Janaiah Kota

Therapeutic microRNA Delivery Suppresses Tumorigenesis in a Murine Liver Cancer Model

Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues. Expression of this miRNA in liver cancer cells in vitro induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of this miRNA in a mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. These findings suggest that delivery of miRNAs that are highly expressed and therefore tolerated in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies.

Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver

miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.

Eteplirsen for the treatment of Duchenne muscular dystrophy

In prior open‐label studies, eteplirsen, a phosphorodiamidate morpholino oligomer, enabled dystrophin production in Duchenne muscular dystrophy (DMD) with genetic mutations amenable to skipping exon 51. The present study used a double‐blind placebo‐controlled protocol to test eteplirsens ability to induce dystrophin production and improve distance walked on the 6‐minute walk test (6MWT).

Gentamicin-induced readthrough of stop codons in duchenne muscular dystrophy

The objective of this study was to establish the feasibility of long‐term gentamicin dosing to achieve stop codon readthrough and produce full‐length dystrophin. Mutation suppression of stop codons, successfully achieved in the mdx mouse using gentamicin, represents an important evolving treatment strategy in Duchenne muscular dystrophy (DMD).

Sustained alpha‐sarcoglycan gene expression after gene transfer in limb‐girdle muscular dystrophy, type 2D

The aim of this study was to attain long‐lasting alpha‐sarcoglycan gene expression in limb‐girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno‐associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK).

Limb-girdle muscular dystrophy type 2D gene therapy restores α-sarcoglycan and associated proteins†‡

α‐Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb‐girdle muscular dystrophy type 2D [LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression.

INHIBITION OF MYOSTATIN WITH EMPHASIS ON FOLLISTATIN AS A THERAPY FOR MUSCLE DISEASE

In most cases, pharmacologic strategies to treat genetic muscle disorders and certain acquired disorders, such as sporadic inclusion body myositis, have produced modest clinical benefits. In these conditions, inhibition of the myostatin pathway represents an alternative strategy to improve functional outcomes. Preclinical data that support this approach clearly demonstrate the potential for blocking the myostatin pathway. Follistatin has emerged as a powerful antagonist of myostatin that can increase muscle mass and strength. Follistatin was first isolated from the ovary and is known to suppress follicle‐stimulating hormone. This raises concerns for potential adverse effects on the hypothalamic–pituitary–gonadal axis and possible reproductive capabilities. In this review we demonstrate a strategy to bypass off‐target effects using an alternatively spliced cDNA of follistatin (FS344) delivered by adeno‐associated virus (AAV) to muscle. The transgene product is a peptide of 315 amino acids that is secreted from the muscle and circulates in the serum, thus avoiding cell‐surface binding sites. Using this approach our translational studies show increased muscle size and strength in species ranging from mice to monkeys. Adverse effects are avoided, and no organ system pathology or change in reproductive capabilities has been seen. These findings provide the impetus to move toward gene therapy clinical trials with delivery of AAV‐FS344 to increase size and function of muscle in patients with neuromuscular disease. Muscle Nerve 39: 283–296, 2009

Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates

A vector delivered into muscles of monkeys generates a natural regulatory molecule, which increases muscle size and strength and may be useful therapeutically. Beyond Mighty Mouse: Building Muscle Mass Strength in Monkeys Patients with progressive neuromuscular disorders all experience the foreboding of the severe disability that awaits them and from which there is little to no relief. Although this class of disorders has multiple genetic and physiological origins, a therapy that directly addresses the debilitating muscle weakness that is the hallmark of these maladies would enhance the lives of millions. Now, in an extension of their previous work in dystrophic mice, Kota et al. describe such a therapeutic approach in preclinical studies performed in nonhuman primates. This treatment mode is applicable to several progressive neuromuscular disorders whether or not scientists have defined their precise genetic defects. The authors used a gene therapy approach to introduce a version of the human gene encoding follistatin into the muscles of the femurs of healthy cynomolgus macaques. Follistatin is a potent inhibitor of myostatin, a signaling molecule that regulates skeletal muscle mass. Follistatin blocks myostatin signaling and augments muscle size and strength safely in mice but, until now, has not been tested in primates. Kota et al. injected a follistatin-producing gene therapy vector into the leg muscles of the monkeys and measured increases in muscle mass and strength. Sustained follistatin expression caused no aberrations in the structures or functions of a variety of organs. This promising progress comes with some caveats. Because healthy monkeys served as subjects for this therapeutic protocol, these findings are not predictive of the outcome in a clinical setting with patients suffering from muscle disorders. In certain genetic neuromuscular diseases, the muscles undergo a repeated cycle of degeneration and regeneration. The vector used in this study does not integrate into the muscle cell genome and thus can be lost from the cells during the degeneration-regeneration cycles. However, the authors point out that the enhancement of muscle size and strength observed in similarly treated dystrophic mice persisted for more than a year even though there was appreciable muscle turnover. More study is needed before follistatin enters the clinic, such as a molecular assessment of gene and vector sequences in multiple tissues. Nonetheless, the work of Kota et al. constitutes proof of principle for the use of myostatin inhibitors to build muscle in primates. Antagonists of myostatin, a blood-borne negative regulator of muscle growth produced in muscle cells, have shown considerable promise for enhancing muscle mass and strength in rodent studies and could serve as potential therapeutic agents for human muscle diseases. One of the most potent of these agents, follistatin, is both safe and effective in mice, but similar tests have not been performed in nonhuman primates. To assess this important criterion for clinical translation, we tested an alternatively spliced form of human follistatin that affects skeletal muscle but that has only minimal effects on nonmuscle cells. When injected into the quadriceps of cynomolgus macaque monkeys, a follistatin isoform expressed from an adeno-associated virus serotype 1 vector, AAV1-FS344, induced pronounced and durable increases in muscle size and strength. Long-term expression of the transgene did not produce any abnormal changes in the morphology or function of key organs, indicating the safety of gene delivery by intramuscular injection of an AAV1 vector. Our results, together with the findings in mice, suggest that therapy with AAV1-FS344 may improve muscle mass and function in patients with certain degenerative muscle disorders.

Pancreatic cancer: Stroma and its current and emerging targeted therapies

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies with a 5-year survival rate of 8%. Dense, fibrotic stroma associated with pancreatic tumors is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Targeting stroma is considered as a potential therapeutic strategy to improve anti-cancer drug efficacy and patient survival. Although numerous stromal depletion therapies have reached the clinic, they add little to overall survival and are often associated with toxicity. Furthermore, increasing evidence suggests the anti-tumor properties of stroma. Its complete ablation enhanced tumor progression and reduced survival. Consequently, efforts are now focused on developing stromal-targeted therapies that normalize the reactive stroma and avoid the extremes: stromal abundance vs. complete depletion. In this review, we summarized the state of current and emerging anti-stromal targeted therapies, with major emphasis on the role of miRNAs in PDAC stroma and their potential use as novel therapeutic agents to modulate PDAC tumor-stromal interactions.

Impaired regeneration in LGMD2A supported by increased Pax7 positive satellite cell content and muscle specific microRNA dysregulation

Introduction: Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, up‐regulation of miR‐1 and miR‐206 facilitates transition from proliferating SCs to differentiating myogenic progenitors. Methods: We examined the histopathological stages, Pax7 SC content, and muscle‐specific microRNA expression in biopsy specimens from well‐characterized LGMD 2A patients to gain insight into disease pathogenesis. Results: Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7‐positive SCs were highest in the fibrotic group and correlated with down‐regulation of miR‐1, miR‐133a, and miR‐206. Conclusions: These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7‐positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis. Muscle Nerve 47: 731–739, 2013