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Stanford BIO 230 - Establishment of HIV-1 resistance in CD4+ T cells

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Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleasesRESULTSDesign of ZFNs targeted against CCR5 (CCR5 ZFN)Entry inhibition of CCR5-tropic HIV-1 by ZFN-targeted disruption Figure 1 ZFN-mediated disruption of CCR5 and protection from HIV-1 infection in GHOST-CCR5 cells.Survival advantage of ZFN-modified CD4+ T cells in vitroFigure 2 In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4+ T-cell line, PM1.Selective advantage of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infectionSpecificity of CCR5 ZFNs in primary CD4+ T cellsFigure 3 Enrichment of CCR5 ZFN-modified primary CD4+ T cells during in vitro HIV-1 challenge.Figure 4 Reduction in viremia and selection for CCR5 ZFN-modified CD4+ T cells in the presence of HIV-1 challenge in vivo.Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivoDISCUSSIONMETHODSCCR5 ZFN construct assemblySurveyor nuclease assayCell cultureEx vivo targeted gene disruptionIn vitro HIV-1 infection challengesIn vivo HIV-1 infection challengesMicroscopyPyrosequencingStatistical analysisRequests for reagentsACKNOWLEDGMENTSAUTHOR CONTRIBUTIONSCOMPETING INTERESTS STATEMENTReferencesEstablishment of HIV-1 resistance in CD4+T cells bygenome editing using zinc-finger nucleasesElena E Perez1,2, Jianbin Wang3, Jeffrey C Miller3, Yann Jouvenot3,4, Kenneth A Kim3, Olga Liu1,Nathaniel Wang3, Gary Lee3, Victor V Bartsevich3, Ya-Li Lee3, Dmitry Y Guschin3, Igor Rupniewski3,Adam J Waite3, Carmine Carpenito1, Richard G Carroll1, Jordan S Orange2, Fyodor D Urnov3,Edward J Rebar3, Dale Ando3, Philip D Gregory3, James L Riley1, Michael C Holmes3& Carl H June1Homozygosity for the naturally occurring D32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection.We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5.Transient expression of CCR5 ZFNs permanently and specifically disrupted ~50% of CCR5 alleles in a pool of primary humanCD4+T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro andin vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4+T cells had lower viral loadsand higher CD4+T-cell counts than mice engrafted with wild-type CD4+T cells, consistent with the potential to reconstituteimmune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4+T-cell population. Thus adoptivetransfer of ex vivo expanded CCR5 ZFN–modified autologous CD4+T cells in HIV patients is an attractive approach for thetreatment of HIV-1 infection.CCR5, a seven-transmembrane chemokine receptor, is the major co-receptor for HIV-1 entry1,2. Since the discovery that the homozygousD32 deletion in CCR5 confers resistance to HIV-13–5, CCR5 has beenintensely studied and validated as a target for HIV therapy6,7. Recently,small-molecule approaches that block the CCR5-HIV interaction haveshown promise in clinical trials8. However, the small-moleculeapproach has resulted in the development of resistance by selectionfor escape mutants, which continue to use CCR5 for viral entry9.These results, taken together with experience from individuals hetero-zygous for the D32 allele, point to the importance of a geneticknockout of CCR5 for phenotypic penetrance and long-term resis-tance to infection rather than its knock-down by approaches basedon small molecules, intrabodies, antisense or RNA interference(RNAi)10–15. Therefore, we sought to permanently disrupt the endo-genous CCR5 and thus make a phenocopy of the D32CCR5 nullgenotype in primary human CD4+T cells by the application ofengineered ZFNs.Previously, we have shown that reconstituting CD4+helper T-cellactivity through adoptive transfer of costimulated CD4+T cells mayaugment natural immunity to HIV-1 infection13. Here we show thatengineered ZFNs targeting human CCR5 efficiently generate a double-strand break at a predetermined site in the CCR5 coding regionupstream of the natural CCR5D32 mutation. The CCR5 ZFNspromote efficient and permanent disruption of CCR5 in primaryhuman CD4+T lymphocytes and confer robust protection againstHIV-1 infection both in v itro and in an in vivo mouse model of HIV-1infection. Combining the two approaches may provide further benefitto patients with HIV-1 in future clinical trials.RESULTSDesign of ZFNs targeted against CCR5 (CCR5 ZFN)We designed and optimized a large series of ZFNs targeted to humanCCR5 using a previously described approach16. For both target sitestwo zinc-finger protein (ZFP) DNA-binding domains, each containingfour zinc-finger motifs (recognizing a total of 24 base pairs), wereassembled from an archive of ZFP DNA-binding modules17,18.TheseZFPs were coupled to the DNA cleavage domain of the type IISrestriction enzyme, FokI, to produce novel ZFNs in which the locationof DNA cleavage is determined by the DNA-binding specificity of theengineered ZFP domains, as previously shown16,17,19.Targetingadouble-strand break to a specific site in the genome with ZFNs hasbeen used to disrupt permanently the genomic sequence surroundingthe ZFN target site in a variety of eukaryotic organisms20,21viaimperfect repair by nonhomologous end joining (NHEJ)22,23.Toexploit this property of double-strand break repair, we elected tofocus our ZFN designs upon the DNA sequence encoding the firsttransmembrane domain (TM1 spans residues Arg31 to Asn57) of theCCR5 co-receptor. We reasoned that this location, upstream of theReceived 22 April; accepted 22 May; published online 29 June 2008; doi:10.1038/nbt14101Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, 421 Curie Blvd., Room 554, BRB II/III, Philadelphia, Pennsylvania19104-6160, USA.2Children’s Hospital of Philadelphia, Division of Allergy and Immunology, Joseph Stokes, Jr. Research Institute, 3615 Civic Center Blvd.,Philadelphia, Pennsylvania 19104-4318, USA.3Sangamo BioSciences, Inc., Point Richmond Tech Center II, 501 Canal Blvd., Suite A100, Richmond, California 94804,USA.4Present address: Process Science Department, Bayer Hematology/Cardiology, 800 Dwight Way, Berkeley, California 94701, USA. Correspondence should beaddressed to C.H.J. ([email protected]).808 VOLUME 26 NUMBER 7 JULY 2008 NATURE BIOTECHNOLOGYARTICLES© 2008 Nature Publishing Group http://www.nature.com/naturebiotechnologyD32


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