Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.

Spinal muscular atrophy (SMA), the most common autosomal recessive neurodegenerative disease affecting children, results in impaired motor neuron function. Despite knowledge of the pathogenic role of decreased survival motor neuron (SMN) protein levels, efforts to increase SMN have not resulted in a treatment for patients. We recently demonstrated that self-complementary adeno-associated virus 9 (scAAV9) can infect approximately 60% of motor neurons when injected intravenously into neonatal mice. Here we use scAAV9-mediated postnatal day 1 vascular gene delivery to replace SMN in SMA pups and rescue motor function, neuromuscular physiology and life span. Treatment on postnatal day 5 results in partial correction, whereas postnatal day 10 treatment has little effect, suggesting a developmental period in which scAAV9 therapy has maximal benefit. Notably, we also show extensive scAAV9-mediated motor neuron transduction after injection into a newborn cynomolgus macaque. This demonstration that scAAV9 traverses the blood-brain barrier in a nonhuman primate emphasizes the clinical potential of scAAV9 gene therapy for SMA.

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.

Follistatin gene delivery enhances muscle growth and strength in nonhuman 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.

Delivery of AAV-IGF-1 to the CNS extends survival in ALS mice through modification of aberrant glial cell activity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Recent work in rodent models of ALS has shown that insulin-like growth factor-1 (IGF-1) slows disease progression when delivered at disease onset. However, IGF-1's mechanism of action along the neuromuscular axis remains unclear. In this study, symptomatic ALS mice received IGF-1 through stereotaxic injection of an IGF-1-expressing viral vector to the deep cerebellar nuclei (DCN), a region of the cerebellum with extensive brain stem and spinal cord connections. We found that delivery of IGF-1 to the central nervous system (CNS) reduced ALS neuropathology, improved muscle strength, and significantly extended life span in ALS mice. To explore the mechanism of action of IGF-1, we used a newly developed in vitro model of ALS. We demonstrate that IGF-1 is potently neuroprotective and attenuates glial cell-mediated release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). Our results show that delivering IGF-1 to the CNS is sufficient to delay disease progression in a mouse model of familial ALS and demonstrate for the first time that IGF-1 attenuates the pathological activity of non-neuronal cells that contribute to disease progression. Our findings highlight an innovative approach for delivering IGF-1 to the CNS.

Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors.

Increasing the size and strength of muscles represents a promising therapeutic strategy for musculoskeletal disorders, and interest has focused on myostatin, a negative regulator of muscle growth. Various myostatin inhibitor approaches have been identified and tested in models of muscle disease with varying efficacies, depending on the age at which myostatin inhibition occurs. Here, we describe a one-time gene administration of myostatin-inhibitor-proteins to enhance muscle mass and strength in normal and dystrophic mouse models for >2 years, even when delivered in aged animals. These results demonstrate a promising therapeutic strategy that warrants consideration for clinical trials in human muscle diseases.

Human bone marrow stromal cells enhance breast cancer cell growth rates in a cell line-dependent manner when evaluated in 3D tumor environments.

Our understanding of the impact that fibroblasts have on cancer cell behavior in vivo has been limited by the complexities of in vivo tumor microenvironments, which contain many distinct cell populations that influence tumor growth and survival. Herein, we describe a novel, three-dimensional (3D), in vitro, fluorometric, Tumor Growth Assay (TGA) that allows for non-invasive measurements of cancer cell expansion in the presence of multiple tumor-associated cell types or soluble factors, while embedded in Cultrex or Matrigel Basement Membrane Extract (BME). Using this assay, we investigated the direct biological impact of primary human bone marrow stromal cells (hMSC) on the growth rates of a panel of metastatic breast cancer cell lines. Human MSC can be readily isolated from bone marrow, a principle site of breast cancer metastasis, and were found to significantly enhance the growth rate of MCF-7 (P-value<0.0001), an estrogen receptor-alpha (ERalpha) positive breast cancer cell line, in a soluble factor-dependent manner. MSC paracrine factors also enhanced the growth of other ERalpha positive breast cancer cell lines including T47D, BT474, and ZR-75-1 (P-value<0.05). In contrast, the ERalpha negative cell line MDA-MB-231 was unaffected by hMSC and the growth rate of another ERalpha negative cell line MDA-MB-468 was elevated in the presence of hMSC, albeit to a lesser extent than MCF-7 or the other ERalpha positive cell lines tested.