Our earlier work includes local gene delivery system, nontoxic water soluble lipopolymer (WSLP), poly(ethylenimine)-co-[N-(2-aminoethyl)ethyleneimine]-co-N-(N-cholesteryloxycarbongl-(2- animoethyl)-ethylenimine) was synthesized. Branched polyethylenimine (PEI MW 1800) and cholesteryl chloroformate were used. We have developed a hypoxia-inducible plasmid construct expressing VEGF, pRTP801-VEGF, to treat myocardial ischemia and infarction. Rabbits underwent ligation of the proximal circumflex coronary artery and were immediately injected with an ischemiainducible VEGF gene (RTP801-VEGF) or a constitutively expressed VEGF gene (SV-VEGF); or no injection therapy using this WSLP. Four weeks following treatment, ligation alone resulted in an infarction of 48 7% of the left ventricle. The constitutively expressed gene constrict, SV-VEGF, reduced infarct size to 32 ± 7%, while the ischemia-inducible gene construct, RTP801-VEGF, reduced infarct size to 13 ± 4%. RTP801-VEGF was associated with decreased in apoptosis and an increase in capillary growth compared to the SV-VEGF construct. The disulfide-linked bioreducible polymer poly(cystaminebisacrylamide-diaminohexane) [CBA-DAH] was synthesized via Michael addition reaction. Primary rat skeletal myoblasts were transfected with poly(CBA-DAH)/pCMV-VEGF165 at the ratio 20:1 (w/w) in serum free media 24 hours prior to implantation. Rats were divided into four treatment groups: 1) thoracotomy sham operation control, 2) ligation only control, 3) ligation and implantation of skeletal myoblasts only, and 4) ligation and implantation of skeletal myoblasts transfected with a VEGF165 plasmid. MRI analysis of the treatment groups reveled a significant recovery of ejection fraction in the VEGF myoblast treatment (61.7%) over myoblasts only (48.0%) and ligation control (48.2%). Not only did the VEGF myoblast treatment improve ejection fraction over myoblasts only treatment and ligation control, but it was not significantly lower than the thoracotomy control value of 67.6%. Apoptotic cell population reveled a significant attenuation of apoptosis in the myoblast only group (21.6%) but a higher attenuation in the VEGF myoblast group (11.9%) compared to ligation controls (27.5%). This indicated that while myoblast implantation alone limits apoptosis in the myocardium, the VEGF myoblast group is producing a significantly higher protective effect. Infarct length (as % LV) was significantly shorter in the VEGF myoblast group (5.4%) compared to ligation control (32.8%) (P<0.05). The myoblast only group produced an average infarct of 14.9%. This work demonstrates that bioreducible polymers can successfully be used to transfect skeletal myoblasts with angiogenic factors. Interestingly, while myoblasts only treatment significantly limited apoptosis in the myocardium, this increase in cell survival did not translate to increased cardiac efficiency as measured by ejection fraction. These results suggest the potential of genetically modified skeletal myoblasts in improving current stem cell therapies and should be further investigated. A new candidate siRNA, SHP1 siRNA was evaluated to present apoptosis of cardiomyocyte. The SRC homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) has been know as a key molecule in apoptosis and a blocker in phosphorylation of Akt. Akt activation could lead cardioprotective effect after ischemia, and cardiomyocyte apoptosis is thought to contribute to the acute myocardial infarction (AMI). Thus, siRNA targeting of SHP-1 gene would be beneficial in the cardiac disease, such as AMI. We studied and used Fas siRNA for treating MI, so we compared SHP-1 siRNA with Fas siRNA as a therapeutic gene to present apoptosis. Three SHP-1 siRNA candidates using ABP (arginine-grafted bioreducible polymer) to select a high-potential siRNA in the rat SHP-1 gene silencing were studied. Of synthesized three siRNAs, SHP-1 siRNA no.2 (sense 5’- GGACAUUUCUUGUGCGUGA-3’) potently suppressed the expression of SHP-1 in H9C2 cells. The real-time PCR result revealed that SHP-1 siRNA no.2 inhibited SHP-1 mRNA production by about 55% (50nM) and 70% (100 nM) compared to the untreated control. On the basis of this result, we selected and used SHP-1 siRNA no.2 for further studies to develop the cardiomyocyte targeted SHP-1 delivery system. To deliver siRNA specifically to cardiomyocytes with a high transfection efficiency, primary cardiomyocytetargeting (PCM) and/or cell-penetrating (Tat) peptides were incorporated into the siRNA. With the addition of plasmid DNA, these peptide-conjugated siRNAs were able to form compact and stable nanosized polyplex particles with bioreducible poly(CBA-DAH). The peptide- modified siRNA polyplexes enhanced the cellular uptake and the gene-silencing capacity of the siRNA in cardiomyocytes without significant immunogenicity or cytotoxicity. These findings demonstrate that the cell-targeting peptide and/or cell-penetrating peptide conjugation of siRNA may be a potentially important strategy for cell-specific gene therapy in gene-mediated disease states. The novel bioreducible polymer was used to transfect rat hearts in vivo immediately after transient myocardial infarction. siRNA towards BNIP3, a potent hypoxia-inducible pro-apoptoic Bcl-2 family member, was delivered directly to the infarcted myocardium of Sprague Dawley rats. siBNIP3 therapy in this model protected rats from loss of heart function as evidenced by global left ventricular ejection fraction and stroke volume. Angiogenesis-based gene therapy has emerged as a promising treatment for myocardial ischemia due to its direct effects on cardiomyocytes. Despite their usefulness, the gene expression needs to be regulated because non-specific expression of the gene may cause severe side effects. In this study, a VEGF plasmid was designed and constructed for post-translational regulation that stabilizes VEGF and facilitates VEGF secretion under hypoxia. In addition, the furin recognition site was included to lead to the secretion of wild-type VEGF. All results reveal that the new VEGF plasmid (b -SP- ODD-VEGF) is useful for hypoxia-specific gene therapy for myocardial ischemia.
Research Interests :
Drug Delivery, Self-Regulating Drug-Delivery, Medical Polymer, Design of Novel Polymer
Awards and Honors :
- Annual Award of the ISBP (1994)
- Controlled Release Society (CRS) Founders Award (1995)
- AAPS Research Achievement Award in Drug Delivery (1995)
- Japanese Biomaterials Research Award (1996)
- American Association of Pharmaceutical Scientists (AAPS) Dale Wurster Award (1998)
- AACP Volwiler Award (2002)
- Rosenblatt Prize (2003)
- elected to two U.S. national academies: the Institute of Medicine (1999) and the National Academy of Engineering (2003)
- Honorary doctorate degree from the University of Twente. (2006)
- Founder and former Co-Chairman for the International Symposium on Recent Advances in Drug Delivery
- Editorial Board of the Journal of Controlled Release, Pharmaceutical Research, Journal of Biomedical Materials Science, and Biomaterials Science journals.
- Fellow in the AAPS, Biomaterials Society, and the AIMBE societies.