A Lentiviral Strategy for Highly Efficient Retrograde Gene Transfer by Pseudotyping with Fusion Envelope Glycoprotein

Abstract

The lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) is used extensively in gene therapy trials of neurological and neurodegenerative diseases. Retrograde axonal transport of viral vectors offers a great advantage to the delivery of genes into neuronal cell bodies that are situated in regions distant from the injection site. Pseudotyping of HIV-1-based vectors with selective variants of rabies virus glycoprotein (RV-G) increases gene transfer via retrograde transport into the central nervous system. Because large-scale application for gene therapy trials requires high titer stocks of the vector, pseudotyping of a lentiviral vector that produces more efficient retrograde transport is needed. In the present study, we developed a novel vector system for highly efficient retrograde gene transfer by pseudotyping an HIV-1 vector with a fusion envelope glycoprotein (termed FuG-B) in which the cytoplasmic domain of RV-G was substituted by the corresponding part of vesicular stomatitis virus glycoprotein. The FuG-B pseudotype shifted the transducing property of the lentiviral vector and enhanced the retrograde transport-mediated gene transfer into different brain regions innervating the striatum with greater efficiency than that of the RV-G pseudotype in mice. In addition, injection of the FuG-B-pseudotyped vector into monkey striatum (caudate and putamen) allowed for highly efficient gene delivery into the nigrostriatal dopamine system, which is a major target for gene therapy of Parkinson's disease. Our strategy provides a powerful tool for the treatment of certain neurological and neurodegenerative diseases by promoting retrograde gene delivery via a lentiviral vector.

Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord

“…Similar findings have been reported using the CVS strains of rabies glycoprotein; [8][9][10] however, to our knowledge this is the first report using the B19 glycoprotein VSVg chimera, and the first to demonstrate longrange retrograde transport from the spinal cord to cortex.…”

“…11 More recent studies by the group of Kobayashi showed that the intravirion domain of the VSV glycoprotein could enhance viral stability and packaging efficiency, whereas the extracellular domain of rabies allowed retrograde transport from striatal or peripheral muscle injections. 8,9,13 In our hands, the B19 glycoprotein-pseudotyped viruses showed similar titers to N2c on HT1080 cells, even though expression of the N2c glycoprotein in the packaging cells was lower when measured by both immunocytochemistry and western blot. Analysis of the signal peptides of B19 and N2c show significant differences, which may account for expression differences (Figure 1a).…”

“…On the basis of the recently published work by Kato et al 8,9 using the rabies CVS-N2c strain (N2c), we generated a new chimeric envelope containing the intra-virion domain of VSVg and the extra-virion and transmembrane domains of the SADB19 (B19), producing the B19/VSVg chimera (Figure 1a). The N2c chimeric construct, N2c/VSVg was similarly generated.…”

“…Instead, two groups have developed chimeric rabies/VSVg envelopes for packaging HIV-1 or equine lentivirus. [8][9][10][11] In these cases, the rabies strains used were challenge virus standard strains CVS24 N2c [8][9][10] or B2c. 11 These subtypes were developed by passaging CVS through suckling mice and isolating variants from the brain (N2c) or kidney (B2c).…”

“…11 Both strains performed similarly following lentiviral chimeric pseudotyping, producing high-titer virus with the capacity for retrograde transport from striatal injections to transduce neurons in the cortex, substantia nigra and thalamus. 8,9,11 Here we compared the previously used CVS-N2c (N2c) chimeric envelope with a chimeric envelope containing the SADB19 (B19) extra-virion and transmembrane domains and the intra-virion domain of VSVg. We tested expression and trafficking to the plasma membrane of the viral packaging cell line and then assessed the transduction capacity of the B19/VSVg-pseudotyped virus from a lumbar spinal cord injection to test its efficiency over long-range retrograde transduction.…”