Abstract
The rhesus macaque autologous transplantation model has been predictive and instructive in better understanding human hematopoiesis and translating novel gene and cell therapies. We have developed a robust CRISPR/Cas9 gene editing platform in this model for a variety of applications. Engraftment of multilineage hematopoietic stem and progenitor cells with up to 90% of edited knockout alleles has been achieved in macaques. We have created a macaque model of age-related clonal hematopoiesis(ARCH) following autologous transplantation of edited HSPC in macaques, demonstrating reproducible clonal expansion of TET2 mutant cells, and gained insights into hematopoietic and cardiovascular manifestations of ARCH, along with using the model to investigate potential therapeutic interventions able to inhibit clonal expansion. We have pursued knockout of CD33 as an approach to protect normal HSPC from CAR-T attack, providing a pathway to safe and effective utilization of CAR-T to target myeloid malignancies. Finally, we have compared approaches to predict off-target effects in normal primary engrafting HSPC, investigating whether <i>in silico</i> algorithm prediction or CircleSeq, a method for selective sequencing of DNA exposed to nucleases <i>in vitro</i> is better able to identify valid off-target sites found in vivo in macaques following autologous transplantation.
Published Version
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