HIV/AIDS

Coding Dimension ID: 
293
Coding Dimension path name: 
HIV/AIDS

ZIinc Finger Nuclease-Based Stem Cell Therapy for AIDS

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01490
ICOC Funds Committed: 
$14 583 187
Disease Focus: 
HIV/AIDS
Immune Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Some years ago it was discovered that patients homozygous for a natural mutation (the Δ32 mutation) in the CCR5 gene are generally resistant to HIV infection by blocking virus entry to a cell. Building on this observation, a study published in 2009 reported a potential "cure" in an AIDS patient with leukemia after receiving a bone marrow transplant from a donor with this Δ32 CCR5 mutation. This approach transferred the hematopoietic stem cells (HSC) residing in the bone marrow from the Δ32 donor, and provided a self-renewable and lifelong source of HIV-resistant immune cells. After transplantation, this patient was able to discontinue all anti-HIV drug treatment, the CD4 count increased, and the viral load dropped to undetectable levels, demonstrating an effective transplantation of protection from HIV and suggesting that this approach could have broad clinical utility. But donors with the Δ32 CCR5 mutation are not generally available, and so how could we engineer an analogous CCR5 negative state in human HSC needed for bone marrow transplantation? A potential answer comes from zinc finger nucleases (ZFNs) which have been demonstrated to efficiently block the activity of a gene by cleaving the human genome at a predetermined site and altering the genetic sequence via an error-prone DNA repair process. This modification of the cellular DNA is permanent and can fully block gene function. Recently, ZFNs have been shown to inactivate CCR5 in primary human CD4 T cells, allowing them to preferentially survive and expand in the presence of HIV. A human clinical trial evaluating this approach is on-going, in which patient T cells are re-infused after ZFN-treatment to block CCR5 expression and possibly provide an HIV-resistant reservoir of CD4 T cells. The CIRM Disease Team proposes an approach to modify a patient’s own HSC to circumvent the need to find matched donors that carry the Δ32 CCR5 mutation and yet provide a renewable and long-lasting source of HIV-resistant cells. Testing of this concept is proposed in selected AIDS lymphoma patients who routinely undergo HSC transplantation. Preliminary results in mice transplanted with ZFN-treated HSC show that ZFN-modified, CCR5-negative HSC are functional and support the reconstitution of the immune system. Importantly, after HIV infection, these mice have results similar to those observed in the human patient: (i) reduced viral loads, (ii) maintenance of CD4 T cells in peripheral tissues; and (iii) a powerful selective advantage for the CCR5 negative immune cells. These data support the development of a ZFN approach to treat AIDS patients by first isolating their HSC, modifying them using CCR5-specific ZFNs, and re-infusing them to reconstitute the immune system with CCR5-negative, HIV-resistant immune cells.
Statement of Benefit to California: 
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. In 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 (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 2007-08, the Fund will decrease to $24M by 2009-10. 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 2011-12. 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. The basic problem is that HIV/AIDS is a life-long infection and our current strategy of treatment requires that medication be taken daily for a lifetime. Thus, there is a real need to develop a strategy of treatment that has the potential to reduce the duration of antiviral chemotherapy. This will have significant impact on the quality of life for persons with HIV/AIDS. In this proposal, a cellular therapy derived from genetically modified blood stem cells will be developed and preclinical studies completed, leading to its first evaluation in patients. As important is the benefit that the first test of this technology will have on the overall field of embryonic stem cell research. The ZFN technology used here will have application to other diseases and hurdles surmounted now will benefit future embryonic cell research.
Progress Report: 
  • During the first year of the project, we have made significant progress in meeting the first milestone of the project: Defining the final process of genetically modifying hematopoietic stem/progenitor cells (HSPC) (Item #14, Milestone M3 of Gantt chart). In addition, initial effort has started in Phase II Scale-up/Pre-clinical testing (G15) and more specifically, in hematopoietic stem/progenitor cell processing development (G16).
  • Some 10 years ago it was discovered that patients homozygous for a natural mutation (the delta 32 mutation) in the CCR5 gene are generally resistant to HIV infection by blocking virus entry to a cell. Building on this observation, a study published in 2009 reported a potential "cure" in an AIDS patient with leukemia after receiving a bone marrow transplant from a donor with this delta 32 CCR5 mutation. This approach transferred the hematopoietic stem/progenitor cells (HSPC) residing in the bone marrow from the delta 32 donor, and provided a self-renewable and lifelong source of HIV-resistant immune cells. After transplantation, this patient was able to discontinue all anti-HIV drug treatment, the CD4 count increased, and the viral load dropped to undetectable levels, demonstrating an effective transplantation of protection from HIV and suggesting that this approach could have broad clinical utility.
  • But donors with the delta 32 CCR5 mutation are not generally available, and so how could we engineer an analogous CCR5 negative state in human HSC to be used for bone marrow transplantation, including a patient’s own HSPC? A potential answer comes from zinc finger nucleases (ZFNs) which have been demonstrated to efficiently block the activity of a gene by cleaving the human genome at a predetermined site and altering the genetic sequence via an error-prone DNA repair process. This modification of the cellular DNA is permanent and can fully block gene function. Recently, ZFNs have been shown to inactivate CCR5 in primary human CD4 T cells, allowing them to preferentially survive and expand in the presence of HIV. A human clinical trial evaluating this approach is on-going, in which patient T cells are re-infused after ZFN-treatment to block CCR5 expression and possibly provide an HIV-resistant reservoir of CD4 T cells.
  • This CIRM Disease Team proposed an approach to modify a patient’s own HSPC to circumvent the need to find matched donors that carry the delta 32 CCR5 mutation and yet provide a renewable and long-lasting source of HIV-resistant cells. Testing of this concept is proposed in selected AIDS lymphoma patients who routinely undergo HSPC transplantation. During the second year of this project, the disease team has made considerable progress and met all the project milestones for year 2. More specifically, the team developed an optimized procedure for efficiently introducing the CCR5-specific ZFNs in HSPC. We showed that these modified cells function normally and retain their “stemness” in tissue culture systems. We also showed these modified cells can be transplanted into mice to reconstitute the immune system. Given HSPC are long lasting stem cells, we have been able to stably detect these cells in mice for over 3 months post-transplantation. The team is in the process of scaling up the cell production procedures to ensure we can generate CCR5-modified HSPC at clinical scale. We are also moving ahead with the remaining pre-clinical safety and efficacy studies required before initiating a clinical trial.
  • It is well known that infection with HIV-1 requires a protein called CCR5, and persons with a natural mutation in this gene (CCR532) are protected from HIV/AIDS. Everyone has two copies of the CCR5 gene, one inherited from their mother and one from their father. People with both copies of CCR5 mutated (CCR532/ CCR532) are highly resistant to becoming infected with HIV-1. If only one copy is abnormal (CCR5/ CCR532), infection can occur but progression of the infection to AIDS is delayed. The only clear cure of HIV-1 infection occurred in a patient with leukemia who received a blood stem cell transplant from a tissue-matched donor whose cells carried the double mutation CCR532/CCR532. After transplantation, this patient was able to stop all anti-HIV medicine, the immune system improved, and the level of HIV-1 in the blood dropped to undetectable levels. Even after more than 4 years off anti-HIV medicine, the patient is considered cured, as there is no evidence of an active HIV-1 infection.
  • This Disease Team proposes to treat blood stem cells from an HIV-1 infected person with a protein that can mutate the CCR5 gene, and then transplant these same cells back into the patient to try and reproduce the effects of the CCR532 mutation by providing a renewable and long-lasting source of HIV-1 resistant cells. This will circumvent the need to find a stem cell donor who happens to carry the CCR532/ CCR532 mutation and is a suitable "perfect match" for tissue transplant. The proteins that will be used in this treatment are called Zinc Finger Nucleases (ZFNs). Preliminary results in mice transplanted with ZFN-treated blood stem cells have shown that the modified cells are functional and produce CCR5 mutant progeny cells - including CD4 T cells that are the natural target of HIV-1. Importantly, after HIV-1 infection, the mice demonstrated reduced viral loads, maintenance of CD4 T cells in peripheral tissues, and a powerful survival advantage for the CCR5-negative cells [Holt et al., Nature Biotechnology 2010; 28: 839-47]. These data support the development of this ZFN approach to treat HIV-1 infected patients by first isolating the subjects own blood stem cells, modifying them using CCR5-specific ZFNs, and then re-infusing them back into the patient to thereby reconstitute the immune system with CCR5-mutant, HIV-1 resistant cells. The Disease Team assembled to accomplish this goal has expertise in stem cell technology [City of Hope], HIV-1 infection in pre-clinical mouse models [University of Southern California], and in ZFN-based clinical trial development [Sangamo BioSciences].
  • In the first two years of study, the Disease Team focused on the use of an existing delivery technology for introducing the ZFNs into blood stem cells. This approach used a type of gene therapy vector called an adenoviral vector, which had been previously used in early stage investigational clinical trials for the modification of patients’ T cells. During this phase of the project, the Disease Team was able to establish a method that allowed the large scale manufacture of ZFN-modified blood stem cells under conditions suitable for a clinical trial. These results were recently published [Li L. et al. Molecular Therapy; advance online publication 16 April 2013]. In year 3 of the study, the Disease Team developed a new method for delivering the ZFNs to the blood stem cells using messenger RNA (mRNA, or SB-728mR). Using a process called electroporation, in a technique that involves exposing a mixture of the blood stem cells and the SB-728mR to a transient electrical field, efficient mutation of the CCR5 gene was achieved. These cells were able to be transplanted into mice, where they engrafted and differentiated to generate human immune cells carrying mutated CCR5 genes. This mRNA-based approach has proven to be robust, well-tolerated and eliminates all viral vector components from the manufacturing process. Thus, electroporation of SB-728mR has now been chosen to move into clinical-scale manufacturing and to support our proposed clinical trial. In Year 4 of the study, the Disease Team will complete the necessary studies to demonstrate the safety of these modified blood stem cells, and submit the required federal and local regulatory documents to support the Phase I clinical trial of this new drug.

Development of RNA-based approaches to stem cell gene therapy for HIV

Funding Type: 
Early Translational II
Grant Number: 
TR2-01771
ICOC Funds Committed: 
$3 124 130
Disease Focus: 
HIV/AIDS
Immune Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
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).
Statement of Benefit to California: 
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.
Progress Report: 
  • 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.

Human Embryonic Stem Cell Therapeutic Strategies to Target HIV Disease

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00149
ICOC Funds Committed: 
$2 516 831
Disease Focus: 
HIV/AIDS
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
AIDS is a disease that currently has no cure. It arises when the human immunodeficiency virus (HIV) infects certain types of blood cells. These cells would normally be used to fight infection, but instead are destroyed by the virus, leading to immunodeficiency. We have recently been able to induce the development of human embryonic stem cells (hESC) into the types of cells that HIV can infect. In addition, we were able to show that a marker gene could be introduced into the hESC, and this gene continued to produce its protein throughout development of the cell into the more mature blood cell types. This sets the stage for testing the possibility of using gene-modified hESC to treat HIV or other immune system diseases. We have 3 different types of anti-HIV genetic approaches that we will test in laboratory models. These will be placed into hESC, and the cells allowed to develop into blood cells. We will then test whether our “therapeutic” genes can inhibit HIV infection in culture. We will also develop novel mouse models that allow development of hESC into blood cells in the body (in vivo). We will test the efficacy of certain of these genetic approaches in these systems, as they should more closely represent the situation in people. These studies will provide proof-of-principle that cells in the immune system can be modified by manipulation of hESC, and may help to develop future therapeutic approaches to combat HIV disease. In addition, these studies will be relevant to other immune system disorders such as autoimmune diseases. It was estimated that by January 31 2005, approximately 151,000 Californians were HIV infected. Furthermore, according to the California HIV Surveillance Report, 1752 new cases of HIV infection (1700 adult and 52 pediatric cases), and 5 deaths were reported between April 1 and September 31, 2006. Current treatment strategies prolong life, but do not cure infection, and are themselves quite toxic. Consequently HIV disease, and improved therapeutic approaches for this disease, are issues of great importance to the people of California.
Statement of Benefit to California: 
Current treatment strategies to halt HIV infection (AIDS) prolong life, but do not cure infection, and are themselves quite toxic. There are over 150,000 Californians infected with the AIDS virus. Consequently HIV disease, and improved therapeutic approaches for this disease, are issues of great importance to the people of California. Our studies will explore the potential of using human embryonic stem cells to fight AIDS and HIV infection. We have shown that human embryonic stem cells can develope into the immune system cells that are destroyed by the AIDS virus. In addition, we are exploring ways to genetically modify these cells (gene therapy) so that they would be protected from infection, and be better able to fight the infection in the body. We hope to eventually use these genetically modified cells to treat HIV infected individuals. If successful, our results may allow HIV infected individuals to discontinue, or greatly reduce the amount of anti-viral drugs that they must now take. This could directly benefit the patients' health, cut the cost of therapy, and allow less productive time lost from work, thus benefitting the State economy as a whole.
Progress Report: 
  • This CIRM grant was designed to explore the potential to genetically manipulate human embryonic stem cells (hESC), to develop new approaches to combat and protect against AIDS virus (HIV) infection. This is an important goal, because even though current anti-HIV medications are quite beneficial, they are expensive, may have unwanted side effects, and must be taken for the remainder of the patient’s life. Stem cell gene therapy approaches may have the ability to provide long-term protective effects for the patient. HIV infects several different types of blood cells, most notably CD4+ T cells and macrophages. Both of these cell types express CD4, the protein that allows the virus to attach to and infect the target cell. Our initial studies optimized macrophage and T cell development from hESC, which should allow us to subsequently introduce our candidate genetic approaches into hESC, and then test the ability of these genes to function in these blood cells derived from hESC. Several different approaches are being tested to maximize the potential for controlling or eliminating HIV from the body of infected individuals, These are in various stages of development. One area that has shown promise, which initially involved the use of hematopoietic stem cells as a prelude to working with hESC, is engineering stem cells such that mature cells that develop from these stem cells can actually attack HIV infected cells. It is our hope that genetically manipulated hESC will allow us to replace cells lost to HIV infection with cells protected against viral infection, and/or to develop strategies that allow the body to defend itself against infection.
  • This CIRM grant was designed to explore the potential to genetically manipulate human embryonic stem cells (hESC), to develop new approaches to combat and protect against AIDS virus (HIV) infection. This is an important goal, because even though current anti-HIV medications are quite beneficial, they are expensive, may have unwanted side effects, and must be taken for the remainder of the patient’s life. Stem cell gene therapy approaches may have the ability to provide long-term protective effects for the patient. HIV infects several different types of blood cells, most notably CD4+ T cells and macrophages. Both of these cell types express CD4, the protein that allows the virus to attach to and infect the target cell. Our initial studies optimized macrophage and T cell development from hESC, which has allowed us to introduce our candidate genetic approaches into hESC, and then test the ability of these genes to function in these blood cells derived from hESC. Several different approaches are being tested, some in hESC and others in hematopoietic stem cells (HSC) to maximize the potential for controlling or eliminating HIV from the body of infected individuals. These are in various stages of development. One area that has shown promise, which initially involved the use of HSC as a prelude to working with hESC, is engineering stem cells such that mature cells that develop from these stem cells can actually attack HIV infected cells. This type of technology could be extended to other chronic infectious diseases or cancers. A second approach involves manipulating cells such that they control virus replication. Development of this latter approach is ongoing. It is our hope that genetically manipulated hESC will allow us to replace cells lost to HIV infection with cells protected against viral infection, and/or to develop strategies that allow the body to defend itself against infection.
  • This CIRM grant was designed to explore the potential of human embryonic stem cells to develop into cells found in the blood, and to adapt these stem cells with genetic approaches to combat infection by the AIDS virus. Over the course of this grant we made significant progress in showing that human embryonic stem cells can develop into several types of blood cells. Importantly this includes T lymphocytes, including cells of the CD4+ T cell lineage and cells of the macrophage lineage, which are the main cell types that can be infected by the AIDS virus. We had originally proposed several genetic strategies to either prevent HIV from productively infecting target cells, or to allow cells to directly attack virally infected target cells. To date we have shown that we can add new genes into human embryonic stem cells, and that we can control their expression in mature T lymphocytes derived from these stem cells, however the process of development of T cells from embryonic stem cells is very difficult and inefficient. We had the most success with one of our three genetic approaches, however to best test the efficacy of this approach, we took a step back and assessed its usefulness using human bone marrow-derived stem cells, which are much easier to convert into mature human T lymphocytes than are human embryonic stem cells. The successful strategy involved the genes for a cell surface molecule, called the T cell receptor (TCR). The TCR is the molecule that normal T lymphocytes use to target and attack foreign or infected cells in the body. These molecules are exquisitely specific for particular foreign particles, and will only recognize a particular pathogenic agent. Thus for example, a TCR specific for measles virus will not detect cells infected with the AIDS virus. We engineered genes encoding a TCR that specifically recognized a part of the AIDS virus that could be detected on infected cells, and inserted these genes into the bone marrow stem cells. Following this, we directed the development of these genetically altered stem cells into T lymphocytes, by implanting them into a mouse that contained human immune tissues required for the development of these cells into a mature state. This strategy allowed the genetically engineered stem cells to develop into CD8+ “killer” T lymphocytes that could detect and kill HIV infected cells. Theses T lymphocytes did not kill normal, uninfected cells, thus we engineered cells that could target and eliminate HIV infected cells. We are continuing to explore this system, and to adapt and improve this method such that it can be employed with human embryonic stem cells.
  • Our project was designed to develop new genetic approaches that could be introduced into stem cells to combat HIV (AIDS virus) infection. We have used this recent time to complete experiments demonstrating that cell surface molecules that direct cytotoxic T cells towards their HIV-infected targets, can be introduced into human blood-forming stem cells. These in turn direct the stem cells to develop into killer T cells that can attack HIV infected cells. We have tested this approach in a new in vivo model, and found it to be highly efficacious. The introduction of this genetic engineering approach allowed the animals to control HIV infection, and preserved the presence of CD4 T cells, which are normally killed by the virus.

HPSC based therapy for HIV disease using RNAi to CCR5.

Funding Type: 
Early Translational from Disease Team Conversion
Grant Number: 
TRX-01431
ICOC Funds Committed: 
$1 505 000
Disease Focus: 
HIV/AIDS
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • 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.

Stem Cell Programming With Chimeric Antigen Receptors to Eradicate HIV Infection

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06845
ICOC Funds Committed: 
$5 303 375
Disease Focus: 
HIV/AIDS
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
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.
Statement of Benefit to California: 
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.

RUNNING TITLE: Stem Cell Gene Therapy for HIV in AIDS Lymphoma Patients

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05327
ICOC Funds Committed: 
$74 195
Disease Focus: 
Blood Cancer
Cancer
HIV/AIDS
oldStatus: 
Closed
Public Abstract: 
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.
Statement of Benefit to California: 
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.
Progress Report: 
  • 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.

Stem cell differentiation to thymic epithelium for inducing tolerance to stem cells

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01702
ICOC Funds Committed: 
$1 314 090
Disease Focus: 
Immune Disease
HIV/AIDS
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
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
Statement of Benefit to California: 
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.
Progress Report: 
  • 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.

Genetic modification of the human genome to resist HIV-1 infection and/or disease progression

Funding Type: 
SEED Grant
Grant Number: 
RS1-00172
ICOC Funds Committed: 
$642 652
Disease Focus: 
HIV/AIDS
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The proposed studies describe the genetic approaches utilizing human embryonic stem cells to suppress and/or eliminate the expression of the human protein CCR5. CCR5 is found on the surface of white blood cells. HIV-1 attaches to CCR5 and uses CCR5 to enter into its target cells. Our approach is to utilize established as well as new non-established approaches to prevent CCR5 from appearing on the surface of the cells. If CCR5 is not present, HIV-1 cannot infect the cells. Interestingly, this concept has already been proven in nature. Approximately 1% of the Caucasian population is genetically deficient for CCR5 and these individuals are resistant to HIV-1 transmission. Their white blood cells, when placed in culture, also resist HIV-1 infection in the laboratory. As such, we believe that our approach can be used to protect high risk individuals from HIV-1 infection as well as impede or stop progression of disease in those individuals already infected.
Statement of Benefit to California: 
According to the Centers for Disease Control, California is second only to New York of individuals living with AIDS. Developing means to stop HIV-1 infection and cure those individuals already infected with HIV-1 is of paramount importance for the state of California.
Progress Report: 
  • The overall goal of this project is to investigate the use of embryonic stem cells and blood stem cells as a potential therapeutic approach for HIV-1 disease. We investigated a process known as RNA interference to block HIV-1 infection. We characterize the properties of cells carrying RNA interference to HIV-1 and developed new tools to facilitate the study.

GENE-MODIFIED HEMATOPOIETIC STEM/PROGENITOR CELL BASED THERAPY FOR HIV DISEASE

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-06893
Investigator: 
ICOC Funds Committed: 
$8 278 722
Disease Focus: 
HIV/AIDS
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • CAL-USA-11 is a Phase I/II human study designed to assess the safety, feasibility, and tolerability of the Cal-1 product in HIV-infected individuals who have previously been on ART but are not currently taking any antiretroviral agent. The objective of the Cal-1 therapy is to increase the number of protected cells in the body of an individual infected with HIV to the point where the virus is incapable of causing harm. This would potentially reduce or eliminate the need for a lifetime of antiretroviral therapy.
  • In 1996, scientists determined that CCR5 is the primary co-receptor by which HIV enters and infects T cells. Most people inherit two normal copies (one from each parent) of the gene that codes for the CCR5 protein. However, about 1% of the European population has a mutation in both of these copies. Because they do not produce any CCR5, these individuals are naturally resistant to HIV infection.
  • This clinical trial is a first-in-human test of Calimmune’s one-time outpatient gene therapy that has been designed to confer a similar genetic resistance to the T cells and hematopoietic stem/progenitor cells of HIV-infected patients. This will be accomplished by reducing CCR5 expression through a process called RNA interference (RNAi), and preventing HIV entry through the use of a membrane-bound fusion inhibitor.
  • As such, our approach seeks to protect target cells from HIV via two distinct mechanisms. The potential benefit of this combined approach is twofold: Because we are treating stem cells along with T cells, we will be creating the potential for the progeny of the stem cells to also exhibit genetic resistance to HIV and therefore repopulate the participant’s immune system; and because we are utilizing a dual therapy, we minimize the possibility of cellular infection via different or mutated HIV strains.
  • The study is enrolling participants at sites in Los Angeles and San Francisco, Calif., under the direction of Principal Investigators Ronald Mitsuyasu, M.D., of UCLA and Jacob P. Lalezari, M.D., of Quest Clinical Research in San Francisco.
  • The first participant was infused with Cal-1 treated T cells and hematopoietic stem/progenitor cells (HSPC) in June 2013. Since then, additional participants have also been infused.
  • The study has three arms. All participants will receive the Cal-1 product. Participants in two of the three study arms will also receive different doses of a drug known as busulfan prior to the infusion, which has the potential to make the therapy more effective.
  • Laboratory assessments performed throughout the course of the study will monitor:
  • • the participants’ general health and level of HIV infection;
  • • the participants’ level of CD4+ T cells;
  • • the presence of Cal-1 modified cells in various cell types in the blood and lymphoid tissue; and
  • • the safety of the approach.
  • The primary objectives of the study are to evaluate:
  • • The safety, feasibility, and tolerability of Cal-1 gene-transduced hematopoietic cell populations.
  • • The safety and tolerability of low- and moderate-dose busulfan as a non-myeloablative conditioning agent as a means to improve engraftment of transduced HSPC.
  • The study is open to men and women ages 18 to 65 who are HIV-infected but do not have any other serious medical conditions. Participants must have been well-controlled on ART in the past, but must not be taking ART currently.
  • Full details of the study are available at:
  • http://www.clinicaltrials.gov/ct2/show/NCT01734850?term=NCT01734850&rank=1

HPSC based therapy for HIV disease using RNAi to CCR5.

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01431
ICOC Funds Committed: 
$9 905 604
Disease Focus: 
HIV/AIDS
Immune Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 
RNA interference is a naturally occurring means to block the function of genes in our body. We propose that RNA interference can be used to block HIV-1 infection and its reproduction within the body. When RNA interference is introduced into a stem cell, its blocking activity will be present throughout the lifetime of the stem cell, theoretically the lifespan of a human being. Thus, in theory an effective stem cell RNA interference therapy will require only a single treatment as opposed to the current lifetime administration of anti-HIV-1 drugs often accompanied by serious side effects. In nature, some individuals carry a genetic mutation that renders them resistant to HIV-1 infection. This mutation prevents HIV-1 from attaching to the white blood cells. Our RNA interference approach will be to mimic this natural situation by blocking the activity of this “co-receptor” within infected individuals by creating a new blood system that carries the RNA interference therapy. This therapy will be developed as a combination with other gene therapeutic reagents to protect the new blood system from HIV infection.
Statement of Benefit to California: 
The need for novel approaches to the treatment of HIV infection has never been greater, because new infections continue to occur at undiminished rates, in California and across the nation, despite decades of prevention efforts. Moreover, the number of people living with HIV is rising steadily, thanks to improved management of HIV infection. As a result, California, which ranks second in the nation in diagnosed cases of HIV infection, behind only New York, has identified 67,500 men, women, and children who carry the virus. (Estimates of the number of Californians who are infected but have yet to be diagnosed range as high as 33,513.) Not all of the state’s HIV-positive residents are currently on therapy, but eventually virtually all of them will be—and many of them will receive their drugs through government-supported programs. In addition, the longer these infected individuals live, the more likely they are to avail themselves of a range of support services that the state pays for. The drugs themselves, which are routinely administered in combination, are initially effective in suppressing viral replication in infected individuals, but their potency diminishes over time, even as the toxic effects of therapy accumulate. California, which is supporting the nation’s second-highest case-load of HIV-positive individuals, can expect to see that number grow at a rate of 5,000-7,000 a year for the foreseeable future. The cost of providing life-sustaining medications and social services to this burgeoning population will also continue to rise, not arithmetically but exponentially—because as increasing numbers of infected individuals fail standard drug regimens, it will be necessary to shift them to newer, more expensive treatments, and as the side effects of therapy become more manifest, it will be necessary to prescribe drugs to combat those toxicities, adding to the overall drug burden for patients and the overall cost of drug therapy for the state. In such circumstances, the prospect of stem-cell based therapy that will require “only a single treatment” is especially compelling. In theory, RNA interference might effectively cure individuals infected with HIV, by blocking the ports through which the virus enters CD4 cells and destroys the body’s immune system. And even if RNA interference proves only partially effective in blocking viral entry, it could significantly suppress viremia while sparing patients the toxicities associated with drug therapy. This would be a boon to infected individuals, but it would also be a benefit to uninfected Californians, because reductions in viremia in infected individuals translate into a reduction in the community burden of HIV infection—and that, in turn, reduces the overall rate of new infections statewide.
Progress Report: 
  • Our overall goal is to file an IND within 4 years for a hematopoietic stem cell based genetic therapy for HIV-1 disease. The concept is that introducing anti-HIV gene therapeutics into hematopoietic stem cells will produce a protected population of T lymphocytes and monocyte/macrophages (the cells specifically infected by HIV) in individuals to decrease viral load and maintain stable T lymphocyte counts. Hemapoietic stem cells are unique in that they are multipotent stem cells that give rise to all the types of blood cells, including T cells and monocytes/macrophages. During the first year we have met each of our key milestones and made significant progress in identifying and testing genetic reagents combined in the context of a lentiviral vector for stable delivery into hematopoietic stem cells. The vector candidates include combinations of gene therapeutics aimed at different stages of HIV replication namely: i) binding to the CCR5 HIV co-receptor (RNA interference to down-regulate CCR5), ii) fusion of the HIV virion to the cell surface (fusion inhibitor), iii) a restriction factor inhibiting translocation of the HIV genomic material from the cell surface to the nucleus (restriction factor) and, iv) inhibition of HIV expression within the cell (RNA interference directed to a key portion of HIV that drives its expression). We are presently identifying the optimal combination and vector/target ratio. We have also tested several reagents designed to increase transduction efficiency of hematopoietic stem cells and have validated assays to examine potential toxicity including genotoxicity of therapeutic vectors. Thus far, we have not seen any general vector-induced toxicity. In order for this gene transfer to be applied to patients, the hemapoietic progenitor stem cell transduction must be scaled up significantly. Experiments are currently in progress maximizing transduction of hemapoietic progenitor stem cells at sufficiently high cell numbers for future therapeutic analysis.
  • HIV-1 therapy requires combinations of reagents in order to effectively suppress HIV-1 replication. We have created several combinations of anti-HIV reagents through genetic engineering, which will eventually be delivered to humans through adult blood stem cells. We have compared the effectiveness and safety of these genetic “vectors” in cell culture and in an advanced mouse model, which allows human blood cells to grow in tissues. In addition, this mouse model allows one to investigate HIV-1 infection within the animals. Through these tests, we narrowed combinations down to those that seem to be the most effective based upon showing no toxicity and possessing the ability to be maintained within human blood cells in the mouse, and resist multiple strains of HIV-1 infection both in cell culture and in the humanized mice.

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