Slide 1Slide 2HIV animationSlide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 controls HIV-1 in vivoCo-receptor CCR5 Permits HIV-1 EntrySlide 18ZFN-mediated disruption of CCR5 in CD34+ HSPCs.Slide 20Protection of human CD4+ T cells in peripheral blood of HIV-infected mice previously engrafted with ZFN-modified CD34+ HSPCs.Effects of HIV-1 infection on human cells in HSPC-engrafted NSG mice.Reasons for CD4+ and CD8+ T-cell Depletion in the ThymusHIV-1 infection selects for disrupted CCR5 alleles.ZFN activity produces heterogeneous mutations in CCR5.Control of HIV-1 replication in mice receiving ZFN -treated CD34+ HSPCsSlide 27ConclusionsAverage = 77.9A=95+ C+ =70-74A-=90-94 C = 64-69B+ = 85-89 C- = 59-63B= 80-84B- = 75-79Special Biology SeminarFriday May 6 at 1 pm in CB386DNA STR* TypingSherri PhillipsForensic Scientist & Western AlumnusThe Washington State PatrolCODIS** Crime Lab* Short Tandem Repeat **Combined DNA Index SystemFriday May 6 at2pm in BI 415After the seminarSherri will meet with interested students to discuss her workas a forensic scientist and how she “found her way” to the CODIS Lab. This will be an informal question & answer sessionHIV animation•Another•Good integration videoHIV-1 reverse transcriptase active site. The p66, p51, p66-thumb and p66-fingers domains are all labeled on the diagram. The white strand indicates the primer and the template strand is in blue. The amino acid residues that are thought to interact with the DNA are highlighted in yellow. The purple arrow shows the direction the enzyme turns in order to catalyze the polymerization of the growing DNA strand. This image also clearly indicates the importance of the p51 in stabilizing the heterodimer. Patel et al. 1995Integration of provirusIntegration. The newly reverse-transcribed double-stranded retroviral DNA genome and a piece of chromosomal DNA are specifically cleaved by the retroviral integrase protein. This is accompanied by a deletion of two base pairs from the retroviral genome and a duplication of four to six base pairs from the host DNA!!!!!! Following retroviral genomic insertion into the cleaved host DNA, the DNA is relegated.(a) Activation of HIV transcription by the interaction of viral protein Tat with DNA-dependent protein kinase (DNA-PK) results in the phosphorylation at Ser131 of Sp1. Phosphorylated Sp1 results in increased transcription of proviral DNA, resulting in an increase in Tat production,. (b) Inhibition of HIV transcription involves O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzing the addition of O-GlcNAC to Sp proteins which blocks their interaction with their binding sites on the LTR, resulting in an inhibition/reduction in HIV transcription.Kilareski et al. Retrovirology 2009 6:118HIV Transcription•Where the idea came from •Science CentralHuman hematopoietic stem/progenitor cells modified Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 controls by zinc-finger nucleases targeted to CCR5 controls HIV-1 HIV-1 in vivoin vivoNathalia Holt, Jianbin Wang, Kenneth Kim, Geoffrey Friedman, Xingchao Wang, Vanessa Taupin, Gay M Crooks, Donald B Kohn, Philip D Gregory, Michael C Holmes & Paula M CannonCo-receptor CCR5 Permits HIV-1 Entry•CCR5 is the major co-receptor used by HIV-1 and is expressed on key T-cell subsets and monocytes.•CCR5Δ32 is a relatively common allele in Western Europe•Confers an innate resistance to HIV-1 infection•CCR5 antagonists have proved to be an effective salvage therapy in patients infected with drug-resistant HIV-1 Kuby Immunology says CCR5 is only expressed on Monocytes, but this is an extremely misleading oversimplification.“Art” by Haley WatermanZFN-mediated disruption of CCR5 in CD34+ HSPCs.•ZFN: Zinc Finger Nuclease–Artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. •Cel 1 nuclease:–Preferentially cleaves DNA at distorted duplexes caused by mismatches. •Used for quantification of the CCR5 disrupted alleles. •NSD Mice:–NOD SCID IFγ-null Mice•Non-obese diabetic severe combined immunodeficiency interferon-gamma null mice.–Lack T, B, and NK cells–Deficient in multiple cytokine signaling pathways–Defects in innate immunity.Image courtesy of Wikipedia: “The only website on the internet guaranteed to be 90% accurate!”Fun Fact: “Cel” in Cel 1 nuclease stands for celery!ZFN-mediated disruption of CCR5 in CD34+ HSPCs.Figure 1 Analysis•a) Representative gel showing extent of CCR5 disruption in CD34+ HSPCs 24 hours after nucleofection–Neg: No gene digestion–Mock: No gene digestion–ZFN: Gene digestion•b) Mean percentage of human CD45+ cells in peripheral blood of mice 8 weeks after transplantation.–No statistical difference between Neg, Mock, and ZFN groups.•c) Fluorescence-activated cell sorting (FACS, or flow cytometry) profiles of human cells of various organs from one ZFN-treated mouse.–Results indistinguishable from that of mice transplanted with unmodified cells.•Holds true for both the location of cells and their frequency in that particular tissue.Protection of human CD4+ T cells in peripheral blood of HIV-infected mice previously engrafted with ZFN-modified CD34+ HSPCs.Figure 2 Analysis•a) FASC readouts showing human CD4+ and CD8+ T-cells in peripheral blood of representative animals from each of three cohorts.–Uninfected: Normal CD4+ T-cell levels–HIV-1 Infected (Negative): Complete T-cell depletion–HIV-1 Infected (ZFN-treated): Normal CD4+ T-cells•b) Ratio of human CD4+ to CD8+ lymphocytes in peripheral blood of individual mice to which were infected with HIV-1. –Measured pre-infection and 6-8 weeks post-infection.–Significant reduction in CD4+/CD8+ T-cell ratio in post-infection untreated mice.Effects of HIV-1 infection on human cells in HSPC-engrafted NSG mice.Figure 3 Analysis•a) FACS analysis of human cells in tissues of representative NSG mice from three cohorts.–SSC means “side scatter” of light.–Bone Marrow: No CD45 cell loss•Not a major organ for HIV-1 infection–Spleen: Reduction of CD4+ T-helper cells post HIV-1 infection w/o ZFN treatment.–Thymus: Complete loss of CD4+ and CD8+ T-cells.–Small intestine: Complete loss of all human lymphocytes w/o ZFN treatment.•b)