Pascal Bigey

Inhibiting myostatin with follistatin improves the success of myoblast transplantation in dystrophic mice.

Duchenne muscular dystrophy is a recessive disease due to a mutation in the dystrophin gene. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the myostatin signal with an antagonist, follistatin. Three different approaches were studied to overexpress follistatin in the muscles of mdx mice transplanted with myoblasts. First, transgenic follistatin/mdx mice were generated; second, a follistatin plasmid was electroporated in mdx muscles, and finally, follistatin was induced in mdx mice muscles by a treatment with a histone deacetylase inhibitor. The three approaches improved the success of the myoblast transplantation. Moreover, fiber hypertrophy was also observed in all muscles, demonstrating that myostatin inhibition by follistatin is a good method to improve myoblast transplantation and muscle function. Myostatin inhibition by follistatin in combination with myoblast transplantation is thus a promising novel therapeutic approach for the treatment of muscle wasting in diseases such as Duchenne muscular dystrophy.

Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation

The clinical application of monoclonal antibodies (mAbs) potentially concerns a wide range of diseases including, among others, viral infections, cancer and autoimmune diseases. Although intravenous infusion appears to be the simplest and most obvious mode of administration, it is very often not applicable to long-term treatments because of the restrictive cost of mAbs certified for human use and the side effects associated with injection of massive doses of antibodies. Gene/cell therapies designed for sustained and, possibly, regulatable in vivo production and systemic delivery of mAbs might permit to advantageously replace it. We have already shown that several such approaches allow month- to year-long ectopic antibody production by non-B cells in living organisms. Those include grafting of ex vivo genetically modified cells of various types, in vivo adenoviral gene transfer and implantation of encapsulated antibody-producing cells. Because intramuscular electrotransfer of naked DNA has already been used for in vivo production of a variety of proteins, we have wanted to test whether it could be adapted to that of ectopic mAbs as well. We report here that this is actually the case since both long-term and regulatable production of an ectopic mAb could be obtained in the mouse taken as a model animal. Although serum antibody concentrations obtained were relatively low, these data are encouraging in the perspective of future therapeutical applications of this technology in mAb-based immunotherapies, especially in developing countries where cost-effective and easily implementable technologies would be required for large-scale applications in the context of severe chronic viral diseases such as HIV and HCV infections.

Non-viral gene therapy for GDNF production in RCS rat: the crucial role of the plasmid dose

Glial cell line-derived neurotrophic factor (GDNF) is one of the candidate molecules among neurotrophic factors proposed for a potential treatment of retinitis pigmentosa (RP). It must be administered repeatedly or through sustained releasing systems to exert prolonged neuroprotective effects. In the dystrophic Royal College of Surgeons (RCS) rat model of RP, we found that endogenous GDNF levels dropped during retinal degeneration time course, opening a therapeutic window for GDNF supplementation. We showed that after a single electrotransfer of 30 μg of GDNF-encoding plasmid in the rat ciliary muscle, GDNF was produced for at least 7 months. Morphometric, electroretinographic and optokinetic analyses highlighted that this continuous release of GDNF delayed photoreceptors (PRs) as well as retinal functions loss until at least 70 days of age in RCS rats. Unexpectedly, increasing the GDNF secretion level accelerated PR degeneration and the loss of electrophysiological responses. This is the first report: (i) demonstrating the efficacy of GDNF delivery through non-viral gene therapy in RP; (ii) establishing the efficacy of intravitreal administration of GDNF in RP associated with a mutation in the retinal pigment epithelium; and (iii) warning against potential toxic effects of GDNF within the eye/retina.

Effects of ciliary muscle plasmid electrotransfer of TNF-α soluble receptor variants in experimental uveitis

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-α (TNF-α) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-α soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-α in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-α soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

Genetic pharmacology: progresses in siRNA delivery and therapeutic applications

In the RNA interference process, the catalytic degradation of an endogenous mRNA results from the Watson-Crick complementary recognition by either a small silencing synthetic double-stranded ribonucleotide (siRNA) or by a small hairpin RNA (shRNA) produced in the cell by transcription from a DNA template. This interference process ideally results in an exquisitely specific mRNA suppression. The present review is dedicated to siRNAs. It describes the mechanism of RNA silencing and the main siRNA delivery techniques, with a focus on siRNA self-complexing to cationic lipids to form nanoparticles, which are called lipoplexes. The addition to lipoplexes of an anionic polymer leads to the ternary formulation APIRL (Anionic-Polymer-Interfering-RNA-Lipoplexes) with increased in vivo stability and biological efficacy. In terms of clinical development, the review focuses on therapeutic applications by intravenous delivery to the liver and inflammatory joints, and to localized siRNA delivery to the ocular sphere.

Optimization of plasmid backbone for gene expression in mammalian cells

Publisher Summary Two major groups of vectors are used for gene therapy: viral and non-viral. Viruses are considered to be very efficient vehicles for gene transfer. However, their use is limited by safety concerns, such as immune response, possible mutagenesis and carcinogenesis, and high production costs. Considering these limitations, the delivery of therapeutic genes to target cells upon direct in vivo administration of non- viral vectors, i.e., plasmids, is of great value for the development of gene therapy. However, the use of plasmids is plagued by poor transfer efficiency, intracellular penetration and nuclear localization, and low expression level. The success of non-viral gene therapy depends on the development of optimized plasmids. This chapter focuses on the importance of the plasmid backbone, which is often not considered in gene therapy experiments. Several features are considered in the chapter: bacterial DNA sequences, nuclear import, and safety. The chapter also describes two examples of vectors. .