405. Enhanced Chondrogenic Potential of Human Bone Marrow Aspirates Upon rAAV-Mediated Overexpression of IGF-I

Musculo-Skeletal Diseases I miR142-3p, a hematopoietic specific microRNA target sequence. Compared with the original AAV microgene vector, the newly engineered vector significantly reduced infiltration of CD4+ and CD8+ T cells in the canine DMD model. Our results suggest that the dual restriction strategy may greatly enhance clinical translation of AAV micro-dystrophin gene therapy.

404. Development of Human Artificial Chromosomes for Gene Cell Therapy of Muscular Dystrophies

Artur A. Isaev,1 Ilya I. Eremin,2 Andrey A. Pulin,2 Roman V. Deev,1,2 Alexey N. Tomilin,3 Michail A. Liskovykh,3 Vladimir Larionov,4 Natalia Kouprina,4 Kseniya Iur’eva,1 Vadim L. Zorin,1,2 Ilya Ya Bozo,1,2 Konstantin V. Kotenko.2 1 Human Stem Cells Institute OJSC, Moscow, Russian Federation; 2 State Research Center - Burnasyan Federal Medical Biophysical Center FMBA of Russia, Moscow, Russian Federation; 3Institute of Cytology of Russian Academy of Sciences, Saint Petersburg, Russian Federation; 4National Cancer Institute NIH, Bethesda. Muscular dystrophies are clinically heterogeneous group of muscle diseases related to different genes defects. The results of defects impair myogenic differentiation and histophysiology of skeletal muscles. One of major forms of disease - limb-girdle muscular dystrophy 2B (LGMD2B), caused by dysferlin gene mutations. Both big size of gene and wide variety of mutations complicate development of genetically engineered drugs. Human artificial chromosomes (HAC) could be used as alternative approach. This vector can contain unlimitedly large DNA region for transfer, is retained during cell division, and does not have carcinogenic and immunogenic properties. HAC matrix was developed earlier by Larionov V. et al. (http:// www.ncbi.nlm.nih.gov/pubmed/22123967). Optimized DYSF gene sequence (optDYSF) from pAd/CMV/V5-DEST vector (kindly provided by A.A. Rizvanov) was transferred into backbone vector also containing loxP sites for further transfer into HAC. Transfer correctness confirmed by sequencing. After that Chinese hamster ovary cells carrying HAC were transfected by backbone vector containing optDYSF. Selection performed in HAT medium. 7 clones were selected and analyzed at day 10. Expression of optDYSF observed in all obtained clones. Episomal HAC localization determined using fluorescent hybridization in sity by colocalization of label at optDYSF and a-satellite DNA. Episomal localization of HAC determined only in 2 clones from 7. Dysferlin production observed in all clones. On other hand described vector requires somatic myogenic vector cell. Human myoblasts could be successfully used for this purpose. The major source of myoblasts - skeletal muscle tissue is not optimal due to limited proliferative potential, especially in patients with muscular dystrophies. Our team revealed presence of myoblasts in gingival mucosa specimens and developed new method of myoblasts obtainment from it. Primary cultures of multipotent mesenchymal stromal cells (MSC) were derived from gingiva biopsy samples. The cells expressed typical mesenchymal stem cells markers and had ability for 3-way differentiation. Myogenic induction of gingiva derived MSC in culture was demonstrated. Gingiva derived MSC are capable to express markers of myogenic differentiation (skeletal actin, sceletal myosin, MyoD1) and form multicellular elongated fibers myotubes. Ability of gingiva derived MSC cultures to differentiate in myogenic direction was preserved during up to 10th passage. Such features make this cell source attractive for using in treatment of inherited muscular degenerative diseases. In our future research we are planning to transfer HAC containing optDYSF gene into clonogenic gingiva derived MSC using microcellmediated chromosome transfer. This method could be used for further development of gene cell therapy of LGMD2B. S160

405. Enhanced Chondrogenic Potential of Human Bone Marrow Aspirates Upon rAAVMediated Overexpression of IGF-I

Janina Frisch,1 Ana Rey-Rico,1 Jagadeesh K. Venkatesan,1 Henning Madry,1 Magali Cucchiarini.1 1 Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany. Genetic modification of human bone marrow aspirates is an attractive strategy as a convenient, one-step treatment of articular cartilage defects. Here, we tested whether overexpression of the mitogenic and pro-anabolic IGF-I factor via rAAV vectors enhances the reparative activities of human bone marrow aspirates. Aspirates obtained from the distal femurs of patients undergoing total knee arthroplasty were directly transduced with an rAAV-hIGF-I vector vs. control rAAV-lacZ transduction and maintained in chondrogenic medium for up to 21 days. Transgene expression was monitored by immunohistochemistry on paraffin-embedded sections of aspirates also stained with toluidine blue and for type-II collagen immunoreactivity. The DNA and proteoglycan contents were monitored by Hoechst 22358 assay and binding to DMMB, respectively. Real-time RT-PCR was performed using the 2-∆∆Ct method to monitor the expression of SOX9, ACAN, COL2A1, COL1A1, and COL10A1, with GAPDH as a control for normalization, by measuring the fold inductions in IGF-I vs. lacZ conditions. Each condition was performed in duplicate in three experiments. The t-test was employed with P ≤ 0.05 considered statistically significant. Effective production of IGF-I was achieved in IGF-I- vs. lacZ-treated aspirates. Enhanced chondrogenic differentiation was observed after 21 days in the aspirates with rAAVhIGF-I, as noted by more robust toluidine blue staining and stronger type-II collagen immunostaining compared with rAAV-lacZ. IGF-I treatment also increased the DNA and proteoglycan contents in the aspirates vs. lacZ. These findings were corroborated by real-time RTPCR analysis showing higher levels of SOX9, ACAN, and COL2A1 expression with IGF-I that also increased the levels of COL1A1 and COL10A1. Overexpression of IGF-I via rAAV is thus capable of stimulating the metabolic and chondrogenic activities in human bone marrow aspirates. Regulation of growth factor expression will be necessary to contain terminal differentiation and hypertrophy for translation of the approach in cartilage defects.

406. The MDX/UTR +/- Mouse Is a Superior Mouse Model for Duchenne Muscular Dystrophy

Thais B. Lessa,1 Chady H. Hakim,2 Carlos E. Ambrósio,3 Dongsheng Duan.2 1 Department of Surgery, University of São Paulo, São Paulo, Brazil; 2Department of Molecular Microbiology and Immunology, University of Missouri, Columbia; 3Department of Veterinary Medicine, University of São Paulo, Pirassununga, Brazil. Duchenne Muscular Dystrophy (DMD) is a severe muscle wasting disease caused by the absence of the dystrophin protein. Dystrophin, the core component of the dystrophin-associated glycoprotein complex, is responsible in stabilizing the sarcolemma during muscle contraction and relaxation. In the absence of dystrophin, muscle becomes fragile and susceptible to damage. This results in progressive muscle degeneration and replacement with fibrotic tissue. The mdx mouse is the most commonly used animal model for DMD. However, it is also considered a poor model because it cannot reproduce the severe dystrophic phenotype seen in patients. Genetic elimination of utrophin, an autosomal homolog of dystrophin, yields a symptomatic double knockout (dko) model. Unfortunately, human patients do not have utrophin deficiency. An utrophin heterozygous mdx (mdx/ utrn+/-) mouse would theoretically be a better model should it show more severe muscle dysfunction than the mdx mouse. To test this Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy