Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications, hurdles and possible optimizations.

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well-tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non-viral gene transfer following hydrodynamic limb vein injection.

Does gene therapies clinical research in rare diseases reflects the competitivity of the country: Example of France.

Rare diseases are chronic, serious and generally genetic conditions affecting a small number of people, and their therapeutic management is a real challenge. They represent a considerable burden for patients, caregivers and society alike. Compared with existing symptomatic treatments, gene therapies represent a promising new approach aimed at treating these diseases by replacing a defective gene, or by abolishing or reviving a gene-derived function. France is considered one of the leading countries in the research and development of drugs for rare diseases, yet the position of French public and private stakeholders in the research and development of gene therapies for rare diseases at global and European level remains unclear. To answer this question, we used the GENOTRIAL FR database developed by OrphanDev to clarify France's involvement and competitiveness in this field. The results show that France is actively involved in gene therapy clinical trials, with a dense international collaboration network and solid expertise. However, the French medical infrastructure is mainly involved in clinical research on gene therapy candidates sponsored by several foreign countries. To a lesser extent, French public and private entities are also developing their own gene therapy candidates for various rare diseases, some of which have already reached advanced clinical phases. In conclusion, a number of technical and financial challenges need to be overcome if France is to maintain its position as a European and world leader and increase its contribution to reducing the economic and social burden of rare diseases by developing revolutionary and effective new therapies.

Gene-based therapies of neuromuscular disorders: an update and the pivotal role of patient organizations in their discovery and implementation.

This review updates the state-of-the art accomplishments of the multifaceted gene-based therapies, which include DNA or RNA as either therapeutic tools or targets for the treatment of neuromuscular diseases. It also provides insights into the key role that patient organizations have played in research and development; in particular, by addressing bottlenecks and generating boundary conditions that have contributed to scientific breakthroughs, and the effectiveness of innovation processes. Several gene therapy methods have reached the clinical stage and are now addressing both specific and classical issues related to this novel technology. Not ready yet for clinical application, genome editing is at its infancy. More rapidly progressing, RNA-based therapeutics, and especially exon skipping, exon inclusion and stop codon readthrough strategies, are about to move to the market. Most importantly, patients were at the forefront of this discovery process, from basic knowledge to innovation and translational research in a rapidly growing field of unmet medical needs. In recent years, Duchenne muscular dystrophy was the fertile ground for new therapeutic concepts that have been extended to other neuromuscular disorders, such as spinal muscular atrophy, myotonic dystrophies or fascioscapulohumeral dystrophy. In line with their longstanding policy, patient organizations will keep working in a proactive manner to bring together all stakeholders with a view to working out truly therapeutic solutions over a long-term perspective.

Microdystrophin Expression as a Surrogate Endpoint for Duchenne Muscular Dystrophy Clinical Trials.

Duchenne muscular dystrophy (DMD) is a serious, rare genetic disease, affecting primarily boys. It is caused by mutations in the DMD gene and is characterized by progressive muscle degeneration that results in loss of function and early death due to respiratory and/or cardiac failure. Although limited treatment options are available, some for only small subsets of the patient population, DMD remains a disease with large unmet medical needs. The adeno-associated virus (AAV) vector is the leading gene delivery system for addressing genetic neuromuscular diseases. Since the gene encoding the full-length dystrophin protein exceeds the packaging capacity of a single AAV vector, gene replacement therapy based on AAV-delivery of shortened, yet, functional microdystrophin genes has emerged as a promising treatment. This article seeks to explain the rationale for use of the accelerated approval pathway to advance AAV microdystrophin gene therapy for DMD. Specifically, we provide support for the use of microdystrophin expression as a surrogate endpoint that could be used in clinical trials to support accelerated approval.

E-Health & Innovation to Overcome Barriers in Neuromuscular Diseases. Report from the 1st eNMD Congress: Nice, France, March 22-23, 2019.

By definition, neuromuscular diseases are rare and fluctuating in terms of symptoms; patients are often lately diagnosed, do not have enough information to understand their condition and be proactive in their management. Usually, insufficient resources or services are available, leading to patients' social burden. From a medical perspective, the rarity of such diseases leads to the unfamiliarity of the medical staff and caregiver and an absence of consensus in disease assessment, treatment, and management. Innovations have to be developed in response to patients' and physicians' unmet needs.It is vital to improve several aspects of patients' quality of life with a better comprehension of their disease, simplify their management and follow-up, help their caregiver, and reduce the social and economic burden for living with a rare debilitating disease. Database construction regrouping patients' data and symptoms according to specific country registration on data privacy will be critical in establishing a clear consensus on neuromuscular disease treatment.Clinicians also need technological innovations to help them recognize neuromuscular diseases, find the best therapeutic approach based on medical consensus, and tools to follow patients' states regularly. Diagnosis also has to be improved by implementing automated systems to analyze a considerable amount of data, representing a significant step forward to accelerate the diagnosis and the patients' follow up. Further, the development of new tools able to precisely measure specific outcomes reliably is of the matter of importance in clinical trials to assess the efficacy of a newly developed compound.In this context, creation of an expert community is essential to communicate and share ideas. To this end, 97 clinicians, healthcare professionals, researchers, and representatives of private companies from 9 different countries met to discuss the new perspective and challenges to develop and implement innovative tools in the field of neuromuscular diseases.

Neuroscience20 (BRAIN20, SPINE20, and MENTAL20) Health Initiative: A Global Consortium Addressing the Human and Economic Burden of Brain, Spine, and Mental Disorders Through Neurotech Innovations and Policies.

Neurological disorders significantly impact the world's economy due to their often chronic and life-threatening nature afflicting individuals which, in turn, creates a global disease burden. The Group of Twenty (G20) member nations, which represent the largest economies globally, should come together to formulate a plan on how to overcome this burden. The Neuroscience-20 (N20) initiative of the Society for Brain Mapping and Therapeutics (SBMT) is at the vanguard of this global collaboration to comprehensively raise awareness about brain, spine, and mental disorders worldwide. This paper aims to provide a comprehensive review of the various brain initiatives worldwide and highlight the need for cooperation and recommend ways to bring down costs associated with the discovery and treatment of neurological disorders. Our systematic search revealed that the cost of neurological and psychiatric disorders to the world economy by 2030 is roughly $16T. The cost to the economy of the United States is $1.5T annually and growing given the impact of COVID-19. We also discovered there is a shortfall of effective collaboration between nations and a lack of resources in developing countries. Current statistical analyses on the cost of neurological disorders to the world economy strongly suggest that there is a great need for investment in neurotechnology and innovation or fast-tracking therapeutics and diagnostics to curb these costs. During the current COVID-19 pandemic, SBMT, through this paper, intends to showcase the importance of worldwide collaborations to reduce the population's economic and health burden, specifically regarding neurological/brain, spine, and mental disorders.

[Gene-based therapies of spinal muscular atrophy: a piece of history of medicine]. / Thérapies géniques de l'amyotrophie spinale infantile - Un morceau d'histoire de la médecine.

It is worth stating that a generation is needed to bring about a new family of drugs. After the deciphering of the genetic cause in 1995, two innovative classes of therapeutics are now available for spinal muscular atrophy (SMA): the repeated administration of antisens oligonucleotides and the one-shot administration of a scAAV9-SMN as a gene therapy. By addressing the genetic mechanisms of the disease, these drugs fundamentally change its course. These major advances in an extremely severe disease, often fatal before the age of 18 months in the type 1 form (50% of patients), pave the way for the treatment of other serious pathologies of the nervous or neuromuscular system, and provide unambiguous evidence of the effectiveness of these new classes of drugs called to address a number of genetic or acquired diseases. These breakthroughs raise also new scientific and technological questions (limited production yields of gene therapy drugs) but also ethical issues (access of patients to these innovative therapies) that resonate beyond this disease alone.

TITLE: Thérapies géniques de l'amyotrophie spinale infantile - Un morceau d'histoire de la médecine. ABSTRACT: On convient de dire qu'une génération est nécessaire pour faire émerger une nouvelle famille de médicaments. L'amyotrophie spinale infantile (SMA), après l'élucidation du gène causal en 1995, dispose depuis peu de deux classes innovantes de thérapeutiques : l'administration répétée d'oligonucléotides antisens et l'administration unique d'une thérapie génique par scAAV9-SMN. En s'adressant aux mécanismes génétiques de la maladie, elles en modifient fondamentalement le cours. Ces avancées majeures dans une maladie extrêmement sévère, mortelle souvent avant l'âge de 18 mois dans les formes de type 1 (50 % des malades), ouvrent la voie pour d'autres pathologies graves du système nerveux ou neuromusculaire, et apportent une preuve déterminante de l'efficacité de ces classes nouvelles de produits appelés à s'adresser à de nombreuses maladies génétiques ou acquises. Elles génèrent aussi de nouvelles questions d'ordre scientifique et technologique (capacités limitées de production des quantités nécessaires en thérapie génique) mais également d'ordre éthique (conditions d'accès des malades à ces thérapies innovantes), qui résonnent au-delà de cette seule maladie.

[Cell therapy in muscular disorders: the future lies in comparisons of progenitors]. / Thérapie cellulaire des maladies musculaires - Un avenir à l'aune d'une comparaison des progéniteurs.

Cell therapy approaches dedicated to the treatment of dystrophinopathies and involving essentially myoblasts and mesoangioblasts have produced mitigated clinical results. If several types of alternative progenitors have been developed, no standardized comparison has been carried out yet to investigate their regenerative efficacy in vivo, at least at a local level. A comparative study has therefore been designed recently aiming at giving a new impetus to this therapeutic field.

TITLE: Thérapie cellulaire des maladies musculaires - Un avenir à l'aune d'une comparaison des progéniteurs. ABSTRACT: Les approches de thérapie cellulaire des dystrophinopathies basées sur l'utilisation de myoblastes ou de mésoangioblastes se sont traduites par des résultats cliniques mitigés. De nombreux candidats cellulaires alternatifs ont été décrits, mais aucune comparaison standardisée n'a pu encore établir leurs efficacités, ne serait-ce qu'en vue d'une régénération musculaire localisée. Une étude comparative a donc été décidée récemment et pourrait permettre de donner un nouvel élan à cette approche.

Muscular gene transfer using nonviral vectors.

Skeletal muscle is a target tissue of choice for the gene therapy of both muscle and non-muscle disorders. Investigations of gene transfer into muscle have progressed considerably from the expression of plasmid reporter genes to the production of therapeutic proteins such as trophic factors, hormones, antigens, ion channels or cytoskeletal proteins. Viral vectors are intrinsically the most efficient vehicles to deliver genes into skeletal muscles. But, because viruses are associated with a variety of problems (such as immune and inflammatory responses, toxicity, limited large scale production yields, limitations in the size of the carried therapeutic genes), nonviral vectors remain a viable alternative. In addition, as nonviral vectors allow to transfer genetic structures of various sizes (including large plasmid DNA carrying full-length coding sequences of the gene of interest), they can be used in various gene therapy approaches. However, given the lack of efficiency of nonviral vectors in experimental studies and in the clinical settings, the overall outcome clearly indicates that improved synthetic vectors and/or delivery techniques are required for successful clinical gene therapy. Today, most of the potential muscle-targeted clinical applications seem geared toward peripheral ischemia (mainly through local injections) and cancer and infectious vaccines, and one locoregional administration of naked DNA in Duchenne muscular dystrophy. This review updates the developments in clinical applications of the various plasmid-based non-viral methods under investigation for the delivery of genes to muscles.

Magnetic resonance imaging-monitored plasmid DNA delivery in primate limb muscle.

The purpose of this work is to investigate the use of magnetic resonance imaging (MRI) to monitor the effects of high-pressure naked plasmid DNA (pDNA) intravascular injections in primate limbs, studying both the distribution of the injected solution in the muscle space, as well as the effects on the vascular system. The distal portion of the four limbs of each of six rhesus monkeys were hydrodynamically injected with naked pDNA, which expressed the luciferase reporter gene. Three-dimensional (3D) T1-weighted gradient echo and 2D multislice T2-weighted fast spin echo (FSE) series were acquired before and immediately after the injection to confirm the volume of solution injected into the limb, and to study the distribution of the injected solution in the individual muscle groups. Time-resolved contrast-enhanced 3D magnetic resonance angiography (MRA) was performed several days before, immediately after, and in a follow-up examination after the pDNA injection to study the effects of the procedure on the primate peripheral vascular system. T1-weighted gradient echo imaging confirmed the delivery of the majority of the solution after successful pDNA injections. T2-weighted FSE imaging demonstrated the distribution of the saline solution in individual muscles in the target limbs, with enhancement showing a weak but significant correlation with the level of gene expression. Time-resolved contrast-enhanced MRA demonstrated effects of the injection procedure on the arterial and venous vascular systems, and the intramuscular compartments; and these effects largely returned to normal on short-term follow-up.

Artificial macropores attract crop roots and enhance plant productivity on compacted soils.

The structure of compacted soils is characterised by decreased (macro-)porosity, which leads to increased mechanical impedance and decreased fluid transport rates, resulting in reduced root growth and crop productivity. Particularly in soils with high mechanical impedance, macropores can be used by roots as pathways of least resistance. This study investigated how different soil physical states relate to whole plant growth and whether roots grow towards spots with favourable soil physical conditions. Experiments were conducted under controlled and field conditions. Soybean (Glycine max L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) were grown on uncompacted soil, compacted soil and compacted soil with artificial macropores. The interactions between roots and artificial macropores were quantified using X-ray computed tomography. Active growth of roots towards artificial macropores was observed for all three species. Roots grew either into macropores (predominantly in maize) or crossed them (predominantly in wheat). The presence of artificial macropores in compacted soil enabled all three species to compensate for decreased early vigour at later developmental stages. These results show that roots sense their physical environment, enabling them to grow towards spots with favourable soil conditions. The different kinds of root-macropore interaction indicated that macropores serve as a path of least resistance and a source of oxygen, both resulting in increased crop productivity on compacted soils.

Perspectives on best practices for gene therapy programs.

With recent successes in gene therapy trials for hemophilia and retinal diseases, the promise and prospects for gene therapy are once again garnering significant attention. To build on this momentum, the National Institute of Neurological Disorders and Stroke and the Muscular Dystrophy Association jointly hosted a workshop in April 2014 on "Best Practices for Gene Therapy Programs," with a focus on neuromuscular disorders. Workshop participants included researchers from academia and industry as well as representatives from the regulatory, legal, and patient advocacy sectors to cover the gamut from preclinical optimization to intellectual property concerns and regulatory approval. The workshop focused on three key issues in the field: (1) establishing adequate scientific premise for clinical trials in gene therapy, (2) addressing regulatory process issues, and (3) intellectual property and commercialization issues as they relate to gene therapy. The outcomes from the discussions at this workshop are intended to provide guidance for researchers and funders in the gene therapy field.

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.

Phase I study of dystrophin plasmid-based gene therapy in Duchenne/Becker muscular dystrophy.

Nine patients with Duchenne or Becker muscular dystrophy were injected via the radialis muscle with a full-length human dystrophin plasmid, either once with 200 or 600 microg of DNA or twice, 2 weeks apart, with 600 microg of DNA. In the biopsies taken 3 weeks after the initial injection, the vector was detected at the injection site in all patients. Immunohistochemistry and nested reverse transcription-polymerase chain reaction indicated dystrophin expression in six of nine patients. The level of expression was low (up to 6% weak, but complete sarcolemmal dystrophin staining, and up to 26% partial sarcolemmal labeling). No side effects were observed, nor any cellular or humoral anti-dystrophin responses. These results suggest that exogenous dystrophin expression can be obtained in Duchenne/Becker patients after intramuscular transfer of plasmid, without adverse effects, hence paving the way for future developments in gene therapy of hereditary muscular diseases.

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

Since the identification of abnormalities in the dystrophin gene as primary cause of Duchenne muscular dystrophy, gene therapy has been seen as an obvious option among various approaches to treat the disease. It is also considered to be especially challenging, as in this context, one must achieve massive transfer of the gene with a sustained lifelong correction of the muscle phenotype. Our goal is to allow large scale transfection of skeletal muscle fibers of Duchenne muscular dystrophy patients with the full-length 11-kb human dystrophin cDNA. Extensive in vitro and in vivo studies, together with safety considerations and the prospects of a very efficient intra-arterial delivery procedure, led us progressively to focus our efforts on plasmid DNA administration. We are now conducting a phase I safety clinical trial which will pave the way for future therapeutic gene therapy trials for Duchenne muscular dystrophy.

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

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.

Naked plasmid DNA for the treatment of muscular dystrophy.

Muscular dystrophy presents a formidable challenge for gene therapy. Major hurdles include the need to correct large masses of tissue (40% of the body weight) with minimal damage to the already inflamed and necrotic muscles, absence of immune rejection of the therapeutic protein, and sustained (if possible, life-long) expression. Plasmid DNA has long been neglected as a candidate vector for this devastating disease, due to a low in vivo transfection efficiency. It, nevertheless, meets many of the prerequisites for a clinically viable treatment: ease of manufacturing, low toxicity, immunologically innocuous (to allow repeated administrations and insensitivity to pre-existing immunity), and accommodation of the large 11-kb dystrophin cDNA. Over the past year, interest has increased with two major breakthroughs: the first gene-based clinical trial for Duchenne muscular dystrophy that involved a human dystrophin plasmid, and a new method of intravascular delivery showing widespread transfection of limb muscles in large animals, including non-human primates. This opens a new avenue for the treatment of Duchenne dystrophy and many applications using gene delivery to skeletal muscle.