caa a22 4500 397574029 CHVBK 20180308164849.0 cr unu---uuuuu 161202e199607 xx s 000 0 eng 10.1093/hmg/5.7.913 doi (NATIONALLICENCE)oxford-10.1093/hmg/5.7.913 Encapsidated Adenovirus Minichromosomes Allow Delivery and Expression of a 14 kb Dystrophin cDNA to Muscle Cells [Elektronische Daten] [Rajendra Kumar-Singh, Jeffrey S. Chamberlain] Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of ∼8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (∼14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus ß packaging signals, a β-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1+E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (>108/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues. © 1996 Oxford University Press Kumar-Singh Rajendra Department of Human Genetics, University of Michigan Medical School, 1150 West Medical Center Drive, Med Sci II, Rm 3726, Ann Arbor, MI 48109-0618, USA aut Chamberlain Jeffrey S. Department of Human Genetics, University of Michigan Medical School, 1150 West Medical Center Drive, Med Sci II, Rm 3726, Ann Arbor, MI 48109-0618, USA aut Human Molecular Genetics Oxford University Press 5/7(1996-07), 913-921 0964-6906 5:7<913 1996 5 hmg https://doi.org/10.1093/hmg/5.7.913 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/hmg/5.7.913 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kumar-Singh Rajendra Department of Human Genetics, University of Michigan Medical School, 1150 West Medical Center Drive, Med Sci II, Rm 3726, Ann Arbor, MI 48109-0618, USA aut NATIONALLICENCE 700 1- Chamberlain Jeffrey S. Department of Human Genetics, University of Michigan Medical School, 1150 West Medical Center Drive, Med Sci II, Rm 3726, Ann Arbor, MI 48109-0618, USA aut NATIONALLICENCE 773 0- Human Molecular Genetics Oxford University Press 5/7(1996-07), 913-921 0964-6906 5:7<913 1996 5 hmg Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford