Plastic Surgery Research Council
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TOPICAL GENE SILENCING: TOWARDS AN OFF-THE-SHELF SOLUTION
Presenter: Maria J Ham, PG Student
Co-Authors: Soares MA; Zhou AT; Ayaz MM; Wetterau M; Lotfi P; Lalezarzadeh F; Wilson SC; Ojo CO; Bandekar A; Sofou S; Ceradini DJ; Saadeh PB
New York University Medical Center

Introduction: Cutaneous gene therapy is limited by difficulty in dermal penetration while resisting drug degradation & off-target effects. Topical siRNA delivery using cationic lipid nanoparticles (CLNs) is attractive due to its miscibility, predictability & ease of application. Here, we determine the optimal formulation of CLNs to therapeutically deliver siRNA for cutaneous conditions.

Methods: 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) & sodium cholate (NaChol) in varying weight:weight (w:w) ratios (6:1, 8:1, 10:1) were used to synthesize CLNs. Particle size, surface charge, & cell viability were measured to characterize the CLNs. CLNs complexed with fluorescent siGLO Red siRNA were used to determine depth of dermal penetration in murine skin. 3T3 fibroblasts treated with CLNs & MAPK1 siRNA in vitro were used to guide optimal dosing for in vivo use.

Results: CLN diameters measured 105-106 nm without & 133-145 nm with siRNA, with narrow distribution between w:w ratios. Surface charge measured 40-60 mV without & 25-35 mV with siRNA, with increased surface charge as a function of increasing w:w ratio, suggesting adequate inhibition of CLN aggregation & improved solubility. Cytotoxicity assay revealed least cell viability with 10:1 CLNs. Fluorescent siRNA applied onto murine skin demonstrated maximum dermal penetration on day 1, greatest with 8:1 but present in all 3 CLNs, without any histological evidence of inflammatory reaction to the CLNs or siRNA during the application process. In vitro study showed greatest silencing of MAPK1 after 2 days with 6:1 & 8:1 CLNs showing 74% & 76% knockdown, respectively (p<0.05). In vivo silencing confirmed maximal effect with 6:1 CLNs with 87% knockdown on day 3 (p<0.05).

Conclusion: We determined that 6:1 is the optimal ratio of DOTAP/NaChol with therapeutic siRNA as a topical delivery method of gene silencing for cutaneous disorders. It offers quick, localized, & non-systemic gene silencing without skin penetration enhancers, providing an efficient means to topical gene therapy that is applicable to the clinical setting.


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