114. Replication-Defective HSV Vector Development for Targeted Gene Delivery

Gene therapy treatment strategies for Huntingtonu0027s disease (HD) depend on a vector that (i) allows targeted delivery of large therapeutic transgenes to specific cell populations and (ii) is replication-defective and non-cytotoxic. Thus, our goal is to develop a replication-defective HSV-based gene therapy vector to deliver therapeutic transgenes to the cells most affected by expression of the mutant Huntington gene (Htt). Strategies for full retargeting of HSV require virus detargeting from its cognate receptors (HVEM and nectin1), recognized by the virus attachment/entry component glycoprotein D (gD), and introduction of a new ligand into gD that allows entry through recognition of the corresponding cellular receptors. To target an HSV vector for entry exclusively into cells expressing the receptor GFRα1 we employed the ligand GDNF. We replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp)GDNF to create a GFRα1 targeting protein, gD(Y38)_GDNF, that can still bind nectin1. Virus expressing gD(Y38)_GDNF was propagated on cells expressing nectin1 and purified virus was shown to enter nectin1-deficient J1.1-2 and B78H1 cells in a GFRα1-dependent manner. U2OS cells engineered to express GFRα1 demonstrated the most robust virus entry and spread, allowing us to create and propagate a fully retargeted virus that can no longer bind nectin1, gD(Δ38)_GDNF. Once the functionality of gD(Δ38)_GDNF was confirmed, we transferred this mutation to a replication-defective backbone lacking essential immediate early gene expression. Complementing cells were screened and those cells that best supported growth of the replication-defective backbone were selected. Virus stocks were propagated and tested for receptor specificity in vitro and experiments are currently underway to assess GFRα1-specific entry in vivo. We anticipate that the use of neuronal-specific targeting ligands, such as GFRα1, in replication-defective backbones will promote homing of non-toxic HSV gene therapy vectors to cells affected in HD.