Efficient Transduction of LEDGF/p75 Mutant Cells by Gain-of-Function HIV-1 Integrase Mutant Viruses.
Controlling the specificity of retroviral DNA integration could improve the safety of gene therapy vectors, and fusions of heterologous chromatin binding modules to the integrase-binding domain from the lentiviral integration host cofactor LEDGF/p75 are a promising retargeting strategy. We previously proposed the utility of integrase mutant lentiviral vectors that are selectively activated by complementary LEDGF/p75 variants, and our initial modifications in HIV-1 integrase and LEDGF/p75 supported about 13% of wild-type vector transduction activity. Here we describe the selection and characterization of the K42E gain-of-function mutation in HIV-1 integrase, which greatly improves the efficiency of this system. Both K42E and initial reverse-charge mutations in integrase negatively impacted reverse transcription and integration, yet when combined together boosted viral transduction efficiency to ~75% of the wild-type vector in a manner dependent on a complementary LEDGF/p75 variant. Although the K42E mutation conferred functional gains to integrase mutant viral reverse transcription and integration, only the integration boost depended on the engineered LEDGF/p75 mutant. We conclude that the specificity of lentiviral retargeting strategies based on heterologous LEDGF/p75 fusion proteins will benefit from our optimized system that utilizes the unique complementation properties of reverse-charge integrase mutant viral and LEDGF/p75 host proteins.
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
Hao
H
Wang
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
Ming-Chieh
MC
Shun
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
Xiang
X
Li
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
Francesca
F
Di Nunzio
Division of Infectious Diseases, Imperial College London, St-Mary's Campus, Norfolk Place, London, UK.
Stephen
S
Hare
Division of Infectious Diseases, Imperial College London, St-Mary's Campus, Norfolk Place, London, UK ; Clare Hall Laboratories, London Research Institute, Cancer Research UK, Hertfordshire, UK.
Peter
P
Cherepanov
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
Alan
A
Engelman