Initiation of a Pre-Clinical Gene Therapy Study in Non-Human Primates Using Lentivectors Targeting Hematopoietic Cells for Correction of Fabry Disease.
Authors: Walia Jagdeep S. , Yoshimitsu Makoto , Silvertown Josh D. , Poeppl Armando , Rasaiah Vanessa I. , Donahue Robert E. , Andrews Robert G. , McCart J. Andrea , Medin Jeffrey A. 
Published: April 2020

Fabry disease is a lysosomal storage disorder (LSD) due to low or absent levels of α-galactosidase A (α-gal A). This results in accumulation of substrate with terminal galactosyl residues such as globotriaosylceramide (Gb3) in lysosomes causing pathology in different organs. Previously, we have demonstrated correction of the deficiency in Fabry mice in numerous gene therapy studies targeting hematopoietic cells. Here we have initiated a pre-clinical gene therapy study in non-human primates (NHPs) targeting Fabry disease. Three rhesus macaques are housed in our animal facility and implantation of telemetric devices and vascular access ports have occurred. We have mobilized hematopoietic stem/progenitor cells from all three animals independently by treatment for 5 days with 10 μg/kg/day of recombinant human granulocyte colony stimulating factor (rhuG-CSF) and 200 μg/kg/day recombinant human stem cell factor (rhuSCF). On the 5th day of mobilization, all animals underwent leukapheresis. To be more clinically relevant, we are using a protocol with an unmodified, commercially available apheresis machine for the rhesus macaques, which can be used for humans of an equivalent weight. From each successful apheresis, we collected approximately 1 x109/kg mobilized peripheral blood mononuclear cells (MoPBMNCs). After collection of MoPBMNCs, we isolated CD34+ cells using an anti-human CD34 antibody (clone 12.8) with a recovery of approximately 15–20 x 106 CD34+ cells per kg body weight of the animal with &gt;80% purity. Collected CD34+ cells are stored in liquid nitrogen. These cells will be prestimulated for 24 hours with huSCF, huFlt3L, huIL-6 and huTPO (kindly provided by Amgen) and will be transduced with a concentrated bicistronic lentivector (LV) that engineers co-expression of huα-gal A and huCD25, a cell surface marker for transduced cells. Our lab has recently shown overexpression of a rhesus form of CD25 in &gt;80% of transduced rhesus BM CD34+ cells mediated by a LV, validating its candidacy as a marker gene. Transduced cells will then be transplanted autologously in the NHPs after myeloablation by irradiation (10Gy) or mild chemotherapy (fludarabine and cyclophosphamide). The irradiation protocol has been optimized and a special plexiglass chamber, with the capacity for inhalational and intravenous anesthesia as well as a space for a HEPA filter, has been prepared for the animal procedures. The transplanted animals will be followed for at least one year and outcomes will be assessed by full measurement of safety parameters, α-gal A activity in plasma and relevant organs along with the real-time PCR and LAM PCR on BM and peripheral blood cells for the persistence of LV. Gb3 levels will also be examined in different organs compared to pre-transplant levels in tissue biopsies. We expect that this preclinical study in NHPs will serve as a roadmap to clinical gene therapy of Fabry disease using LV and provide important safety information for the use of this promising gene delivery system.