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

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

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 two major studies that have been published in Blood and Frontiers in Immunology- both are highly regarded journals in the field. The first paper in Blood describes developing a clonal hematopoiesis (CH) model in rhesus macaques via autologous transplantation of CRISPR/Cas9-mediated gene-edited HSCs, targeting the top human CH genes including TET2, with loss-of-function (LOF) mutations. These animals demonstrated expected phenotypes including expansion of the TET2 LOF clones in the circulation as well as hyperinflammation in bone marrow cells. This model would be very useful for future studies to examine the pathophysiology of CH and to test potential therapeutic interventions. Another paper in Frontiers in Immunology reported the study investigating tissue trafficking kinetics of rhesus macaque natural killer (NK) cells using the very powerful intravascular staining method, developed by Dr. Roederers lab at VRC. Our study demonstrates differential rates of retention in blood and trafficking into tissues between NK cell subsets in a NHP model relevant to human biology, providing valuable insights for designing potential therapeutic applications using these cells. Our program also has made successful transition of the antiquated CBC analyzer to a newer model to continue carrying out its core function of veterinary critical care of the study animals, while minimizing the delay or disruption as well as saving money on consumables as the new analyzer is much more reliable and cheaper to maintain. 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.