Transgene Expression In Heart Research Articles

Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin, FGF2, and SERCA2a into Ischemic Heart.

Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.

Evaluation of Vascular Delivery Methodologies to Enhance rAAV6-mediated Gene Transfer to Canine Striated Musculature

A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector "dwell" time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans.

Downstream utrophin enhancer is required for expression of utrophin in skeletal muscle

Duchenne muscular dystrophy is caused by the absence of the muscle cytoskeletal protein dystrophin. Utrophin is an autosomal homologue of dystrophin, and overexpression of utrophin is expected to compensate for the dystrophin deficit. We previously reported that the 5.4-kb 5'-flanking region of the utrophin gene containing the A-utrophin core promoter did not drive transgene expression in heart and skeletal muscle. To clarify the regulatory mechanism of utrophin expression, we generated a nuclear localization signal-tagged LacZ transgenic (Tg) mouse, in which the LacZ gene was driven by the 129-bp downstream utrophin enhancer (DUE) and the 5.4-kb 5'-flanking region of the utrophin promoter. Two Tg lines were established. The levels of transgene mRNA expression in several tissues were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Cryosections of several tissues were stained with haematoxylin and eosin and X-gal. The transgene expression patterns were consistent with endogenous utrophin in several tissues including heart and skeletal muscle. Transgene expression was also up-regulated more in regenerating muscle than in nonregenerating muscle. Moreover, utrophin expression was augmented in the skeletal muscle of DUE Tg/dystrophin-deficient mdx mice through cross-breeding experiments. We finally established cultures of primary myogenic cells from this Tg mouse and found that utrophin up-regulation during muscle differentiation depends on the DUE motif. Our results showed that DUE is indispensable for utrophin expression in skeletal muscle and heart, and primary myogenic cells from this Tg mice provide a high through-put screening system for drugs that up-regulate utrophin expression.

Cardiac-specific and ligand-inducible target gene expression in transgenic mice

Conditional transgene expression in the heart is a useful approach to explore the physiological basis of the cardiac phenotype. The present study describes the development of a binary transgenic system in which transgene expression in the mouse heart can be turned on/off by administration/withdrawal of an exogenous compound. We generated a transgenic line (αMHC-Glp 65) harboring a mifepristone (RU486)-controlled chimeric transcription factor (Glp 65) under the regulatory control of the cardiac-specific α-myosin heavy chain (αMHC) promoter. In the presence of RU486, Glp 65 expressed in the heart is able to bind to a target gene promoter containing four copies of the 17-mer GAL4 binding site, resulting in ligand-inducible transactivation of the target gene. We tested this system by crossing the transgenic mice, αMHC-Glp 65, with a transgenic line harboring human growth hormone (hGH) target gene. We observed that expression of hGH could be induced in adults as well as in the embryonic hearts of bigenic mice by RU486. The basal hGH expression was very low and the inducible level in the heart was estimated over 800-fold higher versus the basal level after 4 days of administration of RU486 at 500 μg/kg body weight per day at 2–5 months of age. The level of the transgene returned to the basal level within 7 days after withdrawal of RU486. This system can be used to control cardiac-specific expression of transgene in a time- and dose-dependent manner.

Direct comparison of efficiency and stability of gene transfer into the mammalian heart using adeno-associated virus versus adenovirus vectors

ObjectiveRecent gene therapy strategies have relied on the use of adenovirus or plasmid as vehicles for gene delivery to the heart. These approaches have been limited by low transduction frequencies and transient transgene expression. We sought to determine whether adeno-associated virus produces more stable, higher efficiency gene expression in the rodent heart than did previous conventional methods. MethodsTwo recombinant viral constructs were made: an adeno-associated virus containing the lacZ gene under the control of the cytomegalovirus promoter (AAV-lacZ) and an adenovirus expressing lacZ under the control of the same promoter (Adeno-lacZ). Twenty rats were injected (into the ventricular apex) with 1 × 107-8 genomic particles of each virus. Animals were put to death at serial time points and transgene expression quantitated by β-galactosidase activity, myocardial staining, and Western blot protein analysis. ResultsThree months after adeno-associated virus gene transfer, animals demonstrated stable β-galactosidase expression in 60% of cardiomyocytes without evidence of myocardial inflammation/necrosis. The distribution and degree of protein expression and number of positive cells at 3 months were equivalent to transgene expression at 4 weeks. Adeno-associated virus was not detected in organs other than the heart. In contrast, Adeno-lacZ animals displayed transient β-galactosidase activity in 60% of cardiomyocytes, which was undetectable 4 weeks after gene transfer. Adenovirus-treated animals manifest significant myocardial inflammation and had transgene expression in other organs. ConclusionDirect intramyocardial injection of an adeno-associated virus vector programs stable, long-term, cardiac-specific transgene expression in the rodent heart for up to 3 months. Our results suggest adeno-associated virus has significant advantages for long-term transgene expression in the heart compared to adenovirus vectors.