Post-electroporation Membrane Stabilization Improves Adipocyte Survival And Peptide Secretion After Gene Delivery
Mengfan Wu, MD, PhD, Ziyu Chen, MD, PhD, William G. Austen, MD, Shailesh Agarwal, MD.
Harvard Medical School, Boston, MA, USA.
PURPOSE: Our lab has developed a novel cell therapy composed of genetically-modified autologous adipocytes. This process has been developed to enable intra-operative gene delivery via electroporation to obviate incubation times required for chemical or viral transfection. However, post-electroporation adipocyte apoptosis presents a challenge to efficient gene delivery. We hypothesize that delivery of poloxamer-188 (P188), a hydrophobic compound which stabilizes the cell membrane in the setting of electrical injury, can reduce post-electroporation apoptosis and improve overall gene delivery.
METHODS: Adipocytes were isolated from human abdominal subcutaneous fat (n=3) by a mechanical, collagenase-free method. Plasmids encoding peptide inhibitor of bone morphogenetic protein (BMP): CMV-Bmpr1afc was introduced into adipocytes by electroporation (20 ug plasmid DNA, 500 V/5 msec/4 pulses). Adipocytes were incubated with P188 (0.1% vol/vol) either 1) immediately prior to electroporation, 2) immediately after electroporation in culture media (Post-P188), or both before and after (Pre/Post-P188) (n=3). Control adipocytes were not treated with P188 at any time (n=3). mRNA was isolated from adipocytes and qPCR performed 7 days after electroporation to quantify pro-apoptotic (Bid and Casp3) gene expression with normalization (Actb). Culture media was collected weekly from day 7 through 28 after the electroporation, and ELISA were performed to quantify secreted BMPR1A-Fc.
RESULTS: Normalized Bid and Casp3 expression were significantly reduced with Post-P188 when compared with no P188 (Bid: 0.31 v. 1.00, p<0.01; Casp3: 0.44 v. 1.00, p<0.05). Similar results were obtained with Pre/Post-P188 treatment (Bid: 0.31 v. 1.00, p<0.01; Casp3: 0.34 v. 1.00, p<0.05). ELISA detected a significantly higher concentration of secreted BMPR1A-Fc with Post-P188 on day 7 (64.27 ng/ml v. 55.75 ng/ml, p<0.05) and day 14 (58.83 ng/ml v. 39.66 ng/ml, p<0.05). Interestingly, Pre-P188 resulted in reduced peptide secretion at day 7 (44.4 ng/ml v. 55.75 ng/ml, p<0.01) and day 14 (22.09 ng/ml v. 39.66 ng/ml, p<0.05)
CONCLUSION: Our findings provide strong evidence that treatment with P188 can improve adipocyte survival after electroporation-mediated gene delivery. Specifically post-electroporation treatment with P188 results in elevated secretion levels which are sustained through 2 weeks in culture. Reduced peptide secretion observed with pre-electroporation P188 may be due to poor gene delivery into the cell caused by the improved cell membrane stabilization. The inclusion of post-electroporation P188 is likely to improve the efficacy of our desired cell therapy, and may have broader impact in laboratory approaches to cell transfection.
Figure Legend: (A) Increased protein secretion at day 7 and day 14 with post-electroporation P188 treatment when compared with no P188 (control); (B) Reduced apoptotic Bid and Casp3 expression with post-electroporation P188 treatment when compared with no P188 (control)
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