Funding opportunities

Funding Type: 
Disease Team Research I
Grant Number: 
Principle Investigator: 
Funds requested: 
$14 583 187
Funding Recommendations: 
Grant approved: 
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.

Review Summary: 

This proposal aims to develop a treatment for HIV/AIDS that has the potential to render patients' hematopoietic stem cells (HSCs) permanently resistant to HIV infection. The applicant proposes to use novel zinc-finger nuclease (ZFN) technology to disrupt the gene encoding a critical HIV co-receptor, CCR5, in patients’ HSC. The hypothesis is that the CCR5 gene-disrupted HSC and progeny cells, which include those cells normally susceptible to HIV infection, will become resistant to HIV infection. The applicant plans to disrupt the CCR5 gene by ex vivo transduction of mobilized HSC with non-integrating vectors engineered to express ZFN specifically targeted to CCR5.

The applicant proposes a preclinical research and development plan that includes all activities leading to the Investigational New Drug (IND) filing for use of gene-modified autologous HSCs in AIDS patients already undergoing HSC transplantation for lymphoma. The proposed plan consists of 4 coordinated phases addressing process development and regulatory requirements for the proposed cell-based investigational product. Phase I includes optimizing vector transduction conditions and functional evaluation of hematopoietic potential and HIV resistance in vivo. Parallel studies with a backup good manufacturing practice (GMP)-grade viral vector will be performed for comparison. In Phase II the applicants propose to develop clinical scale cGMP cell product manufacture and release methodologies. Metrics used to evaluate the process include process yields, safety profile (in vitro and in vivo) and functional testing (in vivo). Phase III consists of validation studies that include full-scale manufacturing runs. Phase IV runs concurrently with Phase I-III and includes carcinogenicity studies and activities such as pre- IND FDA meeting, and reviews by the RAC (Recombinant DNA Advisory Committee of the Office of Biotechnology Activities of the National Institutes of Health) amongst others. The filing of an IND is scheduled in the 4th year of the grant.

Reviewers agreed that the underlying scientific rationale for the project is very sound. There is published evidence that individuals homozygous for the delta-32 mutation in the CCR5 gene are generally resistant to HIV infection. A recent publication showed convincingly that a patient with leukemia and AIDS who received a bone marrow transplant from a donor homozygous for the same mutation became free of HIV infection. Moreover, small molecule antagonists of CCR5 have proven efficacious as antiretroviral agents, further validating CCR5 as a target for HIV therapy. Thus, reviewers agreed that there is a strong rationale for the anti-CCR5 component of the proposed therapeutic. An additional strength of the proposed strategy is that it results in permanent disruption of CCR5 after only transient expression of the ZFN in contrast to other strategies that require continuous expression of a therapeutic gene or siRNA to show therapeutic effect. Reviewers also praised the initial focus on patients who require autologous HSC transplantation for relapsed AIDS-associated lymphoma, as it targets the intervention to patients in whom marrow ablative therapy is clinically indicated. One reviewer commented that the principal weakness in the rationale is that a substantial fraction (50% or more) of patients with advanced HIV disease carry CXCR4 co-receptor-using viruses that would be unaffected by this agent. Another limitation could be the low yield of HSCs from peripheral blood in patients with advanced HIV disease. However, reviewers recognized the fact that the investigators have considerable experience in autologous transplantation of HIV-infected patients with lymphoma, and no doubt have dealt with this issue before.

Reviewers agreed that if successful, the proposal could have significant impact in that it could alter the course of HIV infection and minimize and potentially eliminate the need for life-long anti-viral chemotherapy with its associated toxicities Furthermore, reviewers noted that, if successful, the proposed zinc-finger nuclease technology might have applications to other diseases.

The reviewers agreed that the preliminary data strongly supported the maturity of the proposed project. The applicants have generated substantial data validating the use of ZFN to knock down expression of CCR5 in human HSC. Experiments in a mouse model for xenotransplantation showed that the modified HSCs appropriately differentiated into various lineages. Additionally the applicants showed that the ZFN-modified HSC generated HIV-resistant progeny, leading to significantly lower HIV1 levels in vivo in these mice, along with normal peripheral blood CD4:CD8 ratios and T-cell retention in lymphoid tissue. One reviewer commented that while elegant, the small size and the relatively short lifespan of mice makes these results somewhat "soft” as the demands on stem cells in mice are infinitely small compared to those in humans. Reviewers noted that the investigators have already successfully navigated many of the regulatory hurdles with the FDA and RAC through previous clinical trials in hematopoietic transplantation in HIV patients. Specifically, safety testing of the proposed vector has been reviewed and approved by RAC and FDA in the past, and can be referenced in the IND application for this product. Reviewers found this experience to provide a significant advantage in that a regulatory framework already exists for the proposed work.

Reviewers considered the project plan to be well thought through and logical. It adequately addressed the activities necessary for an IND filing. Milestones are appropriate and well outlined. Feasible strategies and alternatives are proposed in all areas critical to the successful achievement of the project. The reviewers had some specific comments. They suggested the applicant consider the addition to the plan of stability testing and some form of cell tracking study to provide information regarding cell fate post-administration. Reviewers noted that the process development plan appropriately includes attempting to transition to closed-system, semi-automated cell processing technology, which would greatly facilitate successful clinical-scale manufacturing. Reviewers noted that CCR5 knockout by ZNF might lead to disruption of genes at off target sites which perhaps could pose a carcinogenicity risk. They suggested that the applicants should determine if their proposed carcinogenicity study is of adequate duration given this observation. Despite these considerations, reviewers believed that an IND application within four years is an achievable goal for this project.

Reviewers noted that the PI is an extremely accomplished investigator who is highly regarded in the field of virology, especially as it is associated with hematopoietic cell transplantation. In addition the PI has significant experience in clinical trials using autologous transplantation in HIV patients and understands the complexity of the development process. S/he is highly qualified to lead this endeavor. Moreover, the PI has put in place an outstanding team of collaborators. This proposal represents a 3-way collaboration between investigators at three different institutions and there is already ample evidence of close collaboration. Reviewers found that these collaborations bring significant strength to the proposal. The PI and Co-PI and other key personnel have committed appropriate levels of effort to the proposed project. The leadership plan appears appropriate and includes regular meetings of the team to assess progress and make decisions. An appropriate plan to resolve potential issues and conflicts appears in place. Reviewers found the resources and environment were outstanding and the institutional commitments from each of the three institutions to this translational program very strong. They considered the budget appropriate, given that several different sites are involved in the proposed activities.

Overall, reviewers were very enthusiastic about this proposal given the strong scientific rationale, the unmet need, the strength of the preclinical research and development plan and the strong relevant experience of the team leadership. They were confident that this proposal will very likely lead to a successful IND filing in four years.

  • John Rasko
  • John Wagner