Fraser J. Wilkes

Stable gene transfer to the nervous system using a non-primate lentiviral vector.

We have constructed a non-primate lentiviral vector system based on the equine infectious anaemia virus (EIAV). This system is able to transduce both dividing and non-dividing cells, including primary cultured hippocampal neurons and neurons and glia in the adult rat central nervous system (CNS), at efficiencies comparable with HIV-based vectors. We demonstrate that the only EIAV proteins required for this activity are gag/pol and that the only accessory protein required for vector production is rev. In addition, we show that the pol encoded dUTPase activity that is found in all non-primate lentiviruses is not required. The vectors can be pseudotyped with a range of envelopes including rabies G and MLV 4070A and can be concentrated to high titres. The ability of EIAV to infect mitotically inactive cells makes this vector an attractive alternative to the immunodeficiency viruses for gene therapy.

Analysis of factor VIII mediated suppression of lentiviral vector titres.

Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) are near normal, RNA genome levels are reduced and measurement of integrated copies revealed the virus is severely defective in its ability to transduce target cells. This is due to the absence of sufficient vesicular stomatitis virus glycoprotein (VSV-G) envelope on viral particles derived from cells expressing FVIII. By using an internal tissue-specific promoter, that has low activity in the producer cells, to drive expression of FVIII we have overcome this inhibitory effect allowing us to generate titres approaching those obtained with vector genomes encoding reporter genes. Furthermore, we report that codon optimization of the full-length FVIII gene increased vector titres approximately 10-fold in addition to substantially improving expression per integrated vector copy.

528. VEGF gene therapy with retrogradely transported lentivirus prolongs survival in mouse ALS model

We have developed a minimal lentiviral vector system that mediates efficient gene transfer into mammalian cells. The lentiviral vector system is derived from the non-primate equine infectious anaemia virus (EIAV, LentiVector®). LentiVector® has been shown to mediate long term gene transfer in the central nervous system with efficient expression and minimal toxicity (Mazarakis et al., 2001; Azzouz et al., 2002). We have previously demonstrated that pseudotyping of LentiVector® with the glycoprotein of the ERA strain of the rabies virus confers retrograde axonal transport on these vectors (Mazarakis et al., 2001). Delivery of such vectors by direct injection into muscle of rodent resulted in transduction of cells local to the site of injection. Efficient transduction of motor neurons (MNs) projecting into the injection site was also observed. These results, therefore indicated, that gene therapy approaches involving targeting of LentiVector® to MNs via peripheral administration into muscle may offer a promising strategy for gene therapy in motor neuron diseases (MND).

168. EIAV-based vectors for the treatment of haemophilia A

Effective gene therapy for haemophilia necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, can transduce non-dividing cells at a relevant site and gives long-term expression. Lentiviral-based vector systems are currently the only integrating vector systems that fulfil these criteria for the delivery of Factor VIII. Oxford BioMedicas lentiviral vector system is based on the non-primate lentivirus, Equine Infectious Anaemia Virus (EIAV).