RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

Plasmid pRSVL persisted and expressed luciferase for at least 19 months in mouse skeletal muscle after intramuscular injection. Other injected plasmids also stably expressed long-term suggesting that any plasmid DNA could stably persist and express in muscle. Plasmid DNA was demonstrated by quantitative PCR in some of the muscle DNA samples for at least 19 months after injection. The methylation pattern of the plasmid DNA remained in its bacterial form indicating that the foreign DNA did not replicate in the muscle cells. The electroporation of total cellular DNA from injected muscles into bacteria indicated that the plasmid DNA was extrachromosomal. Chromosomal integration of plasmid DNA was searched for by electroporating the injected muscle DNA into bacteria after restriction enzyme digestion and ligation. No plasmids containing plasmid/chromosome junctions were observed in over 1800 colonies examined. Lack of integration increases the theoretical safety of this gene transfer technique. Long-term stability of plasmid DNA in muscle indicates that muscle is an attractive target tissue for the introduction of extrachromosomal plasmid or viral DNA for the purpose of gene therapy.

Objective 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 two 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. Methods As part of the Inherited Neuropathies Consortium, patients aged 3–20 years with a variety of CMT types were recruited from the USA, UK, Italy and Australia. Initial development stages involved: definition of the construct, item pool generation, peer review and pilot testing. Based on data from 172 patients, a series of validation studies were conducted, including: item and factor analysis, reliability testing, Rasch modeling and sensitivity analysis. Results Seven areas for measurement were identified (strength, dexterity, sensation, gait, balance, power, endurance), and a psychometrically robust 11-item scale constructed (Charcot-Marie-Tooth disease Pediatric Scale: CMTPedS). Rasch analysis supported the viability of the CMTPedS as a unidimensional measure of disability in children with CMT. It showed good overall model fit, no evidence of misfitting items, no person misfit and it was well targeted for children with CMT. Interpretation The CMTPedS is a well-tolerated outcome measure that can be completed in 25-minutes. It is a reliable, valid and sensitive global measure of disability for children with CMT from the age of 3 years.

Duchenne's muscular dystrophy (DMD), which affects one in 3,500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death. One approach to treatment would be to introduce the normal dystrophin gene into diseased muscle cells. When pure plasmid DNA is injected into rodent skeletal or cardiac muscle, the cells express reporter genes. We now show that a 12-kilobase full-length human dystrophin complementary DNA gene and a 6.3-kilobase Becker-like gene can be expressed in cultured cells and in vivo. When the human dystrophin expression plasmids are injected intramuscularly into dystrophin-deficient mdx mice, the human dystrophin proteins are present in the cytoplasm and sarcolemma of approximately 1% of the myofibres. Myofibres expressing human dystrophin contain an increased proportion of peripheral nuclei. The results indicate that transfer of the dystrophin gene into the myofibres of DMD patients could be beneficial, but a larger number of genetically modified myofibres will be necessary for clinical efficacy.

High titre (10(11)-10(12) pfu/ml) suspensions of autonomously replication-defective type 5 human adenovirus (AV) recombinants with different reporter gene inserts (CMV-Luciferase (Lux), CMV-beta-galactosidase (Lac Z), RSV-Lux and RSV-Lac Z) were injected into intact quadriceps muscles of 1-5 day old (Group 1) or 35-45 day old (Group 2) normal mice, as well as regenerating adult mouse muscles (Group 3) and 35 day old mdx muscles (Group 4). The expression of the reporter genes was quantitated 10 days and 2 months later. At 10 days postinjection all reporter gene expression was very high in the neonatally injected (Group 1) muscles. In Group 2 muscles the transduction was markedly less. In Group 3 muscles the gene expression was significantly better than in the Group 2 muscles. In adult mdx muscles (Group 4) where spontaneous regeneration is usually present, the results were similar to those in Group 3 animals. At 2 months post-injection in Group 1 animals, the RSV-Lux expression was even higher than at 10 days postinjection. The cell surface density of alpha v-integrin-containing molecules including the internalization receptor for AV in Groups 1, 2, 3 and 4 showed a positive correlation with AV transducibility. We conclude that adenovirus vector in high titre (10(10) pfu/ml or above) is capable of efficiently transducing only immature muscle cells but not mature muscle fibers in vivo and this appears to correlate with a higher surface density of the available AV internalization receptor in immature muscle cells and lower level in mature muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

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.ConclusionsWhile 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 RegistrationClinicalTrials.gov

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.

We have generated high-titer adenoviral recombinants (AVR) expressing a 6.3-kb partial dystrophin cDNA insert under the control of either the Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter. These AVR preparations were free of both E1-containing AVR and AVR with a nonfunctional dystrophin expression cassette. With these optimal AVR preparations, we have obtained a high degree of short-term (10 days) expression of a truncated (approximately 200 kD) dystrophin in dystrophin-deficient mdx muscles injected in the neonatal period; a lesser degree of expression of dystrophin was found in muscles injected in the young adult age and in old animals. Microscopic indices of muscle damage revealed that the truncated dystrophin provided a significant protection of the transduced muscle fibers. However, by 60 days post-injection, a substantial reduction of the number of dystrophin-positive fibers was noted, even in the neonatally injected muscles, and near-total elimination of dystrophin-positive fibers occurred in muscles injected in the adult age. These effects appeared to be brought about by the activity of CD8+ cytotoxic lymphocytes directed against the transduced cells, leading to their eventual elimination. In severe combined immunodeficiency (SCID) mice, lacking both humoral and cellular immune competence, muscles transduced (either in the neonatal or adult age) by AVR containing a CMV-LacZ expression cassette maintained the early (10 day) transduction level up to 30 days post-injection. Systemic administration of AVR (i.e., into the left ventricle of the heart) led in 5 days to a high number of dystrophin-positive fibers in heart, diaphragm, and intercostal muscles but not in limb muscles.