Two critical components of cell and gene therapy for HIV cure approaches are: (1) the ability to efficiently engineer hematopoietic stem and progenitor cells (HSCs) and (2) to facilitate efficient engraftment of gene- modified, gene-edited cells with as little toxicity as possible. There has been significant progress in technologies to precisely edit / target genes using engineered nucleases such as zinc finger nucleases (ZFNs), TALENs and CRISPR/Cas9. These nucleases create double-stranded breaks at a targeted DNA sequence, whose subsequent repair can then be exploited to achieve the site-specific addition of new genetic material at the locus via homology-directed repair (HDR), an approach that would greatly reduce the risk of insertional mutagenesis associated with any retroviral gene therapy. Likewise, HSC transplantation and evaluation of multilineage long-term repopulating cell engraftment is the only means to assess the gene modification efficiency in HSCs and potential side or off-target effects. We propose to use our nonhuman primate (NHP) autologous transplantation model, which allows us to evaluate engraftment and long-term repopulation of gene-edited cells in an autologous model similar to studies in a clinical setting. To achieve efficient and robust engraftment of gene-edited cells in vivo, conditioning of the host or recipient is required unless modified cells have a significant survival advantage in vivo. The level of gene-modified HSC engraftment is a function of the balance between the number of cells infused and the intensity of the conditioning regimen, which reduces competition from endogenous HSC recovery. When a higher HSC cell dose is infused, less conditioning is required for rapid reconstitution. This high cell dose / low conditioning approach would be beneficial for HIV patients without malignancy as it would reduce the likelihood of conditioning-related toxicity. HSC expansion would provide such an increase in cell dose to effectively compete with the endogenous HSCs, thereby facilitating gene-modified cell engraftment in the context of a limited conditioning regimen. Recently, several novel and effective methods for HSC expansion have been described which include StemRegenin1 (SR1), novel small molecules UM171 and UM729, and an ex vivo vascular niche co-culture system. SR1 is an aryl hydrogen receptor (AhR) suppressor that has recently been shown to expand cord blood cells. UM171 is a novel small molecule that supports robust expansion of human and NHP CD34+ cells which retain engraftment potential. Given that these technologies act on different cellular pathways to balance self-renewal and differentiation, integration of compatible expansion methods has the potential to synergize for greater HSC expansion. Thus, we propose to study combinations of these HSC expansion platforms and then combine the best expansion technology with optimized cell engineering tools to determine whether these expanded, SHIV- resistant cells are able to protect NHPs from infection with an SIV/HIV (SHIV) chimeric virus.
HIV and AIDS continue to be devastating health problems in the United States and worldwide. Highly active antiretroviral therapy (HAART) has greatly helped controlling the disease but cannot lead to a cure. We propose to evaluate novel approaches for HIV cure based on the gene modification and blood stem cell expansion, to be tested in a clinically relevant nonhuman primate model of transplantation and HIV/SHIV.
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