Novel MECP2 gene therapy is effective in a multicenter study using two mouse models of Rett syndrome and is safe in non-human primates.

The AAV9 gene therapy vector presented in this study is safe in mice and non-human primates and highly efficacious without causing overexpression toxicity, a major challenge for clinical translation of Rett syndrome gene therapy vectors to date. Our team designed a new truncated methyl-CpG-binding protein 2 (MECP2) promoter allowing widespread expression of MECP2 in mice and non-human primates after a single injection into the cerebrospinal fluid without causing overexpression symptoms up to 18 months after injection. Additionally, this new vector is highly efficacious at lower doses compared with previous constructs as demonstrated in extensive efficacy studies performed by two independent laboratories in two different Rett syndrome mouse models carrying either a knockout or one of the most frequent human mutations of Mecp2. Overall, data from this multicenter study highlight the efficacy and safety of this gene therapy construct, making it a promising candidate for first-in-human studies to treat Rett syndrome.

Early postnatal administration of an AAV9 gene therapy is safe and efficacious in CLN3 disease.

CLN3 disease, caused by biallelic mutations in the CLN3 gene, is a rare pediatric neurodegenerative disease that has no cure or disease modifying treatment. The development of effective treatments has been hindered by a lack of etiological knowledge, but gene replacement has emerged as a promising therapeutic platform for such disorders. Here, we utilize a mouse model of CLN3 disease to test the safety and efficacy of a cerebrospinal fluid-delivered AAV9 gene therapy with a study design optimized for translatability. In this model, postnatal day one administration of the gene therapy virus resulted in robust expression of human CLN3 throughout the CNS over the 24-month duration of the study. A range of histopathological and behavioral parameters were assayed, with the therapy consistently and persistently rescuing a number of hallmarks of disease while being safe and well-tolerated. Together, the results show great promise for translation of the therapy into the clinic, prompting the launch of a first-in-human clinical trial (NCT03770572).

Follistatin-induced muscle hypertrophy in aged mice improves neuromuscular junction innervation and function.

Sarcopenia, or age-related loss of muscle mass and strength, is an important contributor to loss of physical function in older adults. The pathogenesis of sarcopenia is likely multifactorial, but recently the role of neurological degeneration, such as motor unit loss, has received increased attention. Here, we investigated the longitudinal effects of muscle hypertrophy (via overexpression of human follistatin, a myostatin antagonist) on neuromuscular integrity in C57BL/6J mice between the ages of 24 and 27 months. Following follistatin overexpression (delivered via self-complementary adeno-associated virus subtype 9 injection), muscle weight and torque production were significantly improved. Follistatin treatment resulted in improvements of neuromuscular junction innervation and transmission but had no impact on age-related losses of motor units. These studies demonstrate that follistatin overexpression-induced muscle hypertrophy not only increased muscle weight and torque production but also countered age-related degeneration at the neuromuscular junction in mice.

Voluntary wheel running with and without follistatin overexpression improves NMJ transmission but not motor unit loss in late life of C57BL/6J mice.

Sarcopenia, or pathological loss of muscle mass and strength during aging, is an important contributor to loss of physical function in older adults. Sarcopenia is a multifactorial syndrome associated with intrinsic muscle and upstream neurological dysfunction. Exercise is well-established as an effective intervention for sarcopenia, but less is known about the long-term neurobiological impact of exercise. The goals of this study were to investigate the effects of exercise, alone or in combination with follistatin (FST) overexpression (antagonist of myostatin), on neuromuscular junction transmission and motor unit numbers in mice between the age of 22 and 27 months, ages at which prior studies have demonstrated that some motor unit loss is already evident. C57BL/6J mice underwent baseline assessment and were randomized to housing with or without voluntary running wheels and injection with adeno-associated virus to overexpress FST or vehicle. Groups for comparison included sedentary and running with and without FST. Longitudinal assessments showed significantly increased muscle mass and contractility in the 'running plus FST' group, but running, with and without FST, showed no effect on motor unit degeneration. In contrast, running, with and without FST, demonstrated marked improvement of neuromuscular junction transmission stability.

Sox11 is an Activity-Regulated Gene with Dentate-Gyrus-Specific Expression Upon General Neural Activation.

Neuronal activity initiates transcriptional programs that shape long-term changes in plasticity. Although neuron subtypes differ in their plasticity response, most activity-dependent transcription factors (TFs) are broadly expressed across neuron subtypes and brain regions. Thus, how region- and neuronal subtype-specific plasticity are established on the transcriptional level remains poorly understood. We report that in young adult (i.e., 6-8 weeks old) mice, the developmental TF SOX11 is induced in neurons within 6 h either by electroconvulsive stimulation or by exploration of a novel environment. Strikingly, SOX11 induction was restricted to the dentate gyrus (DG) of the hippocampus. In the novel environment paradigm, SOX11 was observed in a subset of c-FOS expressing neurons (ca. 15%); whereas around 75% of SOX11+ DG granule neurons were c-FOS+, indicating that SOX11 was induced in an activity-dependent fashion in a subset of neurons. Environmental enrichment or virus-mediated overexpression of SOX11 enhanced the excitability of DG granule cells and downregulated the expression of different potassium channel subunits, whereas conditional Sox11/4 knock-out mice presented the opposite phenotype. We propose that Sox11 is regulated in an activity-dependent fashion, which is specific to the DG, and speculate that activity-dependent Sox11 expression may participate in the modulation of DG neuron plasticity.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

Gene Therapy Corrects Brain and Behavioral Pathologies in CLN6-Batten Disease.

CLN6-Batten disease, a form of neuronal ceroid lipofuscinosis is a rare lysosomal storage disorder presenting with gradual declines in motor, visual, and cognitive abilities and early death by 12-15 years of age. We developed a self-complementary adeno-associated virus serotype 9 (scAAV9) vector expressing the human CLN6 gene under the control of a chicken ß-actin (CB) hybrid promoter. Intrathecal delivery of scAAV9.CB.hCLN6 into the cerebrospinal fluid (CSF) of the lumbar spinal cord of 4-year-old non-human primates was safe, well tolerated, and led to efficient targeting throughout the brain and spinal cord. A single intracerebroventricular (i.c.v.) injection at post-natal day 1 in Cln6 mutant mice delivered scAAV9.CB.CLN6 directly into the CSF, and it prevented or drastically reduced all of the pathological hallmarks of Batten disease. Moreover, there were significant improvements in motor performance, learning and memory deficits, and survival in treated Cln6 mutant mice, extending survival from 15 months of age (untreated) to beyond 21 months of age (treated). Additionally, many parameters were similar to wild-type counterparts throughout the lifespan of the treated mice.

Ibuprofen enhances synaptic function and neural progenitors proliferation markers and improves neuropathology and motor coordination in Machado-Joseph disease models.

Machado-Joseph disease or spinocerebellar ataxia type 3 is an inherited neurodegenerative disease associated with an abnormal glutamine over-repetition within the ataxin-3 protein. This mutant ataxin-3 protein affects several cellular pathways, leading to neuroinflammation and neuronal death in specific brain regions resulting in severe clinical manifestations. Presently, there is no therapy able to modify the disease progression. Nevertheless, anti-inflammatory pharmacological intervention has been associated with positive outcomes in other neurodegenerative diseases. Thus, the present work aimed at investigating whether ibuprofen treatment would alleviate Machado-Joseph disease. We found that ibuprofen-treated mouse models presented a significant reduction in the neuroinflammation markers, namely Il1b and TNFa mRNA and IKB-α protein phosphorylation levels. Moreover, these mice exhibited neuronal preservation, cerebellar atrophy reduction, smaller mutant ataxin-3 inclusions and motor performance improvement. Additionally, neural cultures of Machado-Joseph disease patients' induced pluripotent stem cells-derived neural stem cells incubated with ibuprofen showed increased levels of neural progenitors proliferation and synaptic markers such as MSI1, NOTCH1 and SYP. These findings were further confirmed in ibuprofen-treated mice that display increased neural progenitor numbers (Ki67 positive) in the subventricular zone. Furthermore, interestingly, ibuprofen treatment enhanced neurite total length and synaptic function of human neurons. Therefore, our results indicate that ibuprofen reduces neuroinflammation and induces neuroprotection, alleviating Machado-Joseph disease-associated neuropathology and motor impairments. Thus, our findings demonstrate that ibuprofen treatment has the potential to be used as a neuroprotective therapeutic approach in Machado-Joseph disease.

MRI-Guided Focused Ultrasound for Targeted Delivery of rAAV to the Brain.

Recombinant adeno-associated viral (rAAV) vectors are a promising tool for therapeutic gene delivery to the brain. However, the delivery of rAAVs across the blood-brain barrier (BBB) and entry into the brain remains a major challenge for rAAV-based gene therapy. To circumvent this limitation, transcranial MRI-guided focused ultrasound (MRIgFUS) combined with intravenously injected microbubbles has been used to transiently and reversibly increase BBB permeability in targeted brain regions. Systemic administration of rAAVs at the time of sonication with focused ultrasound (FUS) facilitates the passage of rAAVs through the BBB and into the brain parenchyma. We and others have demonstrated that FUS-mediated rAAV delivery to the brain results in efficient transduction and transgene expression in vivo. Using this approach, the dose of intravenously injected rAAV variants that can cross the BBB can be reduced by 100 times, achieving significant transgene expression in the brain parenchyma with reduced peripheral transduction. Moreover, this strategy can be used to deliver rAAV variants that do not cross the BBB from the blood to selected brain regions. Here, we provide a detailed protocol for FUS-induced BBB permeability for targeted rAAV delivery to the brain of adult mice and rats.

Translating SOD1 Gene Silencing toward the Clinic: A Highly Efficacious, Off-Target-free, and Biomarker-Supported Strategy for fALS.

Of familial amyotrophic lateral sclerosis (fALS) cases, 20% are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of RNAi for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge, this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials.

Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy.

BACKGROUND: Spinal muscular atrophy type 1 (SMA1) is a progressive, monogenic motor neuron disease with an onset during infancy that results in failure to achieve motor milestones and in death or the need for mechanical ventilation by 2 years of age. We studied functional replacement of the mutated gene encoding survival motor neuron 1 (SMN1) in this disease. METHODS: Fifteen patients with SMA1 received a single dose of intravenous adeno-associated virus serotype 9 carrying SMN complementary DNA encoding the missing SMN protein. Three of the patients received a low dose (6.7×1013 vg per kilogram of body weight), and 12 received a high dose (2.0×1014 vg per kilogram). The primary outcome was safety. The secondary outcome was the time until death or the need for permanent ventilatory assistance. In exploratory analyses, we compared scores on the CHOP INTEND (Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders) scale of motor function (ranging from 0 to 64, with higher scores indicating better function) in the two cohorts and motor milestones in the high-dose cohort with scores in studies of the natural history of the disease (historical cohorts). RESULTS: As of the data cutoff on August 7, 2017, all 15 patients were alive and event-free at 20 months of age, as compared with a rate of survival of 8% in a historical cohort. In the high-dose cohort, a rapid increase from baseline in the score on the CHOP INTEND scale followed gene delivery, with an increase of 9.8 points at 1 month and 15.4 points at 3 months, as compared with a decline in this score in a historical cohort. Of the 12 patients who had received the high dose, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 walked independently. Elevated serum aminotransferase levels occurred in 4 patients and were attenuated by prednisolone. CONCLUSIONS: In patients with SMA1, a single intravenous infusion of adeno-associated viral vector containing DNA coding for SMN resulted in longer survival, superior achievement of motor milestones, and better motor function than in historical cohorts. Further studies are necessary to confirm the safety and efficacy of this gene therapy. (Funded by AveXis and others; ClinicalTrials.gov number, NCT02122952 .).

HSPB1 mutations causing hereditary neuropathy in humans disrupt non-cell autonomous protection of motor neurons.

Heat shock protein beta-1 (HSPB1), is a ubiquitously expressed, multifunctional protein chaperone. Mutations in HSPB1 result in the development of a late-onset, distal hereditary motor neuropathy type II (dHMN) and axonal Charcot-Marie Tooth disease with sensory involvement (CMT2F). The functional consequences of HSPB1 mutations associated with hereditary neuropathy are unknown. HSPB1 also displays neuroprotective properties in many neuronal disease models, including the motor neuron disease amyotrophic lateral sclerosis (ALS). HSPB1 is upregulated in SOD1-ALS animal models during disease progression, predominately in glial cells. Glial cells are known to contribute to motor neuron loss in ALS through a non-cell autonomous mechanism. In this study, we examined the non-cell autonomous role of wild type and mutant HSPB1 in an astrocyte-motor neuron co-culture model system of ALS. Astrocyte-specific overexpression of wild type HSPB1 was sufficient to attenuate SOD1(G93A) astrocyte-mediated toxicity in motor neurons, whereas, overexpression of mutHSPB1 failed to ameliorate motor neuron toxicity. Expression of a phosphomimetic HSPB1 mutant in SOD1(G93A) astrocytes also reduced toxicity to motor neurons, suggesting that phosphorylation may contribute to HSPB1 mediated-neuroprotection. These data provide evidence that astrocytic HSPB1 expression may play a central role in motor neuron health and maintenance.

NEUROD1 Instructs Neuronal Conversion in Non-Reactive Astrocytes.

Currently, all methods for converting non-neuronal cells into neurons involve injury to the brain; however, whether neuronal transdifferentiation can occur long after the period of insult remains largely unknown. Here, we use the transcription factor NEUROD1, previously shown to convert reactive glial cells to neurons in the cortex, to determine whether astrocyte-to-neuron transdifferentiation can occur under physiological conditions. We utilized adeno-associated virus 9 (AAV9), which crosses the blood-brain barrier without injury, to deliver NEUROD1 to astrocytes through an intravascular route. Interestingly, we found that a small, but significant number of non-reactive astrocytes converted to neurons in the striatum, but not the cortex. Moreover, astrocytes cultured to minimize their proliferative potential also exhibited limited neuronal transdifferentiation with NEUROD1 expression. Our results show that a single transcription factor can induce astrocyte-to-neuron conversion under physiological conditions, potentially facilitating future clinical approaches long after the acute injury phase.

Follistatin Gene Therapy for Sporadic Inclusion Body Myositis Improves Functional Outcomes.

Sporadic inclusion body myositis, a variant of inflammatory myopathy, has features distinct from polymyositis/dermatomyositis. The disease affects men more than women, most commonly after age 50. Clinical features include weakness of the quadriceps, finger flexors, ankle dorsiflexors, and dysphagia. The distribution of weakness is similar to Becker muscular dystrophy, where we previously reported improvement following intramuscular injection of an isoform of follistatin (FS344) by AAV1. For this clinical trial, rAAV1.CMV.huFS344, 6 × 1011 vg/kg, was delivered to the quadriceps muscles of both legs of six sporadic inclusion body myositis subjects. The primary outcome for this trial was distance traveled for the 6-min walk test. The protocol included an exercise regimen for each participant. Performance, annualized to a median 1-year change, improved +56.0 m/year for treated subjects compared to a decline of -25.8 m/year (p = 0.01) in untreated subjects (n = 8), matched for age, gender, and baseline measures. Four of the six treated subjects showed increases ranging from 58-153 m, whereas two were minimally improved (5-23 m). Treatment effects included decreased fibrosis and improved regeneration. These findings show promise for follistatin gene therapy for mild to moderately affected, ambulatory sporadic inclusion body myositis patients. More advanced disease with discernible muscle loss poses challenges.

Additive amelioration of ALS by co-targeting independent pathogenic mechanisms.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which glia are central mediators of motor neuron (MN) death. Since multiple cell types are involved in disease pathogenesis, the objective of this study was to determine the benefit of co-targeting independent pathogenic mechanisms in a familial ALS mouse model. METHODS: Recently, our laboratory identified that ALS microglia induce MN death in an NF-κB-dependent mechanism. We also demonstrated that a single, post-natal, intravenous injection of adeno-associated viral vector serotype 9 encoding a shRNA against mutant SOD1 is able to traverse the blood-brain barrier of ALS mice and reduce SOD1-expression in astrocytes and MNs. Reducing mutant SOD1 in MNs and astrocytes led to a robust increase in survival. To evaluate the benefit of co-targeting multiple cell types in ALS, we combined microglial NF-κB suppression with SOD1 reduction in astrocytes and MNs. RESULTS: Targeting both astrocytes and microglia resulted in an additive increase in survival and motor function by delaying both onset and progression. Strikingly, targeting all three cell types (astrocytes, motor neurons [MNs], and microglia) resulted in an additive increase in lifespan and motor function, with maximum survival reaching 204 days, 67 days longer than the mean survival of untreated control animals. INTERPRETATION: Our data suggest that a combinatorial approach co-targeting different pathogenic mechanisms in independent cell types is a beneficial therapeutic strategy for ALS.

Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.

Gene therapy rescues disease phenotype in a spinal muscular atrophy with respiratory distress type 1 (SMARD1) mouse model.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease affecting children. It is caused by mutations in the IGHMBP2 gene (11q13) and presently has no cure. Recently, adeno-associated virus serotype 9 (AAV9)-mediated gene therapy has been shown to rescue the phenotype of animal models of another lower motor neuron disorder, spinal muscular atrophy 5q, and a clinical trial with this strategy is ongoing. We report rescue of the disease phenotype in a SMARD1 mouse model after therapeutic delivery via systemic injection of an AAV9 construct encoding the wild-type IGHMBP2 to replace the defective gene. AAV9-IGHMBP2 administration restored protein levels and rescued motor function, neuromuscular physiology, and life span (450% increase), ameliorating pathological features in the central nervous system, muscles, and heart. To test this strategy in a human model, we transferred wild-type IGHMBP2 into human SMARD1-induced pluripotent stem cell-derived motor neurons; these cells exhibited increased survival and axonal length in long-term culture. Our data support the translational potential of AAV-mediated gene therapies for SMARD1, opening the door for AAV9-mediated therapy in human clinical trials.

A large animal model of spinal muscular atrophy and correction of phenotype.

OBJECTIVES: Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers. METHODS: Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed. RESULTS: Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology. INTERPRETATION: High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

Transplantation of cerebellar neural stem cells improves motor coordination and neuropathology in Machado-Joseph disease mice.

Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with Machado-Joseph disease exhibit significant motor impairments such as gait ataxia, associated with multiple neuropathological changes including mutant ATXN3 inclusions, marked neuronal loss and atrophy of the cerebellum. Thus, an effective treatment of symptomatic patients with Machado-Joseph disease may require cell replacement, which we investigated in this study. For this purpose, we injected cerebellar neural stem cells into the cerebellum of adult Machado-Joseph disease transgenic mice and assessed the effect on the neuropathology, neuroinflammation mediators and neurotrophic factor levels and motor coordination. We found that upon transplantation into the cerebellum of adult Machado-Joseph disease mice, cerebellar neural stem cells differentiate into neurons, astrocytes and oligodendrocytes. Importantly, cerebellar neural stem cell transplantation mediated a significant and robust alleviation of the motor behaviour impairments, which correlated with preservation from Machado-Joseph disease-associated neuropathology, namely reduction of Purkinje cell loss, reduction of cellular layer shrinkage and mutant ATXN3 aggregates. Additionally, a significant reduction of neuroinflammation and an increase of neurotrophic factors levels was observed, indicating that transplantation of cerebellar neural stem cells also triggers important neuroprotective effects. Thus, cerebellar neural stem cells have the potential to be used as a cell replacement and neuroprotective approach for Machado-Joseph disease therapy.

A phase 1/2a follistatin gene therapy trial for becker muscular dystrophy.

Becker muscular dystrophy (BMD) is a variant of dystrophin deficiency resulting from DMD gene mutations. Phenotype is variable with loss of ambulation in late teenage or late mid-life years. There is currently no treatment for this condition. In this BMD proof-of-principle clinical trial, a potent myostatin antagonist, follistatin (FS), was used to inhibit the myostatin pathway. Extensive preclinical studies, using adeno-associated virus (AAV) to deliver follistatin, demonstrated an increase in strength. For this trial, we used the alternatively spliced FS344 to avoid potential binding to off target sites. AAV1.CMV.FS344 was delivered to six BMD patients by direct bilateral intramuscular quadriceps injections. Cohort 1 included three subjects receiving 3 × 10(11) vg/kg/leg. The distance walked on the 6MWT was the primary outcome measure. Patients 01 and 02 improved 58 meters (m) and 125 m, respectively. Patient 03 showed no change. In Cohort 2, Patients 05 and 06 received 6 × 10(11) vg/kg/leg with improved 6MWT by 108 m and 29 m, whereas, Patient 04 showed no improvement. No adverse effects were encountered. Histological changes corroborated benefit showing reduced endomysial fibrosis, reduced central nucleation, more normal fiber size distribution with muscle hypertrophy, especially at high dose. The results are encouraging for treatment of dystrophin-deficient muscle diseases.