BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codon-optimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100 000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV `silence' and `replace' vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the `silence' and `replace' cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.

Systemic therapy of experimental breast cancer metastases by mutant vesicular stomatitis virus in immune-competent mice.

In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Oncolytic vesicular stomatitis virus (VSV) is a promising novel therapeutic agent for the treatment of cancer. The aim of this study was to evaluate the potential of recombinant VSV containing the M51R mutation in the matrix (M) protein gene administered intravenously as an effective and safe therapeutic agent for treating mice with experimental breast cancer metastases. Recombinant VSV(M51R)-LacZ was generated and characterized in vitro on human and murine breast cancer cells. Breast cancer metastases were established in immune-competent Balb/c mice by intravenous injection of syngeneic 4T1 cells. The vector was infused into the tumor-bearing animals via the tail vein, and productive infection of pulmonary breast cancer lesions was assessed by X-gal stainings of frozen lung sections. To evaluate potential systemic toxicity, histology of major organs and serum chemistries were analyzed. To assess effectiveness, buffer- or vector-treated tumor-bearing mice were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. We found that VSV(M51R)-LacZ efficiently replicated and lysed human breast cancer cells but was partially attenuated in 4T1 cells in vitro. We also demonstrated that its maximum tolerated dose after intravenous infusion in normal Balb/c mice was elevated by at least 100-fold over that of the parental VSV vector containing the wild-type M gene. When VSV(M51R)-LacZ was repeatedly injected intravenously into mice bearing syngeneic 4T1 tumors, the virus was able to infect multiple breast cancer lesions in the lungs without apparent toxicities, which led to significant prolongation of animal survival (P=.003). In conclusion, systemic administration of M mutant VSV is both effective and safe in the treatment of experimental breast cancer metastases in immune-competent mice, suggesting that further development of this approach may have potential for clinical application in patients.

Foxo1 mediates insulin action on apoC-III and triglyceride metabolism.

The apolipoprotein apoC-III plays an important role in plasma triglyceride metabolism. It is predominantly produced in liver, and its hepatic expression is inhibited by insulin. To elucidate the inhibitory mechanism of insulin in apoC-III expression, we delivered forkhead box O1 (Foxo1) cDNA to hepatocytes by adenovirus-mediated gene transfer. Foxo1 stimulated hepatic apoC-III expression and correlated with the ability of Foxo1 to bind to its consensus site in the apoC-III promoter. Deletion or mutation of the Foxo1 binding site abolished insulin response and Foxo1-mediated stimulation. Likewise, Foxo1 also mediated insulin action on intestinal apoC-III expression in enterocytes. Furthermore, elevated Foxo1 production in liver augmented hepatic apoC-III expression, resulting in increased plasma triglyceride levels and impaired fat tolerance in mice. Transgenic mice expressing a constitutively active Foxo1 allele exhibited hypertriglyceridemia. Moreover, we show that hepatic Foxo1 expression becomes deregulated as a result of insulin deficiency or insulin resistance, culminating in significantly elevated Foxo1 production, along with its skewed nuclear distribution, in livers of diabetic NOD or db/db mice. While loss of insulin response is associated with unrestrained apoC-III production and impaired triglyceride metabolism, these data suggest that Foxo1 provides a molecular link between insulin deficiency or resistance and aberrant apoC-III production in the pathogenesis of diabetic hypertriglyceridemia.

Elevated vascular endothelial growth factor production in islets improves islet graft vascularization.

Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, of which only approximately 30% become stably engrafted. Rapid and adequate revascularization of transplanted islets is important for islet survival and function. Delayed and insufficient revascularization can deprive islets of oxygen and nutrients, resulting in islet cell death and early graft failure. To improve islet revascularization, we delivered human vascular endothelial growth factor (VEGF) cDNA to murine islets, followed by transplantation under the renal capsule in diabetic mice. Diabetic animals receiving a marginal mass of 300 islets that were pretransduced with a VEGF vector exhibited near normoglycemia. In contrast, diabetic mice receiving an equivalent number of islets that were transduced with a control vector remained hyperglycemic. Immunohistochemistry with anti-insulin and anti-CD31 antibodies revealed a relatively higher insulin content and greater degree of microvasculature in the VEGF vector-transduced islet grafts, which correlated with significantly improved blood glucose profiles and enhanced insulin secretion in response to glucose challenge in this group of diabetic recipient mice. These results demonstrate that VEGF production in islets stimulates graft angiogenesis and enhances islet revascularization. This mechanism might be explored as a novel strategy to accelerate islet revascularization and improve long-term survival of functional islet mass posttransplantation.

Fat depot-specific expression of adiponectin is impaired in Zucker fatty rats.

Adiposity, particularly increased intra-abdominal fat, is a predisposing factor for the development of insulin resistance in obesity and type 2 diabetes. Visceral fat seems to differ from subcutaneous adipose tissue in adipocytokine production. This fat depot-related difference has been viewed as an important mechanism by which adipose tissue exerts its paracrine/autocrine effects on peripheral tissue in modulating insulin sensitivity. We have studied the relative expression of adiponectin in visceral versus subcutaneous fat in Zucker fatty versus lean rats. Visceral fat, as opposed to subcutaneous fat, exhibited relatively higher levels of adiponectin production in lean animals. However, in Zucker fatty rats, adiponectin expression in visceral fat was suppressed to basal levels, which correlated with significantly reduced plasma adiponectin concentrations and increased insulin resistance. These results suggest that an impaired depot-specific expression of adiponectin is a contributing factor for the development of insulin resistance in Zucker fatty rats.

Inhibition of Foxo1 function is associated with improved fasting glycemia in diabetic mice.

Excessive hepatic glucose production is a contributing factor to fasting hyperglycemia in diabetes. Insulin suppresses hepatic glucose production by inhibiting the expression of two gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase). The forkhead transcription factor Foxo1 has been implicated as a mediator of insulin action in regulating hepatic gluconeogenesis, and a Foxo1 mutant (Foxo1-Delta256), devoid of its carboxyl domain, has been shown to interfere with Foxo1 function and inhibit gluconeogenic gene expression in cultured cells. To study the effect of Foxo1-Delta256 on glucose metabolism in animals, the Foxo1-Delta256 cDNA was delivered to the livers of mice by adenovirus-mediated gene transfer. Hepatic Foxo1-Delta256 production resulted in inhibition of gluconeogenic activity, as evidenced by reduced PEPCK and G-6-Pase expression in the liver. Mice treated with the Foxo1-Delta256 vector exhibited significantly reduced blood glucose levels. In contrast, blood glucose levels in control vector-treated animals remained unchanged, which coincided with the lack of alterations in the expression levels of PEPCK and G-6-Pase. When tested in diabetic db/db mice, hepatic production of Foxo1-Delta256 was shown to reduce fasting hyperglycemia. Furthermore, we showed that hepatic Foxo1 expression was deregulated as a result of insulin resistance in diabetic mice and that Foxo1-Delta256 interfered with Foxo1 function via competitive binding to target promoters. These results demonstrated that functional inhibition of Foxo1, caused by hepatic expression of its mutant, is associated with reduced hepatic gluconeogenic activity and improved fasting glycemia in diabetic mice.