For many envisioned applications of lentivirus vectors as tools in respiratory biology and therapeutic gene delivery, the efficiency of gene transfer must be improved. We previously demonstrated stable, persistent (>11 months) in vivo expression following a single application of a feline immunodeficiency virus (FIV)-based lentivirus vector (GP64-FIV) to murine nasal epithelia. Here we investigate the efficacy of repeated administration of lentivirus vectors to the airways. Using quantitative bioluminescent imaging, we found that consecutive daily dosing achieved a linear increase in gene expression and greatly increased the number of epithelial cells targeted. Surprisingly, reporter gene expression also increased additively following each of seven doses of FIV delivered over consecutive weeks (1 dose/week), without the development of systemic or local neutralizing antibodies. This approach enhanced expression of both reporter and therapeutic transgenes. Transduction efficiency achieved following a single dose of FIV expressing mouse erythropoietin was insufficient to increase hematocrit, whereas seven consecutive daily doses significantly increased hematocrit. These unexpected results contrast strikingly with findings reported for adenovirus vectors. Prolonged gene expression has been observed in vivo following a single dose of virus vector; however, depending on the application, repeated administration of vector may be necessary to achieve stable, therapeutic gene expression.

The practical application of gene transfer as a treatment for genetic diseases such as cystic fibrosis or hemophilia has been hindered, in part, by low efficiencies of vector delivery and transgene expression. We demonstrated that a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the envelope glycoprotein from the baculovirus Autographa californica (GP64) efficiently transduces and persistently expresses a reporter gene in respiratory epithelium in the absence of agents that disrupt cellular tight junction integrity. GP64-pseudotyped FIV also efficiently transduced murine hepatocytes after tail vein delivery. To improve the FIV-based vector, we tested the contribution of a series of modifications to luciferase expression in vitro and in vivo. These modifications included the addition of spleen necrosis virus U5 (SNV U5) and mutation of the major splice donor and gag start codon located in the packaging region of the FIV transgene plasmid. After vector modification, we observed significantly enhanced expression of luciferase in respiratory epithelia after nasal application and in the liver after tail vein delivery. In addition, we observed significantly enhanced human factor VIII production after tail vein delivery. These sequential modifications provide an improved FIV lentivirus platform for gene therapy applications and may be applied to other retroviral vectors.

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