491. Efficient Template-Free Genetic Correction with TALE Nucleases

NUCLEASE MEDIATED GENOME EDITING principle establishing phenotypic correction of hemophilia B, and show that appropriately designed donors may expand this strategy to treat a range of monogenic diseases.

489. Quantifying Rates of Gene Targeting and Off-Target Cleavage of Engineered Nucleases Using SMRT Sequencing

Eli J. Fine,1 Eric Kildebeck,2 Yanni Lin,1 Matthew H. Porteus,2 Thomas J. Cradick,1 Gang Bao.1 1 Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA; 2Pediatrics, Stanford University, Palo Alto, CA. Transcription Activator-Like Effector Nucleases (TALENs) are a powerful tool for genome engineering since they can be designed to target a DNA sequence in a modular fashion for specific DNA cleavage in a given genome. Repair of this lesion through the nonhomologous end-joining (NHEJ) pathway can result in a targeted knockout of a gene of interest. Alternatively, homology directed repair (HDR) using an exogenously supplied DNA template can result in targeted gene addition or modification. TALENs have been used to create model organisms, custom cell lines, and are in development for gene therapy. However, discriminating between similar targets in the same gene family has been a challenge for engineered nucleases; the CCR5 zinc finger nucleases in clinical trials have only a 4:1 preference for cleaving CCR5 over CCR2. Quantifying rates of gene targeting in primary cells is another major challenge due to the low frequency of these events. To date, the commonly used techniques for assessing the efficacy of TALENs are the Surveyor Nuclease Assay and Restriction Fragment Length Polymorphism (RFLP). Both techniques are well suited for detecting modifications at rates as low as a few percent. However, for detecting low-frequency off-target events or low, but clinically relevant, rates of HDR in primary cells, these techniques are not effective. Deep sequencing can be used to detect low-frequency off-target events and the analysis of single cell clones can detect low rates of HDR, but both of these methods are difficult and expensive. To address these challenges, we employed Single Molecule Real-Time (SMRT) sequencing: a 3rd generation platform that offers long read lengths, high fidelity, reasonable sensitivity, and variable sequencing depth at a relatively low cost. We created TALENs targeting the beta-globin gene near the sickle-cell mutation with cleavage rates of 62% in 293T cells. Using SMRT sequencing, we found that these TALENs cleave a highly similar sequence in the delta-globin gene at a rate less than 0.58% (p < 0.05), demonstrating a greater than 100 fold preference for cleaving beta-globin over delta-globin. Using a different set of TALENs, we were also able to detect rates of targeting cDNA to the IL2Rg locus in primary human CD34+ cells of 1%. Cleavage at off-target sites by engineered nucleases is a large concern. Previously, only three TALEN off-target sites had been identified, all through using SELEX as a predictive assay and deep sequencing to confirm cleavage at the off-target site. We used the newly developed PROGNOS algorithm to predict potential off-target sites for TALENs with different repeat variable di-residues to target guanine (NK, NN, and NH) and analyzed the cleavage sites from transfected cells using SMRT sequencing. We identified several off-target sites for these novel nucleases, some with homology to the intended target as low as 76% and others lacking a 5’ pyrimidine. The use of SMRT sequencing as an assay format for genomic modifications induced by engineered nucleases holds great promise for allowing detection of low frequency events.

Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

490. Precise Modification of Human Genes Directed by Single-Stranded DNA Oligonucleotides and TALENs

Shengdar Q. Tsai,1,2 Deepak Reyon,1,2 Cyd Khayter,1 J. Keith Joung.1,2 1 Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA; 2Department of Pathology, Harvard Medical School, Boston, MA.

The capability to direct precise modifications to the genome could broadly enhance biological research, and in the longer term, enable therapies of genetic diseases. Single-stranded DNA oligonucleotides (ssODNs) have been used with engineered nucleases to introduce precise mutations into endogenous genes. While ssODNs have been optimized with zinc finger nucleases (ZFNs), the robustness of ssODN-mediated repair with TAL effector nucleases (TALENs) has not been systematically optimized. We assessed the effect of ssODN homology length on the efficiency of gene alteration using an EGFP reporter repair assay and then used optimal-length ssODNs with TALENs to successfully direct precise nucleotide insertions or substitutions to sites in 13 human endogenous genes. The high frequency of sequence modifications we observed enabled us to isolate precisely modified single-cell clones without the need for selection. This high efficiency of modification coupled with the extended targeting range of TALENs compared to ZFNs make ssODN/TALENdirected repair an attractive approach for simple, rapid, and precise genome modification.

491. Efficient Template-Free Genetic Correction with TALE Nucleases

David G. Ousterout,1 Pablo Perez-Pinera,1 Pratiksha I. Thakore,1 Ami M. Kabadi,1 Matthew T. Brown,1 Kamel Mamchaoui,2 Jacques P. Tremblay,3 Charles A. Gersbach.1,4 1 Biomedical Engineering, Duke University, Durham, NC; 2Institut de Myologie, Paris, France; 3Unité de Recherche de Recherche en Génétique Humaine, Université Laval, Quebec, Canada; 4Institute for Genome Sciences and Policy, Duke University, Durham, NC. Genome editing with engineered nucleases is a powerful approach for correcting genetic mutations. Current strategies exclusively utilize homologous recombination guided by a synthetic DNA repair template. Although this approach is extremely valuable for restoring the complete coding sequence of the affected gene, simply restoring a disrupted reading frame or removing a premature stop codon could ameliorate the symptoms of some genetic diseases. Therefore, frame restoration could be accomplished by utilizing the stochastic, error-prone non-homologous end-joining DNA repair pathway that creates small insertions and deletions of random length at the targeted DNA break point. Here we demonstrate the restoration of dystrophin expression in cells from Duchenne muscular dystrophy (DMD) patients with transcription activator-like effector nucleases (TALENs) in the absence of a DNA template by utilizing homologyindependent repair pathways to correct disrupted reading frames. TALENs were targeted to exon 51 of the dystrophin gene with the potential to correct aberrant reading frames occurring in up to 8.4% of known DMD mutations. First, several combinations of TALEN pairs targeted to exon 51 were screened for optimal activity and minimal cytotoxicity in human cells. Using the optimal TALEN pair, we demonstrated that reading frame restoration after TALEN treatment rescued dystrophin expression in corrected clonal populations of skeletal myoblasts from DMD patients. Furthermore, this TALEN mediated high efficiency gene editing of up to 12.7% of alleles and restored detectable levels of dystrophin protein expression in a bulktreated population of DMD muscle cells without any selection or clonal isolation. This TALEN also modified up to 5.5% of alleles in S189

GENE & CELL THERAPY OF DIABETES, METABOLIC AND GENETIC DISEASES II primary human DMD fibroblasts. Following conversion into muscle cells by MyoD overexpression, dystrophin protein expression was detectable in this unselected cell population as well. We showed that the types of mutations introduced by the designer TALENs reliably introduced frame-correcting insertions and deletions, as well as small, frame-sparing deletions that could be used to remove aberrant stop codons. Finally, we sequenced the whole exomes of several clonal populations of TALEN-modified human DMD cells and showed TALEN gene modification did not produce any off-target events. Our current work aims to demonstrate the successful engraftment of corrected DMD myoblasts in an immunodeficient mouse model as well as expression of human dystrophin that is properly localized to the sarcolemma. Importantly, this strategy is generally applicable to the correction of genetic diseases caused by frameshifts or premature stop codons in nonessential gene regions.

492. Enhancing the Efficiency of Zinc Finger Nuclease Delivery by Integrase-Defective Lentiviral Vectors Using Valproic Acid

Alok V. Joglekar,1 Michelle Ho,1 Libby Stein,2 Sara Sanadiki,1 Roger P. Hollis,1 Donald B. Kohn.1 1 Microbiology, Immunology, Molecular Genetics, University of California, Los Angeles, CA; 2Brown University, Providence, RI.

Integrase defective lentiviral vectors (IDLVs) can be used to deliver Zinc Finger Nucleases (ZFNs) to human cells. However, because their expression per vector genome is often low, the efficiency of ZFN mediated gene modification they support is lower than that obtained with adenoviral vectors or electroporation. The reason(s) for this lower level of gene expression are not fully understood, but could involve the epigenetic silencing of vector genomes, carried out primarily by histone deacetylases (HDACs). In this study, we investigated whether the use of Valproic Acid (VPA), an FDA-approved HDAC inhibitor, would lead to an increase in transgene expression from IDLVs. Preliminary experiments in K562 human erythroleukemia cells demonstrated that transient VPA treatment was able to induce a 4-5 fold increase in transgene expression from IDLVs encoding a GFP reporter. Importantly, we observed a similar 3-fold increase in expression from IDLVs carrying ZFNs targeting the human adenosine deaminase (ADA) gene after treatment of the K562 cells with VPA. The increased ZFN levels correlated with a 2-fold increase in disruption at the ADA gene. Interestingly, VPA treatment was also able to increase IDLV mediated transgene expression in primary human CD34+ cells. These results suggest that transient treatment with VPA could be used to enhance the efficiency of IDLV-mediated genome editing. Moreover, this improved expression level could also be exploited for other applications of IDLVs, such as antigen expression in direct vaccination.

Gene & Cell Therapy of Diabetes, Metabolic and Genetic Diseases II 493. Mechanism of Hematopoietic Stem CellMediated Therapy in Cystinosis

Brian A. Yeagy,1 Jay Sharma,1 Celine J. Rocca,1 Sarah Ur,1 Thomas Whisenant,2 Daniel R. Salomon,2 Stephanie Cherqui.1 1 Department of Pediatrics, University of California, San Diego, La Jolla, CA; 2Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA. Cystinosis is a lysosomal storage disorder which results from a genetic defect in the gene CTNS encoding the lysosomal cystine transporter protein, cystinosin. Cystine accumulates in every tissue compartment and leads to multi-organ failure. Our previous work has shown that transplantation of syngeneic wild-type (Wt) hematopoietic S190

stem cell progeny (HSPC) in the mouse model of cystinosis, Ctns-/mice, was effective in treating cystinosis. Bone marrow-derived cells integrated abundantly into all tissues examined leading to significant cystine decrease and preservation of the kidney. The mechanisms under investigation for stem cell-based treatment are differentiation, trans-differentiation, cell fusion, cross correction, or a combination of these processes. In this context, we developed a new mouse model for cystinosis, the DsRed Ctns-/- mice, ubiquitously expressing the DsRed reporter gene. We then transplanted GFP-expressing HSPC so the differentiated bone marrow-derived cells (DsRed-GFP+) and the fused cells (DsRed+GFP+) could be unequivocally recognized, quantified and sorted. Using confocal microscopy, flow cytometry and DNA array analysis, we showed that stem cells mainly differentiate in tissue-resident-phagocytic cells such as kupffer cells in the liver, microglia cells in the brain, and dendritic cells in the kidney. We also showed that they could play a role in tissue repair by fusing with or by phagocytosing the apoptotic host cells. Using a lentiviral vector driving the expression of the fusion protein cystinosin-eGFP, we also showed that cystinosin could be transferred from CTNS-expressing cells to Ctns-deficient adjacent cells in vitro and in vivo. This transfer led to cystine decrease in Ctns-deficient cells in vitro. We optimized in vitro functional assays involving macrophages to study these two potential mechanisms in the context of cystinosis. We showed that macrophage-mediated phagocytosis was stimulated in the presence of Ctns-/- fibroblasts. Live imaging also showed that transfer of cystinosin-eGFP was performed via nanotube-like structures extending from the macrophages to cystinotic cells. Cystinosin proteins were carried by lysosomes themselves trafficking along microtubules present in the tunneling nanotubes. This work provides new insights into the mechanism of the long-term therapeutic effect of transplanted HSPC in cystinosis. This strategy could potentially be applicable to other genetic diseases for which curative therapy requires the addition of the gene to many cells and multiple tissue compartments, and where the protein involved is an intracellular transmembrane protein.

494. Hematopoietic Stem Cell Gene Therapy for Cystinosis

Sarah Ur,1 Celine J. Rocca,1 Jay Sharma,1 Donald B. Kohn,2 Stephanie Cherqui.1 1 Department of Pediatrics, University of California, San Diego, La Jolla, CA; 2Department of Pediatrics and Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA.

Cystinosis is an autosomal metabolic disease belonging to the family of lysosomal storage disorders. Mutations in the CTNS gene, encoding a lysosomal cystine transporter, lead to cystine accumulation and multi-organ failure such as blindness, myopathy, diabetes and central nervous system defects. Affected individuals also develop proximal tubulopathy and eventually progress to end stage renal failure. Treatment is available with the drug cysteamine to reduce intracellular cystine content. However, cysteamine only delays the progression of the disease. In April 2012, the first clinical trial using stem cells for cystinosis was approved as an allogeneic Hematopoietic Stem and Progenitor Cell (HSPC) transplant to be conducted at University of California, Los Angeles. This trial was based on the preclinical studies we performed in the mouse model of cystinosis, the Ctns-/- mice, which showed that transplantation of HSPCs expressing a functional Ctns gene led to the abundant tissue integration of bone marrow-derived cells, a significant decrease of cystine accumulation and kidney preservation. However, allogeneic transplants are associated with high risks of mortality and morbidity. Thus, our long-term goal is to develop an autologous transplantation strategy of HSPCs genetically modified ex vivo to express a functional CTNS gene as a treatment for cystinosis. Preclinical studies using a Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy