The relentless toll on human life from AIDS demands continued research into new strategies for prevention and treatment. Although current treatment strategies have been effective, it is estimated that between 30 to 45% of patients have developed drug-resistant HIV. In consideration of the long-term costs of Highly Active Anti-Retroviral Therapy (HAART) as well as the steady increase in patients developing drug resistant HIV strains, it has become imperative to develop innovative HIV treatments. The main objective of this multi-institutional collaborative program is to develop novel strategies to restore and rebuild an immune system that is resistant to HIV infection by utilizing genetically modified stem cells with anti-HIV retroviral vectors. Our proposal will result in stem cell sources derived from umbilical cord blood (UCB) or human embryonic stem cell lines (hESC) that can eventually be utilized for transplantation directly into patients. These transplanted HIV resistant cells should be protected and used to generate a durable and HIV-resistant immune system. In the short-term, UCB derived stem cells will provide a viable donor cell source. However, it is anticipated that for a long-term solution, hESC will provide a durable donor cell resource that will not depend on collection from a limited neonatal cell source and may be engineered selectively to develop HIV resistant immune systems. The importance of this approach is that it could potentially eradicate cells with latent as well as active HIV and achieve a complete and sustained immune system restoration in HIV infected patients. Thus, it is imperative to develop technologies for successfully protecting cells from HIV infection and also evaluate whether these technologies alter in any way human embryonic stem cells. This proposal will focus on the development and optimization of genes targeting cellular and HIV messages and proteins, thereby disrupting HIV infection by intervening the viral entry and replication in target cells of the immune system. We will examine the efficacy of anti-HIV lentiviral vectors in both hESC and UCB stem cells in order to determine their anti-HIV activity without interfering with cell proliferation and differentiation into hematopoietic cells. Cells harboring the anti-HIV vectors will be evaluated in humanized animal models for HIV resistance and cell maturation to rebuild the immune system. This collaborative proposal capitalizes on our diverse expertise and strong track record in research with hESC, UCB stem cells and HIV therapy. Our goal is to develop the ability to rebuild and restore a robust immune system in HIV infected patients that have been offered little hope of further treatment upon the development of resistance to HAART. This will also provide better insights for developing novel strategies for other immune disorders.
Statement of Benefit to California:
AIDS was first described by Dr. Gottlieb at UCLA in California. Currently, over 150,000 HIV patients live in the State of California. Despite advances in treatment options and research, HIV still poses a major challenge for the health care of Californians. Although Highly Active Anti-Retroviral Therapy (HAART) is effective in many HIV infected patients, challenges of the continuing emergence of drug-resistant HIV, drug toxicity and incomplete viral suppression in about 30 to 45% of patients warrant the development of innovative HIV therapies. Novel strategies to rebuild the competent and functional immune system will be essential to support HIV infected patients. In addition, insights gained into the repair and regeneration of the immune system in HIV infection will be applicable to other diseases and disorders involving immunodeficiency. Proposed studies focus on the use of human umbilical cord blood derived stem cells and human embryonic stem cell lines as a source of stem cells for regenerative medicine translational research and clinical applications with specific emphasis on AIDS. The potential benefits of the proposed studies are following: (1) increased knowledge of novel approaches of HIV therapy, (2) development of new opportunities for cord blood banking and cell processing business, (3) gaining better insights into the regenerative medicine aspects of immunodeficiency syndromes.
SYNOPSIS: The applicants propose to rebuild an immune system resistant to HIV infection by genetically modifying hematopoietic stem cells with anti-HIV multigene retroviral vectors. Stem cells would either be derived from allogeneic umbilical cord blood or human embryonic stem cells (hESCs), which would eventually be used for clinical transplantation to cure patients with AIDS. Their first specific aim is the development and optimization of anti-HIV multigene retroviral vectors designed to disrupt viral integration and function. The second aim is to evaluate anti-HIV lentiviral vectors both in hESCs and cord blood stem cells. Finally, they would like to evaluate transduced stem cells in a NOD/SCID model.
IMPACT & SIGNIFICANCE: The goal is to build an HIV resistant immune system using pluripotent stem cells. The rationale is that although anti-retroviral drugs and treatment are effective in HIV, challenges of drug-resistant HIV, drug toxicity and incomplete viral suppression in about 30-40% of patients warrant the development of innovative alternative treatments. The idea of gene therapy for preventing cellular entry of HIV is not new. In this application, the investigators propose to combine multiple shRNAs, nucleolar localizing RNAase, and protein based antivirals to target different sites in viral and cellular targets. They have some evidence that a triple combination approach of anti-HIV shRNA, anti-CCR5 ribosome and nucleolar-localizing TAR decoy, and the shRNA and intrabody to CXCR4 in a vector to target CXCR4, a highly expressed chemokine receptor, has provided superior protection compared to vectors containing each anti-viral component alone.
QUALITY OF THE RESEARCH PLAN: The goal of this multinstitutional/collaborative program is to develop novel strategies to restore and rebuild an immune system resistant to HIV infection by utilizing genetically modified stem cells with anti-HIV multigene retroviral vectors. Sources of stem cells include allogeneic umbilical cord blood (UCB) or hESCs. The hypothesis is that transplantation of HIV-1 resistant donor hematopoietic cells may lead to generation of a durable and HIV-resistant immune system. hESCs could provide a durable source with engineered HLA genotypes. Three aims are proposed.
Aim1: development and optimization of anti-HIV multigene lentiviral vectors containing genes targeting both cellular and HIV messages and proteins. Current vector has anti-tat/rev HIV shRNA, anti-CCR5 ribozyme, a nucleolar-localizing TAR decoy, and a reporter EGFP; provides long-term HIV inhibition in primary CD34+ cells. The Principal Investigator (PI) now wishes to test in it hESCs and UCB stem cells.
Aim2: testing of vectors in both hESCs and UCB cells. The PI will transduce undifferentiated hESCs and then separate hematopoietic marked cells after embryoid body (EB) differentiation; for example using macrophages and looking at cytokine response to HIV infection.
Aim3: evaluation of transduced stem cells in humanized animal models (NOD/SCID) for HIV resistance and potential for hematopoiesis (macrophage response, etc.).
One reviewer thought that the most exciting part is Aim 1, the development and optimization of anti-HIV multigene targeting vectors. The second specific aim, evaluating such vectors in hematopoietic stem cells for efficacy, will be difficult, given the absence of a reliable in vitro readout for efficacy. The NOD/SCID studies are unlikely to yield useful information, given the poor or virtually absent engraftment of hESCs in that model with current techniques. Even if, somehow, cells were to engraft, the model would not provide information on long-term engraftment, given the small size of the mouse and the limited proliferative demands on the stem cells.
The PI is Chair, Medical Microbiology and Immunology at UC Davis, and plans collaborations with Scripps and City of Hope for studies on anti-HIV vector molecular biology, humanized animal models and clinical trials; and with UC Davis for studies on HIV molecular biology and hESCs.
STRENGTHS: The principal investigator has a long history of work both in human and simian immunodeficiency viruses although he depends on outside investigators, not supported by this grant, for design and construction of the anti-HIV multigene vectors. The collaboration with Drs. Torbett and Rossi has provided access to appropriate HIV vector platforms for gene delivery. Dr. Li's laboratory has experience with hESCs.
In summary, (1) it is a worthy goal to improve HIV treatments; (2) the PI has a strong background in HIV and blockade; and (3) there is good preliminary data on vector in human CD34+ cells.
WEAKNESSES: The entire proposal is misdirected by focusing on allogeneic hematopoietic stem cells derived from cord blood and hESCs. Both of these cell sources require overcoming the allogeneic barrier for successful transplantation into an HIV infected host. Both carry the risk of graft-versus-host disease owing to histocompatibility disparities. The use of autologous hematopoietic stem cells, that can easily be obtained in large numbers from a given patient, would seem more appropriate.
It is not clear why further effort isn’t directed to establishing efficacy with CD34+ or umbilical cells prior to hESCs. There is no critical analysis of the cell types to be generated by hESC differentiation.
DISCUSSION: This proposal is from a good group, and the topic was interesting but mis-directed. The work could be done just as well with autologous cells from patients. Both reviewers thought that the effort is misdirected by focusing on allogeneic barrier; likely to be rejected. A more appropriate approach is to use patient’s own stem cells.