RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors

Lentiviral vectors derived from the HIV-1 genome offer great promise for gene therapy due to their ability to transduce non- dividing cellsand sustain long-term expression of transgenes. The majority of current lentiviral vectors are pseudotypedwith the vesicular stomatitis viral envelope(VSV-G). VSV-G equips lentiviral vectors with a broad host cell tropism and increased stability. Increased particle stability enables viral supernatants to be concentrated by high-speed centrifugation to enhance their infectivity. Despite its efficacy, VSV-G is cytotoxic - a feature that prohibits the development of stable cell lines that constitutively express this envelope. Therefore, non-toxic envelopeproteins are being investigated. RD114 is an attractive alternative because it also provides increased particle stability and its receptor is widely expressed on hematopoietic stem cells (HSCs). In this study, the packaging efficiency of three envelopeproteins, RD114, RDpro and VSV-G, were evaluated with two lentiviral vectors (TRIP GFP and HPV-402). RDpro is an RD114-HIV chimera designed to pseudotypelentiviral vectors more efficiently. In transient systems, VSV-G generated titersof 10 8 and 10 7 viral particles/mL for TRIP GFP and HPV-402. RDpro possessed titersof 10 7 and 10 6 , while RD114 titerswere one log lower for each vector. Despite having relatively lower titers, RD114 proteins are less toxic; this was demonstrated in the extension of transient transfection reactions from 48 to 96 h. VSV-G transfections are generally limited to 48 h. In regard to gene therapy applications, we show that RDpro supernatants efficiently transduce peripheral blood HSCs. The versatility of RD114 envelopeswas again demonstrated by using a 'mixed' expression system; composed of stably expressed RD114 envelopeproteins to pseudotypelentiviral vectors generated in trans ( titerrange 10 3 ―10 5 ). Our data show that RD114 envelopeproteins are effective and versatile constructs that could prove to be essential components of therapeutic lentiviral gene transfer systems.