HIV continues to be a major public health problem with more than 30 million people infected worldwide. Although small molecule drugs prolong the lives of HIV infected individuals, there is no cure or effective vaccine available. Gene therapy for HIV offers a promising alternative to current treatments due to advantages including the possibility of a one-time treatment, controlled or constitutive anti-HIV gene expression, and long-term viral inhibition upon transduction of blood stem cells with the possibility of creating a "functional cure". HIV is characterized by a prolonged course of infection often spanning several years, thus providing a large window of opportunity for gene therapeutic intervention. All cells susceptible to HIV infection (CD4 T cells, macrophages, and dendritic cells) are derived from hematopoietic stem cells (HSCs). If anti-HIV activity could be introduced into HSCs, this phenotype would be passed on to the differentiated HIV target cells. Current drawbacks to allogeneic and autologous bone marrow transplants for HIV gene therapy with ex vivo engineered HSCs include the possibility of graft rejection and low transduction efficiency which leads to low in vivo cell marking of the transplanted cells.
With the development of induced pluripotent stem cells (iPSCs), however, it is now possible to generate patient-specific pluripotent stem cells from an adult individual. These iPSCs can be maintained in an undifferentiated state and have enormous potential as a source of autologous cells and tissues for therapeutic use. iPSC derived anti-HIV HSCs, generated from patient specific cells, will eliminate the problem of graft rejection. Also, HSCs derived from a single iPSC colony will be clonal thus allowing for complete characterization of the engineered cells including the site of integration of the anti-HIV genes, their functionality, safety, and anti-HIV efficacy. All cells arising from the clonal iPS colony will contain the anti-HIV genes eliminating the problem of low transduction efficiency and in vivo marking of transplanted cells. As iPSCs can be maintained in an undifferentiated state, a continuous supply of HIV resistant cells can be generated. In the proposed research we plan on optimizing the generation of anti-HIV gene expressing iPSCs from multiple cell sources and differentiating these cells into HIV resistant HSCs and immune cells including macrophages and dendritic cells in vitro and T cells, B cell, macrophages, and dendritic cells in vivo in a humanized mouse model. We will characterize the safety, toxicity, functionality, and anti-HIV efficacy of the anti-HIV iPSC derived immune system cells. The data generated from this proposal will bring these novel therapies closer to the clinic.
Statement of Benefit to California:
Currently around 106,000 California residents are infected with HIV while an additional 68,000 have AIDS. California is second only to New York in AIDS cases with no end in sight due to the lack of effective vaccines or a cure. Novel and innovative therapeutic approaches need to be developed to combat this devastating disease. Gene therapy for HIV offers a promising alternative treatment to current antiretroviral drugs with the potential of creating a “functional cure”.
Recently, long term control of viral replication was observed in an HIV-1 infected individual who received a stem cell transplant in Berlin, Germany for acute myeloid leukemia. The transplanted allogeneic stem cells were from an individual who was homozygous for the CCR5 ∆32-bp deletion. This study highlights the importance in developing anti-HIV gene and cellular therapies which are capable of generating HIV-resistant immune system cells. Based on these results, there is hope for developing a novel treatment for HIV infected individuals by generating a continuous supply of HIV resistant autologous cells which could be used for regenerative therapy. With the introduction of iPSC technology, there is now the potential for developing a source of self-renewing pluripotent stem cells capable of indefinite expansion from a patient’s own cells.
All HIV infected individuals, male, female, of any age and race, will be able to benefit from this treatment as we would be using their own cells to generate the HIV-resistant HSCs. The anti-HIV HSCs generated from these iPSC lines can be frozen and stored for future use. This strategy would help overcome the disadvantages of current HIV gene therapy protocols which have obtained minimal in vivo gene marking and the transplantation of a finite number of cells which have not been fully characterized. Also, if subsequent treatments are needed, future transplantations can be administered without having the patient’s cells undergo apheresis, transduction, and re-transplantation due to the continuous supply of HIV resistant immune cells from the patient-specific anti-HIV iPSC lines.
This application for a Development Candidate Award focuses on a gene-modified induced pluripotent stem cell (iPSC)-derived therapy for HIV/AIDS. Recent publications describing a patient with leukemia and HIV have suggested that a functional cure for HIV infection can be achieved following myeloablation and bone marrow transplantation of hematopoietic stem cells (HSCs) from a donor carrying an HIV-resistance gene. In contrast, clinical trials in HIV-lymphoma patients of autologous HSCs genetically modified with HIV-resistance genes demonstrated some engraftment, but not at sufficient levels for clinical benefit, hypothesized to be due to insufficient numbers of genetically modified HSC. The applicant proposes to gene-modify iPSCs, prior to differentiation into HSCs, to enable production of large numbers of gene-modified HSCs. The applicant hypothesizes that transplanting more gene-modified cells will improve engraftment and permit the use of a less toxic, nonmyeloablative conditioning regimen prior to transplant. The applicant proposes three Specific Aims: (1) to evaluate different cell sources for their potential to generate anti-HIV gene expressing iPSCs which can be differentiated into HSCs; (2) to evaluate the potential of these HSCs to differentiate into HIV-resistant immune cells in vitro; and (3) to determine the in vivo engraftment and differentiation potential of these HSCs and test their functionality, safety and efficacy in an animal model of HIV.
Reviewers agreed that this proposal addresses a significant unmet medical need. There are currently no curative therapies for HIV/AIDS and existing treatment options are accompanied by significant toxicity and quality of life issues and do not restore full immune system functionality. However, reviewers had questions about the scientific rationale for the proposed approach. One of the applicant’s primary justifications for using iPSCs is that the failure to achieve sufficient HIV resistance using gene-modified adult HSCs reflects poor viral transduction efficiency. However, data suggest that other biological mechanisms might influence poor engraftment, such as a lack of selective advantage for HIV-resistant HSCs over endogenous HSCs. This issue would be even more problematic with nonmyeloablative conditioning as proposed by the applicant. It is also unknown whether iPSC-derived HSCs have comparable competitive engraftment efficiency to adult gene-modified HSCs. Reviewers also had concerns with the applicant’s plan to generate an “off-the-shelf” HIV therapy by generating enough iPSC lines to cover all human HLA and blood types. They noted that current unrelated donor registries have over 8 million individuals and still result in only three-quarters coverage while cord blood banks have over 2 million units and still can’t provide even 4 out of 6 HLA matching for all potential patients.
The reviewers raised serious concerns about the feasibility of the research plan. They noted that the applicant does not present preliminary data demonstrating the generation of HSCs from iPSCs, a process that is critical for the success of the project. Data are provided with human embryonic stem cells (hESCs), but this cannot necessarily be extrapolated to iPSCs. In addition, the hESCs are cultured with mouse feeder cells carrying potential risk of transmitting endogenous mouse viruses to human cells, which would present a regulatory roadblock for clinical translation. Reviewers were also concerned about the choice of viral vector, which, according to a reference, contains two other transgenes in addition to the two anti-HIV genes specified, all driven by separate promoters. In addition, applicants propose to include a suicide gene in the vector if needed. This complicated vector presents challenges in ensuring that all genes are expressed at sufficient levels. Reviewers also noted a lack of important studies that would advance the proposed cell product to a development candidate stage. For example, there is no prospective plan to identify a dose response, and it is unclear how the final product candidate would be characterized to ensure appropriate in vivo differentiation into the desired cell types. Finally, reviewers were puzzled by the many references to work being done in parallel in the labs of the Principal Investigator (PI) and co-PI and were concerned that it would be needless duplication of effort.
The reviewers described the PI and Co-PI as well qualified to carry out the proposed research. They noted that they both have relevant experience, the PI in lentiviral vector design and the Co-PI in cellular reprogramming. Although, the PI and Co-PI had no obvious development experience to ensure understanding of how to proceed towards a lead candidate, reviewers appreciated the collaborations established with investigators with clinical trial and GMP manufacturing experience and found the assembled research team to be quite strong. They noted that the budget is excessive, including significant equipment purchases that should be obtained through shared resources and large supply budgets.
Overall, while reviewers appreciated the potential impact of an iPSC-derived therapy for HIV/AIDS, they raised significant questions about the proposal’s scientific rationale and feasibility. They did not believe that the current state of the research supported advancement to a development candidate at the end of three years.