Study On CRISPR/Cas9-based HIV-1 Gene Therapy By Editing Co-receptors CXCR4 And CCR5 Simultaneously

CRISPR/Cas9 was firstly developed in 2013,which can be used to edit almost any gene in genomic level in eukaryotic cells.The system was primarily identified as an adaptive immune system in bacteria and archaea,which consists of a target-specific sgRNA and non-specific endonuclease CRISPR-associated 9(Cas9).The DNA target will be edited after cleavage of Cas9,and the Double Strand Breaks will be repaired by homologous directed repair(HDR)or non-homologous end joining repair(NHEJ).In most cases,the NHEJ leads to the nucleotide mutation,insertion or frame shift,thus causes gene mutation.The CRISPR/Cas9 technology has been applied in all areas of medical study,including virus denfense application,for its simple use,low price and high efficiency.Acquired immune deficient syndrome(AIDS)was a world distributing immune disease,which derived from HIV-1 infection.The main therapy method of AIDS in clinic is highly active anti-retroviral therapy(HAART),which was based on the co-mbination of reverse transcriptase(RT)and protease(PR).According to clinical therapeutical effect of HAART,the HIV-1 related disease can be well controlled with acceptable cell counts of CD4+ T cells and low HIV-1 load in serum.However,the limitation of HAART,such as long term medicine taking,high cost as well as side effects makes it inevitable to consider other alternatives,such as gene therapy.During the first step of HIV-1 infection life cycle,the HIV must bind the CD4 and the coreceptor-CXCR4 or CCR5 in host cell surface,and then the HIV-1 will fuse into the cell.According to this receptor binding,CRISPR/Cas9 mediated single gene editing of CXCR4 or CCR5 has been reported,and indeed,the edited cells show resistance to specific HIV-1 infection.Although,the HIV-1 enter cells by CCR5 at early phase,the CXCR4 may be used as coreceptor at later stage,which can be observed in some HIV-1 infected patients,so single editing about HIV-1 coreceptor may not be enough and appropr:iate.The main content of the study in this thesis is about simultaneous editing of coreceptor CXCR4 and CCR5 with CRISPR/Cas9 technology,and then observing the resistant efficiency with different HIV-1 types.The results show that the edited cells can resist HIV-1x4 and HIV-1R5 infection simultaneously,and the knockout of CXCR4 and CCR5 presents no cellular toxicity.Furthermore,the study has also verified that the edited cells can be enriched when challenged with different HIV-1 coinfection(HIV-1NL4-3 and HIV-1YU-2),and take a selective advantage over un-edited cells.Taken together,the study of HIV-1 co-receptor editing simultaneously with CRISPR/Cas9 has confirmed that the technology could be applied to protect infection of different HIV-1 types,which has supplied a novel possibility of HIV-1 therapy.