Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system

Maunder, H. E.; Wright, J. Fraser; Kolli, B. R.; Vieira, Catarina; Mkandawire, Tapoka T.; Tatoris, S.; Kennedy, Vicky; Iqball, Sharifah; Devarajan, G.; Ellis, Scott; Lad, Yatish; Clarkson, N. G.; Mitrophanous, Kyriacos; Farley, Daniel Colin

doi:10.1038/ncomms14834

Cited by 16 publications

(15 citation statements)

References 49 publications

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“…The resulting low HTI/Gag ratio (1:3) weakened the HTI negative effect and allowed for higher IDLV-HTI recovery. A further increase in IDLV-HTI production was obtained by using the tbs/ TRAP system, 48,49 originally described by Nie and Htun, 48 who provided evidence that Bacillus subtilis TRAP may repress the translation of mRNA containing the tbs in transiently transfected mammalian cells. Cloning of tbs upstream the HTI open reading frame (ORF) in the transfer vector and subsequent production of IDLV-tbsHTI using the tbs/TRAP system allowed for a further increase of recovered vector compared with IDLV produced using a lower HTI/Gag ratio.…”

Section: Discussionmentioning

confidence: 95%

“…In particular, we harnessed the Bacillus subtilis Tryptophan RNA-binding Attenuation Protein (TRAP) and a TRAP-binding sequence (tbs), inserted upstream of the HTI sequence, which acts to repress translation of HTI. 48,49 To validate this system, we constructed a plasmid containing the tbs sequence upstream the HTI-mCherry fusion protein (ptbsHTI-mCherry). The plasmid was co-transfected in 293T Lenti-X cells with plasmid expressing SIV-Gag, with or without pTRAP, for CLSM analysis (Figure 5A).…”

Section: Inhibition Of Hti Synthesis During Idlv Production Attenuates the Hti Dominant-negative Effectmentioning

confidence: 99%

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Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice

Gallinaro

Borghi

Pirillo

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Cellular immune responses play a fundamental role in controlling viral replication and AIDS progression in human immunodeficiency virus (HIV)-infected subjects and in simian immunodeficiency virus (SIV)-infected macaques. Integrase defective lentiviral vector (IDLV) represents a promising vaccine candidate, inducing functional and durable immune responses in mice and non-human primates. Here, we designed HIV-and SIV-based IDLVs to express the HIVACAT T cell immunogen (HTI), a mosaic antigen designed to cover vulnerable sites in HIV-1 Gag, Pol, Vif, and Nef. We observed that HTI expression during lentiviral vector production interfered profoundly with IDLV particles release because of sequestration of both HIV-and SIV-Gag proteins in the cytoplasm of the vector-producing cells. However, modifications in IDLV design and vector production procedures greatly improved recovery of both HIV-and SIV-based IDLV-HTI. Immunization experiments in BALB/c mice showed that both IDLVs elicited HTI-specific T cell responses. However, immunization with HIV-based IDLV elicited also a T cell response toward exogenous HIV proteins in IDLV particles, suggesting that SIV-based IDLV may be a preferable platform to assess the induction of transgene-specific immune responses against rationally designed HIV structural antigens. These data support the further evaluation of IDLV as an effective platform of T cell immunogens for the development of an effective HIV vaccine.

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Section: Discussionmentioning

confidence: 95%

Section: Inhibition Of Hti Synthesis During Idlv Production Attenuates the Hti Dominant-negative Effectmentioning

confidence: 99%

Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice

Gallinaro

Borghi

Pirillo

et al. 2020

Molecular Therapy - Methods & Clinical Development

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“…To facilitate the translational success emerging with in-vivo gene transfer, considerable focus must be brought to bear on the cost-of-goods associated with manufacturing vectors. This will require efficient scaling of vector production, for example by developing vector production from stable cell lines [105,106] to reduce the cost and procedural burden of transient transfection that is the backbone of current viral vector approaches [107]. …”

Section: Expert Opinionmentioning

confidence: 99%

Lessons learned from lung and liver in-vivo gene therapy: implications for the future

Haasteren

Hyde

Gill

2018

Expert Opinion on Biological Therapy

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Introduction: Ex-vivo gene therapy has had significant clinical impact over the last couple of years and in-vivo gene therapy products are being approved for clinical use. Gene therapy and gene editing approaches have huge potential to treat genetic disease and chronic illness. Areas covered: This article provides a review of in-vivo approaches for gene therapy in the lung and liver, exploiting non-viral and viral vectors with varying serotypes and pseudotypes to target-specific cells. Antibody responses inhibiting viral vectors continue to constrain effective repeat administration. Lessons learned from ex-vivo gene therapy and genome editing are also discussed. Expert opinion: The fields of lung and liver in-vivo gene therapy are thriving and a comparison highlights obstacles and opportunities for both. Overcoming immunological issues associated with repeated administration of viral vectors remains a key challenge. The addition of targeted small molecules in combination with viral vectors may offer one solution. A substantial bottleneck to the widespread adoption of in-vivo gene therapy is how to ensure sufficient capacity for clinical-grade vector production. In the future, the exploitation of gene editing approaches for in-vivo disease treatment may facilitate the resurgence of non-viral gene transfer approaches, which tend to be eclipsed by more efficient viral vectors.

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“…We anticipate that the platform should be compatible with strategies for transgene suppression in producer cells such as the recently reported TRiP System. 23 …”

Section: Discussionmentioning

confidence: 99%

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Chen

Pallant

Sampson

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

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Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system

Cited by 16 publications

References 49 publications

Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice

Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice

Lessons learned from lung and liver in-vivo gene therapy: implications for the future

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

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