Engineering of a stable retroviral gene delivery vector by directed evolution

Halong N. Vu; Joshua D. Ramsey; Daniel W. Pack

doi:10.1038/sj.mt.6300350

Engineering of a stable retroviral gene delivery vector by directed evolution

Halong N. Vu, Joshua D. Ramsey, Daniel W. Pack

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

Abstract

The lack of safe and effective delivery vectors continues to be a critical limitation facing human gene therapy. Viruses offer excellent efficiency but can be difficult and expensive to produce and purify. For example, the production and efficiency of murine leukemia virus (MLV) are limited by its inherent instability; the half-life of infectivity is 5-8 hours at 37 °C. In order to generate a stable MLV, we randomly mutated the virus genome and selected for infectivity after prolonged incubation at 37 °C. After seven rounds of incubation and infection, we isolated a pool of MLV variants with double the half-life of wild-type MLV. Remarkably, a single mutation in the viral protease (PR), G119E, was responsible for the enhanced stability. Saturation mutagenesis at residue 119 revealed variants with half-lives of ∼24 hours at 37 °C. Double mutants combining the changes at position 119 of the PR and substitutions in the PR substrate-binding pocket exhibited half-lives of up to ∼40 hours. MLV variants provided two- to fourfold higher viral titers and exhibited increased stability with various wild-type envelope proteins. The improved stability of the variant MLVs will provide more facile virus production and increased transduction efficiency.

Original language	English
Pages (from-to)	308-314
Number of pages	7
Journal	Molecular Therapy
Volume	16
Issue number	2
DOIs	https://doi.org/10.1038/sj.mt.6300350
State	Published - Feb 2008

Bibliographical note

Funding Information:
We thank Paula Cannon (University of Southern California, Los Angeles, CA) for pMo(4070A) and pMo(10A1), Richard Mulligan for pMD.M EcoEnv, Alan Rein for pA14, and Jing-Kuan Yee (NCI, Frederick, MD) for pCMVgag-pol DNA plasmids. We thank Laura Guest (Keck Center, University of Illinois, Urbana, IL) for her sequencing expertise at the Keck Center, Jerome Baudry (University of Illinois, Urbana, IL) for helpful discussions and assistance in protein structure, and Sandy McMasters at the SCS Cell Media Facility. We are grateful for the financial support from the Roy J. Carver Charitable Trust.

Funding

We thank Paula Cannon (University of Southern California, Los Angeles, CA) for pMo(4070A) and pMo(10A1), Richard Mulligan for pMD.M EcoEnv, Alan Rein for pA14, and Jing-Kuan Yee (NCI, Frederick, MD) for pCMVgag-pol DNA plasmids. We thank Laura Guest (Keck Center, University of Illinois, Urbana, IL) for her sequencing expertise at the Keck Center, Jerome Baudry (University of Illinois, Urbana, IL) for helpful discussions and assistance in protein structure, and Sandy McMasters at the SCS Cell Media Facility. We are grateful for the financial support from the Roy J. Carver Charitable Trust.

Funders	Funder number
Roy J. Carver Charitable Trust

ASJC Scopus subject areas

Molecular Medicine
Molecular Biology
Genetics
Pharmacology
Drug Discovery

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Cite this

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title = "Engineering of a stable retroviral gene delivery vector by directed evolution",

abstract = "The lack of safe and effective delivery vectors continues to be a critical limitation facing human gene therapy. Viruses offer excellent efficiency but can be difficult and expensive to produce and purify. For example, the production and efficiency of murine leukemia virus (MLV) are limited by its inherent instability; the half-life of infectivity is 5-8 hours at 37 °C. In order to generate a stable MLV, we randomly mutated the virus genome and selected for infectivity after prolonged incubation at 37 °C. After seven rounds of incubation and infection, we isolated a pool of MLV variants with double the half-life of wild-type MLV. Remarkably, a single mutation in the viral protease (PR), G119E, was responsible for the enhanced stability. Saturation mutagenesis at residue 119 revealed variants with half-lives of ∼24 hours at 37 °C. Double mutants combining the changes at position 119 of the PR and substitutions in the PR substrate-binding pocket exhibited half-lives of up to ∼40 hours. MLV variants provided two- to fourfold higher viral titers and exhibited increased stability with various wild-type envelope proteins. The improved stability of the variant MLVs will provide more facile virus production and increased transduction efficiency.",

author = "Vu, {Halong N.} and Ramsey, {Joshua D.} and Pack, {Daniel W.}",

note = "Funding Information: We thank Paula Cannon (University of Southern California, Los Angeles, CA) for pMo(4070A) and pMo(10A1), Richard Mulligan for pMD.M EcoEnv, Alan Rein for pA14, and Jing-Kuan Yee (NCI, Frederick, MD) for pCMVgag-pol DNA plasmids. We thank Laura Guest (Keck Center, University of Illinois, Urbana, IL) for her sequencing expertise at the Keck Center, Jerome Baudry (University of Illinois, Urbana, IL) for helpful discussions and assistance in protein structure, and Sandy McMasters at the SCS Cell Media Facility. We are grateful for the financial support from the Roy J. Carver Charitable Trust.",

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Engineering of a stable retroviral gene delivery vector by directed evolution

Abstract

Bibliographical note

Funding

ASJC Scopus subject areas

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