Retroviral Mediated Gene Transfer Into 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. Our efforts over the past year have resulted in several publications. We reported a lentiviral vector-based approach involving BCL11A interference with co-expression of truncated erythropoietin receptor, as an alternative gene therapy strategy for sickle cell disease (SCD). We also published two preclinical studies on evaluating various gene-editing strategies for SCD. One study involved disrupting BCL11A enhancer to result in fetal -globin induction in a NHP model. The other report demonstrated that HDR-mediated SCD gene correction strategy achieved therapeutic-level gene correction in NHP. In addition, to evaluate the mRNA transfection approach for NADPH oxidase correction in chronic granulomatous disease, we accessed the safety and persistence following the infusion of eGFP-mRNAtransfected autologous rhesus leukocyte apheresis cells. We also conducted a preclinical study investigating predictive values of off-target prediction methodologies, which is the critical step to further increase the safety of clinical development of CRISPR/Cas9 based gene-editing. Our NHP competitive transplantation model was successfully used in two studies- one to evaluate relative engraftment kinetics and durability following HSC expansion on human umbilical vein endothelial cells and the other study to compare different delivery routes (intravenous delivery vs intrabone injection) of HSCs in combination with imaging techniques. On-going studies include testing various antibody-conditioning methods as non-toxic transplant conditioning regimens to permit efficient engraftment of genetically modified rhesus HSCs. We also have continued studies utilizing electroporation and CRISPR/Cas9 to develop preclinical models to better understand various hematological diseases. 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.