Gene Therapy Strategies Based On GPI-anchored Bifunctional HIV Entry Inhibitors

Human immunodeficiency virus(HIV)has always been an important pathogen that severely threatening global public health since its discovery in 1983.The development of prevention and treatment strategy for HIV infection has been continuously explored.With nearly 40 years efforts,a safe and effective vaccine for HIV is still elusive.And highly active antiretroviral therapy(HAART)remains the gold standard for controlling viral infections.Although HAART has achieved the unquestionable success in controlling viral replication,it cannot eradicate the HIV reservoirs,while a lifelong treatment is needed to prevent the occurrence of HIV-associated immunodeficiency.Its limitations such as long-term toxicity and resistance,become evident and thus make alternative therapeutic approaches that is effective and less-toxic become one priority in fighting against HIV infection.Currently,the research focus has been on the effective therapy that can functionally cure HIV infection.The successful cure or functional cure of the "Berlin patient" and "London patient" provides strong proof-of-principle that gene therapy might achieve HIV cure and,in this context,creation of genetically engineered HIV-resistant cells with broad resistance would be a powerful strategy.So far,multiple CCR5-targeting approaches have been developed to generate HIV-resistant cells by reducing or eliminating cell surface expression of CCR5.Although CCR5-targeting strategies are not easy to induce resistance,they are ineffective in protecting modified cells from X4-and R5X4-tropic virus infection.Therefore,a gene therapy that generate HIV-resistant cells with broad resistance should be highly imperative.In this study,we focus on generating HIV-resistant cells with broad resistance by expressing the different viral entry inhibitors targeting distinct viral Env sites on the cell surface vial a glycosylphosphatidylinositol(GPI).First,by expressing a gp41-targeting short-peptide HIV fusion inhibitor 2P23 on the target cell surface through a glycosylphosphatidylinositol(GPI)anchor,we constructed the GPI-2P23 and demonstrated that its expression was primarily directed to the lipid raft using co-focusing experiment,and did not affect the expression levels of CD4,CCR5,and CXCR4 on the cell surface using FACS experiment.We testified that GPI-2P23 could fully protected the target cell from infections of HIV-1,HIV-2,and SIV,and efficiently blocked the viral Env-mediated cell-cell fusion and cell-cell HIV-1 transmission.Additionally,GPI-2P23-modified CD4+T cells(CEMss-CCR5)were highly resistant to R5-and X4-tropic HIV-1 infections,and displayed a survival advantages under HIV-1 replication.During the development of the potent newly-designed GPI-anchored fusion inhibitor with different target site,we explored their underlying mechanism and had three findings.First,pocket-binding domain(PBD)motif was critical for GPI-anchored fusion inhibitors in inhibiting HIV;second,linker was not required for GPI-anchored fusion inhibitors.Third,it was found for the first time that GPI-anchored fusion inhibitors had capability to interfere with viral Env processing and viral infectivity.It was also found that GPI-anchored fusion inhibitors,especially GPI-CP41,harboring pocket-binding domain(PBD)motif had the stronger interference ability.In a recent report,we have identified and characterized GPI-10E8(target to the gp41)and GPI-m36.4(target to the gp120)with remarkable potency and breadth,and these two GPI-anchored entry inhibitors and GPI-CP41 possess the different target site.Finally,to develop membrane-anchoring bispecific entry inhibitors,fusion inhibitor(CP41)and neutralizing antibodies(10E8 or m36.4)were incorporated into a single-component molecule,and expressed on the target cell surface by means of a glycosylphosphatidylinositol(GPI).It was verified by virus-infection and viral Env-mediated cell-cell fusion assay that GPI-10E8-L-CP41,GPI-CP41-L-10E8,and GPI-m36.4-L-CP41 were indeed membrane-anchored bispecific entry inhibitors.In conclusion,we employed HIV entry inhibitors targeting distinct viral Env sites to successfully construct the membrane-anchored bispecific entry inhibitor,which has the capacity to produce HIV-resistant cells with highly broad viral resistance,thus has the potential for gene therapy of HIV infection.