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Efficient Topical Gene Silencing via Self-Assembling Engineered Lipoproteoplex
Anna T. Zhou, BA1, William Rifkin, BS1, Haresh More, MTech2, Nikita Srivastava, BS2, Muhammad H. Junejo, BS1, Rohini L. Kadle, BS1, Camille Kim, BS1, Nicholas D. Brownstone, BA1, Jin K. Montclare, PhD2, Pierre B. Saadeh, MD1, Piul S. Rabbani, PhD1, Daniel J. Ceradini, MD1, Rita A. Sartor, BA1.
1New York University, New York, NY, USA, 2Polytechnic Institute of New York University, New York, NY, USA.
A major limiting factor in the development of topical siRNA therapeutics is the formulation of safe and effective drug delivery systems. A recently engineered cartilage oligomeric matrix protein coiled coil (COMPcc) based supercharged coiled-coil protein (CSP) complexes siRNA via cationic residues and has a central pore capable of encapsulating drugs. Here, we investigate a self-assembling lipoproteoplex consisting of CSP packaged into cationic lipid nanoparticles (CLNs) for in vitro and in vivo gene silencing.
We optimized the CSP to siRNA ratio using electrophoretic mobility shift and PicoGreen intercalation assays. CSP cytotoxicity was evaluated in vitro using a lactate dehydrogenase cytotoxicity assay. We then prepared lipoproteoplex by incorporating varying CSP:siRNA ratios into a standardized CLN and treated 3T3 fibroblasts to assess gene expression changes. Cellular internalization of fluorescent siRNA (siGLO) packaged in lipoproteoplex was visualized with fluorescent microscopy. In vivo, we applied lipoproteoplex with siGLO or siRNA to the skin of C57BL/6 mice. We then measured biodistribution via IVIS imaging, dermal penetration using histological sections, and gene expression in treated skin and internal organs 0-5 days post-treatment.
The CSP to siRNA ratio of 1:1 (w/w) yielded favorable siRNA complexing conditions, allowing for balanced binding and release of siRNA from CSP. Cytotoxicity assays showed 98.5 ± .6% cell viability when exposed to CSP alone. A concurrent cytotoxicity assay showed an insignificant increase in cell death (95% CI 4.5 ± 7.8%) when CSP was added to CLNs compared to CLNs alone. In vitro functional assays showed 54.5% mRNA knockdown in cells treated with lipoproteoplex containing a 1:1 CSP:siRNA ratio (CLN:1:1), which corresponds to transfection efficiency of 11/13 cells (84 ± 14%) as seen with siGLO cellular internalization imaging. In vivo imaging of mouse skin treated with CLN:1:1 containing siGLO showed evenly distributed fluorescence spanning 93% of the treated area at 3 days-post treatment. Dermal penetration showed siGLO penetration through the stratum corneum and epidermal layers into the underlying dermis as seen upon histological examination. While there was minimal mRNA knockdown at 3 days, effective gene silencing was attained by 5 days post-treatment, showing 30.5% mRNA knockdown compared to sham treated mice. There were minimal gene expression changes in the liver at 3 or 5 days post-treatment indicating minimal off-target effects.
Our results suggest that a 1:1 CSP:siRNA ratio in lipoproteoplex achieves efficient gene silencing in vitro and in vivo. The additional benefits of minimal cytotoxicity and minimal off-target effects pose the optimized lipoproteoplex formulation as a dynamic and promising novel topical gene delivery system. Finally, successful dermal penetration without the need for pretreatment facilitates translation into clinical use.
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