Abstract
The C-X-C chemokine receptor type 4 (CXCR4) is one of the major co-receptors for human immunodeficiency virus type 1 (HIV-1) entry and is considered an important therapeutic target. However, its function in maintaining the development of hematopoietic stem cells (HSC) makes it difficult to be used for HIV-1 gene therapy with HSC transplantation. A previous report showed that the natural CXCR4 P191A mutant inhibits HIV-1 infection without any defect in HSC differentiation, which could provide a basis for the development of new approaches for HIV-1 gene therapy. In the present study, we used CRISPR-Cas9 combined with the piggyBac transposon technologies to efficiently induce the expression of the CXCR4 P191A mutant in an HIV-1 reporter cell line, leading to no detectable exogenous sequences. In addition, no off-target effects were detected in the genome-edited cells. The decline of HIV-1 replication in biallelic CXCR4 gene-edited cells suggests that individuals equipped with homologous recombination of the CXCR4 P191A mutant could prevent or reduce HIV-1 infection. This study provides an effective approach to create a CXCR4 mutation with HIV-1 infection inhibition function and without leaving any genetic footprint inside cells, thereby shedding light on an application in HIV-1 gene therapy and avoiding side effects caused by deficiency or destruction of CXCR4 function.
Highlights
Human immunodeficiency virus type 1 (HIV-1) can utilize the primary cellular receptor CD4 and co-receptor C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) to enter into cells by membrane fusion[1]
Upon examination of the sequences 69-bp upstream of the P191A mutation, we found that replacing the leucine codon sequence CTG with the leucine codon TTA allows the piggyBac transposon cassette be excised and released from the host genome[31] without affecting the amino acid sequence of CXCR4 (Fig. 1a)
After transfection with transposase plasmid into TZM-bl cells, the piggyBac transposon containing the selection cassette inserted at the TTAA site can be excised from the genome, leaving the CXCR4 P191A mutation behind (Fig. 1d,e)
Summary
Human immunodeficiency virus type 1 (HIV-1) can utilize the primary cellular receptor CD4 and co-receptor C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) to enter into cells by membrane fusion[1]. For HIV-1 patient treatment, in addition to the highly active antiretroviral therapy (HAART) that effectively blocks HIV-1 replication, CCR5 and CXCR4 disruption by genome engineering technologies is considered a potential strategy to inhibit viral infection[6,7]. We have reported that silencing CXCR4 expression with shRNA and introducing a CXCR4 (P191A) mutant with a lentiviral vector inhibits HIV-1 replication by approximately 60% without impairing CXCR4-mediated downstream signal transduction[22] This approach allowed for extra insertion of lentiviral vector in genome, which may cause tumorigenesis as a side effect. By combining a CRISPR-Cas[9] strategy and piggyBac transposon system, we achieved efficient replacement of wild-type CXCR4 with a CXCR4 P191A homozygous mutant in an HIV-1 reporter cell line and observed significant inhibition of HIV-1 infection. Our approach provided a potential gene correction method for treatment of genetic disorders in the future
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