Optimization of gene transfer and gene editing safety and efficacy focusing on the NHP model

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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 transfer to primitive stem and progenitor cells, and on understanding and enhancing safety of established and new vector systems. We retrieve and analyze clonal contributions to peripheral blood populations following transplantation of CD34+ transduced progenitor cells. We have applied our genetic barcoding technology to map contributions of thousands of individual hematopoietic stem and progenitor cell clones, and investigated whether clonal expansion as an early measure of genotoxicity can be assessed in a high throughput manner using this approach. Relevant preclinical model for assessing genotoxicity prior to clinical trials are an unmet need, since in vitro assays and murine models have not been predictive. The quantitative assessment of oligoclonality in vivo, via our highly sensitive and quantitative barcoding approach, allows relevant comparisons between vectors. In animals followed for 1-7.5 years, we have now clonally tracked the behavior of almost 200,000 lentivirally-transduced HSPC over time, and whether the vector contains strong (SFFV), medium (MSCV) or weak promoter/enhancers, with a single exception we have not seen clonal expansion or other evidence of genotoxicity. This direct comparative work was published recently (Yabe et al MTCMD, 2019) However recently we have now reported the first clear evidence of genotoxicity utilizing a lentiviral vector to transduce HSPC in a human or primate. A rhesus macaque receiving lentivirally-barcoded cells, with a vector containing a moderately strong enhancer, developed markedly abnormal neoplastic hematopoiesis, with profound thrombocytopenia, eosinophilia, and most strikingly an erythroid expansion with very high levels of circulating nucleated red blood cells. These abnormal cell populations were shown to be clonal by barcode retrieval, and the clone contains 9 independent barcoded insertions. We have retrieved the insertion sites, and analysis of which site or sites is most likely responsible for the syndrome has identified at least two genes over expressed and aberrantly spliced that likely contributed to the phenotype, specifically the transcription factor PLAG1 and the cytokine Stem Cell Factor. This important information has been presented a several international meetings, generated interest and concern at the FDA, and was recently published (Espinoza et al, Molecular Therapy, 2019). Given the potential for genotoxicity with random integration of lentiviral vectors, and other drawbacks of 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 and creating indels via non-homologous end joining repair. We have successfully engrafted 9 animals with gene-edited cells, with long-term engraftment at levels of up to 90% for cells that have 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). This work was published last FY, but this FY we have 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. 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. Recently we have developed a macaque gene editing model for RUNX1 deficiency and this is being used to understand the biology of the condition and the feasbility of gene therapies. 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 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 have identified persistent CD33 off-target sites in vivo and are now mapping these clones over time and in various lineages. This work was presented in the Plenary Session at ASGCT 2020.