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 & patents

Abstract

My research group has worked for over 30 years in the laboratory and in the clinic to develop safe and effective gene addition and gene correction therapies directed at hematopoietic stem and progenitor cells (HSPC). In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing 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 to create disease models and to develop gene editing therapies targeting HSPC. We have optimized CRISPR/Cas9 gene editing of rhesus CD34+ HSPC, initially knocking out loci via CRISPR/CAs-induced non-homologous end joining repair, creating loss-of function indels. We have successfully engrafted 17 animals with gene-edited cells, with long-term levels of up to 70-90% for blood cells with targeted indels. We created a model to investigate whether clonal expansion in paroxysmal nocturnal hemoglobinuria is intrinsic to HSPC via targeting of the PIG-A locus (Shin et al, Blood, 2019). We knocked out CD33 in neutrophils produced from edited HSPC as an approach to make marrow resistant to CAR-T cells targeting CD33 in acute myeloid leukemia, demonstrating no change in any aspect of myeloid cell development or function following CD33 knockout, and demonstrating the utility of this approach to safely treat myeloid leukemias with CAR-T cells (Kim, Yu et al Cell, 2018) and we have now set up a rhesus macaque CAR-T model and will move forward to deliver CD33 CAR-T to macaques with and without engrafted CD33KO HSPC created by CRISPR/Cas gene editing to evaluate whether the CD33 KO HSPC protect the animals from CAR-T-associated myeloablation. 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, adn this year we have shown that treatment with tociluzumab reverses clonal expansion due to TET2 deficiency in this model in some animals. We hypothesize that clonal hematopoiesis accompanied by an inflammatory phenotype could be associated with severe COVID-19 disease, and we are now investigating this using our macaque clonal hematopoiesis mode, 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. 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. Thus far in two animals mutant cells predominate, a concerning finding for gene therapies of this condition. A model is being developed for Diamond-Blackfan anemia as well to be used to identify new therapies and better understand this bone marrow failure disorder. 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)