Genetic Modification and Novel Cell Therapies in Non-Human Primate Hematopoietic Cells

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

For successful gene transfer to primitive hematopoietic cells, several requirements need to be achieved. These include identification of the desired target cell population, identification of the appropriate vector to be used, and achieving desired levels of gene expression. To date, successful gene transfer in human subjects remains problematic. To address these problems as well as important safety issues, studies in non-human primates (NHP) are being undertaken to optimize gene transfer to NHP hematopoietic cells prior to human clinical studies. Vectors that have been evaluated include self-inactivating retroviral vectors, and lentiviral vectors constructed to optimally transduce rhesus hematopoietic stem cells (HSCs). These vectors have been constructed to express reporter genes, such as the enhanced green fluorescent protein (eGFP), or therapeutic genes, such as hemoglobin. Innovative technologies including gene editing, such as CRISPR/Cas9, are also being evaluated in transplants involving rhesus HSCs. In the past year, we completed four major studies that have been published in highly regarded journals, including Blood and Molecular Therapy Nucleic Acids. First, we reported the preclinical CRISPR RUNX1 gene editing model that successfully recapitulated hematologic abnormalities observed in human RUNX1 deficiency, which has not been previously achieved in other preclinical models. Second, we used a genetic barcoded HSC autologous transplantation model to investigate the impact of busulfan conditioning, which is less toxic and more clinically relevant compared to traditional total body irradiation, on hematopoietic reconstitution in NHP. This study provides the first direct comparison of the impact of conditioning on clonality and will serve as a platform for further comparisons with novel conditioning approaches such as stem cell-targeted cytotoxic antibodies. Third, we investigated the existence of immunity to SpCas9 and SaCas9 in control and transplanted animals, and demonstrated pre-existing immunity does not impair the engraftment of CRISPR-Cas9-edited cells in NHPs conditioned with busulfan or radiation, providing an optimistic outlook for the use of ex vivo CRISPR/Cas9-edited cells. Finally, in collaboration with the Childrens Hospital of Philadelphia, we reported a proof-of-concept study using the b-globin locus as a model to suggest that forced redirection of gene-regulatory elements may be used to alter gene expression and therefore could be a useful tool for treatment of genetic disorders. In addition, we published a protocol paper to provide detailed methodologies for lentiviral vector transduction of rhesus macaque HSCs which is the critical element for successful transplantation. We also have improved our cell processing procedure, resulting in significant increase in cell yield following apheresis. On-going studies include testing various antibody-conditioning methods as non-toxic transplant conditioning regimens to permit efficient engraftment of genetically modified rhesus HSCs. Efforts continue to be made to improve the level of gene marking, confine gene expression to specific cell types, such as red blood cells, evaluate immune reconstitution following transplant, the contribution of genetically marked cells to reconstitution post-transplantation. Studies are in progress aimed to improve HSC collection efficiency using a new apheresis machine; improve HSC transduction efficiency; and further delineate the nature and clonality of populations contributing to reconstitution using genetic tracking methodologies. Collaborative studies both within the intramural and extramural programs continue to be initiated to determine the validity of the technology and its safety and efficacy in the NHP model system.