Genes & CellsGenes & Cells2313-18292500-2562Human Stem Cells Institute12156710.23868/gc121567Formation of the recombinant adenovirus encoding codon-optimized dysferlin gene and analysisof the recombinant protein expression in cell culture in vitroStarostinaI GKazan (Volga region) federal university, Kazan-SolovyevaV VKazan (Volga region) federal university, Kazan-ShevchenkoK GHuman Stem Cells Institute, Moscow-DeevR VHuman Stem Cells Institute, Moscow-IsaevA AHuman Stem Cells Institute, Moscow-RizvanovA AKazan (Volga region) federal university, Kazan-Kazan (Volga region) federal university, KazanHuman Stem Cells Institute, Moscow1509201273252811012023Copyright © 2012, Eco-Vector2012Dysferlinopathies belong to neuromuscular diseases
associated with aberrant expression and/or function of
dysferlin protein in skeletal muscle, which is caused by
mutations in the dysf (dystrophy-associated fer-1-like, DYSF)
gene. Because of the large size of the codon-optimized dysf
coding region (6243 bp), adenoviral vectors are suitable for
the creation of genetic constructs, which are capable of
delivering a large amount of recombinant genetic information
into both dividing and non-dividing cells, as well as provide a
high level of transgene expression.
We generated a recombinant adenovirus serotype
5 encoding a codon-optimized gene for human dysferlin (Ad5-
Dysf) and analysed recombinant protein expression in vitro in
HEK-293T cell line.Recombinant adenovirusdysferlincodon optimizationgene therapyрекомбинантный аденовирусдисферлинкодонная оптимизациягенная терапия[Bushby K.M. Making sense of the limb-girdle muscular dystrophies. Brain 1999; 122: 1403-20.][Liu J., Aoki M., Illa I. et al. Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat. Genet. 1998; 20(1): 31-6.][Bashir R., Strachan T., Keers S. et al., A gene for autosomal recessive limb-girdle muscular dystrophy maps to chromosome 2p. Hum. Mol. Genet. 1994; 3(3): 455-7.][Therrien C., Dodig D., Karpati G. et al. Mutation impact on dysferlin inferred from database analysis and computer-based structural predictions. J. Neurol. Sci. 2006; 250(1-2): 71-8.][Fujita E., Kouroku Y., Isoai A. et al., Two endoplasmic reticulumassociated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/ lysosome ERAD(II). Hum. Mol. Genet. 2007; 16(6): 618-29.][Davis D.B., Doherty K.R., Delmonte A.J. et al. Calcium-sensitive phospholipid binding properties of normal and mutant ferlin C2 domains.][J. Biol. Chem. 2002; 277(25): 22883-8.][Bansal D., Miyake K., Vogel S.S. et al., Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 2003; 423(6936): 168-72.][Lennon N.J., Kho A., Bacskai B.J. et al., Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing. J. Biol. Chem. 2003; 278(50): 50466-73.][Bashir R., Britton S., Strachan T. et al. A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limbgirdle muscular dystrophy type 2B. Nat. Genet. 1998; 20(1): 37-42.][Aoki M., Liu J., Richard I. et al. Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy. Neurology 2001; 57(2): 271-8.][Guglieri M., Magri F., D'Angelo M.G. et al. Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients. Hum. Mutat. 2008; 29(2): 258-66.][Vetrini F., Ng P. Gene therapy with helper-dependent adenoviral vectors: current advances and future perspectives. Viruses 2010; 2(9): 1886-917.][Dudley R.W., Lu Y., Gilbert R. et al. Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector. Hum. Gene Ther. 2004; 15(2): 145-56.][Jiang Z., Feingold E., Kochanek S. et al. Systemic delivery of a high-capacity adenoviral vector expressing mouse CTLA4Ig improves skeletal muscle gene therapy. Mol. Ther. 2002; 6(3): 369-76.][Roth J.A., Cristiano R.J. Gene therapy for cancer: what have we done and where are we going? J. Natl. Cancer Inst. 1997; 89(1): 21-39.][Cohen S.N., Chang A.C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. PNAS USA 1972; 69(8): 2110-4.][Sambrook J., Russell D.W. Molecular cloning: a laboratory manual. 3 ed. Cold Spring Harbor Laboratory Press; 2001.][Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-5.][Graham F.L., Smiley J., Russell W.C. et al. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 1977; 36(1): 59-74.][Harrison T., Graham F., Williams J. Host-range mutants of adenovirus type 5 defective for growth in HeLa cells. Virology 1977; 77(1): 319-29.]