain their ability to support engraftment of the mice with human T-cells and monocytes. This means that we are able to make a large number of mice with which to screen genetic therapies for HIV. During this period, Dr. Rossi's group has created a 5 new anti-HIV viral vectors that can be used to genetically modify HSPC and impart HIV resistance to the T-cells and monocytes that are derived from these HSPC. Assays performed in culture dishes indicate that 3 of these vectors are very potent HIV inhibitors. Addionally, the vectors contain a gene that will allow us to selectively enrich for those HSPC that carry the gene under specific culture conditions. Thus, we can produce a population highly enriched for disease resistant HSPC. We are now beginning testing of anti-HIV vectors in our mouse model to establish which would be the best for moving towards clinical trials. The ultimate goal of the study is to define the best clinical candidate and we on track for meeting that goal within the timeframe of the grant.
- In the second year of the CIRM ETR2-01771 project entitled “Development of RNA-based approaches to stem cell gene therapy for HIV” we have made significant progress in the development of a robust animal model of human hematopoietic stem and progenitor cell (HSPC) engraftment, completed the cloning and in vitro testing of the candidate second generation anti-HIV lentiviral vectors and begun testing HIV infectivity of humanized NSG mice. Additionally, we have initiated transplantation of NSG mice with gene modified HSPC to evaluate the level of gene modified CD4+ T-cells and monocytes in vivo. This work will culminate in HIV challenge assays in the final year of the grant and selection of the second generation candidate therapeutic to be used in our clinical development program.
- We are working on a cure for HIV using blood stem cell transplantation of HIV-infected individuals. We have completed the development of a second generation of anti-HIV genes with potent anti-viral activity and reduced toxicity from our first generation products. We have demonstrated that we can modify adult human blood stem cells with these vectors and that macrophage and T-cell progeny of these modified cells are resistant to HIV infection and spread. We are currently screening the genes to identify the next drug development candidate using a humanized mouse model developed in our laboratory. The completion of these experiments will allow us to immediately move towards clinical testing. This approach to HIV therapy is likley to replace drug therapy and may result in a functional or sterilizing cure for HIV.
- During the final reporting period for this grant we have completed in vivo testing of our second generation anti-HIV constructs. We have identified a construct (SGLV4) that has an improved safety profile compared to our first generation vectors (less toxic) and superior anti-HIV activity including inhibition of viral replication and protection of CD4+ T-cells and monocytes as demosntrated in our in vivo animal model system. The use of this vector to genetically modify heamtopoietic stem cells may lead to a functional cure for HIV through the provision of an HIV-resistant immune system that will clear virus through normal immune mediated mechanisms. We will move this vector construct into pre-clinical development testing as funds become available through CIRM or other sources (NIH).
The thymus is an organ that plays a key role in controlling immune responses and immune tolerance. The thymus promotes immune tolerance by deleting and removing self-reactive T cells from the immune system. In addition, the thymus also helps drive the production of important suppressor T cell populations like regulatory T cells that also control immune tolerance. Thus, strategies that expand and improve thymic function could be critical in improving transplantation of tissues derived from embryonic stem cells. The thymus consists of a supporting network of thymic epithelial cells that help bone marrow derived T cell precursors mature and differentiate into fully functional T lymphocytes. Despite their importance, there has been little progress in methods to grow and expand out the supportive thymic epithelial network. This project will explore strategies to grow and expand out functional thymic epithelial cells from human embryonic stem cells using a multi-step culturing technique. These expanded thymic epithelial cells will be characterized and tested for the ability to support T cell development and differentiation. Finally, the expanded thymic epithelial cells will be put into transplantation models in humanized mice to test their ability to improve and enhance the acceptance of transplanted tissues. These studies offer enormous potential for promoting graft-specific immune tolerance in that embryonic stem cells could be differentiated into both a replacement tissue and into functional thymus
The work in this proposal is designed to help improve the effectiveness of stem cell treatments by preventing immunological rejection of transplanted tissue derived from stem cells. Although significant progress and promise has been shown to use stem cells to regenerate damaged organs for the treatment of a wide variety of diseases, an important barrier to bringing this to the clinic is the potential of the immune system to reject or damage this regenerated tissue. Currently, there are efforts underway to use stem cells to treat diseases that have a wide impact on the health of Californians, including diabetes, Parkinson’s disease, Alzheimer’s disease, retinal eye diseases, and musculoskeletal diseases to name just a few.
The work proposed here will help improve treatment for these diseases by improving the ability to put a break on the immune system to reject or destroy cells or tissues that are derived from stem cells for the treatment of these diseases. Here we will improve methods to grow and expand an important organ that controls the ability of the immune system to be “tolerant” of transplanted tissues called the thymus. If methods to grow and expand the thymus from stem cells can be done, this would represent a significant advance in improving stem cell therapies. Thus, the impact of this work could have a broad impact on a large number of the disease treatments that involve stem cells.
- Through the support of the CIRM we have initiated our efforts to differentiate and grow thymic epithelial cells from human embryonic stem cells. Over the last year we have been culturing human ES cells under different conditions and have examined marker expression in the cells for similarity to thymic epithelium. We have made significant progress, with a number of known markers being upregulated in our cells. We plan to continue similar efforts over the next year and expand our experiments to test whether these cells can induce immune tolerance. These findings are critical for the CIRM in that they may be an important way to keep the immune system in check and prevent immune rejection when ES cell derived material is transplanted.
- Over the last year we have made significant progress on ability to differentiate stem cells towards a cell lineage that can help contribute to a functional thymus. We have been able to induce a number of key markers of thymic progenitor cells and are also making progress on experiments to confirm the functionality of the differentiated cells. Taken together, these results could have a broad and significant impact on the field of regenerative medicine due to the ability of the thymus to control immune tolerance.
- Over the last year we have made significant progress on ability to differentiate stem cells towards a cell lineage that can help contribute to a functional thymus. We have been able to induce a number of key markers of thymic progenitor cells and are also making progress on experiments to confirm the functionality of the differentiated cells. Taken together, these results could have a broad and significant impact on the field of regenerative medicine due to the ability of the thymus to control immune tolerance.
- Over the last year we have made significant progress on ability to differentiate stem cells towards a cell lineage that can help contribute to a functional thymus. We have developed methods to differentiate several different stem cell sources into functional thymic progenitors. Our progress will help us further refine new methods to modulate immune tolerance.