INTRACELLULAR DNA DELIVERY USING POLYMERIC NANOPARTICLES FOR LYSOSOMAL STORAGE DISORDERS GENE THERAPY

INTRODUCTION: Gene therapy can be currently performed by viral or non-viral vectors. Although viral ones have already come to a clinical translation, concerns remain about both safety and the industrial applicability of this system. We therefore started the study of a transposon-based gene transfer system. We used nanoparticles (NPs) as vector to carry the transposon within the blood stream, then in cells. These had to be able to hold large plasmids, such as transposon. The NPs must then be able to reach the tissue most affected by the genetic disorder. Coating the NPs with specific peptides, they acquire the ability to accumulate in the target tissue, in our case the Central Nervous System (CNS) penetrating the blood-brain barrier (BBB). We started to develop a non-viral gene therapy approach in order to treat a neurological disease, globoid cell leukodystrophy in (GLD), an inherited, rapidly fatal disorder affecting myelin. METHODS: We tested the ability of a transposon-based system called Sleeping Beauty (SB), to convey the luciferase marker gene in the control cells. We then verified the ability of NPS of chitosan to contain large plasmids. We then verified the quality (size, shape) of the NPS obtained and their ability to integrate the gene of interest (GALC) within the cellular genome. Lastly, we verified the ability of a heptameric synthetic glycopeptide, of opioid origin, g7, to convey inside the CNS NPs of Poly Lactic-Co-Glycolic Acid (PLGA). We injected PLGA NPs coated with g7 in tail vein\u2019s Balb/c mice and followed through fluorescence spectroscopy their distribution. RESULTS: We verified the high SB transduction efficiency on control cells by their expression of luciferase marker gene. We then assessed the copies number of the integrated gene. We demonstrated the high transduction efficiency of the transgene within the genome of fibroblasts grown from biopsies of patients with GLD. We assessed the quality and features of chitosan-SB NPs. We could observe that g7 coated PLGA NPs accumulate in the CNS 10 minutes after infusion, with accumulation even and up to 2 hours. DISCUSSION: SB demonstrated high efficiency in stable transduction of control cells. So we thought to encapsulate SB in chitosan NPs. These have proven to be highly efficient in stably transducing deficient fibroblasts. We can confirm that g7 is today the best way to address the NPs to the CNS. CONCLUSIONS: Future studies will test the ability of g7 to coat the NPs of chitosan, verify its efficiency in conveying the chitosan NPs to the CNS as well as they did for PLGA NPs. Finally, by cloning into SB the gene whose mutation causes GLD, can be assessed the therapeutic efficacy of the system in the mouse model of the disease