CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

WEDNESDAY, FEBRUARY 25, 2015 Session P-F5 Poster Session

Poster Hall

2:30 pm– 4:00 pm Gene Editing

400 Targeted Disruption of Essential HIV-1 Proviral Genes by Rare-Cutting Endonucleases Harshana S. De Silva Feelixge; Nixon Niyonzima; Harlan L. Pietz; Martine Aubert; Dan Stone; Keith R. Jerome Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA, US

Background: HAART is remarkably successful in suppressing HIV viral load to nearly undetectable levels. However, HAART does not reduce the size of the viral reservoir residing mainly in long-lived memory T cells. Lifelong adherence to HAART is paramount in controlling viral loads. If interruption of therapy occurs, HIV rebounds to pre-HAART viral loads within weeks. In recent years much attention has been focused on developing newmethods of gene therapy that have the potential to contribute to HIV cure. Here we evaluate the generation of disruptive mutations within essential HIV-1 proviral genes in order to make the latent HIV pool non-functional. If successful HIV gene disruption can be achieved, reactivation from latency would yield non-viable progeny virus preventing further reseeding of new target cells. Methods: We developed four zinc finger nucleases (ZFNs) engineered to bind distinct HIV pol target sites within protease, reverse transcriptase and integrase. The ZFNs were tested in sup-T1 cells for activity against integrated HIV. Results: All four ZFNs showed activity in a yeast cleavage assay and against a plasmid-derived HIV genome in HEK293 cells. ZFNs were then delivered to the CD4+ sup-T1 T cell line containing integrated defective HIV-1 provirus (DHIV) using scAAV1 vectors. Two of four ZFNs were able to disrupt integrated DHIV provirus at levels up to 28% in sequences within reverse transcriptase and integrase. To increase the levels of gene disruption cells were treated with the end processing exonuclease Trex2 in combination with HIV-targeted ZFNs. Cells treated with Trex2 showed up to a 2-fold increase in levels of provirus disruption for both ZFNs active against integrated provirus. Treated cells showed reduced production of virus, and molecular cloning and expression of ZFN-induced mutations demonstrated that they efficiently disrupted the ability of HIV to replicate. To further improve disruptive mutagenic events, we also tested another class of rare-cutting endonucleases, megaTALs, which consist of a Transcription-activator like effector (TALE) domain fused to a homing endonuclease. MegaTALs containing 5.5, 6.5 or 7.5 TALE repeats were all able to cleave a plasmid-derived target sequence within the catalytic core domain (CCD) of HIV-1 integrase in HEK293 cells at equivalent levels. Conclusions: Our data demonstrate the potential of genome editing as a curative therapy for latent HIV infection. Efforts are underway to further increase the frequency of target site disruptions. 401 Enhancing Anti-HIV Gene Therapy: Combining MegaTAL Nuclease Gene EditingWith Selection Cassettes Biswajit Paul 2 ; Alexander Astrakhan 3 ; PatrickYounan 2 ; Blythe D. Sather 4 ; Jordan Jarjour 3 ; Guillermo Romano 2 ; John P. Kowalski 2 ; Iram Khan 4 ; David J. Rawlings 4 ; Hans-Peter Kiem 2 1 Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA, US; 2 Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA, US; 3 BluebirdBio, Cambridge, MA, US; 4 Seattle Children’s Research Institute, Seattle, WA, US Background: Human Immunodeficiency Virus (HIV) infection remains a substantial health problemworldwide. The human C-C chemokine receptor 5 (CCR5) gene, which encodes a co-receptor required for HIV entry into CD4 + T-cells, is a promising alternative therapeutic target. Early clinical trials using CCR5-disrupting zinc finger nucleases in patients have demonstrated sustained functional control of HIV during antiretroviral treatment interruption. However, two limitations of current gene editing required to achieve therapeutic benefit remain unaddressed. These are (a) the need for higher levels of CCR5-disruption in long-termmemory cells and (b) preferential selection of gene modified cells protected from subsequent infection during transplant. Methods: The CCR5-targeting megaTAL is a novel nuclease architecture that combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator- like (TAL) effector array to achieve efficient site-specific cleavage. We are coupling megaTAL nuclease treatment with drug selection in order to disrupt the CCR5 locus, and select modified CD4+ T-cells to achieve therapeutically relevant levels of HIV-protected cells. The mutant P140K-O6-methylguanine-DNA-methyltransferase (MGMT-P140K) construct delivers resistance against the drug O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea at 50uM O6BG/ 10uM BCNU. The mutant human dihydrofolate reductase (DHFR) construct renders cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX) at 0.05uM. For optimal cell viability we deliver nucleases via mRNA and selection- constructs via adeno-associated virus (AAV). Results: Electroporation with megaTAL mRNA demonstrated robust CCR5 disruption: 95% in GHOST-Hi5 cell lines and 70-90% in human CD4 + T-cells. Gene-modified human T-cells were transplanted into NOD/SCID/ γ c-null ‘humanized’ mice and subsequently challenged with HIV-1 infection. CCR5-null modified cells preferentially survived during active HIV infection in vivo (100 fold increase). Primary T-cells transduced with a MGMT-P140K cassette at 60% efficiency showed two-fold expansion over 48-hours in vitro . Preliminary data in Jurkat cell lines transduced with a Tyr-22-DHFR cassette at 86% efficiency showed two-fold expansion over 72-hours.

Poster Abstracts

Conclusions: The CCR5-megaTAL nuclease platform produces the highest level of gene-modified CD4 + T-cells reported to date and protects these cells from subsequent HIV infection in vivo at significant levels.

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CROI 2015