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1.  Gene transfer of arginine kinase to skeletal muscle using adeno-associated virus 
Gene therapy  2014;21(4):387-392.
In this study we tested the feasibility of non-invasively measuring phosphoarginine (PArg) after gene delivery of arginine kinase (AK) using an adeno-associated virus (AAV) to murine hindlimbs. This was achieved by evaluating the time course, regional distribution, and metabolic flux of PArg using 31 phosphorus magnetic resonance spectroscopy (31P-MRS). AK gene was injected into the gastrocnemius of the left hindlimb of C57Bl10 mice (age 5wk, male) using self-complementary AAV, type 2/8 with desmin promoter. Non-localized 31P-MRS data were acquired over nine months after injection using 11.1-T and 17.6-T Bruker Avance spectrometers. In addition, 31P 2-D chemical shift imaging and saturation transfer experiments were performed to examine the spatial distribution and metabolic flux of PArg, respectively. PArg was evident in each injected mouse hindlimb after gene delivery, increased until 28 weeks, and remained elevated for at least nine months (p<.05). Furthermore, PArg was primarily localized to the injected posterior hindimb region with the metabolite being in exchange with ATP. Overall, the results show the viability of AAV gene transfer of AK gene to skeletal muscle, and provide support of PArg as a reporter that can be utilized to non-invasively monitor the transduction of genes for therapeutic interventions.
PMCID: PMC3975678  PMID: 24572791
31Phosphorus magnetic resonance spectroscopy (31P-MRS); gene reporter; arginine kinase; phosphoarginine; creatine kinase; phosphocreatine; skeletal muscle
2.  Enhancing the Utility of Adeno-Associated Virus Gene Transfer through Inducible Tissue-Specific Expression 
Human Gene Therapy Methods  2013;24(4):270-278.
The ability to regulate both the timing and specificity of gene expression mediated by viral vectors will be important in maximizing its utility. We describe the development of an adeno-associated virus (AAV)-based vector with tissue-specific gene regulation, using the ARGENT dimerizer-inducible system. This two-vector system based on AAV serotype 9 consists of one vector encoding a combination of reporter genes from which expression is directed by a ubiquitous, inducible promoter and a second vector encoding transcription factor domains under the control of either a heart- or liver-specific promoter, which are activated with a small molecule. Administration of the vectors via either systemic or intrapericardial injection demonstrated that the vector system is capable of mediating gene expression that is tissue specific, regulatable, and reproducible over induction cycles. Somatic gene transfer in vivo is being considered in therapeutic applications, although its most substantial value will be in basic applications such as target validation and development of animal models.
Chen and colleagues describe the development of an AAV-based vector with tissue-specific gene regulation, using the ARGENT dimerizer-inducible system. They demonstrate that administration of these vectors via either systemic or intrapericardial injection leads to gene expression that is tissue specific, regulatable, and reproducible over induction cycles.
PMCID: PMC3753727  PMID: 23895325
3.  Gene Transfer in Skeletal and Cardiac Muscle Using Recombinant Adeno-Associated Virus 
Current protocols in microbiology  2013;0 14:Unit-14D.3.
Adeno-associated virus (AAV) is a DNA virus with a small (~4.7kb) single-stranded genome. It is a naturally replication-defective parvovirus of the dependovirus group. Recombinant AAV (rAAV), for use as a gene transfer vector, is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. Only the viral inverted terminal repeats (ITRs) are required in cis for replication and packaging during production. The ITRs are also necessary and sufficient for vector genome processing and persistence during transduction. The tissue tropism of the rAAV vector is determined by the AAV capsid. In this unit we will discuss several methods to deliver rAAV in order to transduce cardiac and/or skeletal muscle, including: intravenous delivery, intramuscular delivery, isolated limb infusion, intrapericardial injection in neonatal mice, and left ventricular wall injection in adult rats.
PMCID: PMC3641885  PMID: 23408131
Adeno-associated virus; gene therapy; heart; skeletal muscle; vector delivery
4.  Long-Term Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Golden Retriever Muscular Dystrophy 
Human Gene Therapy  2011;22(12):1499-1509.
Duchenne muscular dystrophy (DMD) is a lethal, X-linked recessive disease affecting 1 in 3,500 newborn boys for which there is no effective treatment or cure. One novel strategy that has therapeutic potential for DMD is inhibition of myostatin, a negative regulator of skeletal muscle mass that may also promote fibrosis. Therefore, our goal in this study was to evaluate systemic myostatin inhibition in the golden retriever model of DMD (GRMD). GRMD canines underwent liver-directed gene transfer of a self-complementary adeno-associated virus type 8 vector designed to express a secreted dominant-negative myostatin peptide (n=4) and were compared with age-matched, untreated GRMD controls (n=3). Dogs were followed with serial magnetic resonance imaging (MRI) for 13 months to assess cross-sectional area and volume of skeletal muscle, then euthanized so that tissue could be harvested for morphological and histological analysis. We found that systemic myostatin inhibition resulted in increased muscle mass in GRMD dogs as assessed by MRI and confirmed at tissue harvest. We also found that hypertrophy of type IIA fibers was largely responsible for the increased muscle mass and that reductions in serum creatine kinase and muscle fibrosis were associated with long-term myostatin inhibition in GRMD. This is the first report describing the effects of long-term, systemic myostatin inhibition in a large-animal model of DMD, and we believe that the simple and effective nature of our liver-directed gene-transfer strategy makes it an ideal candidate for evaluation as a novel therapeutic approach for DMD patients.
Bish and colleagues evaluate the therapeutic potential of systemic myostatin inhibition in the golden retriever model of Duchenne muscular dystrophy. Canines underwent liver-directed gene transfer of a self-complementary adeno-associated virus type 8 vector expressing a secreted dominant-negative myostatin peptide. Myostatin inhibition resulted in increased muscle mass, largely due to hypertrophy of type IIA fibers, as well as in reduced fibrosis and serum creatine kinase.
PMCID: PMC3237695  PMID: 21787232
5.  Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines 
Human Gene Therapy  2011;22(8):969-977.
Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways.
In this preclinical study, Bish and colleagues report that adeno-associated virus serotype 6 (AAV6)-mediated expression of short hairpin RNA (shRNA) directed against phospholamban (PLB), a regulator of heart failure (HF), is effective at knocking down PLB expression. Yet, safety assessments revealed that healthy canines treated with shRNA, but not empty AAV6 capsid, experienced serum cardiac troponin I elevation, cardiac dysfunction, and alteration of cardiac microRNA expression, suggesting that this approach may not be a feasible therapeutic strategy.
PMCID: PMC3159526  PMID: 21542669
6.  Percutaneous transendocardial delivery of self-complementary adeno-associated virus 6 achieves global cardiac gene transfer in canines 
Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Prior strategies have employed cardio-pulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus or plasmid DNA. While single stranded adeno-associated virus vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented by using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated virus vectors to the canine myocardium. Four vectors were evaluated—single stranded adeno-associated virus 9, self-complementary adeno-associated virus 9, self-complementary adeno-associated virus 8, self-complementary adeno-associated virus 6—so that comparison could be made between single stranded and self complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that self-complementary adeno-associated virus is superior to single stranded adeno-associated virus and that adeno-associated virus 6 is superior to other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15 to 4000 times more abundant in the heart than in any other organ for self-complementary adeno-associated virus 6. Percutaneous transendocardial injection of self-complementary adeno-associated virus 6 is a safe, effective method for achieving efficient cardiac gene transfer.
PMCID: PMC3241935  PMID: 18813281
7.  Myostatin Is Elevated in Congenital Heart Disease and After Mechanical Unloading 
PLoS ONE  2011;6(9):e23818.
Myostatin is a negative regulator of skeletal muscle mass whose activity is upregulated in adult heart failure (HF); however, its role in congenital heart disease (CHD) is unknown.
We studied myostatin and IGF-1 expression via Western blot in cardiac tissue at varying degrees of myocardial dysfunction and after biventricular support in CHD by collecting myocardial biopsies from four patient cohorts: A) adult subjects with no known cardiopulmonary disease (left ventricle, LV), (Adult Normal), (n = 5); B) pediatric subjects undergoing congenital cardiac surgery with normal RV size and function (right ventricular outflow tract, RVOT), (n = 3); C) pediatric subjects with worsening but hemodynamically stable LV failure [LV and right ventricle (LV, RV,)] with biopsy collected at the time of orthotopic heart transplant (OHT), (n = 7); and D) pediatric subjects with decompensated bi-ventricular failure on BiVAD support with biopsy collected at OHT (LV, RV, BiVAD), (n = 3).
The duration of HF was longest in OHT patients compared to BIVAD. The duration of BiVAD support was 4.3±1.9 days. Myostatin expression was significantly increased in LV-OHT compared to RV-OHT and RVOT, and was increased more than double in decompensated biventricular HF (BiVAD) compared to both OHT and RVOT. An increased myostatin/IGF-1 ratio was associated with ventricular dysfunction.
Myostatin expression in increased in CHD, and the myostatin/IGF-1 ratio increases as ventricular function deteriorates. Future investigation is necessary to determine if restoration of the physiologic myostatin/IGF-1 ratio has therapeutic potential in HF.
PMCID: PMC3172210  PMID: 21931616
8.  Chronic Losartan Administration Reduces Mortality and Preserves Cardiac but Not Skeletal Muscle Function in Dystrophic Mice 
PLoS ONE  2011;6(6):e20856.
Duchenne muscular dystrophy (DMD) is a degenerative disorder affecting skeletal and cardiac muscle for which there is no effective therapy. Angiotension receptor blockade (ARB) has excellent therapeutic potential in DMD based on recent data demonstrating attenuation of skeletal muscle disease progression during 6–9 months of therapy in the mdx mouse model of DMD. Since cardiac-related death is major cause of mortality in DMD, it is important to evaluate the effect of any novel treatment on the heart. Therefore, we evaluated the long-term impact of ARB on both the skeletal muscle and cardiac phenotype of the mdx mouse. Mdx mice received either losartan (0.6 g/L) (n = 8) or standard drinking water (n = 9) for two years, after which echocardiography was performed to assess cardiac function. Skeletal muscle weight, morphology, and function were assessed. Fibrosis was evaluated in the diaphragm and heart by Trichrome stain and by determination of tissue hydroxyproline content. By the study endpoint, 88% of treated mice were alive compared to only 44% of untreated (p = 0.05). No difference in skeletal muscle morphology, function, or fibrosis was noted in losartan-treated animals. Cardiac function was significantly preserved with losartan treatment, with a trend towards reduction in cardiac fibrosis. We saw no impact on the skeletal muscle disease progression, suggesting that other pathways that trigger fibrosis dominate over angiotensin II in skeletal muscle long term, unlike the situation in the heart. Our study suggests that ARB may be an important prophylactic treatment for DMD-associated cardiomyopathy, but will not impact skeletal muscle disease.
PMCID: PMC3120761  PMID: 21731628
9.  Molecular Analysis of Vector Genome Structures After Liver Transduction by Conventional and Self-Complementary Adeno-Associated Viral Serotype Vectors in Murine and Nonhuman Primate Models 
Human Gene Therapy  2010;21(6):750-761.
The authors report results from a systematic molecular analysis comparing the liver transduction efficiency of single-stranded and self-complementary AAV vectors in mice and nonhuman primates at early and late stages of gene transfer.
Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs. These included treatment with exonucleases with specific substrate preferences, single-cutter restriction enzyme digestion and polarity-specific hybridization-based vector genome mapping, and bacteriophage ϕ29 DNA polymerase-mediated and double-stranded circular template-specific rescue of persisted circular genomes. In mouse liver, vector genomes of both genome formats seemed to persist primarily as episomal circular forms, but sc vectors converted into circular forms more rapidly and efficiently. However, the overall differences in vector genome abundance and structure in the liver between ss and sc vectors could not account for the remarkable differences in transduction. Molecular structures of persistent genomes of both ss and sc vectors were significantly more heterogeneous in macaque liver, with noticeable structural rearrangements that warrant further characterizations.
PMCID: PMC2938357  PMID: 20113166
10.  Myostatin activation in patients with advanced heart failure and after mechanical unloading 
European Journal of Heart Failure  2010;12(5):444-453.
Myostatin inhibits myoblast differentiation/proliferation and may play a role in heart failure (HF) and reverse remodelling after left ventricular assist device (LVAD) support. This study sought to characterize myostatin expression and activation in advanced HF before and after LVAD support.
Methods and results
Left ventricular tissue pairs were collected at LVAD implantation (core) and at cardiac transplantation/LVAD explantation in patients with advanced ischaemic (ICM—ischaemic cardiomyopathy) and non-ischaemic (DCM—dilated cardiomyopathy) HF. Normal cardiac tissue (control) was obtained from hearts not placed for transplantation. Serum was collected independently from patients with stable DCM HF and from healthy controls. Full-length and cleaved propeptide myostatin levels were quantified by western blot analysis. Dilated cardiomyopathy propeptide levels at core were significantly higher than control and significantly increased after LVAD support. Ischaemic cardiomyopathy propeptide levels were higher than control, but did not change after LVAD support. No changes in full-length levels were seen. Serum myostatin levels were significantly higher in DCM HF patients than in healthy controls.
This is the first clinical evidence that myostatin activation is increased in HF. Myostatin may affect cardiac hypertrophy and may mediate regression of cellular hypertrophy after mechanical unloading.
PMCID: PMC2857990  PMID: 20348550
Protein; Myocyte regulation; Dilated cardiomyopathy; Mechanical circulation
11.  Myostatin Is Upregulated Following Stress in an Erk-Dependent Manner and Negatively Regulates Cardiomyocyte Growth in Culture and in a Mouse Model 
PLoS ONE  2010;5(4):e10230.
Myostatin is well established as a negative regulator of skeletal muscle growth, but its role in the heart is controversial. Our goal in this study was to characterize myostatin regulation following cardiomyocyte stress and to examine the role of myostatin in the regulation of cardiomyocyte size. Neonatal cardiomyocytes were cultured and stressed with phenylephrine. Adenovirus was used to overexpress myostatin or dominant negative myostatin in culture. Adeno-associated virus was used to overexpress myostatin or dominant negative myostatin in mice. Myostatin is upregulated following cardiomyocyte stress in an Erk-dependent manner that is associated with increased nuclear translocation and DNA binding activity of MEF-2. Myostatin overexpression leads to decreased and myostatin inhibition to increased cardiac growth both in vitro and in vivo due to modulation of Akt and NFAT3 pathways. Myostatin is a negative regulator of cardiac growth, and further studies are warranted to investigate the role of myostatin in the healthy and failing heart.
PMCID: PMC2856679  PMID: 20419100
12.  Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice 
PLoS ONE  2010;5(2):e9176.
Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies.
Methodology/Principal Findings
Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner.
Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle.
PMCID: PMC2820101  PMID: 20161803
13.  Adeno-Associated Virus (AAV) Serotype 9 Provides Global Cardiac Gene Transfer Superior to AAV1, AAV6, AAV7, and AAV8 in the Mouse and Rat 
Human Gene Therapy  2008;19(12):1359-1368.
Heart disease is the leading cause of morbidity and mortality. Cardiac gene transfer may serve as a novel therapeutic approach. This investigation was undertaken to compare cardiac tropisms of adeno-associated virus (AAV) serotypes 1, 6, 7, 8, and 9. Neonatal mice were injected with 2.5 × 1011 genome copies (GC) of AAV serotype 1, 6, 7, 8, or 9 expressing LacZ under the control of the constitutive chicken β-actin promoter with cytomegalovirus enhancer promoter via intrapericardial injection and monitored for up to 1 year. Adult rats were injected with 5 × 1011 GC of the AAV vectors via direct cardiac injection and monitored for 1 month. Cardiac distribution of LacZ expression was assessed by X-Gal histochemistry, and β-galactosidase activity was quantified in a chemiluminescence assay. Cardiac functional data and biodistribution data were also collected in the rat. AAV9 provided global cardiac gene transfer stable for up to 1 year that was superior to other serotypes. LacZ expression was relatively cardiac specific, and cardiac function was unaffected by gene transfer. AAV9 provides high-level, stable expression in the mouse and rat heart and may provide a simple alternative to the creation of cardiac-specific transgenic mice. AAV9 should be used in rodent cardiac studies and may be the vector of choice for clinical trials of cardiac gene transfer.
PMCID: PMC2940566  PMID: 18795839

Results 1-13 (13)