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

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Summary: Clinical and basic laboratory studies are directed at developing efficient and safe gene transduction and ex vivo manipulation strategies for hematopoietic cells, including stem and progenitor cells, and using genetic marking techniques to answer important questions about in vivo hematopoiesis. 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. Given the occurence of leukemia in patients receiving gene therapy for severe immunodeficiencies with retrovirally-transduced hematopoietic stem cells, we have performed large scale sequencing of retroviral insertion sites in rhesus macaques transplanted with cells transduced either with MLV, HIV or SIV vectors, and we continue to follow animals transplanted up to 18 years ago with transduced CD34+ cells, a unique resource for predicting the long-term safety and utility of retroviral gene transfer. 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 over one year, inclusion of even a very strong enhancer within a lentiviral vector did not result in clonal expansion mapped over thousands of individual transduced HSPC, reassuring regarding the use of these vectors in human clinical trials, however longer followup is required. The lack of appropriate rodent models for some human acquired and congenital diseases has limited investigations of pathophysiology and treatment. There are few natural disease models in monkeys. We have begun to apply several genome engineering approaches to develop rhesus macaque models for human diseases with no appropriate rodent models, specifically paroxysmal nocturnal hemoglobinuria, a serious hematologic disease with many questions remaining regarding the pathophysiology of clonal dominance of PNH stem cells and the pathways resulting in thrombosis, the most common cause of morbidity and mortality in PNH patients. Using the iCas/CRSPR approach, we have in vitro evidence for knockout of the rhesus PIG-A gene (the mutated gene in PNH), and we have recently begun in vivo transplants with PIGA-edited cells. We will utilize mRNA transfection to deliver the knockout construct. We have also utilizes this technology to create a primate model of DNMT3 deficiency in rhesus HSPC. This gene is commonly mutated in human myeloid leukemias, as well as in older adults with clonal hematopoiesis but no leukemia. Before moving into transplantation studies, we intensely investigated and optimized the approach to editing rhesus and human HSPC. We discovered that the presence of Cas9 induces apoptosis, and HSPC in particular only tolerate very short-term expression of this nuclease. We achieved high gene editing efficiency with low toxicity via optimized transfection of Cas9 protein and guide RNAs.