328. Engineering Zinc Finger Nucleases for Targeted Gene Editing in Zebrafish

GENE REGULATION: VECTOR PLATFORM STUDIES 326. Stimulation of Gene Correction by HairpinInduced Double Strand Breaks Matthew L. Hirsch, Curtis F. Hewitt, Richard J. Samulski. Gene Therapy Center, University of North Carolina, Chapel Hill, NC.

Human genetic engineering is becoming a feasible approach to cure a variety of genetic disorders. However, the efficiency of recombination using a homologous substrate, such as a plasmid, a single-strand oligonucleotide or a viral genome is generally low. Reports have demonstrated that a specific double-strand break (DSB) near the genetic defect stimulates homologous recombination (HR) by several orders of magnitude. Thus, efforts have been directed towards the production of custom endonucleases that cleave a unique sequence within the human chromosome. Another way to achieve specific DSBs, described in yeast, is via “fragile” DNA structures that are processed to a DSB by DNA repair enzymes. Here, this finding is expanded to a human context using the inverted terminal repeat sequence (ITR) of adeno-associated virus (AAV). A reporter system was established in which the AAV ITR was inserted into a defective gfp coding sequence (cds) and used as the target for HR. Single-strand oligonucleotides with 40 nucleotide homologous arms to the gfp cds flanking the insertion served as the repair substrate. This reporter harboring the AAV ITR sequence demonstrated stimulation of gene correction to levels approaching the efficiency of reported zinc-finger nucleases. In contrast, a non-significantly structured DNA insertion of the same size demonstrated no Gfp+ cells. Increased phosphorylation of H2AX in the presence of the reporter containing the AAV ITR suggests that the stimulation of gene correction is via DSB repair. Our initial attempt to increase the efficiency of gfp correction by synthesizing a third stem-loop on the suspected T-shaped AAV ITR resulted in an additional 2.5-fold increase. Analysis of additional viral, human chromosome and engineered hairpins in the same context demonstrates that differently structured cis elements are likely processed to DSBs at different frequencies, reflected by a broad range of gene correction efficiencies. Such an understanding of DNA structure-induced HR will not only help in the determination of human chromosome sites to target for gene repair, but may also provide insight into the mechanism of genome rearrangements associated with oncogenesis.

327. Genome-Wide Analysis of Off-Target Events Induced by Zinc Finger Nucleases in Zebrafish

Ankit Gupta,1 Xiangdong Meng,1,2 Lihua J. Zhu,1 Nathan D. Lawson,1 Scot A. Wolfe.1 1 University of Massachusetts Medical School, Worcester, MA; 2 Sangamo BioSciences Inc., Richmond, CA. Genome editing is a powerful tool for studying gene function and has potential to be utilized therapeutically via gene therapy. Recently, Zinc Finger Nucleases (ZFNs) were successfully used for introducing mutations in cell lines as well as in whole organisms including Drosophila, C. elegans and zebrafish. ZFNs are artificial nucleases consisting of two domains: a DNA binding zinc finger protein domain and the endonuclease domain from FokI. ZFNs create DNA double strand breaks (DSB) at specific sites that stimulate DNA repair either by non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ is an error prone repair pathway that can produce mutations at the DSB site. HR on the other hand requires an additional donor DNA template with homology to the DNA flanking the target site to facilitate recombinational repair. Thus, ZFNs can potentially be used to create tailor-made alternations in a genome from an exogenously supplied DNA template. However the current enthusiasm for the use of this technology in gene therapy applications is tempered by the fact that the current generation of ZFNs generate Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

lesions at off-target sites as well as at the desired target. Dimerization of the nuclease domain of the ZFN is required to generate a DSB. Therefore two ZFNs, which recognize 9-12 base pairs of DNA each, are necessary for the activity. Together they recognize a total of 1824 base pairs which should define a unique site within the human genome. Nonetheless, off-target breaks also happen, although at lower frequencies. Understanding the basis of ZFN activity at off-target sites within the genome will be essential for taking the technology a step toward the therapeutic gene therapy. Previous studies characterized off-target lesions generated by a ZFN by sequencing a few potential off-target sites with the closest homology to the target site. Although this approach provides information on the rates of lesions at a subset of potential off-target sites, it does not provide a comprehensive view of sites frequented by ZFNs. To comprehensively investigate the off-target effects of the ZFNs we are assessing the genome-wide occupancies of the ZFNs in zebrafish embryos using chromatin immunoprecipitation followed by deep sequencing. This analysis will provide information about the in vivo specificity of each ZFN for comparison with the specificity that has been determined using a bacterial one-hybrid system. This in vivo analysis should also aid in designing more specific ZFNs. Genomic sites that are appreciably occupied by ZFNs will be sequenced by solexa to characterize the lesion frequency, which will assess the relationship between the occupancy of the ZFN sites and its DSB activity. Identifying the relationship between the specificity and affinity of a ZFN and its fidelity in genome editing represents an important step in developing these tools not only for making new disease models, but also for its eventual use as gene therapy reagents.

328. Engineering Zinc Finger Nucleases for Targeted Gene Editing in Zebrafish

Joseph C. McNulty,1 Stephanie W. Chu,1,3 Marcus B. Noyes,1,3 Xiangdong Meng,1,4 Matthew Buffardi,1 Thomas Smith,1,2 Nathan D. Lawson,1,2 Scot A. Wolfe.1,3 1 Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA; 2Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA; 3Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA; 4 Sangamo Biosciences Inc., Point Richmond, CA. Gene suppression techniques such as RNAi and antisense morpholino injection are powerful and accessible means to manipulate gene networks in vivo. While the transient behavior of these approaches is suitable for many basic research purposes, they are not useful for the generation of new genetic variants. Creating specific germ-line transmissible variations in vertebrate organisms implies the ability to create mutations that are targeted, stable and well-tolerated --- all central requirements for therapeutic applications of any gene editing technology. There is consequently a strong drive to develop methods that enable specific genomic editing at frequencies high enough to be transmitted through the germ line in model vertebrate organisms. Zinc finger nucleases (ZFNs) have emerged as a powerful technique for the creation of targeted double stranded breaks in both cell culture and model organisms. The repair of these breaks by error-prone non-homologous end joining or homologous recombination can be leveraged to create genetic knock-outs or “knock-ins” if a suitable donor DNA is also supplied. To this end, we are working to develop ZFNs to an array of targets in zebrafish. The use of ZFNs in zebrafish for the generation of gene knockouts has been the subject of several recent publications. To date, however, success in using these techniques has depended strongly on specialized expertise in engineering zinc fingers to bind a chosen DNA sequence, either in-house or outsourced for a considerable monetary cost. Here we present a rapid modular assembly method for the creation of zinc finger domains with chosen DNA binding S127

GENE REGULATION: VECTOR PLATFORM STUDIES specificities, and demonstrate its use in the creation of gene knockouts in zebrafish. Critically, the genetic modifications are efficient enough to be passed through the germ line, sidestepping the need for highly optimized zinc finger sets. We also present the development of a platform to determine favorable conditions for HR experiments in the zebrafish embryo. Efficient homologous recombination has been generally limited to cultured cell lines that are relatively tolerant of the HR experimental conditions. Recent notable success has been reported using D. melanogaster strains lacking a functional DNA Ligase IV gene, thereby shutting down the non-homologous endjoining repair pathway; however similar mutants are non-viable in many organisms, including zebrafish. While our results are still preliminary, we hope to leverage information gained in this system to attempt HR experiments in a wide range of zebrafish gene targets. Successful germ-line transmission of targeted HR in zebrafish will enable us to strongly advance our goals of developing new human disease models and improve our understanding of parameters that affect ZFN-mediated gene conversion for application in future gene therapy endeavors.

329. Cytostatic Drugs Enhance AAV-Mediated Gene Targeting

Shamim H. Rahman,1 Katharina Gellhaus,1 Regine Heilbronn,1 Toni Cathomen.1 1 Virology (CBF), Charité Medical School, Berlin, Germany.

Vectors based on adeno-associated virus (AAV) are efficient tools to correct genetic mutations by gene targeting. Gene targeting is based on components of the homologous recombination (HR) pathway, which are mainly available during the G2 phase of the cell cycle. We have reported earlier that insertion of a DNA double strand break (DSB) in the target locus can stimulate gene targeting up to 1000fold, most likely by activating cellular HR repair mechanisms. Here, we generated three human cell lines that carry a copy of a mutated eGFP gene flanked by recognition sites for the meganuclease I-SceI and zinc-finger nucleases (ZFNs). After treatment with different cytostatic drugs, cells were co-transduced with nuclease encoding AAV vectors and HR donor vectors to rescue eGFP expression by gene targeting. The cell cycle profile, the extent of cytotoxicity and the frequency of gene targeting were assessed by flow cytometry. We show that a transient cell cycle arrest before the creation of a DSB in the target locus increased AAV-mediated gene targeting up to 6-fold. Partially, this effect could be attributed to an increase in AAV transduction. Dependent on the cell line and the AAV vector dose, we reached gene editing frequencies of up to 34%. Because the frequency of AAV random integration was not significantly increased, as determined by quantitative real-time PCR, treatment of cells with the chemicals let to a net increase in the targeting ratio. In summary, cytostatic drugs can be used to enhance AAV-mediated gene targeting in different cell lines. The combined beneficial effects of site-specific DSBs and transient cell cycle arrest allowed us to lower the vector dose - and hence random integration - without compromising on the gene targeting efficacy.

330. The Role of Double-Stranded Breaks in Generating Large Regions of LOH Maja Zavaljevski,1 Sophia Tran,1 David W. Russell.1 1 Hematology, University of Washington School of Medicine, Seattle, WA.

Loss of heterozygosity (LOH) can lead to oncogenesis and manifestations of recessive disorders. A method for selective induction of LOH in large regions of the human genome would provide the ability to screen those regions for harmful recessive mutations and tumor suppressor genes. It would also allow us to convert the HLA locus to homozygosity in cells destined for S128

transplantation, thereby matching more recipients. We have developed a system to generate cells with large regions of LOH on the short arm of human chromosome 6 which includes the HLA locus. Using an AAV gene targeting vector, a positive/negative selection marker (hygromycin phosphotransferase-HSV thymidine kinase: HyTK) is inserted at the HMGA1 locus centromeric to the HLA region. Selection in hygromycin is used to recover targeted clones, and selection in gancyclovir (GCV) is used to recover cells that undergo LOH at the HMGA1 locus and thereby lose the HyTK gene. If LOH is a result of a single mitotic recombination event, the whole region telomeric to HMGA1 will also be converted to homozygosity. Using this system, we have generated one hES cell clone that underwent LOH from ∼3 Mb from the centromere to the telomere on the short arm of chromosome 6. However, these large-scale LOH events are relatively rare and we are currently evaluating methods to increase their frequency. Double-stranded breaks (DSBs) can increase the frequency of LOH locally and we hypothesized that they can also do so for large regions. To test this, we used an AAV gene targeting vector to introduce the I-SceI recognition site at a position ∼2 Mb centromeric to the HMGA1 locus. Using the HT1080 cell line, we generated several clones that contain both the HyTK marker at HMGA1 and the I-SceI site. We expressed I-SceI in those cells and monitored the frequency with which they generated GCVr clones. Additionally, we analyze each GCVr clone to verify and map LOH events. Following cleavage with I-SceI, we have recovered a clone with LOH in a region that originates in a 320 kb window around the DSB site, as determined by SNP analysis. This LOH region extends at least 2.2 Mb toward the telomere and includes the HMGA1 locus. We have also recovered GCVr clones that show no sign of LOH and presumably had a mutation in the TK gene. These results indicate that one possible outcome of DSBs is LOH that originates from the break and extends megabases away. We are currently conducting experiments to recover and characterize additional GCVr clones in order to determine more definitively what effect double-stranded breaks have on the frequency and nature of large-scale LOH events, and also whether DSB induction affects homozygosity in other chromosomal regions.

331. Regulation of Cell Surface Expression of Nucleolin by Phosphorylation by Cyclin Dependent Kinase Cdk1

Xuguang Chen,1,2 Pamela B. Davis.1 1 Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, OH; 2Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH.

Nucleolin, a conserved eukaryotic protein with multifaceted cellular functions, is expressed on the surface of certain cell types, and serves as a receptor for DNA nanoparticles, a nonviral gene transfer vector. However, the detailed mechanism of its cell surface residence without a transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor is not clear to date. We previously discovered that nucleolin binds to DNA nanoparticles directly, and shares similar intracellular trafficking pattern with the nanoparticles. Manipulations of cell surface nucleolin correlate positively with the efficiency of transfection by DNA nanoparticles. Serum starvation reduces both cell surface nucleolin and transfection by the nanoparticles. Since removal of serum affects cell cycle progression, we hypothesize that the expression of nucleolin is regulated by cell cycle dependent kinase, most likely cdc2/Cdk1, which has been shown to phosphorylate nucleolin and regulate its nuclear/cytoplasmic shuttling. To dissect the sequence element that mediates its cell surface expression, we constructed serial deletions of nucleolin fused with GFP epitope tag at the C-terminus. C-terminal truncated nucleolin constructs with the N-terminal 69 aa are expressed on the cell surface, and Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy