Gene Therapy (1997) 4, 465–472  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer N Larochelle 1 , H Lochmu ¨ ller 1,2 , J Zhao 1 , A Jani 1 , P Hallauer 1 , KEM Hastings 1 , B Massie 3 , S Prescott 1 , BJ Petrof 4 , G Karpati 1 and J Nalbantoglu 1 1 Neuromuscular Research Group, Montreal Neurological Institute; 3 Biotechnology Research Institute, National Council of Canada, Montreal, Quebec; and 4 Respiratory Division, Royal Victoria Hospital, and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada Replication-defective (E1+ E3-deleted) human adenovirus muscle-specific transgene expression was demonstrated vectors are a promising means of therapeutic gene delivery in immunodeficient mice after local injection of AV into to skeletal muscle cells. Since the tropism of adenovirus muscles at an early age. In nonmuscle tissues (brain, liver, is nonselective, muscle-specific expression of systemically kidney, lung), the transgene expression was extremely low administered vectors can only be achieved by the use of even though in these tissues in situ polymerase chain reac- a tissue-specific promoter/enhancer that is small enough tion showed as high an infectivity of the cells by the AV as to fit the insert capacity of the vector. We have generated in muscle. The relatively small size, the good efficiency and two replication-defective adenovirus recombinants (AV) in the muscle specificity of the MCK promoter would make it which the reporter gene (either firefly luciferase or E. coli - ideal to drive the 6.3 kb (truncated) dystrophin cDNA in galactosidase) was driven by a truncated (1.35 kb) muscle first generation AV (with a limited (8 kb) insert capacity) creatine kinase (MCK) promoter/enhancer or by the fast designed for gene therapy of Duchenne muscular troponin I (TnI) promoter/enhancer. Highly efficient and dystrophy. Keywords: muscle creatine kinase; promoter; adenovirus; gene therapy; muscle-specific expression adenoviral vector must be used which contains the Introduction therapeutic protein-coding sequence driven by Gene therapy for genetic diseases consists of introducing promoter/enhancer elements that support adequate a normal gene into target cells to provide its normal pro- expression levels in the target tissue. 1 tein product and correct the deleterious consequences of One area of focus in gene therapy research has been the genetic mutation (reviewed by Karpati and Acsadi). 1 dystrophin gene replacement for dystrophin deficiency In gene therapy regimens, tissue-specific expression of states. Replication-defective adenoviral vectors have been the transgene is highly desirable as it would minimize shown to be an effective means of introducing dystrophin possible toxicity and immunogenicity in nontarget cells. cDNA into skeletal muscle fibers of dystrophin-deficient In principle, this can be achieved either by limiting vector mdx mice by direct injection or a systemic route of admin- delivery to the cell type of interest, or by engineering istration. 1 Since adenovirus readily infects many cell tissue-specific gene regulatory elements into nonselective types, selective or preferential expression of dystrophin vectors to drive transgene expression. in skeletal muscle (after the systemic route of A variety of vectors for the delivery of therapeutic administration) is not possible. Therefore, the use of genes into target cells have been considered. Particularly muscle-specific promoter/enhancer is the method of promising is the human adenovirus type 5 (reviewed by choice to assure muscle-specific expression of dystrophin Kozarsky and Wilson). 2 The adenoviral genome is well using adenoviral vectors. characterized, large portions of viral DNAs can be substi- Many muscle-specific genes have been cloned and their tuted by other sequences, the recombinants are relatively regulatory sequences are being mapped and charac- stable, the recombinant virus can be grown to high titer terized. These include -skeletal actin, 3–5 -cardiac and no human malignancy is associated with this virus. 2 actin, 6–8 troponin I, 9 myosin light chain 2, 10 myosin heavy For gene therapy applications, a replication-deficient chain 11 and muscle creatine kinase (MCK). 15–17 Both muscle creatine kinase and troponin I have high expression levels in muscle and their promoter/enhancer Correspondence: J Nalbantoglu, Montreal Neurological Institute, 3801 regions are small enough to make them suitable for University Street, Montreal, Quebec, H3A 2B4, Canada inclusion in first generation adenoviral vectors. 2 Present address: Genzentrum, Institut fu ¨ r Biochemie, Ludwig-Maximili- High-level expression of MCK is restricted to differen- ans Universitat, Munich, Germany Received 26 September 1996; accepted 13 January 1997 tiated skeletal and cardiac muscle. 12 In undifferentiated