Generation Of Hutat2:Fc Knock-in Monocytes To Resist HIV-Tat-induced Neurotoxicity Using CRISPR/Cas9

Background: The wide application of HAART(highly active antiretroviral therapy)has led to a substancial change in the course of HIV infection,from an acute process that is complicated with fatal opportunistic infections to a chronic process that can be well managed.However,with the prolongation of survival time and promotion of life-quality in HIV infected patients,HIV infection-associated complications,especially for HAND(HIV-associated neurocognitive disorder),were attracted much more attention.Among all the factors that contribute to the pathogenesis of HAND,HIV-Tat(transactivator of transcription)is considered to be crucial: On the one hand,HIV-Tat directly induces the inflamatory injury in neurons and gliocyte;on the other hand,HIV-Tat activates HIV transcription and translation,indirectly facilitating the spread of virus and process of HAND.Thus,any attempts to neutralize HIV-Tat in the brain are on the benefit side for the treatment of HAND.Hutat2:Fc(humanized anti-HIV-Tat2 sc Fv:Fc fusion protein),notable for its high affinity with HIV-Tat,may provide a potential way for HAND therapy.Thus,a highly effective cell-based delivery system is imperative tobe developedto achievethis strategy.Under the encephalitiscaused by HIV infection,monocytes from the peripheral blood can be recruited to traverse the BBB(blood-brain barrier)and aggregated around the sites of inflammation,which endows monocytes with an attracting potential to carry the therapeutic gene into the CNS(central nervous system).Our present study applied the CRISPR/Cas9 system to knock the large functional Hutat2:Fc DNA fragment into the genome of primary monocytes to resist HIV-Tat-induced neurotoxicity.Methods: First of all,six sg RNAs(single guide RNA)were designed targeting the AAVS1(adeno-associated Virus integration site 1)locus and the most active candidate was selected.The donor plasmid was generated by cloning the Hutat2:Fc,GFP and Puro genes into the PTA2 vector.Additionally,~1.5kb homology arms were ligated on both sides of the target fragments.The best ratio of sg RNA and donor plasmids was determined in He La cell line.Then the optimized experimental condition was carried out in purified peripheral blood monocytes.Hutat2:Fc was successfully integrased into AAVS1 locus in He La,293 T,U937 and monocytes.Secondly,the distribution of GFP and Hutat2:Fc were examined by immunofluorescence.Thirdly,the percentage of relative neurons survival rate was quantified in primary mouse cortical neurons in vitro to reflect the protective effects of Hutat2:Fc secreted in each medium.The levels of HIV-DNA and p24 were determined to show the effects of Hutat2:Fc on the replication and spread of virusby HIVBa-L challenge assay.The effects of Hutat2:Fc on T-cells homeostasiswere examined through the distribution of the subsets as well as the expression of exhausting markers in both CD4+ and CD8+ T cells.Finally,a panel of experiments were performed to compare the novel CRISPR/Cas9 technology with the traditional lentivirus mediated gene transfermethod in monocytes.The transduced efficiency and the expression of Hutat2:Fc was determined in KI-M(CRISPR/Cas9 knock-in monocytes),LT-M(lentivirus transfected monocytes)and LT-MDM(lentivirus transfected monocyte derived macrophages)by immunofluorescence and Western blotting.The expression of 15 factors that were tightly related to the functionality of monocytes were evaluated by q-PCR in non-trasduced monocytes,KI-M,LT-M and LT-MDM.Furthermore,the influence of different transduced strategies on migrating ability of monocytes was also examined by transendothelial migration assay.Results: Among sg RNA1-6,sg RNA2 was most active.Without selection,~10% of He La cells exhibited the knock-out efficiencies when transduced by 2?g sg RNA2.The highest KI efficiencies obtained were ~35% in He La cells with the optimized ratio of 1:4 after selection with Puro,while ~10% in primary human monocytes without any selection.Under the guidance of signal peptide,Hutat2:Fc could be stably expressed in cytoplasm and released in the supernatant.The secretion of Hutat2:Fc could gradually accumulate in the medium and reach the peak.Besides,the secretion of Hutat2:Fc in each transduced cell type was able to resist HIV-Tat-induced neurotoxicity,restrict the replication and spread of HIVBA-L and maintain the homeostasis of lymphocytes.Moreover,the protective effects of the medium were positive related to the integrating rates and expression of Hutat2:Fc in each cell type.What's more,compared with the traditional lentivirus infection,CRISPR/Cas9 had the advantage of hardly causing any side-effects,especially for migrating ability,in monocytes despite inferior in transduced efficiency.Conclusions: CRISPR/Cas9 is effective to accurately knock the Hutat2:Fc into AAVS1 locus in monocytes.Hutat2:Fc was stably expressed and released in KI-M and showed excellent bioactivity to protect neurons and PBMC without negative influence on the normal functionality of monocytes.In summary,our present study provides a promising potential strategy for the HAND therapy in the future.