Ongoing Research Studies
Surgical strategies for the treatment of laryngeal paralysis are limited by the muscle atrophy associated with denervation. Moreover, attempts at reinnervation have not affected significant change in surgical outcome. To address this clinical problem, we have developed a rat laryngeal paralysis model to explore avenues for management of peripheral nerve injuries using both non-viral and viral gene transfer for the delivery of human insulin-like growth factor (hIGF-1) and other growth factors.
Applied to laryngeal paralysis, hIGF-I gene therapy provides opportunity for augmentation of surgical treatment modalities by prevention or reversal of muscle atrophy, enhanced nerve sprouting and reinnervation. Gene expression, muscle fiber size, motor endplate length and nerve to motor endplate contact are evaluated to assess biologic effect of gene transfer. At the molecular level, myosin heavy chain (MHC) composition is characterized using SDS-PAGE and Western Blot to determine the effects of growth factor on MHC expression; and, 2D gel electrophoresis is used to detect shifts in the muscle proteome.
Initial studies using muscle specific non-viral vector containing the a-actin promoter and hIGF-I gene have produced significant increase in muscle fiber diameter, significant decrease in motor endplate length and significant increase in percentage of endplates with nerve contact. In vivo muscle electroporation as an alternative to non-viral gene is also under evaluation.
Electroporation provides a means for simple, low tech, low toxicity, in vivo cell transfection. Electroporation has potential clinical use in the outpatient setting for gene therapy in patients with laryngeal paralysis and other peripheral nerve injuries.
Preliminary studies with adenovirus vectors indicate a high degree of gene transfer efficiency; however, potential toxicity due to host immune response and non-specific targeting of tissue may limit clinical application. Tissue specific gene expression using site-specific promoters has been reported with both viral and non-viral vectors.
Tissue specific gene transfer, however, is more difficult to accomplish and requires cellular recognition at the vector level. Re-targeting of viral vectors to recognize tissue specific antigens other than the CAR receptor is one method to achieve tissue specific gene transfer. Preliminary studies have been initiated to identify muscle cell surface markers as potential candidates for re-targeting of adenovirus vectors. Muscle specific surface proteins are identified using two-dimensional gel electrophoresis and mass spectrometry for peptide analysis. Specific targeting of laryngeal muscle may then be accomplished using re-targeted adenovirus vectors recognizing specific muscle surface protein.
