HIV/AIDS

Coding Dimension ID: 
293
Coding Dimension path name: 
HIV/AIDS
Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01702
Investigator: 
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.
  • 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.
Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00149
Investigator: 
Type: 
PI
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.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00172
Investigator: 
Name: 
Type: 
PI
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.
Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01490
Investigator: 
Name: 
Institution: 
Type: 
PI
Institution: 
Type: 
Co-PI
Type: 
Co-PI
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.
  • Infection with HIV-1 requires entry of the virus into cells by binding to a receptor protein called CCR5. Persons with a natural mutation in the gene encoding CCR5 (CCR532) are protected from HIV-1 infection and AIDS. Everyone has two copies of this 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 with HIV 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 6 years off anti-HIV medicine, the patient is considered cured, as there is no evidence of an active HIV-1 infection.
  • This Disease Team proposed 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. The idea was to try and reproduce the effects of the CCR532 mutation by providing a renewable and long-lasting source of these HIV-1 resistant cells. This approach gets around the need to find a stem cell donor who happens to carry the CCR532/ CCR532 double 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 showed 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 supported 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].
  • 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, 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. This method was tested in mice and again showed evidence of protection from HIV-1. Electroporation of SB-728mR was then moved into clinical-scale manufacturing to support our proposed clinical trial. In Year 4 of the study, the Disease Team completed studies demonstrating the safety of these modified blood stem cells, and obtained the required federal and local approvals to initiate the first-in-human testing of the CCR5-modified blood stem cell therapy. The Phase I clinical trial, which is sponsored by City of Hope and funded by Sangamo BioSciences and CIRM under its Strategic Partnership funding mechanism, is planned to start accrual in the fourth quarter of 2014.
Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01431
Investigator: 
Name: 
Type: 
PI
Institution: 
Type: 
Co-PI
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.

Pages