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ORIGINAL RESEARCH ARTICLE

Nonviral Vector-Based Gene Transfection of Primary Human Skeletal Myoblasts

Lei Ye^*, Husnain Kh Haider, Wahidah Bte Esa^*, Peter K. Law, Wei Zhang, LiPing Su, Yong Zhang^|| and Eugene K. W. Sim^¶,1

^* National University Medical Institutes, National University of Singapore, Singapore 117597; Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267; Cell Transplants Singapore Pte. Ltd., Singapore 117684; National Heart Center, Singapore 168752; ^|| Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 117574; and ^¶ Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 and Gleneagles JPMC Cardiac Center, Brunei Darussalam BN2311

To whom requests for reprints should be addressed at ¹ Department of Surgery, National University of Singapore, 10 Medical Drive, Singapore 117597. E-mail: sursimkw{at}nus.edu.sg

Low-level transgene efficiency is one of the main obstacles in ex vivo nonviral vector–mediated gene transfer into primary human skeletal myoblasts (hSkMs). We optimized the cholesterol:N-[1-(2, 3-dioleoyloxy)propyl]-N, N, N-trimethylammonium methylsulfate liposome (CD liposome) and 22-kDa polyethylenimine (PEI22)– and 25-kDa polyethylenimine (PEI25)–mediated transfection of primary hSkMs for angiogenic gene delivery. We found that transfection efficiency and cell viability of three nonviral vectors were cell passage dependent: early cell passages of hSkMs had higher transfection efficiencies with poor cell viabilities, whereas later cell passages of hSkMs had lower transfection efficiencies with better cell viabilities. Trypsinization improved the transfection efficiency by 20% to 60% compared with adherent hSkMs. Optimum gene transfection efficiency was found with passage 6 trypsinized hSkMs: transfection efficiency with CD lipoplexes was 6.99 ± 0.13%, PEI22 polyplexes was 18.58 ± 1.57%, and PEI25 polyplexes was 13.32 ± 0.88%. When pEGFP (a plasmid encoding the enhanced green fluorescent protein) was replaced with a vector containing human vascular endothelial growth factor 165 (phVEGF₁₆₅), the optimized gene transfection conditions resulted in hVEGF₁₆₅ expression up to Day 18 with a peak level at Day 2 after transfection. This study demonstrated that therapeutic angiogenic gene transfer through CD or PEI is feasible and safe after optimization. It could be a potential strategy for treatment of

Key Words: angiogenesis • gene transfer • nonviral vector • skeletal myoblasts