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Analysis of factor VIII mediated suppression of lentiviral vector titres.
Abstract: 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.
Publication Date: 2007-11-29 PubMed ID: 18046428DOI: 10.1038/sj.gt.3303080Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article focuses on exploring a vector system for gene therapy for haemophilia A that can evade pre-existing immune responses, has sufficient coding capacity, offers long-term expression, and potentially targets non-dividing cells. It discovers that factor VIII (FVIII) protein inhibits the production of functional viral particles when jointly expressed with the Equine Infectious Anaemia Virus (EIAV) vector system. It worked towards overcoming this inhibitory effect by using an internal tissue-specific promoter, discovering in the process that codon optimization of the full-length FVIII gene increased vector capacities approximately 10-fold as well as improving expression per integrated vector copy.
Exploring a Vector System for Hemophilia A Gene Therapy
- The researchers’ main focus is to find a vector system for effective gene therapy for hemophilia A. This vector system needs to have several properties: it should be immune to any pre-existing immune response, it should have sufficient coding capacity, it should offer a long-term expression, and preferably could target non-dividing cells. According to the authors, vecor systems based on lentiviruses such as the Equine Infectious Anaemia Virus (EIAV) meet these criteria for the delivery of Factor VIII (FVIII).
Role of Factor VIII (FVIII)
- The investigators found that B-domain deleted FVIII protein disrupts functional viral particle production when co-expressed with the EIAV vector system. Despite particle numbers being similar to normal (as measured by reverse transcriptase activity), RNA genome levels were reduced. Furthermore, a severe defect in the ability of the virus to transduce target cells was observed due to insufficient envelope from vesicular stomatitis virus glycoprotein (VSV-G) on derived viral particles from cells expressing FVIII.
Overcoming Inhibitory Effects and Improving Vector Titres
- To overcome the inhibitory effect of FVIII on functional viral particle production, the team used an internal tissue-specific promoter with low activity in producer cells to drive expression of FVIII. This strategy allowed them to bring the titres close to the levels obtained with vector genomes encoding reporter genes.
- In addition, the team found that codon optimization of the full-length FVIII gene substantially improved expression per integrated vector copy and increased vector titres about tenfold.
Cite This Article
APA
Radcliffe PA, Sion CJ, Wilkes FJ, Custard EJ, Beard GL, Kingsman SM, Mitrophanous KA.
(2007).
Analysis of factor VIII mediated suppression of lentiviral vector titres.
Gene Ther, 15(4), 289-297.
https://doi.org/10.1038/sj.gt.3303080 Publication
Researcher Affiliations
- Oxford BioMedica (UK) Ltd, Medawar Centre, The Oxford Science Park, Oxford, UK. p.radcliffe@oxfordbiomedica.co.uk
MeSH Terms
- Cell Line
- Codon
- Factor VIII / genetics
- Genetic Therapy
- Genetic Vectors
- Hemophilia A / therapy
- Humans
- Infectious Anemia Virus, Equine / genetics
- Polymerase Chain Reaction
- Promoter Regions, Genetic
Citations
This article has been cited 10 times.- Greig JA, Smith MK, Nordin JML, Goode T, Chroscinski EA, Buza EL, Schmidt N, Kattenhorn LM, Wadsworth S, Wilson JM. Determining the Minimally Effective Dose of a Clinical Candidate Adeno-Associated Virus Vector in a Mouse Model of Hemophilia A. Hum Gene Ther 2022 Apr;33(7-8):421-431.
- Deverman BE, Ravina BM, Bankiewicz KS, Paul SM, Sah DWY. Gene therapy for neurological disorders: progress and prospects. Nat Rev Drug Discov 2018 Sep;17(9):641-659.
- Maunder HE, Wright J, Kolli BR, Vieira CR, Mkandawire TT, Tatoris S, Kennedy V, Iqball S, Devarajan G, Ellis S, Lad Y, Clarkson NG, Mitrophanous KA, Farley DC. Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system. Nat Commun 2017 Mar 27;8:14834.
- Nathwani AC, Nienhuis AW, Davidoff AM. Our journey to successful gene therapy for hemophilia B. Hum Gene Ther 2014 Nov;25(11):923-6.
- Roberts SA, Dong B, Firrman JA, Moore AR, Sang N, Xiao W. Engineering Factor Viii for Hemophilia Gene Therapy. J Genet Syndr Gene Ther 2011 Dec 21;1.
- McIntosh J, Lenting PJ, Rosales C, Lee D, Rabbanian S, Raj D, Patel N, Tuddenham EG, Christophe OD, McVey JH, Waddington S, Nienhuis AW, Gray JT, Fagone P, Mingozzi F, Zhou SZ, High KA, Cancio M, Ng CY, Zhou J, Morton CL, Davidoff AM, Nathwani AC. Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant. Blood 2013 Apr 25;121(17):3335-44.
- Zielonka J, Bravo IG, Marino D, Conrad E, Perković M, Battenberg M, Cichutek K, Münk C. Restriction of equine infectious anemia virus by equine APOBEC3 cytidine deaminases. J Virol 2009 Aug;83(15):7547-59.
- Kutner RH, Zhang XY, Reiser J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 2009;4(4):495-505.
- Kok CY, MacLean LM, Rao R, Tsurusaki S, Kizana E. Promoter Optimization Circumvents Bcl-2 Transgene-Mediated Suppression of Lentiviral Vector Production. Biomolecules 2023 Sep 16;13(9).
- Greig JA, Nordin JML, White JW, Wang Q, Bote E, Goode T, Calcedo R, Wadsworth S, Wang L, Wilson JM. Optimized Adeno-Associated Viral-Mediated Human Factor VIII Gene Therapy in Cynomolgus Macaques. Hum Gene Ther 2018 Jul 23;29(12):1364-75.
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