Efficient correction of Fabry mice and patient cells mediated by lentiviral transduction of hematopoietic stem/progenitor cells

Abstract

A deficiency in a-galactosidase A (a-gal A) activity causes Fabry disease. Virus-based delivery of genes can correct cells and establish a sustained supply of therapeutic proteins. Recombinant lentiviral vectors (LVs) show promise in this context. We first demonstrate LV-mediated marking of peripheral blood (PB) cells by transduction/transplantation of hematopoietic stem/progenitor cells. Stable enGFP expression was observed in PB for 37 weeks. Next, we transplanted Fabry mice with bone marrow mononuclear cells (BMMNCs) transduced a single time with a LV encoding the human a-gal A cDNA. Sustained expression of functional a-gal A in Fabry mice was observed over 24 weeks. Plasma a-gal A activity from treated Fabry mice was two-fold higher than wild-type controls. Increased a-gal A activity, often to supra-normal levels, and reduction of globotriaosylceramide, a glycolipid that accumulates in Fabry disease, was observed in all organs assessed. In secondary bone marrow transplantations, Fabry mice showed multilineage marking of PB, splenocytes and BMMNCs, along with therapeutic levels of agal A activity in plasma and organs over 20 weeks. Lastly, we transduced mobilized PB CD34 + cells from a Fabry patient and observed corresponding enzymatic increases. Thus a single LV-mediated transduction of primitive hematopoietic cells can result in sustained correction for Fabry disease.

Chimeric Vessel Tissue Engineering Driven by Endothelialized Modules in Immunosuppressed Sprague-Dawley Rats

“…Briefly, vesicular stomatitis virus glycoprotein-pseudotyped LV including the pHR 0 -cPPT-EF-GW-SIN plasmid (containing GFP) were generated by transient transfection of 293T cells as described before. 7,8 For cell transduction, RAEC were infected with recombinant LV-GFP at a multiplicity of infection (MOI) of 10 in the presence of 8 mg/mL protamine sulfate. Sixteen to 18 h postinfection, the supernatant was removed and GFP-RAEC were cultured with a fresh medium for at least 2 days before use.…”

α-Galactosidase A-Tat Fusion Enhances Storage Reduction in Hearts and Kidneys of Fabry Mice

“…pHR'cPPT-EF1α-α-gal A-WPRE-SIN (LV/α-gal A), and control recombinant LV encoding enGFP (LV/enGFP) have been described previously (11). Primers from 1 to 6, indicated in Supplementary Table 1, were used to generate the α-gal A-Tat fusion protein by mutagenesis using the QuickChange Kit (Stratagene, La Jolla, CA, USA).…”

“…Twenty-six wks after LV administration, mice were euthanized to evaluate α-gal A activity in extracts of organs from treated and control mice. Gb3 levels were analyzed by high performance liquid chromatography (HPLC) (11). Insufficient bone marrow cells could be harvested to evaluate both α-gal A specific activity and Gb3 levels; therefore, we analyzed only α-gal A specific activity for those samples.…”

“…Gene therapy could obviate one of these issues at least as it has the potential to cure the disorder by a single intervention. We have been focused on developing gene therapy for Fabry disease (7)(8)(9)(10)(11).…”

Neonatal gene transfer using lentiviral vector for murine Pompe disease: long-term expression and glycogen reduction

“…33 The LV system had been used as a therapeutic approach for gene therapy for MPS I, 19,34 MPS III 35 and Fabry disease. 36 Recently, it was reported that a LV-mediated system was able to provide sufficient GAA expression in recipient myoblasts in vitro 20 and induce immunotolerance in mice that underwent ERT. 37 As a first attempt using a LV, we decided to place the gene expression under the control of an ubiquitous CMV promoter.…”