In vivo evaluation of an EIAV vector for the systemic genetic delivery of therapeutic antibodies

Abstract

Lentiviral-based vectors hold great promise as gene delivery vehicles for the treatment of a wide variety of diseases. We have previously reported the development of a nonprimate lentiviral vector system based on the equine infectious anaemia virus (EIAV), which is able to efficiently transduce dividing and nondividing cells both in vitro and in vivo. Here, we report on the application of EIAV vectors for the systemic delivery of an antibody fusion protein designed for the treatment of cancer. The therapeutic potential of a single chain antibody against the tumour-associated antigen, 5T4, fused to immune enhancer moieties has been demonstrated in vitro and here we evaluate the genetic delivery of a 5T4 scFv fused to B7.1 (scFvB7) using an EIAV vector. The kinetics and concentration of protein produced following both intravenous (i.v.) and intramuscular (i.m.) administration was determined in immune competent adult mice. In addition, the immune response to the EIAV vector and the transgene were determined. Here, we show that a single injection of EIAV expressing scFv-B7 can give rise to concentrations of protein in the range of 1-5 mg/ml that persist in the sera for more than 50 days. After a second injection, concentrations of scFv-B7.1 rose as high as 20 mg/ml and levels greater than 2 mg/ml were present in the sera of all mice injected i.v. after 210 days despite the detection of antibodies against both the transgene and viral envelope for the duration of this study. These results demonstrate the potential of EIAV as a gene therapy vector for long-term production of therapeutic recombinant proteins. Gene Therapy (2005) 12, 988-998.

Real-time quantitative PCR for the design of lentiviral vector analytical assays

“…Nevertheless, different groups have already initiated that kind of expertise, where the common measurement analyses for determining which organ is targeted are based either on reporter protein expression profiles, [79][80][81] or on the detection of proviral DNA elements by using standard PCR, 80 fluorescence in situ hybridization, 82 or qPCR amplification method. [83][84][85][86][87][88][89][90][91][92][93] As equivalent to the in vitro strategy (cf Measurement of proviral DNA genomes within transduced target cells), most of these qPCR-based assays measured the number of proviral copies per target cell by normalizing it to the number of endogenous gene amplicons. In Table 3 are summarized primers and probes dedicated for parallel or duplex amplifications of lentiviral DNA proviruses and fragment DNAs derived from animal genomes.…”

“…In other reports that have also based their biodistribution studies upon the real-time PCR technique, intravenous (i.v.) injection of VSV/G-pseudotyped lentiviral particles, whether they are derived from HIV-1, 92,93 or EIAV, 91 have led to a better transduction signal in the liver and spleen.…”

Lentiviral gene transfer to reduce atherosclerosis progression by long-term CC-chemokine inhibition

“…Indeed, other studies have required up to 10-fold more lentivirus particles to achieve sufficient biologically significant transduction. [31][32][33] Thus, our study shows that hydrodynamic delivery is a useful technique to improve efficacy and reduce viral dose in lentiviral gene transfer. Along with the increase viral transduction following hydrodynamic delivery, the tropism of lentiviral vectors for the liver in vivo drives efficient production and release of the soluble 35K molecule in this anatomical site.…”

Enhanced pseudotyping efficiency of HIV-1 lentiviral vectors by a rabies/vesicular stomatitis virus chimeric envelope glycoprotein

“…Large-scale preparations were concentrated as described by Lamikanra et al 49 Briefly, vector containing media from 12 transfected 150 mm plates was pooled and centrifuged overnight (Beckman, F500 rotor, 6000 g). The pellet was resuspended in PBS and repelleted by ultracentrifugation (Beckman, SW-32 Ti rotor, 68 500 g, 1.5 h) and resuspended over several hours in TSSM formulation buffer (20 mM Tromethamine, 100 mM NaCl, 10 mg ml -1 sucrose and 10 mg ml -1 mannitol).…”