Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.

Parkinson's disease (PD) is a neurodegenerative disease affecting 1% of the population over 55 years, whose cardinal symptoms include rigidity, resting tremor, bradykinesia and impaired postural reflexes. PD is characterised by a selective loss of dopaminergic neurons in the substantia nigra, creating dopamine deficiency in the target projection area: the striatum. We and others have demonstrated that expression of the dopamine biosynthetic enzymes tyrosine hydroxyalse (TH), aromatic L-amino acid decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1), in the striatum, results in a significant improvement in motor function in animal models of the disease (Azzouz et al., J Neurosci. 2002 22:10302-12; Shen et al., Hum Gene Ther 2000 11:1509-19; Muramatsu et. al., Hum Gene Ther 2002 13:345-354). We have generated an optimised EIAV tricistronic vector LentiVector® (EIAV-TRIC), with an improved expression and safety profile, that can express all the three enzymes in transduced striatal neurons, both in vitro and in vivo. Stereotactic delivery of these vectors in the sensorimotor putamen of MPTP treated macaques with severe parkinsonism, led to significant motor recovery and improvement in clinical rating scales. This was specific to the EIAV-TRIC treated macaques, for similar treatment with a vector expressing a marker gene failed to yield any such improvement. Our results indicate that intraputamenal delivery of dopamine synthesising genes by LentiVector®, can achieve functional rescue in a primate PD model, thus opening the potential for its use in gene therapy of late stage PD patients.

The ability of lentiviral-based vectors to integrate into non-dividing cells such as neuronal cells has been exploited to correct a number of disease models. Using vectors based on the equine infectious anaemia virus (EIAV) we have previously reported the correction of an animal model of Parkinson's disease (Azzouz et al. J Neurosci. 2002 Dec 1;22(23):10302–12). To use such vectors in the clinic each manufactured lot must be tested and demonstrated to be free of replication competent lentivirus (RCL). We have designed the vectors in such a way to limit the possibility of homologous recombination taking place; the vector genome contains only 824 nt of EIAV sequence with no coding regions; all the accessory genes have been removed entirely from the system; the gag/pol sequence has been codon optimised and a heterologous envelope is used (VSV-G). However, there is still the formal possibility of some form of recombination leading to the generation of an RCL. We have therefore developed an RCL assay based on the property common to all members of the Retroviradae family, namely the presence of reverse transcriptase (RT). RT activity can be detected by a sensitive assay: Product Enhanced Reverse Transcriptase (PERT). The test article is passaged on human cells to amplify any replicating entity and then a PERT assay is carried out on the supernatant. A typical set of assay results will be presented.

Conference Abstract| October 01 1999 DEVELOPMENT OF RETROVIRAL TRANSDUCED HAEMATOPOIETIC STEM CELLS TOWARD A NOVEL APPROACH TO CANCER GENE THERAPY JS Freeth; JS Freeth 1Oxford BioMedica (UK) Ltd, The Oxford Science Park, OXFORD, OX4 4GA Search for other works by this author on: This Site PubMed Google Scholar S. Iqball; S. Iqball 1Oxford BioMedica (UK) Ltd, The Oxford Science Park, OXFORD, OX4 4GA Search for other works by this author on: This Site PubMed Google Scholar J. Rohll; J. Rohll 1Oxford BioMedica (UK) Ltd, The Oxford Science Park, OXFORD, OX4 4GA Search for other works by this author on: This Site PubMed Google Scholar S. Watt; S. Watt 1Oxford BioMedica (UK) Ltd, The Oxford Science Park, OXFORD, OX4 4GA Search for other works by this author on: This Site PubMed Google Scholar CE Lewis; CE Lewis 1Oxford BioMedica (UK) Ltd, The Oxford Science Park, OXFORD, OX4 4GA Search for other works by this author on: This Site PubMed Google Scholar SM Kingsman; SM Kingsman 1Oxford BioMedica (UK) Ltd, The Oxford Science Park, OXFORD, OX4 4GA Search for other works by this author on: This Site PubMed Google Scholar S. Naylor S. Naylor 1Oxford BioMedica (UK) Ltd, The Oxford Science Park, OXFORD, OX4 4GA Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1999) 27 (5): A148. https://doi.org/10.1042/bst027a148c Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation JS Freeth, S. Iqball, J. Rohll, S. Watt, CE Lewis, SM Kingsman, S. Naylor; DEVELOPMENT OF RETROVIRAL TRANSDUCED HAEMATOPOIETIC STEM CELLS TOWARD A NOVEL APPROACH TO CANCER GENE THERAPY. Biochem Soc Trans 1 October 1999; 27 (5): A148. doi: https://doi.org/10.1042/bst027a148c Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1999 Biochemical Society1999 Article PDF first page preview Close Modal You do not currently have access to this content.