Early Translational II
HIV infection is a major public health problem in California and abroad. Current treatments do not cure HIV infection and require lifelong medications that are expensive and often associated with serious side effects. To address the clear need for improved treatments for HIV infection, we propose to develop a cell therapy based approach. Specifically, we aim to modify a gene that encodes a cell surface protein (CCR5) that is crucial for HIV entry into the cell, yet non-essential for normal cell function. HIV causes AIDS by infecting various types of immune cells that each express CCR5 and arise from a common parental cell type called a hematopoietic stem cell (HSC). Studies have shown that HIV replication is substantially reduced if the virus is prevented from binding to CCR5. In fact, rare individuals who have naturally mutated CCR5 genes are highly resistant to HIV infection. Thus, we propose that HIV-infected individuals would benefit from having cells with CCR5 mutations, similar to those found in naturally resistant individuals. This CCR5 mutation need only be present in HSC, as these cells will then develop into a variety of immune cell types having the CCR5 mutation. A challenge for this therapeutic approach is mutating the CCR5 gene in HSC, because these cells are rare (<1 per microliter of blood) and they are difficult to permanently genetically modify. However, HSC can be converted into induced pluripotent stem (iPS) cells in the laboratory. The resulting iPS cells can then be grown to appreciable numbers to allow for CCR5 mutation procedures. The CCR5-mutant iPS cells can then be converted back into HSC and re-administered to the HIV-infected patient. Our cell therapy approach is supported by a recent clinical model where an HIV-infected patient received HSC from an uninfected individual who had CCR5-mutant cells. Now three years following this procedure, the HIV-infected patient shows no evidence of HIV-replication, despite the absence of antiretroviral drugs. Importantly, individuals who naturally have these CCR5-mutant HIV-resistant cells are rare (<1% of the population). Moreover, such transplantations of cells can only take place between genetically compatible individuals. Therefore, while this clinical model is encouraging for future directions such as proposed here, it is not directly applicable to the majority of HIV-infected individuals. Our cell therapy approach addresses the limitations of the clinical model and could be directly beneficial to a much larger number of HIV-infected individuals.
Statement of Benefit to California:
HIV infection is a major public health problem in California and abroad. Current treatments do not cure HIV infection and require lifelong medications that are expensive and often associated with serious side effects. To address the clear need for improved treatments for HIV infection, we propose to develop a cell therapy based approach. Successfully applied, this therapy should 1) be of clinical benefit to HIV-infected individuals in California, 2) lower the risk of HIV infection for Californians, and 3) reduce to health care expenditures incurred by the state of California and its citizens. Moreover, this research will help California maintain its place as a leader in HIV research and medical research in general.
The disease target of this Development Candidate Feasibility Award application is AIDS and the target product consists of autologous induced pluripotent stem cells (iPSC)-derived hematopoietic stem cells (HSC) genetically engineered to be HIV-resistant. The goal is to derive iPSC from the patient’s peripheral blood cells, to genetically modify them so that both alleles of the gene encoding the HIV co-receptor CCR5 are replaced with a naturally occurring mutant version of that gene, and to differentiate them into CD34+ HSC for transfer back into the patient. The CCR5 mutation delta32 is known to confer resistance to infection by CCR5-tropic HIV. Therefore, reconstitution of the immune system with CCR5 delta32 homozygous HSC is anticipated to lead to a functional cure for AIDS, since all newly generated hematopoietic cell derivatives would be protected from HIV infection. The specific aims for this study are to work out CCR5 gene targeting and hematopoietic differentiation using existing iPSC, and to test HIV resistance of the resulting cells in vitro (Aim 1), to repeat these studies with iPSC generated from HIV-infected individuals using integrase-mediated introduction of reprogramming genes into blood cells, followed by recombinase-mediated removal of the reprogramming genes (Aim 2), and to evaluate the hematopoietic potential and level of HIV resistance of CCR5 mutant iPSC derivatives in vivo using two mouse models (Aim 3). Reviewers judged the overall objectives of the proposed studies to be straightforward and the scientific rationale to be sound, since interfering with CCR5 expression or function is well known to confer HIV resistance in vitro. Importantly, HIV has remained undetectable in a HIV-infected patient who discontinued anti-retroviral therapy after receiving an allogeneic CD34+ peripheral blood stem cell transplant from a CCR5 delta32 homozygous donor. However, the application of a CCR5-based approach to autologous stem cell therapy is not novel, as others are pursuing CCR5 modification strategies using autologous HSC. Reviewers acknowledged that the use of iPSC as starting material could offer advantages such as clonal selection of appropriately modified cells, since this would avoid the known problem of low transduction efficiency by gene transfer vectors into adult HSC. The potential for unlimited iPSC proliferation and thus, in theory, production of unlimited numbers of HSC is also an advantage of the proposed approach. Some reviewers felt that the use of the natural CCR5 mutation is an advantage when compared to gene knock down or knock out approaches, since strong data exist that expression of the delta32 CCR5 protein confers HIV resistance. The applicant also failed to discuss possible immunogenicity of the mutated CCR5 protein, as it will be foreign to the patient. Reviewers agreed that if successful, the proposed study could have a major impact for AIDS patients by providing a new treatment approach that would avoid the detrimental side effects of conventional drug therapy. Some reviewers considered the anticipated high cost of this patient-specific treatment a limitation, whereas others pointed out that the savings on direct and indirect costs associated with current life-long anti-retroviral therapies would outweigh this burden. Reviewers identified major limitations with the feasibility of this proposal. Although the applicant included some relevant preliminary data, this information did not substantiate an ability to overcome important technical hurdles. Reviewers did appreciate the choice of reprogramming method, since the chosen approach results in much more limited transgene integration patterns than do conventional viral vectors. This approach was well supported by preliminary data, but no data were provided for the technically challenging BAC-mediated homologous recombination in human pluripotent stem cells, which includes sequential targeting at both CCR5 alleles. A critical technical challenge in these studies is the ability to differentiate human iPSC into HSCs that can efficiently and functionally repopulate murine recipients in vivo. This has not been achieved to date, and preliminary data supporting the feasibility of overcoming this hurdle were limited. The proposed alternate approach was viewed as extremely challenging and likely to encounter serious regulatory issues due to the possibility of introducing animal components during the proposed culture method. The applicants provide no data supporting the feasibility of this backup plan. Finally, they did not address potential engraftment problems for the mutated iPSC-derived HSC and did not discuss the conditioning regimen required for their therapeutic intervention. The principal investigator is a highly recognized leader in the HIV field who is very well funded, has received numerous awards, and has published prolifically in high quality journals. A key collaborator is a highly accomplished expert in hemoglobin regulation and hemoglobin disorders. The key investigators are devoting appropriate effort to the proposed studies and have been extremely productive collaborators for many years; this is expected to continue. The assembled team provides much of the technical and scientific expertise required to execute these studies. However, reviewers noted that the application would have benefited from inclusion of an investigator with expertise in HSC differentiation from human pluripotent stem cells. The applicant institution provides an excellent environment for the proposed studies. Overall, the reviewers appreciated the potential impact of the proposed studies on an important unmet medical need in AIDS treatment, but also emphasized that similar, competing approaches are already underway. Although the applicant team includes preeminent investigators, the feasibility of the project was seriously questioned, as major technical hurdles were not supported by preliminary data.