Gyula Acsadi

Increased Survival and Function of SOD1 Mice After Glial Cell-Derived Neurotrophic Factor Gene Therapy

Amyotrophic lateral sclerosis (ALS) is caused by a progressive degeneration of motor neurons. The cause of sporadic ALS is not known, but 1-2% of all cases are familial and caused by mutations in the copper-zinc superoxide dismutase (SOD1) gene. Transgenic SOD1 mice serve as a transgenic mouse model for these cases. Glial cell-derived neurotrophic factor (GDNF) has a potent trophic effect on motor neurons. Clinical trials in which growth factors have been systemically administered to ALS patients have not been effective, owing in part to the short half-life of these factors and their low concentrations at target sites. Gene transfer of therapeutic factors to motor neurons and/or their target cells, such as muscle, may overcome these problems. Previously, we and others have shown that intramuscularly administered adenovirus vector (AVR) results in foreign gene expression not only in muscle cells, but also in relevant motor neurons in the spinal cord, because of retrograde axonal transport. In this study we utilized an AVR to introduce GDNF into muscles of neonatal SOD1 mice. We showed that AVR-mediated GDNF expression delayed the onset of disease by 7 +/- 8 days (mean +/- SD), prolonged survival by 17 +/- 10 days, and delayed the decline in motor functions (as determined on a rotating rod) by 7-14 days. These results demonstrate that gene delivery to muscle and motor neurons has the potential to treat devastating neurodegenerative diseases such as ALS.

Phase II open label study of valproic acid in spinal muscular atrophy

Preliminary in vitro and in vivo studies with valproic acid (VPA) in cell lines and patients with spinal muscular atrophy (SMA) demonstrate increased expression of SMN, supporting the possibility of therapeutic benefit. We performed an open label trial of VPA in 42 subjects with SMA to assess safety and explore potential outcome measures to help guide design of future controlled clinical trials. Subjects included 2 SMA type I ages 2–3 years, 29 SMA type II ages 2–14 years and 11 type III ages 2–31 years, recruited from a natural history study. VPA was well-tolerated and without evident hepatotoxicity. Carnitine depletion was frequent and temporally associated with increased weakness in two subjects. Exploratory outcome measures included assessment of gross motor function via the modified Hammersmith Functional Motor Scale (MHFMS), electrophysiologic measures of innervation including maximum ulnar compound muscle action potential (CMAP) amplitudes and motor unit number estimation (MUNE), body composition and bone density via dual-energy X-ray absorptiometry (DEXA), and quantitative blood SMN mRNA levels. Clear decline in motor function occurred in several subjects in association with weight gain; mean fat mass increased without a corresponding increase in lean mass. We observed an increased mean score on the MHFMS scale in 27 subjects with SMA type II (p≤0.001); however, significant improvement was almost entirely restricted to participants <5 years of age. Full length SMN levels were unchanged and Δ7SMN levels were significantly reduced for 2 of 3 treatment visits. In contrast, bone mineral density (p≤0.0036) and maximum ulnar CMAP scores (p≤0.0001) increased significantly. Conclusions While VPA appears safe and well-tolerated in this initial pilot trial, these data suggest that weight gain and carnitine depletion are likely to be significant confounding factors in clinical trials. This study highlights potential strengths and limitations of various candidate outcome measures and underscores the need for additional controlled clinical trials with VPA targeting more restricted cohorts of subjects. Trial Registration ClinicalTrials.gov

SMA CARNI-VAL Trial Part I: Double-Blind, Randomized, Placebo-Controlled Trial of L-Carnitine and Valproic Acid in Spinal Muscular Atrophy

Background Valproic acid (VPA) has demonstrated potential as a therapeutic candidate for spinal muscular atrophy (SMA) in vitro and in vivo. Methods Two cohorts of subjects were enrolled in the SMA CARNIVAL TRIAL, a non-ambulatory group of “sitters” (cohort 1) and an ambulatory group of “walkers” (cohort 2). Here, we present results for cohort 1: a multicenter phase II randomized double-blind intention-to-treat protocol in non-ambulatory SMA subjects 2–8 years of age. Sixty-one subjects were randomized 1∶1 to placebo or treatment for the first six months; all received active treatment the subsequent six months. The primary outcome was change in the modified Hammersmith Functional Motor Scale (MHFMS) score following six months of treatment. Secondary outcomes included safety and adverse event data, and change in MHFMS score for twelve versus six months of active treatment, body composition, quantitative SMN mRNA levels, maximum ulnar CMAP amplitudes, myometry and PFT measures. Results At 6 months, there was no difference in change from the baseline MHFMS score between treatment and placebo groups (difference = 0.643, 95% CI = −1.22–2.51). Adverse events occurred in >80% of subjects and were more common in the treatment group. Excessive weight gain was the most frequent drug-related adverse event, and increased fat mass was negatively related to change in MHFMS values (p = 0.0409). Post-hoc analysis found that children ages two to three years that received 12 months treatment, when adjusted for baseline weight, had significantly improved MHFMS scores (p = 0.03) compared to those who received placebo the first six months. A linear regression analysis limited to the influence of age demonstrates young age as a significant factor in improved MHFMS scores (p = 0.007). Conclusions This study demonstrated no benefit from six months treatment with VPA and L-carnitine in a young non-ambulatory cohort of subjects with SMA. Weight gain, age and treatment duration were significant confounding variables that should be considered in the design of future trials. Trial Registry Clinicaltrials.gov NCT00227266

Laminin α2 muscular dystrophy: Genotype/phenotype studies of 22 patients

Objective: To determine the number of primary laminin α2 gene mutations and to conduct genotype/phenotype correlation in a cohort of lamininα2-deficient congenital muscular dystrophy patients. Background: Congenital muscular dystrophies (CMD) are a heterogenous group of muscle disorders characterized by early onset muscular dystrophy and a variable involvement of the CNS. Laminin α2 deficiency has been reported in about 40 to 50% of cases of the occidental, classic type of CMD.1,2 Laminin α2 is a muscle specific isoform of laminin localized to the basal lamina of muscle fibers, where it is thought to interact with myofiber membrane receptor, such as integrins, and possibly dystrophin-associated glycoproteins.3,4 Methods: Seventy-five CMD patients were tested for laminin α2 expression by immunofluorescence and immunoblot. The entire 10 kb laminin α2 coding sequence of 22 completely laminin α2-deficient patients was screened for causative mutations by reverse transcription (RT)-PCR/single strand conformational polymorphisms (SSCP) analysis and protein truncation test(PTT) analysis followed by automatic sequencing of patient cDNA. Clinical data from the laminin α2-deficient patients were collected. Results: Thirty laminin α2-negative patients were identified (40% of CMD patients tested) and 22 of them were screened for laminin α2 mutations. Clinical features of laminin α2-deficient patients were similar, with severe floppiness at birth, delay in achievement of motor milestones, and MRI findings of white matter changes with normal intelligence. Loss-of-function mutations were identified in 95% (21/22) of the patients studied. SSCP analysis detected laminin α2 gene mutations in about 50% of the mutant chromosomes; PTT successfully identified 75% of the mutations. A two base pair deletion mutation at position 2,096-2,097 bp was present in 23% of the patients analyzed. Conclusions: Our data suggest that the large majority of laminin α2-deficient patients show laminin α2 gene mutations.

Mutations in BICD2 Cause Dominant Congenital Spinal Muscular Atrophy and Hereditary Spastic Paraplegia

Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons.

SMA CARNI-VAL TRIAL PART II: A Prospective, Single-Armed Trial of L-Carnitine and Valproic Acid in Ambulatory Children with Spinal Muscular Atrophy

Background Multiple lines of evidence have suggested that valproic acid (VPA) might benefit patients with spinal muscular atrophy (SMA). The SMA CARNIVAL TRIAL was a two part prospective trial to evaluate oral VPA and l-carnitine in SMA children. Part 1 targeted non-ambulatory children ages 2–8 in a 12 month cross over design. We report here Part 2, a twelve month prospective, open-label trial of VPA and L-carnitine in ambulatory SMA children. Methods This study involved 33 genetically proven type 3 SMA subjects ages 3–17 years. Subjects underwent two baseline assessments over 4–6 weeks and then were placed on VPA and L-carnitine for 12 months. Assessments were performed at baseline, 3, 6 and 12 months. Primary outcomes included safety, adverse events and the change at 6 and 12 months in motor function assessed using the Modified Hammersmith Functional Motor Scale Extend (MHFMS-Extend), timed motor tests and fine motor modules. Secondary outcomes included changes in ulnar compound muscle action potential amplitudes (CMAP), handheld dynamometry, pulmonary function, and Pediatric Quality of Life Inventory scores. Results Twenty-eight subjects completed the study. VPA and carnitine were generally well tolerated. Although adverse events occurred in 85% of subjects, they were usually mild and transient. Weight gain of 20% above body weight occurred in 17% of subjects. There was no significant change in any primary outcome at six or 12 months. Some pulmonary function measures showed improvement at one year as expected with normal growth. CMAP significantly improved suggesting a modest biologic effect not clinically meaningful. Conclusions This study, coupled with the CARNIVAL Part 1 study, indicate that VPA is not effective in improving strength or function in SMA children. The outcomes used in this study are feasible and reliable, and can be employed in future trials in SMA. Trial Regsitration Clinicaltrials.gov NCT00227266

Validation of the Charcot–Marie–Tooth disease pediatric scale as an outcome measure of disability

Charcot–Marie–Tooth disease (CMT) is a common heritable peripheral neuropathy. There is no treatment for any form of CMT, although clinical trials are increasingly occurring. Patients usually develop symptoms during the first 2 decades of life, but there are no established outcome measures of disease severity or response to treatment. We identified a set of items that represent a range of impairment levels and conducted a series of validation studies to build a patient‐centered multi‐item rating scale of disability for children with CMT.

Prevalence of Congenital Myopathies in a Representative Pediatric United States Population

The prevalence of congenital myopathies in the United States has not been examined. To address this, we determined the point prevalence of congenital myopathies in a well‐defined pediatric population from Southeastern Michigan. The overall point prevalence was 1:26,000. Mutations in RYR1 were the most common cause of congenital myopathies at 1:90,000. Our data broadly agrees with estimates from previous European studies and provides the first estimate of the prevalence of congenital myopathies in the United States. Ann Neurol 2011;70:662–665

Mitochondrial dysfunction in a neural cell model of spinal muscular atrophy

Mutations of the survival motor neuron (SMN) gene in spinal muscular atrophy (SMA) lead to anterior horn cell death. The cause is unknown, but motor neurons depend substantially on mitochondrial oxidative phosphorylation (OxPhos) for normal function. Therefore, mitochondrial parameters were analyzed in an SMA cell culture model using small interfering RNA (siRNA) transfection that decreased Smn expression in NSC‐34 cells to disease levels. Smn siRNA knock‐down resulted in 35% and 66% reduced Smn protein levels 48 and 72 hr posttransfection, respectively. ATP levels were reduced by 14% and 26% at 48 and 72 hr posttransfection, respectively, suggesting decreased ATP production or increased energy demand in neural cells. Smn knock‐down resulted in increased mitochondrial membrane potential and increased free radical production. Changes in activity of cytochrome c oxidase (CcO), a key OxPhos component, were observed at 72 hr with a 26% increase in oxygen consumption. This suggests a compensatory activation of the aerobic pathway, resulting in increased mitochondrial membrane potentials, a condition known to lead to the observed increase in free radical production. Further testing suggested that changes in ATP at 24 hr precede observable indices of cell injury at 48 hr. We propose that energy paucity and increased mitochondrial free radical production lead to accumulated cell damage and eventual cell death in Smn‐depleted neural cells. Mitochondrial dysfunction may therefore be important in SMA pathology and may represent a new therapeutic target.

Invited Review. The potential for gene therapy in duchenne muscular dystrophy and other genetic muscle diseases

Dystrophin cDNAs have been introduced into skeletal muscle fibers of dystrophin‐deficient mice (mdx) through direct DNA injection in plasmid expression vectors and by replication‐defective recombinant adenovirus vectors. The introduced genes appear to protect those muscle fibers from necrosis in which they become expressed. By direct injection of dystrophin cDNA in plasmid expression vector, only 1–2% of adult mdx muscle fibers of the injected muscle expressed dystrophin. On the other hand, by recombinant adenovirus injection into very young mdx muscle, a better efficiency has been reported. We have discussed several putative and proven factors that may contribute to the thus far demonstrated relatively low efficiency of dystrophin gene transfer. These include poor uptake of gene constructs by muscle fibers, degradation of the injected DNA, and poor access of gene constructs to the nuclear compartment. Neutralization or elimination of these factors could improve the efficiency of gene transfer so that it might, in the future, qualify as an effective therapy for DMD and some other genetic diseases of muscle.© 1993 John Wiley & Sons, Inc.