Updates are coming! In July 2016 we received new funding from NIH. Check back soon for details on our new projects!
Project 1: Hematopoietic Cell Transplant: Platform for Purging the Latent HIV Reservoir
Leader: Ann Woolfrey, MD, Fred Hutchinson Cancer Research Center · Seattle, Washington
The overall aim of Project 1 is to clarify the relative contributions of the preparative regimen and the donor graft required for purging the latent HIV reservoir. Specifically, this project will seek to determine the dose-intensity of conditioning that produces a meaningful reduction in the quantity of latent viral infection. Dose-intensity will be studied methodically in a well-established nonhuman primate model (autologous HCT), as well as in human subjects undergoing autologous or allogeneic HCT for treatment of hematologic malignancy. Once determined, the ideal dose level will be applicable to development of either allogeneic or autologous HIV resistant transplant procedures. The second main focus will be to determine whether allogeneic grafts provide a benefit over autologous grafts, when given in the setting of complete viral suppression. Once the optimum cell source is determined, we will use this knowledge in combination with that gained from Project 3 to develop a clinical protocol for delivery of gene-modified HIV-resistant cells.
Project 2: ZFN-Modified Stem Cells for HIV Eradication
Leader: Philip Gregory, PhD, Sangamo Biosciences · Richmond, California
Project 2 will seek to develop and test a novel approach for the eradication of HIV-1 by endowing the patient’s own cells with stable resistance to infection. This strategy involves the disruption of a gene that is required for HIV entry into cells, the CCR5 co-receptor, directly in the genome of the patient’s hematopoietic stem cells (HSC). Such ablation is achieved by invoking a DNA repair pathway known as non-homologous end-joining (NHEJ) to heal a double-strand break (DSB) introduced in vivo at the CCR5 locus by an engineered DNA cleavage enzyme called a zinc finger nuclease (ZFN). NHEJ is known to generate loss-of-function alleles, and since humans carrying a naturally occurring loss-of-function allele of CCR5 (CCR5Δ32) are resistant to HIV infection, CCR5 is a validated target for development of HIV therapeutics. Therapeutic application of the ZFN technology will focus on disrupting CCR5 in hematopoietic stem cells (HSCs) obtained from the bone marrow of affected patients and then introducing the HIV-resistant cells back into the donor, where the progeny of the disrupted cells will yield and HIV-protected compartment of the immune system. This compartment could lead to effective immune responses to HIV as exemplified by long-term non-progressor HIV patients who are heterozygous for the CCR5Δ32 allele, or the eradication of HIV-1 completely as observed in an HIV-infected leukemia patient receiving a bone marrow transplant from a donor homozygous for the CCR5Δ32 allele. Thus critical HIV patient data supports the potential of a CCR5 negative bone marrow transplant to eradicate HIV-1.
Project 3: CCR5 Targeting to Control HIV/SHIV in the M. nemestrina nonhuman primate model
Leader: Hans-Peter Kiem, MD, Fred Hutchinson Cancer Research Center · Seattle, Washington
The project will evaluate strategies to eliminate HIV and latent reservoirs in preclinical models of HIV/SHIV infection, by genetic modification of hematopoietic stem cells (HSCs) with zinc finger nucleases targeting the CCR5 locus to eliminate CCR5 expression and to create SHIV/HIV-resistant cells. We will study the effect of CCR5 -/- repopulating cells on the latent reservoir using a clinically relevant nonhuman primate AIDS model. This model will allow us to carefully and thoroughly analyze the impact of CCR5 -/- hematopoietic reconstitution on the control of HIV/SHIV. We will evaluate methods to efficiently modify HSCs, determine the engraftment potential of ZFN-modified HSCs and the level of ZFN-modified repopulating cells necessary for efficient control of HIV. Given the high clinical relevance of the SHIV macaque model, these studies will be readily translatable to the understanding of latency in HIV/AIDS patients.
Project 4: Targeted Disruption of Integrated SHIV by Engineered Homing Endonucleases
Leader: Keith Jerome, MD, PhD, Fred Hutchinson Cancer Research Center · Seattle, Washington
This project seeks to directly address the problem of the long-lived HIV reservoirs consisting of cells containing integrated proviral DNA. There have been suggestions of clearing this reservoir by inducing viral reactivation, presumably leading directly to death of latently infected cells or to their recognition and clearance by the immune system. To date, such approaches have not been successful. Even if all infected cells could be reactivated and eliminated, it is unclear what effects this would have on the immune function of the host—it is possible that systemic reactivation of HIV and widespread destruction of immune cells might lead to more severe clinical disease or even death. The ideal solution to the problem of HIV latency would be the ability to eliminate or otherwise disable integrated HIV without inducing death of infected cells. This goal of project is to achieve this via the use of novel, highly specific enzymes known as homing endonucleases (HEs). HEs are proteins specifically targeting long (14-40 base pair) sequences in double stranded DNA. Upon recognition of their target sites, HEs induce DNA double strand breaks at the sites, which in mammalian cells are most commonly repaired via non-homologous end joining (NHEJ). NHEJ repair is error prone, and typically results in deletions surrounding the cleavage site ranging from 1 base to several kilobases.
In latent HIV infections, proviral DNA integrated within the host genome is the major and longest-lasting form of the virus. We hypothesize that integrated provirus can be targeted for attack and cleavage by HEs, and that subsequent repair by NHEJ will delete genetic material surrounding the break, rendering the virus incapable of replication or pathogenesis. To test this hypothesis, we will leverage existing collaborations to develop HEs recognizing SHIV sequences. At the same time, we will evaluate innovative strategies for maximizing the DNA cleavage activity of HEs. Finally, we will test the ability of HEs to effectively eliminate provirus from CD4+ T-cells and CD34+ stem cells in a nonhuman primate model of HIV infection.
Project 5: Aptamer and Dendrimer Delivery of Zn Finger Nuclease and Homing Endonuclease mRNA and cDNA
Leader: John Rossi, PhD, Beckman Research Institute of the City of Hope · Duarte, California
This project will develop and optimize delivery strategies by which ZFNs and HEs can be delivered to their target cells ex vivo, and perhaps ultimately, in vivo. Therapeutic strategies designed to combat HIV/AIDS have primarily relied upon small molecule drugs. Current highly active antiretroviral therapy (HAART) treatment for HIV-1 has been therapeutically effective in the majority of patients. However, drug resistance and toxicity remain a concern, with some individuals not responding to therapy. Alternative therapeutic strategies need to be developed to overcome these limitations.
The research proposed in this project takes advantage of recent developments in targeted disruption of genetic information via the use of sequence specific Zn finger nucleases and homing endonucleases. With this technology it is possible to disrupt coding or regulatory regions rendering genes non-functional. By introducing into cells site specific nucleases that selectively target HIV sequences, inactivation of viral transcription will lead to lowering of viral loads, and possibly can be a strategy to purge HIV-1 reservoirs from infected individuals.