572. Antibiotic-Free Nonviral pFAR4 Vector Displays Efficient Transgene Delivery in Mouse and Human Cells

Abstract

New generations of plasmid vectors used for non-viral gene therapy have a reduced size, thus allowing an increase in transfection efficiency and expression levels. We designed a novel vector, called pFAR4, which is Free of Antibiotic Resistance markers. In bacteria, the production of pFAR4 derivatives relies on a plasmid-borne function that suppresses a nonsense mutation introduced into an essential Escherichia coli gene.Thanks to its reduced size, pFAR4 appears to be an efficient gene vector as it displays a superior transgene expression level either after transfection of cultured B16 melanoma cells or electrotransfer into skin or tumors. Furthermore, hydrodynamic delivery into liver revealed that pFAR4 allowed a prolonged expression level, in comparison with kanamycin resistant plasmids that promote transgene silencing.We took advantage of the pFAR4 superiority in liver to assess a novel therapeutic approach for the Mucopolysaccharidosis type IIIA (MPS-IIIA) or Sanfilippo A syndrome. MPS-IIIA is a lysosomal storage genetic disease which results from the deficiency of the N-sulfoglucosamine sulfohydrolase (SGSH) protein, a sulfamidase required for the degradation of heparan sulfate glycosaminoglycans (GAGs). The accumulation of these macromolecules leads to somatic organ pathologies, severe neurodegeneration and patients’ death. In MPS-IIIA mice, hydrodynamic delivery of a pFAR4 derivative expressing the deficient enzyme from a liver-specific promoter allowed high and prolonged serum levels of sulfamidase protein that was efficiently taken-up by neighboring organs after its endocytosis by mannose 6-phosphate receptors-expressing cells. This led to the correction of GAG accumulation in all peripheral organs as well as in the brain, albeit at early stages of the disease. Improvement of our approach is provided by fusing the SGSH protein with peptides mediating transcytocis across the blood-brain barrier.Having established that pFAR4 is a potent expression vector in a non-integrative approach, our next step consisted in combining the pFAR4 technology and the Sleeping Beauty (SB) transposon system, which is composed of a transposase that excises the transgene-containing transposon from a donor plasmid and mediates its integration into the host cells’ genome. The reduced size of pFAR4 allowed an efficient delivery of SB components into human cells. Our current goal is to take the pFAR4 and Sleeping Beauty technologies to the clinic, in the context of an ex vivo gene therapy approach involving the transfection of autologous primary pigment epithelial cells for the treatment of neovascular age-related macular degeneration disease, in two phase I/II clinical trials.