title: Novel minicircle vector for gene therapy in murine myocardial infarction.
abstract: BACKGROUND
  Conventional plasmids for gene therapy produce low-level and short-term gene expression. In this study, we develop a novel nonviral vector that robustly and persistently expresses the hypoxia-inducible factor-1 alpha (HIF-1alpha) therapeutic gene in the heart, leading to functional benefits after myocardial infarction.
  
  
  RESULTS
  We first created minicircles (MC) carrying double-fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) for noninvasive measurement of transfection efficiency. Mouse C2C12 myoblasts and normal FVB/N mice were used for in vitro and in vivo confirmation, respectively. Bioluminescence imaging showed stable MC gene expression in the heart for >12 weeks and the activity level was 5.6+/-1.2-fold stronger than regular plasmid at day 4 (P<0.01). Next, we created MC carrying HIF-1alpha (MC-HIF-1alpha) therapeutic gene for treatment of myocardial infarction. Adult FVB/N mice underwent left anterior descending ligation and were injected intramyocardially with: (1) MC-HIF-1alpha; (2) regular plasmid carrying HIF-1alpha (PL-HIF-1alpha) as positive control; and (3) PBS as negative control (n=10/group). Echocardiographic study showed a significantly greater improvement of left ventricular ejection fraction in the MC group (51.3%+/-3.6%) compared to regular plasmid group (42.3%+/-4.1%) and saline group (30.5%+/-2.8%) at week 4 (P<0.05 for both). Histology demonstrated increased neoangiogenesis in both treatment groups. Finally, Western blot showed MC express >50% higher HIF-1alpha level than regular plasmid.
  
  
  CONCLUSIONS
  Taken together, this is the first study to our knowledge to demonstrate that MC can significantly improve transfection efficiency, duration of transgene expression, and cardiac contractility. Given the serious drawbacks associated with most viral vectors, we believe this novel nonviral vector can be of great value for cardiac gene therapy protocols.
authors.affiliation: Department of Radiology, Stanford University School of Medicine, CA 94305-5344, USA.
authors.forename: Mei
authors.initials: M
authors.lastname: Huang
authors.forename: ZhiYing
authors.initials: Z
authors.lastname: Chen
authors.forename: Shijun
authors.initials: S
authors.lastname: Hu
authors.forename: Fangjun
authors.initials: F
authors.lastname: Jia
authors.forename: Zongjin
authors.initials: Z
authors.lastname: Li
authors.forename: Grant
authors.initials: G
authors.lastname: Hoyt
authors.forename: Robert C
authors.initials: RC
authors.lastname: Robbins
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004278:DNA, Superhelical / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics*; D051379:Mice; D009203:Myocardial Infarction / Q000628:therapy*; D010957:Plasmids
chemical_list: D004278:DNA, Superhelical; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit