Serge Braun

Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy.

Our previous studies have demonstrated that the intraarterial delivery of naked plasmid DNA leads to high levels of foreign gene expression throughout the muscles of the targeted limb. Although the procedure was first developed in rats and then extended to nonhuman primates, the present study has successfully implemented the procedure in normal mice and the mdx mouse model for Duchenne muscular dystrophy. After intraarterial delivery of plasmid DNA expressing the normal, full-length mouse dystrophin from either the cytomegalovirus promoter or a muscle-specific human desmin gene control region, mdx mouse muscle stably expressed dystrophin in 1-5% of the myofibers of the injected hind limb for at least 6 months. This expression generated an antibody response but no apparent cellular response.

Neurotrophins increase motoneurons' ability to innervate skeletal muscle fibers in rat spinal cord--human muscle cocultures.

Neurotrophins, nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-5 (NT-5) and brain-derived neurotrophic factor (BDNF), were studied in vitro in a coculture model of human skeletal muscle myotubes and rat embryo spinal cord explants, which enables the different steps of functional innervation to be followed, including neurite outgrowth, synapse formation and induction of contractile activity. We found that NT-3, NT-5, BDNF, but not NGF simultaneously induced a significant increase in the number and length of neurites emerging from spinal cord explants, the number of endplates per muscle fiber, and the area of innervated muscle fibers around each spinal cord explant. These results suggest that neurotrophins NT-3, NT-5 and BDNF enhance spinal cord motoneurons potential of innervation.

Muscle could be the therapeutic target in SMA treatment

A nerve‐muscle coculture model (human muscle cells innervated by embryonic rat spinal cord) was used to explore the pathogenesis of spinal muscular atrophy (SMA). Previous studies showed that myofibers from donors with SMA type I or SMA type II (but not SMA type III) undergo a characteristic degeneration 1–3 weeks after innervation (Braun et al. [1995] Lancet 345:694–695). To determine which cells are involved in degeneration, we cloned satellite cells and fibroblasts derived from muscle biopsies of normal (healthy) donors and donors with SMA. We show that fibroblasts are required for successful innervation, that fibroblasts from normal and SMA donors contribute equally well to the establishment of cocultures, and that only SMA satellite cells are responsible for the degeneration of innervated cocultures. We succeeded in preventing the degeneration of cloned satellite cells from SMA donors by adding 50% cloned satellite cells from normal donors to the culture to make heteromyotubes. In mixed cocultures, after innervation, we did not observe degeneration. This result suggests that survival of the cocultures depends on a message derived from the muscle cells. Consequently, we propose that therapeutic approaches for SMA that could repair (or compensate for) the genetic defect in muscle cells (which are otherwise much more accessible for gene therapy than neurons) might prevent motoneuron degeneration. The role of muscle cells in the establishment and the degeneration of neuromuscular junctions deserves further attention and investigation. J. Neurosci. Res. 53:663–669, 1998.

Current protocol of a research phase I clinical trial of full-length dystrophin plasmid DNA in Duchenne/Becker muscular dystrophies

A phase I open clinical study on gene therapy in Duchenne and Becker muscular dystrophy, without direct individual benefit for the patient, is being performed at the Pitié-Salpêtrière Hospital, Paris. The aims of this project are: (a) to determine the tolerance and the safety of the intramuscular administration of dystrophin cDNA and (b) to study the quality of the gene transfer in vivo in human patients affected by Duchenne and Becker muscular dystrophy. This clinical trial is conducted sequentially and includes three cohorts of three patients each. Patients must be at least 15 years of age. Diagnosis of Duchenne and Becker muscular dystrophy was confirmed by molecular analysis of the dystrophin gene and for each patient the abnormal expression of dystrophin was confirmed, in skeletal muscle, with antibodies directed against the deleted part of the dystrophin. This phase I study is scheduled to be completed by the end of 2002.

Evaluation of hydrodynamic limb vein injections in nonhuman primates.

The administration route is emerging as a critical aspect of nonviral and viral vector delivery to muscle, so as to enable gene therapy for disorders such as muscular dystrophy. Although direct intramuscular routes were used initially, intravascular routes are garnering interest because of their ability to target multiple muscles at once and to increase the efficiency of delivery and expression. For the delivery of naked plasmid DNA, our group has developed a hydrodynamic, limb vein procedure that entails placing a tourniquet over the proximal part of the target limb to block all blood flow and injecting the gene vector rapidly in a large volume so as to enable the gene vector to be extravasated and to access the myofibers. The present study was conducted in part to optimize the procedure in preparation for a human clinical study. Various injection parameters such as the effect of papaverine preinjection, tourniquet inflation pressure and duration, and rate of injection were evaluated in rats and nonhuman primates. In addition, the safety of the procedure was further established by determining the effect of the procedure on the neuromuscular and vascular systems. The results from these studies provide additional evidence that the procedure is well tolerated and they provide a foundation on which to formulate the procedure for a human clinical study.

Functional Efficacy of Dystrophin Expression from Plasmids Delivered to mdx Mice by Hydrodynamic Limb Vein Injection

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight. The HLV gene delivery procedure was tolerated well by the dystrophic mice and repeat injections could be performed over an extended period of time. Multiple gene deliveries additively increased the amount of dystrophin protein and also increased the percentages of dystrophin-expressing myofibers. Plasmids expressing dystrophin from a cytomegalovirus (CMV) promoter construct containing the HMG1 intron provided stable dystrophin expression for the life of the mouse and provided significant benefit to the limbs. EBD staining showed that dystrophin gene delivery preserved myofibers in the CMV-HMGi-mDys-injected leg by 2.5- to 5-fold in large groups of muscles and by 2.5-fold throughout the injected legs, compared with the contralateral control legs injected with a nonexpressing plasmid. A similar degree of protection was measured in young adult mice evaluated soon after the last gene delivery and in aged mice injected over an extended period of time. This degree of protection resulted from 18 to 20% of the normal level of dystrophin protein, with 11-16% dystrophin-expressing myofibers. These studies show promise for the use of HLV injections to deliver therapeutic doses of full-length dystrophin-expressing plasmids for long-lasting protection of skeletal muscles in patients with DMD.

In vitro and in vivo effects of glucocorticoids on gene transfer to skeletal muscle.

As a pharmacological approach to potentially improve gene transfer efficiency into skeletal muscle cells, glucocorticoids were shown here to allow efficient transfection of cultured and mouse human myoblasts, human pulmonary A549 cells, but not dog myoblasts, independently of the transfection protocol, the reporter gene and the transcription promoter employed. Transduction with adenovirus was also increased by dexamethasone. Pretreatment of cells 48 h prior to transfection was most effective and was shown to be concentration-dependent. This effect is mediated by binding to the glucocorticoid receptor, but not by glucocorticoid responsive elements present in the vectors. The acute dexamethasone effect could be due to increased plasmid entry into the cells as suggested by Southern blot, whereas the sustained increase of luciferase activity in dexamethasone-treated cultures may be related to intracellular mechanisms following cell entry. In mice in vivo, a similar increase of luciferase activity upon glucocorticoid treatment was found.

Muscle Damage After Delivery of Naked Plasmid DNA into Skeletal Muscles Is Batch Dependent

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

Stimulating effects of prednisolone on acetylcholine receptor expression and myogenesis in primary culture of newborn rat muscle cells

Prednisolone at concentrations of 10(-5) to 10(-8) mol/l, added to 3-day (day D + 2) tissue cultures of newborn rat myogenic cells at the time myoblasts are beginning to fuse, increases the level of myotube acetylcholine receptor expression at the 8th day (day D + 7) of culture. This effect is associated with increases in the number and size of the formed myotubes, not with a changed affinity of the receptor for its ligand, and is very probably mediated by one or more extracellular proteins the synthesis of which is induced early by the presence of prednisolone.

Dose response in rodents and nonhuman primates after hydrodynamic limb vein delivery of naked plasmid DNA.

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 μg/g and maximal expression at approximately 400 μg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.