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

Linked publications & trials

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 vector systems. This project is closely related to HL006063-12, but given the size and scope of the studies, we have separated investigations of basic hematopoietic biology and immunology into HL006063-12 and include method optimization and disease modeling in this report. 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 indels. We are studying the most predictive approaches to identify and detect off-target effects of CRISPR/Cas9 in HSPC and their progeny in our engrafted rhesus macaques. We have completed the first comprehensive analysis of on target versus off target editing of HSPC, at sites identified by in silico algorithms versus In vitro site ID via CircleSeq, in a relevant macaque animal model. We document that in silico algorithms miss relevant sites found in blood cells following editing in vivo and thus that the combination of CircleSeq and in silico approaches is advantageous (Aljanahi et al Mol Ther, 2021) We have also focused on investigating the quantitative adverse impact of gene 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). We hypothesized that clonal hematopoiesis accompanied by an inflammatory phenotype could be associated with severe COVID-19 disease, and carried out pilot studies investigating this using our macaque clonal hematopoiesis model, comparing outcomes of SARS-CoV-2 infection in clonal hematopoiesis versus control animals, documenting higher levels of virus in tissue and shed in the lungs. We have also carried out a large scale targeted sequencing study of rhesus macaque blood cells from cohorts of aged animals, use deep error-corrected sequencing to look at 56 clonal hematopoiesis genes initially identified in aging humans. We have uncovered for the first time a natural animal model of clonal hematopoiesis, showing exactly the same genes mutated as in humans, in contrast to lack of such mutations in rodent models (Shin et al, Blood, 2022). We extended these studies to human cohorts in terms of analyzing the relationship between COVID-19 severity and the presence of clonal hematopoiesis, and in the largest and most definitive study to date, did not demonstrate an impact on COVID-19 severity (Zhou et al, Blood, 2022). We have also developed a gene editing macaque model for RUNX1 deficiency in order to better understand the biology of the inherited marrow failure/leukemia predisposition syndrome and to assess the feasibility of gene therapies in correcting the phenotype, asking whether mutant vs normal cells predominate over time in a chimeric state. Mutant cells predominate, a concerning finding for gene therapies of this condition. This model also recapitulates the platelet and HSPC phenotype of human RUNX1 deficiency, in contrast to murine models.