Objective: To compare the biological effects of single vs multiple treatment of rat denervated laryngeal muscle with human insulinlike growth factor 1 (hIGF1) gene therapy. Experimental Methods or Design: A musclespecific nonviral vector containing the ␣-actin promoter and hIGF1 gene formulated with polyvinyl polymers was injected into denervated adult rat thyroarytenoid muscle. The effects on animals given a single injection (n = 16) vs those given multiple injections (n = 14) vs control

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... (n = 18) were evaluated. Twenty-eight days after the first injection, gene expression, muscle fiber size, motor endplate length, and nerve-to-motor endplate contact were evaluated. Results: Gene expression, detected by reverse transcriptase polymerase chain reaction for hIGF1 messenger RNA, occurred in 13 (81%) of 16 animals receiving single injections and 14 (100%) of 14 animals receiving multiple injections. Compared with controls, hIGF1transfected animals in both single-and multiple-injection groups had a significant increase in the lesser diameter of muscle fiber, a significant decrease in motor endplate length, and a significant increase in the percentage of endplates with nerve contact (P Ͻ.05 for all). There was no statistical difference between single-and multiple-injection groups. Conclusions: Applied to laryngeal paralysis, hIGF1 gene therapy provides an opportunity to augment surgical treatment modalities by the prevention or reversal of muscle atrophy, and enhancement of nerve sprouting and muscle reinnervation. Although the percentage of denervated muscles demonstrating hIGF1 expression was increased following multiple injections, no difference was observed in the biological response compared with that in the single-injection treatment groups. Further investigation will be conducted to assess longterm benefits and physiological responses and to define the limitations of this potentially valuable therapeutic strategy.