Follistatin N terminus differentially regulates muscle size and fat in vivo

Delivery of follistatin (FST) represents a promising strategy for both muscular dystrophies and diabetes, as FST is a robust antagonist of myostatin and activin, which are critical regulators of skeletal muscle and adipose tissues. FST is a multi-domain protein, and deciphering the function of different domains will facilitate novel designs for FST-based therapy. Our study aims to investigate the role of the N-terminal domain (ND) of FST in regulating muscle and fat mass in vivo. Different FST constructs were created and packaged into the adeno-associated viral vector (AAV). Overexpression of wild-type FST in normal mice greatly increased muscle mass while decreasing fat accumulation, whereas overexpression of an N terminus mutant or N terminus-deleted FST had no effect on muscle mass but moderately decreased fat mass. In contrast, FST-I-I containing the complete N terminus and double domain I without domain II and III had no effect on fat but increased skeletal muscle mass. The effects of different constructs on differentiated C2C12 myotubes were consistent with the in vivo finding. We hypothesized that ND was critical for myostatin blockade, mediating the increase in muscle mass, and was less pivotal for activin binding, which accounts for the decrease in the fat tissue. An in vitro TGF-beta1-responsive reporter assay revealed that FST-I-I and N terminus-mutated or -deleted FST showed differential responses to blockade of activin and myostatin. Our study provided direct in vivo evidence for a role of the ND of FST, shedding light on future potential molecular designs for FST-based gene therapy

Single Tyrosine Mutation in AAV8 and AAV9 Capsids Is Insufficient to Enhance Gene Delivery to Skeletal Muscle and Heart

Site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of adeno-associated viral (AAV) capsids have been reported as a simple method to greatly enhance gene transfer in vitro and in vivo. To determine whether the Y-to-F mutation could also enhance AAV8 and AAV9 gene transfer in skeletal muscle and heart to facilitate muscular dystrophy gene therapy, we investigated four capsid mutants of AAV8 (Y447F or Y733F) and AAV9 (Y446F or Y731F). The mutants and their wild-type control AAV8 and AAV9 capsids were used to package reporter genes (luciferase or β-galactosidase) resulting in similar vector yields. To evaluate gene delivery efficiencies, especially in muscle and heart, the vectors were compared side by side in a series of experiments in vivo in two different strains of mice, the outbred ICR and the inbred C57BL/6. Because AAV8 and AAV9 are among the most effective in systemic gene delivery, we first examined the mutant and wild-type vectors in neonatal mice by intraperitoneal injection, or in adult mice by intravenous injection. To our surprise, no statistically significant differences in transgene expression were observed between the mutant and wild-type vectors, regardless of the reporter genes, vector doses, and the ages and strains of mice used. In addition, quantitative analyses of vector DNA copy number in various tissues from mice treated with mutant and wild-type vectors also showed similar results. Finally, direct intramuscular injection of the above-described vectors with the luciferase gene into the hind limb muscles revealed the same levels of gene expression between mutant and wild-type vectors. Our results thus demonstrate that a single mutation of Y447F or Y733F on capsids of AAV8, and of Y446F or Y731F on AAV9, is insufficient to enhance gene delivery to the skeletal muscle and heart

K137R Mutation on Adeno-Associated Viral Capsids Had Minimal Effect on Enhancing Gene Delivery In Vivo

The adeno-associated viral (AAV) vector has emerged as an attractive vector for gene therapy applications. Development of AAV vectors with enhanced gene transduction efficiency is important to ease the burden of AAV production and minimize potential immune responses. Rational mutations on AAV capsids have gained attention as a simple method of enhancing AAV transduction efficiency. A single-amino acid mutation, K137R, on AAV1 and AAV8 was recently reported to increase liver transgene expression by 5–10-fold. To determine whether the same mutation on other AAV serotypes would result in similar gene enhancement effects, K137R mutants were generated on AAV7, AAV8, and AAV9, and their effects were evaluated in vivo. Two reporter genes were utilized: the nuclear LacZ gene driven by the cytomegalovirus promoter and the luciferase gene driven by the CB promoter. Surprisingly, we found no difference in luciferase gene expression in the liver or other tissues using either the wild-type AAV8 capsid or AAV8-K137R. LacZ gene expression in the liver by AAV8-K137R was about onefold higher than that of wild-type AAV8. However, no difference was found in other tissues, such as skeletal muscle and cardiac muscle. In addition, no difference was found in transgene expression with either AAV7-K137R or AAV9-K137R mutants. Our results indicated that the K137R mutation on AAV7, AAV8, and AAV9 had minimal to no effect on transduction efficiency in vivo

Persistence, Localization, and External Control of Transgene Expression After Single Injection of Adeno-Associated Virus into Injured Joints

A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra-articular soft tissues when AAV is injected preinjury. Two AAV injection time points, postinjury and preinjury, were investigated in osteochondral defect and anterior cruciate ligament transection models of joint injury. Rats injected with AAV tetracycline response element (TRE)–luciferase received oral doxycycline for 7 days. Luciferase expression was evaluated longitudinally for 6 months. Transgene expression was persistent and controllable with oral doxycycline for 6 months in all groups. However, the location of transgene expression was different: postinjury AAV-injected knees had luciferase expression highly localized to the cartilage, while preinjury AAV-injected knees had more widespread signal from intra-articular soft tissues. The differential transgene localization between preinjury and postinjury injection can be used to optimize treatment strategies. Highly localized postinjury injection appears advantageous for treatments targeting repair cells. The more generalized and controllable reservoir of transgene expression following AAV injection before anterior cruciate ligament transection (ACLT) suggests an intriguing concept for prophylactic delivery of joint protective factors to individuals at high risk for early osteoarthritis (OA). Successful external control of intra-articular transgene expression provides an added margin of safety for these potential clinical applications

Adeno-associated Virus 9 Mediated FKRP Gene Therapy Restores Functional Glycosylation of α-dystroglycan and Improves Muscle Functions

Mutations in the FKRP gene are associated with a wide range of muscular dystrophies from mild limb-girdle muscular dystrophy (LGMD) 2I to severe Walker–Warburg syndrome and muscle-eye-brain disease. The characteristic biochemical feature of these diseases is the hypoglycosylation of α-dystroglycan (α-DG). Currently there is no effective treatment available. In this study, we examined the adeno-associated virus serotype 9 vector (AAV9)-mediated gene therapy in the FKRP mutant mouse model with a proline to leucine missense mutation (P448L). Our results showed that intraperitoneal administration of AAV9-FKRP resulted in systemic FKRP expression in all striated muscles examined with the highest levels in cardiac muscle. Consistent with our previous observations, FKRP protein is localized in the Golgi apparatus in myofibers. Expression of FKRP consequently restored functional glycosylation of α-DG in the skeletal and cardiac muscles. Significant improvement in dystrophic pathology, serum creatine kinase levels and muscle function was observed. Only limited FKRP transgene expression was detected in kidney and liver with no detectable toxicity. Our results provided evidence for the utility of AAV-mediated gene replacement therapy for FKRP-related muscular dystrophies

Adeno-Associated Virus Serotype 6 Capsid Tyrosine-to-Phenylalanine Mutations Improve Gene Transfer to Skeletal Muscle

Adeno-associated viral (AAV) vectors are the most efficient in vivo gene transfer tools for gene therapy applications. Efforts have been made to translate encouraging results in small animal models to human patients. However, the need for large quantities of vector for clinical application remains a great challenge. Developing novel AAV vectors with enhanced infectivity may reduce the high vector dose requirement for many applications such as gene therapy for muscular dystrophy. Selective mutation of AAV capsid surface-exposed tyrosine (Y) is a novel strategy to improve transduction efficiency. AAV6 has been considered one of the most robust muscle gene delivery vehicles. Here, we hypothesize that AAV6 transduction efficiency can be further enhanced by mutating surface Y to phenylalanine (F). We found that mutants AAV6-Y445F and AAV6-Y731F, especially the former, achieved more efficient gene transfer than the original AAV6 after intramuscular administration to mice. Expression of both firefly luciferase and alkaline phosphatase reporter genes increased up to 8-fold and DNA copy numbers in muscle increased up to 6-fold. Our results suggest that tyrosine-mutant AAV6 vectors may represent powerful tools for testing muscle gene therapy in animal models and potentially in humans

Overcoming Insulin Insufficiency by Forced Follistatin Expression in β -cells of db/db Mice

Diabetes poses a substantial burden to society as it can lead to serious complications and premature death. The number of cases continues to increase worldwide. Two major causes of diabetes are insulin resistance and insulin insufficiency. Currently, there are few antidiabetic drugs available that can preserve or protect β-cell function to overcome insulin insufficiency in diabetes. We describe a therapeutic strategy to preserve β-cell function by overexpression of follistatin (FST) using an AAV vector (AAV8-Ins-FST) in diabetic mouse model. Overexpression of FST in the pancreas of db/db mouse increased β-cell islet mass, decreased fasting glucose level, alleviated diabetic symptoms, and essentially doubled lifespan of the treated mice. The observed islet enlargement was attributed to β-cell proliferation as a result of bioneutralization of myostatin and activin by FST. Overall, our study indicates overexpression of FST in the diabetic pancreas preserves β-cell function by promoting β-cell proliferation, opening up a new therapeutic avenue for the treatment of diabetes

Fermentation Products of Paenibacillus bovis sp. nov. BD3526 Alleviates the Symptoms of Type 2 Diabetes Mellitus in GK Rats

Gut microbiota is closely related to type 2 diabetes mellitus (T2DM). The gut microbiota of patients with T2DM is significantly different from that of healthy subjects in terms of bacterial composition and diversity. Here, we used the fermentation products of Paenibacillus bovis sp. nov. BD3526 to study the disease progression of T2DM in Goto-kakisaki (GK) rats. We found that the symptoms in GK rats fed the fermentation products of BD3526 were significantly improved. The 16S rRNA sequencing showed that the fermentation products of BD3526 had strong effects on the gut microbiota by increasing the content of Akkermansia. In addition, the interaction of the genus in the gut of the BD3526 group also significantly changed. Additional cytokine detection revealed that the fermentation products of BD3526 can reduce the inflammatory factors in the intestinal mucus of GK rats and thereby inhibit the inflammatory response and ameliorate the symptoms of T2DM

Muscle and Heart Function Restoration in a Limb Girdle Muscular Dystrophy 2I (LGMD2I) Mouse Model by Systemic FKRP Gene Delivery

Mutations in fukutin-related protein (FKRP) gene cause a wide spectrum of disease phenotypes including the mild limb-girdle muscular dystrophy 2I (LGMD2I), the severe Walker-Warburg syndrome, and muscle-eye-brain disease. FKRP deficiency results in α-dystroglycan (α-DG) hypoglycosylation in the muscle and heart, which is a biochemical hallmark of dystroglycanopathies. To study gene replacement therapy, we generated and characterized a new mouse model of LGMD2I harboring the human mutation leucine 276 to isoleucine (L276I) in the mouse alleles. The homozygous knock-in mice (L276IKI) mimic the classic late onset phenotype of LGMD2I in both skeletal and cardiac muscles. Systemic delivery of human FKRP gene by AAV9 vector in the L276IKI mice, at either neonatal age or at the age of 9 months, rendered body wide FKRP expression and restored glycosylation of α-DG in both skeletal and cardiac muscles. FKRP gene therapy ameliorated dystrophic pathology and cardiomyopathy such as muscle degeneration, fibrosis, and myofiber membrane leakage, resulting in restoration of muscle and heart contractile functions. Thus, these results demonstrated that the treatment based on FKRP gene replacement was effective

Enhancing Muscle Membrane Repair by Gene Delivery of MG53 Ameliorates Muscular Dystrophy and Heart Failure in δ-Sarcoglycan-deficient Hamsters

Muscular dystrophies (MDs) are caused by genetic mutations in over 30 different genes, many of which encode for proteins essential for the integrity of muscle cell structure and membrane. Their deficiencies cause the muscle vulnerable to mechanical and biochemical damages, leading to membrane leakage, dystrophic pathology, and eventual loss of muscle cells. Recent studies report that MG53, a muscle-specific TRIM-family protein, plays an essential role in sarcolemmal membrane repair. Here, we show that systemic delivery and muscle-specific overexpression of human MG53 gene by recombinant adeno-associated virus (AAV) vectors enhanced membrane repair, ameliorated pathology, and improved muscle and heart functions in δ-sarcoglycan (δ-SG)-deficient TO-2 hamsters, an animal model of MD and congestive heart failure. In addition, MG53 overexpression increased dysferlin level and facilitated its trafficking to muscle membrane through participation of caveolin-3. MG53 also protected muscle cells by activating cell survival kinases, such as Akt, extracellular signal-regulated kinases (ERK1/2), and glycogen synthase kinase-3β (GSK-3β) and inhibiting proapoptotic protein Bax. Our results suggest that enhancing the muscle membrane repair machinery could be a novel therapeutic approach for MD and cardiomyopathy, as demonstrated here in the limb girdle MD (LGMD) 2F model