Lentivirus-based gene transfer systems represent a promising gene delivery technology, as they integrate into the genome of the target cell and mediate sustained expression of the transferred gene. Advanced Vision Therapies, Inc. (AVT) has developed a proprietary lentiviral vector system based on the bovine immunodeficiency virus (BIV), an animal lentivirus not associated with human disease. The BIV vectors combine the transduction efficiency of the HIV-based vectors with the safety advantages of animal-based lentiviral vector systems. Importantly, BlV-vector mediated delivery of an anti-angiogenic transgene efficiently blocked retinal neovascularization in a relevant rodent model, suggesting that the AVT vector is suitable for clinical applications. Lentiviruses are routinely pseudotyped with heterologous viral envelopes to broaden vector tropism. The most widely used envelope is derived from the vesciular stomatitis virus glycoprotein (VSV-G). However, VSV-G has several limitations including cytotoxicity and inactivation by human complement. Therefore, a variety of alternative envelopes have been explored for use in pseudotyping lentiviral vectors including the baculovirus gp64. AVT was recently successful in the generation of a gp64 envelope protein-pseudotyped BIV vector. High vectors titers were obtained, the vectors were stable, and importantly, gp64 can be constitutively expressed in cells without toxicity (an important consideration in the development of a BIV producer cell line). However, gp64 is inactivated by human complement. In this Phase I application, AVT will explore the use of a novel envelope derived from the Thogoto virus. Thogoto virus is transferred to human blood through ticks, and therefore, may display resistance to human complement. Interestingly, the Thogoto gp75 envelope glycoprotein displays significant homology to the baculovirus gp64 envelope glycoprotein, potentially due to the tropism of Thogoto for arthropods and human hosts. AVT has successfully generated a high titer BIV vector pseudotyped with the Thogoto virus gp74 envelope glycoprotein. This Phase I project will focus on the further evaluation of this novel vector. There are 3 specific aims for this Phase I project.
Specific Aim 1 : Evaluation of cellular tropism of Thogoto virus gp75- pseudotyped BIV vectors. A panel of cell lines and primary cells will be evaluated for transduction efficiency using Thogoto gp75-pseudotyped GFP.
Specific Aim 2 : Evaluation of Thogoto-pseudotyped BIV vectors in rodents. Both VSV-G and baculovirus gp64 envelope glycoprotein- pseduotyped vectors specifically transduce RPE cells following a subretinal injection in rodents. The tropism of the Thogoto-pseudotyped vector will be evaluated in rodents.
Specific Aim 3 : Determination of the human complement resistance of the Thogoto-pseudotyped BIV vector. Phase II studies will further evaluate BIV vector physical properties, including stability, Thogoto gp75 cytotoxicity, and vector purification strategies. ? ? ? ?
