Optimization of genetic modification of HSCs in the NHP model and creation of relevant preclinical models of human disease and therapies

National Heart, Lung, And Blood Institute

Investigators

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Abstract

My research group has worked for over 32 years in the laboratory and in the clinic to develop safe and effective gene therapies directed at hematopoietic stem and progenitor cells (HSPC). In the rhesus macaque model, shown to be the only predictive assay for human clinical results, we have focused on optimizing both lentiviral gene addition and gene editing therapies targeting hematopoietic stem and progenitor cells, and on understanding and enhancing the safety of established and new gene therapy systems. Given the potential for genotoxicity with random integration of lentiviral vectors, and other drawbacks of semi-random gene addition as compared to targeted gene correction approaches, we have utilized the rhesus macaque to explore CRISPR/Cas9 genome editing and more recently base editing to create disease models and to develop gene editing therapies targeting HSPC. We have optimized CRISPR/Cas9 gene editing and base editing of rhesus CD34+ HSPC, initially knocking out loci via CRISPR/CAs-induced non-homologous end joining repair, creating loss-of function indels, and now focusing on improving the safety and efficacy of HDR-mediated gene correction and of single mutation-directed base editing. We have successfully engrafted 22 animals with gene-edited cells, with long-term levels of up to 70-90% for blood cells with targeted NHEJ indels. We have focused on investigating the quantitative adverse impact of gene editing and base editing on the engraftment and long-term function of HSPCs in the macaque model. Using quantitative barcoding together with gene editing, we have demonstrated marked loss of functional HSPC numbers with both NHEJ but even more markedly HDR editing, and thus far less adverse impact on HSPCs with base editing, which does not result in double stranded DNA breaks. We have created a robust macaque model of clonal hematopoiesis by targeting DNMT3, TET2 and ASXL1 with CRISPR/Cas9 mediated editing to create loss of function mutations. We have shown marked clonal expansion of TET2 mutated clones in three animals, and less marked expansion of DNMT2 or ASXL1 edited clones, and we have documented a highly inflammatory phenotype for TET2 mutant myeloid cells, relevant to the increased risk of cardiovascular disease in CHIP patients. We have multiple ongoing studies to investigate the biology of clonal expansion in these animals, and have shown that treatment with tociluzumab reverses or slows clonal expansion due to TET2 deficiency in this model (Shin et al, Blood, 2022). A recent report linked clonal hematopoiesis to surprisingly a decreased risk of Alzheimer's disease, and postulated that CH myeloid cells were more potent in entering or functioning in the brain to prevent accumulation of amyloid or tau plaques. We have utilized our macaque CH model and control barcoded non-CH animals to investigate TET2 or other CH mutations results in higher replacement of microglial cells in the brain by analyzing purified macaque microglial cells for CH mutations compared to levels in blood myeloid cells. We have not found enhancement of microglial replacement by HSPC-derived cells in the setting of CH, although we have found an inflammatory signature in microglial cells from the CH animals. Finally we have investigated the efficacy and safety of a CD45-directed antibody drug conjugate as conditioning prior to transplanation of lentivirally-transduced or gene-edited rhesus macaque HSPCs. While this agent allowed robust engraftment of HSPCs and stable gene marking or gene editing, rejection of HSPCs and their progeny expressing a foreign protein (GFP) eventually occured, indicating lack of tolerance, however, decline in marked cells was interrupted by treatment with corticosteroids. Attempts to increase the dose of CD45-ADC to improve tolerance and engraftment resulted in fatal pulmonary toxicity and a hyperinflammatory syndrome, calling into question the ability to use this agent clinically.