583. Nonionic Block Copolymers Guide Macrophages To Transfect Skeletal Muscle Cells Upon Direct i. m. Injections

Abstract

Purpose: Skeletal muscle offers a clinically feasible site for i. m. delivery of genes for therapeutic proteins expression and prophylactic DNA vaccine purposes. Though much research has been focused on development of cationic lipids and polymers for encapsulating plasmid DNA (pDNA) for in vitro transfections, direct i. m. injection of pDNA still results in higher muscle transfection levels. In comparison to existing technologies such as viral vectors there still is a need to further improve muscle transfection levels with pDNA injections. Non-ionic block copolymers, Pluronic P85 and SP1017 (mixture of L61 and F127), when mixed with pDNA, enhance skeletal muscle transfection in euthymic (BALB/c and C57BL/6) mouse and not athymic (BALB/c nu/nu) mouse. Therefore, we studied role of immune cells in muscle transfection.Methods and Results: pDNA alone or an admix of pDNA+Pluronic were injected into tibialis anterior (TA) muscle in presence or absence of local inflammation to demonstrate the effect of immune cells in muscle transfection. Local-inflammation drastically enhanced DNA, RNA and gene expression levels when pDNA was co-delivered with P85 or SP1017 and not pDNA alone. Pluronics enhanced in vitro gene expression in MPs to upto 4 orders in magnitude and not muscle cells. Moreover, adoptive transfer of ex vivo transfected MPs with GFP DNA to ischemic mice resulted in GFP expression in muscle fibers along with MPs. Therefore, in vitro muscle inflammation model was developed by co-culturing DNA transfected macrophages (MPs) with myotubes (MTs) to see the effect of P85 on gene transfer to MTs. Treatment of co-culture (pDNA transfected MPs with MTs) with P85 resulted in increase in total and muscle specific gene expression ca. 30 and 120 times respectively, confirming pDNA transfer from MPs to MTs. Moreover, in vivo depletion of MPs resulted in significant decrease in gene expression confirming the role of MPs in Pluronic based muscle transfection. Finally, pre-injection with P85 on day 1.5 and not day 5 at subsequent sites of DNA injections, recruited MPs to the site of injections and further enhanced the gene expression levels of muscles injected with pDNA+P85 and not pDNA alone. View Large Image | Download PowerPoint SlideConclusions: Pluronic block copolymers utilize immune cells, specifically MPs, to deliver genes to otherwise very hard to transfect skeletal muscle fibers and hence constitute a novel class of biopharmaceuticals or a platform technology for in vivo gene delivery.