Date of Award

Summer 8-14-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pathology & Microbiology

First Advisor

Dr. Alexander V. Kabanov

Abstract

Non-ionic amphiphilic block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), Pluronics, arranged in a tri-block structure PEO-PPO-PEO, have raised a considerable interest in skeletal muscle Gene Therapy. Previous studies have demonstrated that co-administration of Pluronics with naked plasmid DNA (pDNA) by direct i.m. injection enhanced transgene expression not only in muscle but also in distal lymphoid organs (spleen and lymph nodes) and this response was strain-dependent; not observed in athymic (BALB/c nu/nu) mouse; suggesting a role of immune cells in gene transfer to skeletal muscles. Therefore, we first evaluated the role of inflammation and inflammatory cells, on muscle transfection. We showed that local inflammation in murine hind limb ischemia model (MHLIM) drastically increased DNA, RNA and transgene expression levels in muscles injected with pDNA/Pluronic. Ischemic muscles showed high number of GFP+ muscle fibers with GFP expression co-localized with desmin+ muscle fibers and CD11b+ macrophages (MØs). This suggested that MØs assist muscle transfection with pDNA/Pluronic. Moreover, adoptively transferred MØs were shown to pass the transgene to inflamed muscle cells. Hence, we developed an in vitro model of inflammation by co-culturing transfected MØs with myotubes (MTs) and used constitutive (cmv-luciferase) or muscle specific (desmin-luciferase) reporter gene expression to show that Pluronic P85 enhances the horizontal pDNA transfer from MØ to MTs. Systemic inflammation (MHLIM and peritonitis) also increased the transgene expression with pDNA/Pluronic but not pDNA alone. Second, we carried out an immunological profiling of adjuvant (P85/LPS/Alum) induced cellular recruitment and showed that P85 helps modulate the response towards MØs predominated recruitment. Moreover, the contribution of MØ predominance in assisting gene transfer after i.m. injection of x pDNA/P85 was reinforced by in vivo MØ depletion that resulted in near complete attenuation in gene expression to the level of naked pDNA alone. Finally, MØ recruitment response was harnessed to further increase muscle transfection by preinjecting P85 at the subsequent sites of pDNA/P85 injection resulting in improved muscle transfection. This dissertation study provides a key evidence about the role of MØs in assisting gene transfer in pDNA/Pluronic delivery approach. Altogether, we introduce Pluronics as simple and commercially available excipients to enhance gene transfer using pDNA/Pluronic admix compositions; are recognized pharmaceutical excipients in US and British Pharmacopoeia and therefore have great potential in human gene therapy clinical trials in healthy and disease pathologies that often involves inflammation.

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