Preclinical development of gene-edited human hematopoietic stem cells

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The overall goal of this collaborative project is to develop a “Defend and Destroy” strategy for the functional cure of HIV infection in which a patient's CD4+ T cells are rendered resistant to HIV infection (“Defend”) while simultaneously strategies are applied to eliminate viral reservoirs (“Destroy”). The “Defend” portion of this strategy is based upon the “Berlin Patient,” who was cured of HIV infection upon receipt of a bone marrow transplant from a donor harboring a null mutation in the CCR5 gene, which encodes a co-receptor essential for HIV infection. Although transplantation with CCR5 null hematopoietic stem cells (HSCs) is theoretically an attractive treatment strategy for HIV, in practice allogeneic bone marrow transplants from CCR5-null donors are not a viable strategy for treating HIV in the general population due to limited donor availability and the risks associated with the procedure, particularly graft vs. host disease. Here, we propose to harness the power of the CRISPR-Cas9 genome editing system to introduce null mutations in CCR5 in a patient's own hematopoietic stem cells (HSCs), thereby allowing for an autologous transplant and a functional cure of HIV without many of the risks associated with an allogeneic transplant. The success of the proposed strategy hinges upon the recently discovered CRISPR-Cas9 genome editing system, which is a highly customizable and has the on-target efficiency needed to mutate both copies of CCR5 in a high percentage of the target cell population. As the first step towards full clinical translation of the “Defend and Destroy” strategy, the company CRISPR Therapeutics Ltd, the private-sector partner in this application, will transition strategies for disrupting CCR5 in HSCs developed in the academic laboratory (see Project 1) towards clinical application through the execution of two specific aims: 1) Development of a process for genetically modifying human HSCs compliant with applicable regulations governing cell-based medicinal products; and 2) Analysis of pharmacology, biodistribution, toxicology and off-target effects of genetically modified HSCs. The accomplishment of these two aims will ready this technology for a first-in-humans clinical trial, and will provide the basis for a long-term functional cure of HIV. More broadly, these studies will establish a basic clinic-ready platform upon which other elements of the overall strategy such as non-toxic conditioning regimens (Project 3) and strategies to enhance HSC homing or destruction of viral reservoirs (Projects 1 and 2) can be added. More broadly, the methods developed here for conducting GMP-compliant gene editing in HSCs will have widespread applicability to a variety of genetic diseases of the blood.