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

Abstract

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

Generation of human TRIM5α mutants with high HIV-1 restriction activity

“…64 pNL4-3, pNL-GFP, pMD-G, pCIG3N, pCIG3B, pCNCG, pDR8.9, pTRIP CMVÀGFP , pCL-Eco, pROD NefÀGFP , pSIV macÀGFP , pONY3.1 and pONY8.0 have all been extensively described before 44,[65][66][67][68][69][70][71][72] …”

Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy

“…19,20 Cells were grown in DMEM supplemented with 10% fetal calf serum and transfected with plasmid vectors to transiently produce infectious virus particles as previously described. 21 Virus titration was performed on D17 canine cells and human HT1080 cells by seeding cells onto 12-well culture dishes at 8 Â 10 4 cells/well followed by the addition of serially diluted virus.…”

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

“…24 The EIAV vectors used are based on the minimal EIAV vector system, produced using the three-plasmid transfection system comprising a gag/pol expression plasmid, a plasmid expressing the VSV-G envelope protein and the genome plasmid. 18 The genome plasmid, pSMART2, is based on the pONY8.0 series of plasmids, 25 but in addition contains a central polypurine tract (cPPT) upstream of the internal cytomegalovirus (CMV) immediate-early enhancer/promoter and the woodchuck hepatitis post-transcriptional regulatory element (WPRE) downstream of the transgene (Figure 1). Both these elements were included to increase viral titre and protein expression, respectively.…”

“…18,20 Vectors were produced in 10 layer cell factories (Nalgene) each seeded with 5 Â 10 8 HEK293T in DMEM-10 (Sigma Aldrich) containing 10% FBS, 1% MEM nonessential amino acids, 2 mM glutamine and 10 U/ml penicillin, 10 mg/ml streptomycin (DMEM-10). The next day, the genome plasmid was cotransfected into the human kidney cell line, with the plasmid encoding the envelope protein VSV-G (pRV67), EIAV vector for systemic delivery of proteins A Lamikanra et al together with either pONY3.1 that encodes Gag/Pol.…”

“…EIAV infects all Equidae causing anaemia but no immunodeficiency and there has been no documented case of EIAV infecting humans. A recombinant EIAV vector containing the minimal requirements for genetic delivery has previously been described 17,18 and has been used for gene transfer in the CNS to correct a rat model of Parkinson's disease. 19 More recently, long-term treatment of diabetes insipidis via administration of EIAV expressing arginine vasopressin (AVP) into the supraoptic nuclei of the hypothalamus has been demonstrated.…”