117. AAV-TRISPR – A Novel Versatile AAV Vector Kit for Combinatorial CRISPR and RNAi Expression

Gene Targeting and Gene Correction I and MPT1 mast cells. The mast cell line BR was the exception, showing relatively high levels of infection. Q-RT-PCR results showed low level of integrins in MPT1, 17-71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. Interestingly, CAR mRNA levels were comparatively higher in MPT1, 17-71, OSW, and PBMC cells. These data indicate that lack of integrins and not CAR is the primary reason for the inability of Ad5G/L infection to infect cells of lymphocyte origin. The defect in cell surface integrins must be addressed if Adenoviral vectors are to be used to transfer genes to lymphocytes or tumors in cells of lymphocyte origin.

Gene Targeting and Gene Correction I 115. Correction of the Sickle-Cell Disease Mutation in Human Hematopoietic Stem/Progenitor Cells

Megan D. Hoban,1 Matthew C. Mendel,2 Zulema Romero,1 Michael L. Kaufman,1 Alok V. Joglekar,1 Michelle Ho,1 Dianne Lumaquin,1 David Gray,1 Georgia R. Lill,1 Aaron R. Cooper,3 Fabrizia Urbinati,1 Shantha Senadheera,1 Allen Zhu,2 Pei-Qi Liu,2 David E. Paschon,2 Lei Zhang,2 Edward J. Rebar,2 Andrew Wilber,4 Xiaoyan Wang,5 Philip D. Gregory,2 Michael C. Holmes,2 Andreas Reik,2 Roger P. Hollis,1 Donald B. Kohn,1 Gregory J. Cost.2 1 1Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA; 2 Sangamo BioSciences Inc., Richmond, CA; 3Molecular Biology Interdepartmental PhD Program, University of California, Los Angeles, Los Angeles, CA; 44Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL; 5Department of General Internal Medicine and Health Services Research, University of California, Los Angeles, Los Angeles, CA.

Sickle-cell disease (SCD) is characterized by a single point mutation in the seventh codon of the beta-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells (HSCs) would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrated efficient targeted cleavage at the betaglobin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector [IDLV] or a DNA oligonucleotide), high levels of gene modification were achieved in CD34+ hematopoietic stem and progenitor cells (HSPCs). Modified cells maintained their ability to engraft NOD/SCID/IL2rgnull (NSG) mice and to produce cells from multiple lineages. Importantly, ZFN-driven gene correction in CD34+ cells from the bone marrow of sickle patients resulted in the production of wild-type hemoglobin tetramers.

116. MegaTAL Nucleases Outperform TALENs in Promoting Homology-Directed-Gene Modification in Primary Human T Cells

Gabrielle Curinga,1 Guillermo Romano,1 Blythe Sather,1 Karen Sommer,1 Malika Hale,1 Iram Khan,1 Swati Singh,1 Yumei Song,1 Kamila Gwiazda,1 Andy Scharenberg,1,2 David J. Rawlings.1,2 1 Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Childrens Research Institute, Seattle, WA; 2Department of Pediatrics of Immunology, University of Washington School of Medicine, Seattle, WA.

Advances in rare cleaving nuclease technologies have dramatically increased the potential for seamless manipulation of virtually any gene (i.e. gene editing). Among these platforms, TALENs and Cas9-based nucleases have become broadly applied owing to ease in customizing their DNA recognition properties. In contrast, S48

LAGLIDADG homing endonucleases (LHE) are more challenging to engineer, yet exhibit superior physical attributes, DNA recognition and hydrolysis properties. Recent advances have overcome hurdles in LHE engineering leading to the ability to more rapidly customize LHEs as therapeutic nucleases. In addition, LHEs can be fused to TAL effector arrays to create chimeric proteins [referred to here as megaTALs (MTAL)] that exhibit further enhancements in activity. We previously developed a CCR5-specific MTAL as a potential HIV therapeutic. In parallel, we generated high-activity TALEN pairs targeting the identical region in CCR5. In the current study, we directly compared the ability of these nuclease platforms to induce homology directed repair (HDR) in primary human T-cells. We first verified that both reagents facilitated efficient disruption of the CCR5 locus. We directly compared activity in primary CD4 T cells using mRNA nuclease delivery. Indel frequency was estimated using both the T7 endonuclease assay and a re-cleavage assay (RCA; where genomic DNA surrounding the cleavage site is amplified and digested in vitro with the original LHE used to create the MTAL). NHEJ rates were 65-82% for the TALEN vs. 44-61% (6 experiments) for the MTAL using these respective assays. Next, we coupled mRNA-mediated nuclease expression with AAV delivery of a donor template. We codelivered mRNA with a range of doses of an AAV-CCR5-GFP donor (containing 1.3kb CCR5 homology arms and an internal MND-GFP expression cassette). Cell viability was not significantly different; and control experiments using AAV BFP (lacking homology arms) demonstrated that infection and expression rates were equivalent. Strikingly, the proportion of cells with sustained GFP expression (at day 16) was up to 5-fold higher in MTAL treated cells (across a range of AAV doses; n=6, independent donors); with the greatest differences observed when access to donor template was limiting. Molecular analysis verified HDR in both test groups. While the mechanism for these differences remains incompletely defined, preliminary comparisons of HR:NHEJ ratios using single cell analysis suggest that alternative DNA repair pathways may be preferentially utilized by the 3’ vs 5’ overhangs generated by LHE vs Fok1 nucleases, respectively. Taken together, our findings suggest that the MTAL platform and the co-delivery method described here may provide significant clinical utility in future editing applications in human hematopoietic cells.

117. AAV-TRISPR - A Novel Versatile AAV Vector Kit for Combinatorial CRISPR and RNAi Expression

Florian Schmidt,1 Joel Beaudouin,2 Kathleen Börner,1,3 Dirk Grimm.1 1 Heidelberg University Hospital, Heidelberg, Germany; 2German Cancer Research Center Heidelberg, Heidelberg, Germany; 3 German Center for Infection Research (DZIF), Heidelberg, Germany. The CRISPR/Cas9 system currently revolutionizes the gene therapy field due to the unique ease and efficiency with which it can be exploited for targeted DNA editing in mammalian cells. It only requires a small g(uide)RNA that directs the Cas9 protein to a specific DNA sequence which is then cleaved by Cas9. In the absence of a homologous exogenous template, the ensuing double-stranded DNA break is repaired by non-homologous end joining, an error-prone cellular mechanism frequently resulting in insertions or deletions that disrupt the original sequence. While useful in principle to perturb the expression of disease-associated genes, rapidly evolving targets such as human viruses will require concurrent expression of multiple gRNAs to thwart adverse mutational escape. To fill in this gap, we created novel highly customizable AAV vectors permitting multiplexing of up to three gRNAs in a single self-complementary vector backbone. Notably, our constructs readily accommodate and deliver minimal expression cassettes only consisting of gRNA(s) Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

Gene Targeting and Gene Correction I and promoter(s) with total sizes as little as 250 bp, which reduces the amount of ectopic DNA and improves vector safety. We demonstrated the power of our vectors by their use for simultaneous knock-out of three key players in apoptosis signaling, exemplifying their usefulness for dissection of cellular pathways. In addition, we engineered corresponding AAV backbones for shRNA expression, to be able to eventually juxtapose targeting on the DNA (CRISPR) and the RNA (RNAi) level in a single vector. Therefore, we made all constructs compatible with Golden Gate Assembly which minimizes work while maximizing flexibility, in turn paving the way for highthroughput cloning and screening of combinatorial RNAi/CRISPR libraries. Notably, the vectors offer three different RNA polymerase III promoters - U6, H1 or 7SK - for expression of gRNA or shRNA, thus allowing users to fine-tune g/shRNA levels and to alleviate toxic effects from over-expression. Finally, we also assembled a library of promoters for Cas9 expression comprising viral, synthetic and mammalian variants, to further expand the options for customization of our AAV/TRISPR system according to specific targets and needs. Indeed, as described in more detail in a separate abstract (Börner et al.), the selection of optimal gRNA/Cas9 vector designs even permits potent disruption of very challenging targets, such as integrated proviral HIV-1 DNA in T cells. Likewise, the use of proper regulatory elements is crucial for potent in vivo CRISPR-mediated targeting of endogenous genes in the mouse liver, as noted for the miR-122 microRNA. Considering the high degree of versatility of our vector designs, as well as the additional options for in vivo re-targeting through AAV capsid engineering, we expect our novel AAV-TRISPR kit for combinatorial gRNA/shRNA expression to substantially foster future efforts to co-exploit RNAi and CRISPR, and to benefit a multitude of human gene therapy applications.

118. Inhibition of Hepatitis B Virus Replication In Vivo Following Delivery of Antiviral TALENs With Recombinant Adeno-Associated Viral Vectors

Samantha Nicholson,1 Buhle Moyo,1 Kristie Bloom,2 Claudio Mussolino,2 Toni Cathomen,2 Koichi Watashi,3 Abdullah Ely,1 Patrick Arbuthnot.1 1 Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; 2Institute for Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; 3Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Hepatitis B virus (HBV) is hyperendemic to sub-Saharan Africa where there are approximately 600 000 deaths per year associated with chronic HBV infection. HBV may have long periods of dormancy in carriers of the virus, which is dependent on persistence of the nuclear replication intermediate covalently closed DNA (cccDNA). Current HBV therapies, including nucleot(s)ide analogs, target the actively transcribed virus. The cccDNA is not cleared and may act as a reservoir for re-initiation of viral replication following treatment withdrawal. This poses a significant challenge for the successful treatment of chronic HBV sufferers, and makes the cccDNA intermediate an important target for novel HBV treatments. Employing gene editing technology to disable cccDNA has emerged as a promising novel therapeutic strategy. TALENs targeting the surface and the core viral open reading frames, previously described by our group, demonstrated efficacy against HBV targets. To advance this technology to therapeutic application, efficient delivery and evaluation of the TALENs’ safety and target specificity in vivo and in vitro need to be established. This study aimed to evaluate the safety and efficacy of TALENs delivered by adeno-associated viral (AAV) vectors in vivo and in vitro. The HBV-infectable HepG2-hNTCP-C4 cell line was used to establish that the TALENs could disable cccDNA. Capacity Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

for capsid modification, low immunogenicity and good hepatotropic transduction efficiency are favorable features that were considered in selecting these vectors. However, the delivery of TALENs by AAVs is technically complicated, as sequences encoding the two subunits of the complete TALENs exceed the capacity of single stranded AAVs. Consequently, we generated AAVs that encode each of the TALEN subunits. To constitute the complete TALENs, pairs of vectors were thus administered to mice or cultured cells. In vitro testing of AAVTALENs was undertaken using the HepG2-hNTCP-C4 cell line. These cells present a novel tool for testing anti-HBV therapeutics as they overexpress the human sodium taurocholate co-transporting polypeptide (hNTCP) gene allowing them to be infected by HBV. The viral life-cycle is accurately recapitulated and enables assessment of effects of therapeutic agents on cccDNA. Since viral gene expression is dependent on transcription from the cccDNA intermediate in these cells, measurement of markers of HBV replication may be used as an indicator of cccDNA function. This study evaluated the potential of AAV delivered TALENs in vivo and in vitro by testing their activity and evaluating off-target activity, immunogenicity and liver toxicity. The results provide evidence for the utility of applying AAVs to the delivery of anti-HBV TALENs and offers further support for the feasibility of employing TALENs to treat chronic HBV infection.

119. HIV-1 Proviral DNA Excision and Deactivation Using a CRISPR/Cas9 “Nickase” Targeted to the 5’ and 3’ LTR

Tristan Scott,1 Sheena Saayman,2 Daniel Lazar,2 Patrick Arbuthnot,1 Isidor Rigoutsos,3 Kevin Morris,2,4 Fatah Kashanchi,5 Marco Weinberg.1,2 1 Molecular Medicine and Heamatology, University of the Witwatersrand, Johannesburg, South Africa; 2Molecular and Experimental Medicine, The Scripps Research Institute, San Diego; 3Computational Medicine Center, Thomas Jefferson University, Philadelphia; 4Medicine Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia; 5Mircobiology, George Mason University, Washington, DC. Permanent integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected HIV cells capable of reactivation. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. Therapies aimed at targeting the latent virus offer a promising approach to a “functional cure” of HIV/AIDS. Several gene editing technologies, including ZFNs, TALENs, Tre-recombinases and RNA-guided CRISPR/Cas9 tools have been used to disrupt the HIV-1 genome and suppress viral gene expression and replication in latently-infected cells. While CRISPR/Cas9 represents a powerful and facile DNA editing technology, toxicities associated with the Cas9 nuclease activity remains a concern. Here we explore the use of a “nickase” Cas9 (nCas9), which has a mutated RuvC I catalytic domain, and requires the combination of two adjacent small guide RNAs (sgRNAs) for on-target editing and specificity. We initially identified 9 sgRNA sequences targeted to conserved sites within the HIV TAR element, found on both the 5’ and 3’ LTRs. Using integrated and episomal LTR-driven luciferase reporters, we identified functional sgRNA “nickase pairs” capable of inducing an 80% reduction in gene expression, as well as excision of the proviral DNA. Using HIV-1 latency models, plasmids expressing nCas9 and the sgRNA pairs were nucleofected into HIV-1 integrated cell lines J1.1, CHME5 and U1 and treated with the histone deacetylase inhibitor trichostatin A (TSA) to activate infection. All treated cells showed significant reduction (up to 80% for J1.1) in infectious viral output. Cleavage was further verified using ChIP assays to detect DNA damage response proteins recruited to the targeted locus in all cell lines. Lastly, using a custom S49