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RC1-00100-1: Functional Genomic Analysis of Chemically Defined Human Embryonic Stem Cells

Recommendation: Recommended for funding
Scientific Score: 73

First Year Funds Requested: $629,208.00
Total Funds Requested: $2,516,831.00

Public Abstract (provided by applicant)

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 (provided by applicant)

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.

Review

SYNOPSIS: This proposal aims to exploit the applicant’s ability to differentiate human Embryonic Stem Cells (hESCs) into macrophage and T lineage blood cells to study possible therapies for HIV infection. The study will evaluate 3 possible gene therapy approaches and work towards optimizing in vitro and in vivo models to test these therapies.

IMPACT & SIGNIFICANCE: This proposal describes a novel and significant use of hESCs for HIV research. The proposed studies are driven by the fact that current therapeutic approaches to combat AIDS/HIV disease are not curative and have considerable toxicities. If successful, the research will provide new and important information about the differentiation of hESCs to blood lineage cells, and the use of such hESC derivatives for therapeutic screening. The proposal makes use of novel reagents and protocols that were developed in the applicants’ lab, and thus is highly innovative and significant. The proposed optimization of in vivo hematopoietic engraftment strategies for hESC derivatives will be highly significant both for this research and for other studies of blood-related diseases.

The underlying idea of this proposal is to introduce therapeutic genes into hESC to derive genetically manipulated HIV target cells which have resistance to infection and also generate the production of cells that have the potential to inhibit activation of latent HIV. The applicants believe that this approach has advantages over other approaches in which the patient’s own stem cells are being manipulated. Specifically, they believe that the hESCs could be selected such that there would be no potential for deleterious vector-mediated interactions such as have been encountered in human gene therapy trials that eventually resulted in malignant transformation of cells. If the investigators are successful in generating hESCs with the potential of generating HIV-resistant progeny, they would still face the hurdle of documenting HIV resistance in vivo and ensuring transplantability of these cells so that long-term hematopoiesis is sustained.

Reviewers agree that the studies proposed are significant. The studies will provide proof-of-principle evidence that cells in the immune system can be modified by manipulation of hESC, and thus can potentially impact treatment strategies for other immune system disorders in addition to AIDS/HIV.

QUALITY OF THE RESEARCH PLAN: Overall, the proposed studies with direct focus on HIV disease as a model system are elegantly described in this proposal with intent to develop the potential for a therapeutic modality based on hESCs. Convincing and relevant preliminary data are provided. Three specific aims are proposed: Optimize introduction of therapeutic genes into hESC, to derive genetically manipulated HIV target cell; Test the efficacy of the genetic anti-HIV approaches in vitro; Develop improved in vivo models to assess the efficacy of these genetic approaches against HIV infection in vivo. The results of the research plan have considerable promise for novel gene therapeutic approaches for HIV and other types of immune system disorders.

In the first Aim, the applicant will introduce 3 candidate HIV therapeutic genes into hESCs. The choice of these genes and approaches is well-supported by preliminary data demonstrating their efficacy in clinical and pre-clinical trials. These gene-modified hESCs will be differentiated to macrophage or T lineage cells using novel protocols developed in the laboratory, for which appropriate preliminary data is provided. Based on the preliminary data, the reviewers believe that the first specific aim of optimizing introduction of therapeutic genes into hESC is doable.

In Aims 2 and 3, the applicant will use in vitro and in vivo assays to test the efficacy of these genetic manipulations in protecting cells against HIV infection. These studies will exploit the applicant’s significant expertise in HIV research, and build on established assays in the lab. They will also extend available approaches for directing differentiation of hESCs and for studying hematopoietic lineage differentiation and function using hESC models. The second specific aim will focus on the ability of cells to kill infected cells and on the ability of hESC progeny to affect HIV activation. This also seems doable. The problem arises with Aim 3, the development of in vivo models to assess efficacy against HIV infection. A reviewer was unable to see a promising approach to overcome the problem of engraftability of hESCs or their differentiated progeny in any of the mouse models to be used.

STRENGTHS: Reviewers agree that this is a coherent, logical, well-written research plan with compelling and relevant preliminary data. There is appropriate discussion of potential risks and alternative approaches in the experimental plan. The proposed timeline for the studies is thought to be appropriate by a reviewer.

The investigative team is also cited as a strength of this proposal. The Principal Investigator (PI) has a proven record of extensive expertise in the field of HIV pathogenesis & mouse/human chimeric modeling of HIV infection in vivo. The PI proposes collaborations with investigators who have extensive experience in technologies necessary for achieving the goals of the proposal and with whom s/he has collaborated successfully in the past.

The use of hESCs is a novel approach to investigate a significant human disease; this is also considered a strength of the application.

In sum, the PI is highly experienced in the field of HIV and has been able to put together a strong team of collaborators. They have strong preliminary data. One reviewer states s/he has “no doubt” that the first two specific aims can be accomplished.

WEAKNESSES: One weakness of the proposal is that the investigator is relatively new to hESCs and relatively inexperienced in the field of hematopoietic differentiation of hESC.

There is some risk that anti-HIV therapeutic strategies will not be effective. Furthermore, for eventual translation to human therapy, the in vivo (in immunodeficient mice) strategy will not be appropriate.

A major weakness cited by one reviewer is the lack of documented engraftability of hESCs and their progeny even in xenogenetic NOD/SCID mouse models, which have been shown to support the growth of progeny from adult hematopoietic stem cells. It is not clear from the application how exactly the investigators want to get around this problem. Further, the PI’s belief that s/he will be able to “select” genetically manipulated hESC-derived hematopoietic cells such that there would be no deleterious vector-mediated interactions (such as seen in the French gene therapy trial using the patients’ autologous hematopoietic stem cells) is not well founded. It is currently unknown which vector interactions are deleterious and which are non-deleterious.

The proposal is considered to be ambitious, and a reviewer is uncertain that the alternative approaches to currently risky experiments proposed in this application will be successful. The proposed robust in vivo mouse models are probably therapeutically advantageous but one reviewer questions feasibility of the proposal, especially within the planned time-line (and possibly more so given the pending move to a new building).

DISCUSSION: This proposal generated a great deal of lively discussion. One reviewer described it as the best of the applications s/he reviewed, entailing a very interesting and innovative use of hESC which is backed up by good advanced preliminary data. The applicants have shown that they could establish a system to generate hESC-derived progeny in vivo – the efficiency is low but this reviewer is convinced that the investigators could generate enough cells to use in proposed studies. The applicant has made a real effort to utilize the strengths of hESCs and adapt in the context of an HIV program. Although clearly there is a risk that these anti-HIV approaches will not be effective, the choices for candidate therapy research are well-justified.

Another reviewer describes a major bottleneck of this proposal is the limited documentation of engraftability of hESC and their progeny in models which have been shown to support growth from adult hematopoietic stem cells; it is unclear how to get around this with hESC-derived cells – why not simply use genetically modified autologous adult haematopoietic cells instead of hESCs? One reason suggested is that genetic modification of adult cells has in some cases resulted in leukemias, although it was mentioned that many other published studies do not report a resulting leukemia. A reviewer states that there is no good evidence that the transplanted cells will be immuno-privileged, and believes the investigators are naïve if they think that HLAs are not expressed in young cells. Transgene expression could be very immunologically stimulatory. That problem is thought to be easier to address in genetically-modified autologous cells, than is the immune response that will result from the likely mismatch of HLA and the very immunologically strong minor histocompatibility antigens in a hESC-derived therapy. Another reviewer pointed out this is a component of the proposal(i.e. to improve the efficiency of engraftability), and that this research is a platform to provide a novel application of hESCs to test more rapidly candidate therapies, rather than as an immediate route to transplantation. Reviewers point out that there is a current promising clinical trial in Phase II using autologous stem cell intervention which may give some benefit. Discussants generally are convinced of the overall merits of this proposal given the compelling preliminary data.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None