Abstract
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.
Highlights
Many lentiviral vectors used for gene therapy are derived from human immunodeficiency virus type 1 (HIV-1)
P LV21 contains the Rous sarcoma virus enhancer/promoter[17], the HIV-1 5’ R-U5-leader region that contains sequences required for genomic RNA dimerization and encapsidation as well as splice donor 1 (SD1)[8,22,23], a 364 nucleotide HIV-1 gag sequence with a frameshift mutation[8], an 858 nt HIV-1 env sequence[8] that contains the 351 nt RRE6,24 and the splice acceptor used for most fully spliced viral RNAs (SA7)[23], the HIV-1 central polypurine tract that increases transduction efficiency[25], a CMV-GFP reporter sequence, the WPRE10 and the HIV-1 3’ LTR with a 400-nucleotide deletion that abolished its promoter activity[18]
We have shown that when nt 22–378 in gag were deleted in the context of full-length HIV1, production of infectious vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped virus was similar to wild-type HIV-134
Summary
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. Because Rev is required for Gag-Pol expression and vector titre, this protein was expressed in trans allowing a packaging construct containing only the gag and pol genes plus the cis-acting Rev-response element (RRE) These modifications reduced biosafety concerns, including the potential to produce replication-competent lentivirus (RCL)[17,18]. A large portion of the HIV-1 3’ U3 enhancer/promoter sequence was deleted in the vector genome to create self-inactivating (SIN) vectors[18] This prevents transfer of this sequence to the 5’ end during reverse transcription, thereby eliminating the enhancer and promoter elements in the integrated genome and reducing the potential for activating expression of surrounding genes in target cells
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