scholarly journals Regeneration of Subcutaneous Cartilage in a Swine Model Using Autologous Auricular Chondrocytes and Electrospun Nanofiber Membranes Under Conditions of Varying Gelatin/PCL Ratios

2021 ◽  
Vol 9 ◽  
Author(s):  
Rui Zheng ◽  
Xiaoyun Wang ◽  
Jixin Xue ◽  
Lin Yao ◽  
Gaoyang Wu ◽  
...  
Keyword(s):  
Cartilage Regeneration ◽  
Inflammatory Cells ◽  
Large Animal Model ◽  
Swine Model ◽  
Large Animal ◽  
Nude Mouse Model ◽  
Cartilage Formation ◽  
Nanofiber Membranes ◽  
Large Animals

The scarcity of ideal biocompatible scaffolds makes the regeneration of cartilage in the subcutaneous environment of large animals difficult. We have previously reported the successful regeneration of good-quality cartilage in a nude mouse model using the electrospun gelatin/polycaprolactone (GT/PCL) nanofiber membranes. The GT/PCL ratios were varied to generate different sets of membranes to conduct the experiments. However, it is unknown whether these GT/PCL membranes can support the process of cartilage regeneration in an immunocompetent large animal model. We seeded swine auricular chondrocytes onto different GT/PCL nanofiber membranes (GT:PCL = 30:70, 50:50, and 70:30) under the sandwich cell-seeding mode. Prior to subcutaneously implanting the samples into an autologous host, they were cultured in vitro over a period of 2 weeks. The results revealed that the nanofiber membranes with different GT/PCL ratios could support the process of subcutaneous cartilage regeneration in an autologous swine model. The maximum extent of homogeneity in the cartilage tissues was achieved when the G5P5 (GT: PC = 50: 50) group was used for the regeneration of cartilage. The formed homogeneous cartilage tissues were characterized by the maximum cartilage formation ratio. The extents of the ingrowth of the fibrous tissues realized and the extents of infiltration of inflammatory cells achieved were found to be the minimum in this case. Quantitative analyses were conducted to determine the wet weight, cartilage-specific extracellular matrix content, and Young’s modulus. The results indicated that the optimal extent of cartilage formation was observed in the G5P5 group. These results indicated that the GT/PCL nanofiber membranes could serve as a potential scaffold for supporting subcutaneous cartilage regeneration under clinical settings. An optimum GT/PCL ratio can promote cartilage formation.

Beneficial Therapeutic Approach of Acellular PLGA Implants Coupled With Rehabilitation Exercise for Osteochondral Repair: A Proof of Concept Study in a Minipig Model

2020 ◽  
Vol 48 (11) ◽  
pp. 2796-2807
Author(s):  
Chih-Chan Lin ◽  
Chih-Jou Chu ◽  
Pei-Hsi Chou ◽  
Chun-Hao Liang ◽  
Peir-In Liang ◽  
...  
Keyword(s):  
Treadmill Exercise ◽  
Total Duration ◽  
Cartilage Regeneration ◽  
Inflammatory Cells ◽  
Large Animal Model ◽  
Large Animal ◽  
Trochlear Groove ◽  
Plga Scaffold ◽  
Medial Condyle ◽  
Osteochondral Repair

Background: Osteochondral (OC) repair presents a significant challenge to clinicians. However, whether the use of acellular spongy poly(lactic-co-glycolic acid) (PLGA) scaffolding plus treadmill exercise as a rehabilitation program regenerates OC defects in a large-animal model has yet to be determined. Hypothesis: PLGA scaffolding plus treadmill exercise may offer improved OC repair for both high and low weightbearing regions in a minipig model. Study Design: Controlled laboratory study. Methods: A total of 9 mature minipigs (18 knees) were randomly divided into the treadmill exercise (TRE) group or sedentary (SED) group. All pigs received critically sized OC defects in a higher weightbearing region of the medial condyle and a lower weightbearing region of the trochlear groove. In each minipig, a PLGA scaffold was placed in the defect of the right knee (PLGA subgroup), and the defect of the left knee was untreated (empty defect [ED] subgroup). The TRE group performed exercises in 3 phases: warm-up, 3 km/h for 5 minutes; main exercise, 4 km/h for 20 minutes; and cool-down, 3 km/h for 5 minutes. The total duration was about 30 minutes whenever possible. The SED group was allowed free cage activity. Results: At 6 months, the TRE-PLGA group showed the highest gross morphology scores and regenerated a smooth articular surface covered with new hyaline-like tissue, while the defects of the other groups remained and contained nontransparent tissue. Histologically, the TRE-PLGA group also revealed sound OC integration, chondrocyte-like cells embedded in lacunae, abundant glycosaminoglycans, a sound collagen structure, and modest inflammatory cells with an inflammatory response (ie, tumor necrosis factor–α, interleukin-6). In addition, in the medial condyle region, the TRE-PLGA group (31.80 ± 3.03) had the highest total histological scores (TRE-ED: 20.20 ± 5.76; SED-PLGA: 10.25 ± 6.24; SED-ED: 11.75 ± 6.50; P = .004). In the trochlear groove region, the TRE-PLGA group (30.20 ± 6.42) displayed significantly higher total histological scores (TRE-ED: 19.60 ± 7.00; SED-PLGA: 10.00 ± 5.42; SED-ED: 11.25 ± 5.25; P = .006). In contrast, the SED-PLGA and SED-ED groups revealed an irregular surface with abrasion, fibrotic tissue with an empty void and inflammatory cells, disorganized collagen fibers, and less glycosaminoglycan deposition. Micro–computed tomography analysis revealed that the TRE-PLGA group had integrated OC interfaces with continued remodeling in the subchondral bone. Furthermore, comparing the 2 defect regions, no statistically significant differences in cartilage regeneration were detected, indicating the suitability of this regenerative approach for both high and low weightbearing regions. Conclusion: Implanting an acellular PLGA scaffold plus treadmill exercise promoted articular cartilage regeneration for both high and low weightbearing regions in minipigs. Clinical Relevance: This study suggests the use of a cell-free porous PLGA scaffold and treadmill exercise rehabilitation as an alternative therapeutic strategy for OC repair in a large-animal knee joint model. This combined effect may pave the way for biomaterials and exercise regimens in the application of OC repair.

scholarly journals A patient-like swine model of gastrointestinal fibrotic strictures for advancing therapeutics

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ling Li ◽  
Mohamad I. Itani ◽  
Kevan J. Salimian ◽  
Yue Li ◽  
Olaya Brewer Gutierrez ◽  
...  
Keyword(s):  
Argon Plasma Coagulation ◽  
Argon Plasma ◽  
Large Animal Model ◽  
Swine Model ◽  
Large Animal ◽  
High Grade ◽  
Gi Tract ◽  
Endoscopic Techniques ◽  
Approved Drugs

AbstractGastrointestinal (GI) strictures are difficult to treat in a variety of disease processes. Currently, there are no Food and Drug Administration (FDA) approved drugs for fibrosis in the GI tract. One of the limitations to developing anti-fibrotic drugs has been the lack of a reproducible, relatively inexpensive, large animal model of fibrosis-driven luminal stricture. This study aimed to evaluate the feasibility of creating a model of luminal GI tract strictures. Argon plasma coagulation (APC) was applied circumferentially in porcine esophagi in vivo. Follow-up endoscopy (EGD) was performed at day 14 after the APC procedure. We noted high grade, benign esophageal strictures (n = 8). All 8 strictures resembled luminal GI fibrotic strictures in humans. These strictures were characterized, and then successfully dilated. A repeat EGD was performed at day 28 after the APC procedure and found evidence of recurrent, high grade, fibrotic, strictures at all 8 locations in all pigs. Pigs were sacrificed and gross and histologic analyses performed. Histologic examination showed extensive fibrosis, with significant collagen deposition in the lamina propria and submucosa, as well as extensive inflammatory infiltrates within the strictures. In conclusion, we report a porcine model of luminal GI fibrotic stricture that has the potential to assist with developing novel anti-fibrotic therapies as well as endoscopic techniques to address recurring fibrotic strictures in humans.

scholarly journals Review: Tissue Engineering of Small-Diameter Vascular Grafts and Their In Vivo Evaluation in Large Animals and Humans

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 713
Author(s):  
Shu Fang ◽  
Ditte Gry Ellman ◽  
Ditte Caroline Andersen
Keyword(s):  
Animal Models ◽  
Vascular Grafts ◽  
Small Diameter ◽  
Large Animal ◽  
Large Animal Models ◽  
Graft Outcome ◽  
Limited Success ◽  
Large Animals

To date, a wide range of materials, from synthetic to natural or a mixture of these, has been explored, modified, and examined as small-diameter tissue-engineered vascular grafts (SD-TEVGs) for tissue regeneration either in vitro or in vivo. However, very limited success has been achieved due to mechanical failure, thrombogenicity or intimal hyperplasia, and improvements of the SD-TEVG design are thus required. Here, in vivo studies investigating novel and relative long (10 times of the inner diameter) SD-TEVGs in large animal models and humans are identified and discussed, with emphasis on graft outcome based on model- and graft-related conditions. Only a few types of synthetic polymer-based SD-TEVGs have been evaluated in large-animal models and reflect limited success. However, some polymers, such as polycaprolactone (PCL), show favorable biocompatibility and potential to be further modified and improved in the form of hybrid grafts. Natural polymer- and cell-secreted extracellular matrix (ECM)-based SD-TEVGs tested in large animals still fail due to a weak strength or thrombogenicity. Similarly, native ECM-based SD-TEVGs and in-vitro-developed hybrid SD-TEVGs that contain xenogeneic molecules or matrix seem related to a harmful graft outcome. In contrast, allogeneic native ECM-based SD-TEVGs, in-vitro-developed hybrid SD-TEVGs with allogeneic banked human cells or isolated autologous stem cells, and in-body tissue architecture (IBTA)-based SD-TEVGs seem to be promising for the future, since they are suitable in dimension, mechanical strength, biocompatibility, and availability.

scholarly journals Xenogenic Implantation of Equine Synovial Fluid-Derived Mesenchymal Stem Cells Leads to Articular Cartilage Regeneration

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Mohammed Zayed ◽  
Steven Newby ◽  
Nabil Misk ◽  
Robert Donnell ◽  
Madhu Dhar
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Articular Cartilage ◽  
Synovial Fluid ◽  
Cartilage Repair ◽  
Cartilage Regeneration ◽  
Large Animal

Horses are widely used as large animal preclinical models for cartilage repair studies, and hence, there is an interest in using equine synovial fluid-derived mesenchymal stem cells (SFMSCs) in research and clinical applications. Since, we have previously reported that similar to bone marrow-derived MSCs (BMMSCs), SFMSCs may also exhibit donor-to-donor variations in their stem cell properties; the current study was carried out as a proof-of-concept study, to compare the in vivo potential of equine BMMSCs and SFMSCs in articular cartilage repair. MSCs from these two sources were isolated from the same equine donor. In vitro analyses confirmed a significant increase in COMP expression in SFMSCs at day 14. The cells were then encapsulated in neutral agarose scaffold constructs and were implanted into two mm diameter full-thickness articular cartilage defect in trochlear grooves of the rat femur. MSCs were fluorescently labeled, and one week after treatment, the knee joints were evaluated for the presence of MSCs to the injured site and at 12 weeks were evaluated macroscopically, histologically, and then by immunofluorescence for healing of the defect. The macroscopic and histological evaluations showed better healing of the articular cartilage in the MSCs’ treated knee than in the control. Interestingly, SFMSC-treated knees showed a significantly higher Col II expression, suggesting the presence of hyaline cartilage in the healed defect. Data suggests that equine SFMSCs may be a viable option for treating osteochondral defects; however, their stem cell properties require prior testing before application.

scholarly journals Light-degradable hydrogels as dynamic triggers for gastrointestinal applications

2020 ◽  
Vol 6 (3) ◽  
pp. eaay0065 ◽  
Author(s):  
Ritu Raman ◽  
Tiffany Hua ◽  
Declan Gwynne ◽  
Joy Collins ◽  
Siddartha Tamang ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Implantable Devices ◽  
Large Animal Model ◽  
Large Animal ◽  
Gi Tract ◽  
Biomedical Device ◽  
Precise Tool ◽  
And Performance

Triggerable materials capable of being degraded by selective stimuli stand to transform our capacity to precisely control biomedical device activity and performance while reducing the need for invasive interventions. Here, we describe the development of a modular and tunable light-triggerable hydrogel system capable of interfacing with implantable devices. We apply these materials to two applications in the gastrointestinal (GI) tract: a bariatric balloon and an esophageal stent. We demonstrate biocompatibility and on-demand triggering of the material in vitro, ex vivo, and in vivo. Moreover, we characterize performance of the system in a porcine large animal model with an accompanying ingestible LED. Light-triggerable hydrogels have the potential to be applied broadly throughout the GI tract and other anatomic areas. By demonstrating the first use of light-degradable hydrogels in vivo, we provide biomedical engineers and clinicians with a previously unavailable, safe, dynamically deliverable, and precise tool to design dynamically actuated implantable devices.

1 GENERATION OF A STABLE TRANSGENIC SWINE MODEL FOR CELL TRACKING AND CHROMOSOME DYNAMIC STUDIES

2016 ◽  
Vol 28 (2) ◽  
pp. 130
Author(s):  
R. Sper ◽  
S. Simpson ◽  
X. Zhang ◽  
B. Collins ◽  
J. Piedrahita
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Animal Model ◽  
Regenerative Medicine ◽  
Cancer Biology ◽  
Large Animal Model ◽  
Large Animal ◽  
Fetal Fibroblasts ◽  
Transgene Transmission

Transgenic pigs are an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic, and physiological similarities with humans. The development of a transgenic murine model with a fusion of green fluorescent protein (GFP) to histone 2B protein (H2B, protein of nucleosome core) resulted in an easier and more convenient method for tracking cell migration and engraftment levels after transplantation as well as a way to better understand the complexity of molecular regulation within cell cycle/division, cancer biology, and chromosome dynamics. Up to now the development of a stable transgenic large animal model expressing H2B-GFP has not been described. Our objective was to develop the first transgenic porcine H2B-GFP model via CRISPR-CAS9 mediated recombination and somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were cotransfected with CRISPR-CAS9 designed to target the 3′ untranslated region of ACTB locus and a targeting vector containing 1Kb homology arms to ACTB flanking an IRES-H2B-GFP transgene. Four days after transfection GFP cells were fluorescence activated cell sorted. Single cell colonies were generated and analysed by PCR, and heterozygous colonies were used as donor cells for SCNT. The custom designed CRISPR-CAS9 knockin system demonstrated a 2.4% knockin efficiency. From positive cells, 119 SCNT embryos were generated and transferred to a recipient gilt resulting in three positive founder boars (P1 generation). Boars show normal fertility (pregnancies obtained via AI of wild type sows). Generated P1 clones were viable and fertile with a transgene transmission rate of 55.8% (in concordance with Mendel’s law upon chi-square test with P = 0.05). Intranuclear H2B-GFP expression was confirmed via fluorescence microscopy on 8-day in vitro cultured SCNT blastocysts and a variety of tissues (heart, kidney, brain, bladder, skeletal muscle, stomach, skin, and so on) and primary cultured cells (chondrocytes, bone marrow derived, adipocyte derived, neural stem cells, and so on) from P1 cloned boars and F1 42-day fetuses and viable piglets. In addition, chromosome segregation could be easily identified during cell cycle division in in vitro cultured stem cells. Custom designed CRISPR-CAS 9 are able to drive homologous recombination in the ACTB locus in porcine fetal fibroblasts, allowing the generation of the first described viable H2B-GFP porcine model via SCNT. Generated clones and F1 generation expressed H2B-GFP ubiquitously, and transgene transmission rates were with concordance of Mendel’s law. This novel large animal model represents an improved platform for regenerative medicine and chromosome dynamic and cancer biology studies.

361 GROWTH HORMONE RECEPTOR MUTANT PIGS PRODUCED BY USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR) AND CRISPR-ASSOCIATED SYSTEMS IN IN VITRO-PRODUCED ZYGOTES

2015 ◽  
Vol 27 (1) ◽  
pp. 269 ◽  
Author(s):  
M. Kurome ◽  
M. Dahlhoff ◽  
S. Bultmann ◽  
S. Krebs ◽  
H. Blum ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Hormone Receptor ◽  
Growth Hormone Receptor ◽  
Large Animal Model ◽  
Large Animal ◽  
Single Mutation ◽  
Wild Type ◽  
Ghr Gene ◽  
Normal Fertilization

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology is considered as an efficient strategy for generating gene edited large animals, such as pigs. Compared to somatic cell nuclear transfer, this new technology offers a relatively simple way to generate mutant pigs by direct injection of RNA into the cytoplasm of zygotes. Moreover, the use of in vitro produced zygotes would provide a highly effective and practical method for the production of porcine disease models for biomedical research. Here we examined the production efficiency of growth hormone receptor (GHR) mutant pigs by the combination of the CRISPR/Cas system and in vitro produced zygotes. In vitro maturation (IVM) of oocytes was performed as described previously (Kurome et al., Meth. Mol. Biol., in press). In all experiments, the same batch of frozen sperm was used. After IVM, around 20 oocytes with expanded cumulus cells were incubated with 5 × 104 spermatozoa in a 100-μL drop of porcine fertilization medium for 7 h. In vitro-produced embryos were assessed by the ratio of normal fertilization (eggs with 2 pronuclei) and blastocyst formation at Day 7. The Cas9 mRNA and a single guide RNA, recognising a short sequence of 20 base pairs in exon 3 of the GHR gene, were injected directly into the cytoplasm of the embryos 8.5 to 9.5 h after IVF. Injected embryos were transferred laparoscopically to recipient pigs, and 86.4% (57/66) of sperm-penetrated oocytes (66/96) exhibited normal fertilization. Incidence of polyspermy was relatively low (9/66, 13.6%). Developmental ability of in vitro-produced embryos to the blastocyst stage was 17.4% (24/138). In total, 426 RNA-injected embryos were transferred into 2 recipients, one of which became pregnant and gave birth to 8 piglets. All piglets were clinically healthy and developed normally. In 3 out of 8 piglets (37.5%), mutations were introduced. Next-generation sequencing revealed that all of them were mosaics: one with a single mutation (22% wild-type/78% mutant) and 2 piglets with 2 different mutations (80% wild-type/2% mutant_1/18% mutant_2 and 94% wild-type/4% mutant_1/2% mutant_2). Four out of 5 mutations caused a frameshift in the GHR gene. Our study reports for the first time generation of GHR mutant pigs by the use of the CRISPR/Cas system in in vitro-produced zygotes. Because all GHR mutant offspring were mosaic, Cas9 activation probably occurred after the 1-cell stage under our experimental conditions. The founder animal with the highest proportion of mutant GHR alleles will be used for breeding to establish a large animal model for Laron syndrome.This work is supported by the German Research Council (TR-CRC 127).

scholarly journals A systematic evaluation on reporting quality of modern studies on pulmonary heart valve implantation in large animals

2020 ◽  
Vol 31 (4) ◽  
pp. 437-445
Author(s):  
Marcelle Uiterwijk ◽  
Annemijn Vis ◽  
Iris de Brouwer ◽  
Debora van Urk ◽  
Jolanda Kluin
Keyword(s):  
Heart Valve ◽  
Heart Valves ◽  
Animal Experiments ◽  
Reporting Quality ◽  
Systematic Evaluation ◽  
Large Animal Model ◽  
Large Animal ◽  
Before And After ◽  
Large Animals

Abstract OBJECTIVES Before new heart valves can be implanted safely in humans, animal experiments have to be performed. These animal experiments have to be clearly designed, analysed and reported to assess the accuracy and importance of the findings. We aimed to provide an overview of the reporting and methodological quality of preclinical heart valve research. METHODS We conducted a systematic literature search on biological and mechanical pulmonary valve implantations in large animals. We used the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines to score the quality of reporting in each article. We compared the scores before and after the introduction of the ARRIVE guidelines (2010). RESULTS We screened 348 articles, of which 31 articles were included. The included articles reported a mean of 54.7% adequately scored ARRIVE items (95% confidence interval 52.2–57.3%). We did not identify a difference in reporting quality (54.7% vs 54.8%) between articles published before and after 2010. We found an unclear (lack of description) risk of selection bias, performance bias and detection bias. CONCLUSIONS The reporting quality of studies that implanted bioprosthetic or mechanical valves in the pulmonary position in the large animal model is not on the desired level. The introduction of the ARRIVE guidelines in 2010 did not improve the reporting quality in this field of research. Hereby, we want to emphasize the importance of clearly describing the methods and transparently reporting the results in animal experiments. This is of great importance for the safe translation of new heart valves to the clinic. Clinical trial registration number PROSPERO (CRD42019147895).

ZFN-Mediated Gene Targeting at the Albumin Locus in Liver Results in Therapeutic Levels of Human FIX in Mice and Non-Human Primates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 200-200 ◽  
Author(s):  
Thomas Wechsler ◽  
Kathleen E. Meyer ◽  
S. Kaye Spratt ◽  
Judith Greengard ◽  
Yolanda Santiago ◽  
...  
Keyword(s):  
Hemophilia B ◽  
Large Animal Model ◽  
Zinc Finger Nucleases ◽  
Clotting Factor ◽  
Large Animal ◽  
Spontaneous Bleeding ◽  
Virus Serotype ◽  
Donor Template

Abstract Hemophilia is an attractive target for gene therapy, since activity levels as low as 1% to 2% of normal are beneficial and levels of ~5% prevent spontaneous bleeding. Our goal was to provide a single treatment that permanently enables hepatic production of therapeutic levels of hFIX activity to decrease or potentially eliminate the need for prophylactic treatment in hemophilia B patients. We performed targeted in vivo genome editing using 1) two zinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus, and 2) a human F9 donor template construct. The ZFNs and donor template are encoded on separate hepatotropic adeno-associated virus serotype 2/6 (AAV2/6) vectors injected intravenously, resulting in targeted insertion of a corrected copy of the hF9 gene into the albumin locus in a proportion of liver hepatocytes. The albumin locus was selected as a "safe harbor" as production of this most abundant plasma protein exceeds 10 g/day, and moderate reductions in those levels are well-tolerated. These genome edited hepatocytes produce normal hFIX in therapeutic quantities, rather than albumin, driven by the highly active albumin enhancer/promoter, to treat hemophilia B; the genetic modification is expected to be sustained even in the face of hepatocyte turnover, making this approach attractive for treating young children with hemophilia before the appearance of significant organ damage. Transformed and primary human hepatocytes transduced in vitro with AAV2/6 encoding human albumin ZFNs and a promoterless hF9 transgene were shown to secrete hFIX. Extensive molecular analyses demonstrated that this was due to targeted integration of the hF9 transgene at the albumin locus and splicing of this gene into the albumin transcript. By employing AAV2/6 delivery of murine-specific ZFNs in vivo, stable levels of hFIX were observed in blood of mice injected with the albumin ZFNs and hF9 transgene donor. C57BL/6 mice were administered vehicle (n=20) or AAV2/6 vectors (n=25) encoding mouse surrogate reagents at 1.0 x1013 vector genome (vg)/kg via tail vein injection. ELISA analysis of plasma hFIX in the treated mice showed peak levels of 50-1053 ng/mL that were sustained for the duration of the 6-month study. Analysis of FIX activity from mouse plasma confirmed bioactivity commensurate with expression levels. Next, we report the feasibility of this approach in non-human primates (NHPs), showing that a single intravenous co-infusion of AAV2/6 vectors encoding the NHP targeted albumin-specific ZFNs and a human F9 donor at 1.2x1013 vg/kg (n=5/group) resulted in >50 ng/mL (>1% of normal) in this large animal model. The use of higher AAV2/6 doses (up to 1.5x1014 vg/kg) yielded plasma hFIX levels up to 1000 ng/ml (or 20% of normal) in several animals and up to 2000 ng/ml (or 50% of normal) in a single animal, for the duration of the study (3 months). The treatment was well tolerated in mice and NHPs, with no significant toxicological findings related to AAV2/6 ZFN + donor treatment in either species at therapeutic doses. Together, these data support a clinical trial to determine if a single co-administration of ZFN and donor AAV vectors is sufficient to enable therapeutic and potentially lifelong production of the clotting factor for the treatment of Hemophilia B. Disclosures Wechsler: Sangamo BioSciences: Employment. Meyer:Sangamo Biosciences Inc: Employment. Spratt:Sangamo Biosciences Inc: Employment. Greengard:Sangamo Biosciences Inc: Employment. Santiago:Sangamo Biosciences Inc: Employment. Sproul:Sangamo Biosciences Inc: Employment. Surosky:Sangamo Biosciences Inc: Employment. Paschon:Sangamo Biosciences Inc: Employment. Dubois-Stringfellow:Sangamo Biosciences Inc: Employment. Ando:Sangamo Biosciences Inc: Employment. Nichol:Sangamo Biosciences Inc: Employment. Rebar:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment.

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