Novel Minicircle Vector for Gene Therapy in Murine Myocardial Infarction

Department of Radiology, Stanford University School of Medicine, CA 94305-5344, USA.
Circulation (Impact Factor: 14.43). 09/2009; 120(11 Suppl):S230-7. DOI: 10.1161/CIRCULATIONAHA.108.841155
Source: PubMed


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.
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.
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.

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    • "Recently, several groups have used minicircles as tools for transgenic expression in vivo based on these advantages. Some of them delivered minicircles encoding natural proteins, such as the hypoxia-inducible factor-1 alpha (HIF-1α), alpha-l-iduronidase (IDUA), and interferon gamma (IFNγ), into animals to examine the efficiency of minicircles for use in gene therapy91011. Minicircles encoding shRNAs or microRNAs have also been reported as good tools for gene therapy1213. "
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    • "Minicircle (MC) DNA vectors are small, supercoiled molecules devoid of plasmid bacterial sequences. They are believed to be superior to standard plasmid DNA as gene delivery vehicles due to observations of more persistent and increased transgene expression both in vitro and in vivo.36,37,38,39,40,41,42 MC DNA molecules can be obtained through a phage integrase ΦC31-mediated site-specific recombination between attB and attP sites, leading to excision of the intervening bacterial backbone sequence, which is then digested by the I-SceI endonuclease and degraded. "
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