HIV/AIDS

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

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.

Physical and biological mechanisms of hMSC induction in the cartilage microenvironment

Funding Type: 
Basic Biology II
Grant Number: 
DR1-06893
Investigator: 
ICOC Funds Committed: 
$0
Disease Focus: 
HIV/AIDS
Stem Cell Use: 
Adult Stem Cell
Public Abstract: 
Within the shake of a hand, one can tell that bone is hard, skin is soft, and muscle is in between. The physical properties of each of these tissues are important for their distinct functions in the body. At a much smaller scale, a cell uses its sense of ‘touch’ to determine the physical properties of its surroundings. In this way, cells can discern if they reside in a bone, skin, or muscle microenvironment. Not only do cells sense the stiffness of their environment, but they also respond to it. The physical properties of the matrix microenvironment direct key cellular decisions including cell division, migration, and cell fate selection. For a stem cell, this information is a valuable cue that instructs it to select a cell fate that matches the physical surroundings. Experimentally, a stem cell becomes a bone cell when grown on a stiff matrix but it becomes a brain cell on a soft one. In the body, a multitude of physical and biochemical cues cooperate to direct intricate cell fate decisions. Relative to well-studied biochemical cues, we are just beginning to understand how cells sense and respond to physical cues. Despite their importance in stem cell biology, we know almost nothing about how physical cues interact with biochemical cues to instruct stem cells to select a specific fate. Our research seeks to understand how stem cells integrate physical and biochemical cues to select a cell fate. We discovered a specific combination of physical and biochemical cues that, when combined, drive adult human mesenchymal stem cells to select a cartilage cell fate. Culture of these stem cells on a cartilage-like matrix stiffness greatly intensifies their response to biochemical signals to induce cartilage production. By investigating the molecular basis of this response, we have novel insights into the way cells integrate physical and biochemical cues. Building on this foundation, we will uncover new mechanisms by which stem cells sense and respond to their surroundings to select a specific fate. While this research is important for understanding cartilage cell fate selection, these fundamental mechanisms will inform many aspects of stem cell biology, including the maintenance of pluripotency and the selection of multiple cell fates. Although our primary focus is the discovery of basic cellular mechanisms, our model system has important clinical relevance. More than 6 million Californians suffer from osteoarthritis, a disease characterized by the loss of cartilage physical properties. Understanding the interaction of physical and biochemical cues in cartilage may elucidate the cause of osteoarthritis while advancing new therapies to treat it. Already, our research suggests that the combination of biochemical and physical cues enhances the utility of human mesenchymal stem cells for cartilage repair. The results of our work will be applied to promote development of a stem cell-based therapy for cartilage repair.
Statement of Benefit to California: 
This project investigates the cues that direct human mesenchymal stem cells to select a cartilage cell fate. Despite the fact that this stem cell source is very attractive for stem cell-based cartilage repair, several obstacles have limited its clinical application. Our research has identified a novel combination of biochemical and physical cues that overcomes a number of these obstacles. Although we still do not understand how these cues exert their beneficial effects, this strategy has significant therapeutic potential. By investigating the mechanisms by which these cues promote cartilage cell differentiation, this research may advance the translation of these findings to a clinical setting, which would have significant impact on the state of California. Approximately 6 million adults in California, or 27% of the population have some form of arthritis. This disease costs California nearly $32 billion each year, with an estimated $23.2 billion spent on direct medical care and $8.3 billion due to lost wages. Osteoarthritis is a disabling disease that limits the ability to engage in the regular physical activity that prevents obesity, diabetes, and cardiovascular disease. Consequently, successful development of improved arthritis therapies will benefit the health of a significant portion of the California population. In addition to the health of Californians, cell-based therapies for arthritis and other musculoskeletal conditions provide a huge commercial opportunity for California industry. Support from Proposition 71 increases the likelihood that such therapies are developed in partnership with California companies. Clearly their economic success will provide employment opportunities for Californians, tax revenue for the state, and help maintain California as a world leader in biotechnology research and development. Finally, by investigating the cellular response to physical cues, this work has implications for other stem cell based-therapies as well as for biomaterials design. Physical cues that promote a specific cell fate decision can be engineered into novel biomaterials that are used to deliver stem cell-based therapies to any target tissue. Again, these advances have the potential to improve the health of California citizens and to create commercial opportunities for California biotechnology companies.
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

Liver Disease Team

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01490
ICOC Funds Committed: 
$0
Disease Focus: 
HIV/AIDS
Immune Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
Public Abstract: 
Because there is still considerable morbidity and mortality associated with the process of whole liver transplantation, and because more than a thousand people die each year while on the liver transplantation list, and tens of thousands more never get on the list because of the lack of available livers, it is evident that improved and safer liver transplantation would be valuable, as would approaches that provide for an increased number of transplantations in a timely manner. A technology that might address these issues is the development of a human liver cell line that can be employed in liver cell transplantation or in a bioartificial liver. Developing such a cell line from human embryonic stem cells (hESC) would provide a valuable tool for pharmacology studies, as well as for use in cell-based therapeutics. The objective of this proposal is to focus a team effort to determine which differentiated hESC will be the most effective liver-like cells in cell culture and in animal studies, and to then use the best cells in clinical trials of cell transplantation in patients with advanced liver disease. In the proposed studies, the team will differentiate hESC so that they act like liver cells in culture. Once it has been established that the cells are acting like liver cells by producing normal human liver proteins, and that they do not result in tumors, the cells will be assessed in clinically-relevant models using techniques that can then be adapted to future human clinical trials. One of the ways cells can be evaluated is to label the cells which will provide a means to monitor them with various imaging systems. The intent in these studies is to determine which will be the most effective cells to use in human clinical trials. Once this is determined, the best cells can then be employed in human patients. If the studies are successfully undertaken, we will have established a clinically useful and viable liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with whole liver transplantation. Moreover, all people who have liver failure or an inherited liver disease could be treated, because there would be an unlimited supply of liver cells.
Statement of Benefit to California: 
In California, as in all parts of the US, there are not enough livers available for transplantation for all the people who need them. The result is that many more people die of liver failure than is necessary. One way to improve this situation is the transplantation of liver cells rather than whole organ transplantation. We are attempting to develop liver cell lines from stem cells that will act like normal liver cells. If the cells that we develop function well and do not act like cancer cells in culture, the cells will be assessed in clinically-relevant models using techniques that can then be adapted to future human clinical trials. In our studies, we will compare human embryonic stem cells with other stem cells to determine which will be the most effective cells to transplant into people. Finally, we will employ the best cells in clinical trials in humans. If the studies are successfully undertaken, we will have established a clinically useful and viable liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with whole liver transplantation. Moreover, all people who have liver failure or an inherited liver disease could be treated, because there would be an unlimited supply of liver cells.
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.

Develop a cell replacement therapy for Parkinson’s disease using human embryonic stem cells

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01490
ICOC Funds Committed: 
$0
Disease Focus: 
HIV/AIDS
Immune Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
Public Abstract: 
Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated. All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation. We believe that human embryonic stem cells (hESCs) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing hESC differentiation into dopaminergic neurons. Parallel efforts by clinicians have identified processes to implant the cells safely and to follow their behavior in humans in a safe non-invasive fashion. Equally important, useful animal models for testing cell therapy have been developed. We therefore believe that the time is right to mount a coordinated team effort such as the one we have proposed to approval from the FDA to treat PD using dopaminergic neurons obtained from hESCs. For this proposal we have built a California team with both scientists and clinicians that have the potential to translate a promising idea (a cell therapy for PD) to an IND submission. Our goals include: 1) Identifying a clinically compliant hESC line capable of differentiating into midbrain dopaminergic neurons; 2) Developing protocols for generating and purifying dopaminergic neurons on a large scale; 3) Transferring the protocols to a Good Manufacture Practice (GMP) facility and making clinical grade lots; 4) Testing the quality of the cells in suitable PD animal models (rodents and large animal models); 5) Collecting the data to submit to the FDA for permission to conduct a clinical trial. This application to treat a currently non-curable disease (PD) meets CIRM's primary goal for Disease Team Research Awards and we believe our efforts will help take cell-based therapy for PD to the clinic.
Statement of Benefit to California: 
Parkinson’s disease affects more than a million patients United States with a large fraction being present in California. California, which is the home of the Parkinson’s Institute and several Parkinson’s related foundations and patient advocacy groups, has been at the forefront of this research and a large number of California based scientists supported by these foundations and CIRM have contributed to significant breakthroughs in this field. We have assembled a California based team of scientists and clinicians that aim to develop a cell replacement therapy for this currently non-curable disorder. We believe that this proposal which will hire more than thirty employees in California includes the basic elements that are required for the translation of basic research to clinical research. We believe these experiments not only provide a blueprint for moving Parkinson’s disease towards the clinic for people suffering with the disorder but also a generalized blueprint for the development of stem cell therapy for multiple neurological disorders including motor neuron diseases and spinal cord injury. The tools and reagents that we develop will be made widely available to Californian researchers and we will select California-based companies for commercialization of such therapies. We hope that California-based physicians will be at the forefront of developing this promising avenue of research. We expect that the money expended on this research will benefit the Californian research community and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
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.

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