HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

TY - JOUR

T1 - HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

AU - Sertkaya, Helin

AU - Ficarelli, Mattia

AU - Sweeney, Nathan P.

AU - Parker, Hannah

AU - Vink, Conrad A.

AU - Swanson, Chad M.

N1 - Funding Information: We thank the members of Chad Swanson’s, Michael Malim’s and Stuart Neil’s labs for helpful discussions. We also thank Caroline Goujon and Michael Malim for generously providing reagents. The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 Hybridoma (183-H12-5C) (Cat# 1513) from Dr. Bruce Chesebro. These studies were funded by Medical Research Council grant MR/M019756/1 and MR/S000844/1 to CMS. MR/S000844/1 is part of the EDCTP2 programme supported by the European Union. MF is supported by the UK Medical Research Council (MR/ R50225X/1) and is a King’s College London member of the MRC Doctoral Training Partnership in Biomedical Sciences. HS is supported by the BBSRC Industrial CASE Partnerships (ICP) training grant (BB/P504609/1) with GSK. This work was also supported by the Department of Health via a National Institute for Health Research Comprehensive Biomedical Research Centre award to Guy’s and St. Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. Publisher Copyright: © 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/12

Y1 - 2021/12

N2 - Many lentiviral vectors used for gene therapy are derived from HIV-1. An optimal vector genome would include only the viral sequences required for transduction efficiency and gene expression to minimize the amount of foreign sequence inserted into a patient’s genome. However, it remains unclear whether all of the HIV-1 sequence in vector genomes is essential. To determine which viral sequences are required, we performed a systematic deletion analysis, which showed that most of the gag region and over 50% of the env region could be deleted. Because the splicing profile for lentiviral vectors is poorly characterized, we used long-read sequencing to determine canonical and cryptic splice site usage. Deleting specific regions of env sequence reduced the number of splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining a large deletion in gag with repositioning the Rev-response element downstream of the 3’ R to prevent its reverse transcription showed that 1201 nucleotides of HIV-1 sequence can be removed from the integrated vector genome without substantially compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve safety and transfer less viral sequence into a patient’s DNA.

AB - Many lentiviral vectors used for gene therapy are derived from HIV-1. An optimal vector genome would include only the viral sequences required for transduction efficiency and gene expression to minimize the amount of foreign sequence inserted into a patient’s genome. However, it remains unclear whether all of the HIV-1 sequence in vector genomes is essential. To determine which viral sequences are required, we performed a systematic deletion analysis, which showed that most of the gag region and over 50% of the env region could be deleted. Because the splicing profile for lentiviral vectors is poorly characterized, we used long-read sequencing to determine canonical and cryptic splice site usage. Deleting specific regions of env sequence reduced the number of splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining a large deletion in gag with repositioning the Rev-response element downstream of the 3’ R to prevent its reverse transcription showed that 1201 nucleotides of HIV-1 sequence can be removed from the integrated vector genome without substantially compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve safety and transfer less viral sequence into a patient’s DNA.

UR - http://www.scopus.com/inward/record.url?scp=85107570656&partnerID=8YFLogxK

U2 - 10.1038/s41598-021-91309-w

DO - 10.1038/s41598-021-91309-w

M3 - Article

C2 - 34103612

AN - SCOPUS:85107570656

SN - 2045-2322

VL - 11

JO - Scientific Reports

JF - Scientific Reports

IS - 1

M1 - 12067

ER -