title: Hybrid retroviral vector with MCK enhancers inserted in LTR for stable and specific expression of human factor IX in skeletal muscle.
abstract: BACKGROUND
  Retroviral vectors have been widely used to introduce foreign into various target cells in vitro, thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo.
  
  
  METHODS
  FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK, Me2) were inserted in forward or reverse orientation at NheI site of 3' long terminal repeat (LTR), resulting in two hybrid vectors, which were designated as LMe2IXm2SN(F) and LMe2IXm2SN(R), respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice.
  
  
  RESULTS
  Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24 h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2.
  
  
  CONCLUSIONS
  LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo, and MCK enhancer should be positioned in the same orientation as that of LTR promoter.
authors.affiliation: Institute of Genetics, Fudan University, Shanghai 200433, China.
authors.forename: Jian-min
authors.initials: JM
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Hou
authors.forename: Xin-fang
authors.initials: XF
authors.lastname: Qiu
authors.forename: Kotoku
authors.initials: K
authors.lastname: Kurachi
authors.forename: Jing-lun
authors.initials: JL
authors.lastname: Xue
mesh_terms: D000818:Animals; D003402:Creatine Kinase / Q000235:genetics*; D052283:Creatine Kinase, MM Form; D004742:Enhancer Elements, Genetic; D005164:Factor IX / Q000032:analysis / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology*; D005821:Genetic Techniques; D005822:Genetic Vectors; D006824:Hybridization, Genetic; D007527:Isoenzymes / Q000235:genetics*; D051379:Mice; D016513:Mice, SCID; D012190:Retroviridae / Q000235:genetics*; D020079:Terminal Repeat Sequences
chemical_list: D007527:Isoenzymes; D005164:Factor IX; D003402:Creatine Kinase; D052283:Creatine Kinase, MM Form