Lentivector Transduction Improves Outcomes Over Transplantation of Human HSCs Alone in NOD/SCID/Fabry Mice

Abstract

Fabry disease is a lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A) activity that results in progressive globotriaosylceramide (Gb(3)) deposition. We created a fully congenic nonobese diabetic (NOD)/severe combined immunodeficiency (SCID)/Fabry murine line to facilitate the in vivo assessment of human cell-directed therapies for Fabry disease. This pure line was generated after 11 generations of backcrosses and was found, as expected, to have a reduced immune compartment and background α-gal A activity. Next, we transplanted normal human CD34(+) cells transduced with a control (lentiviral vector-enhanced green fluorescent protein (LV-eGFP)) or a therapeutic bicistronic LV (LV-α-gal A/internal ribosome entry site (IRES)/hCD25). While both experimental groups showed similar engraftment levels, only the therapeutic group displayed a significant increase in plasma α-gal A activity. Gb(3) quantification at 12 weeks revealed metabolic correction in the spleen, lung, and liver for both groups. Importantly, only in the therapeutically-transduced cohort was a significant Gb(3) reduction found in the heart and kidney, key target organs for the amelioration of Fabry disease in humans.

Lentivector Iterations and Pre-Clinical Scale-Up/Toxicity Testing: Targeting Mobilized CD34 + Cells for Correction of Fabry Disease

“…The cDNA of the ΔLNGFR/TMPK component encodes a fusion form of the truncated human low-affinity nerve growth factor receptor (ΔLNGFR) combined with mutant TMPK. ΔLNGFR serves as a cell surface protein marker, and the mutant TMPK induces cell death by downstream-phosphorylating the prodrug 3′-azido-3′-deoxythymidine (AZT) to AZT-DP and, thus, is a “suicide gene” when transduced cells need to be eliminated for any reason 15 . Furthermore, the cDNAs of both the ΔLNGFR/TMPK element and the human GLA sequence were codon-optimized to enhance expression in human cells.…”

“…Last year, oral pharmacological chaperone therapy was approved by the European Commission to treat Fabry patients in the European Union (EU) with an amenable mutation of α-gal A 14 . Our group and others have been developing gene therapy for this disorder for nearly 20 years 15, 16, 17, 18, 19, 20, 21, 22, 23. We previously showed that “metabolic cooperativity” or “cross-correction” occurs for Fabry disease.…”

Relative Distribution of Gb 3 Isoforms/Analogs in Nod/Scid/Fabry Mice Tissues Determined by Tandem Mass Spectrometry

“…This solution is stable at 4°C for at least 2 months. [23]. For this study, we analyzed tissues and biological fluids from 24-week-old male NSF mice (n = 9), male NS control mice (n = 2), female NSF mice (n = 9) and female NS control mice (n = 3).…”

“…In Canada, a clinical trial of gene therapy (FACTs project) for Fabry patients is in progress using recombiRelative distribution of Gb 3 isoforms/analogs in NOD/SCID/Fabry mice tissues determined by tandem mass spectrometry future science group nant lentiviruses targeting hematopoietic stem cells for the sustained systemic correction of the enzyme deficiency [23]. Animal models have been extensively used for the study of human genetic diseases, including for Fabry disease.…”

“…In fact, mouse models have been developed for biochemical, behavioral and neurophysiological Fabry disease studies [24][25][26][27][28][29]. A mouse model has been developed for the FACTs project (JA Medin) using a nonobese diabetic (NOD)/severe combined immune deficiency (SCID)/Fabry mouse (NSF) [23]. This mouse model shows marked deficiency of α-gal A enzyme activity, but no clinical manifestations of the disease.…”