Norma Perez

Tetracycline Transcriptional Silencer Tightly Controls Transgene Expression After In Vivo Intramuscular Electrotransfer: Application to Interleukin 10 Therapy in Experimental Arthritis

The doxycycline (Dox)-inducible reverse tetracycline transactivator (rtTA) is often used to control gene expression. However, the Tet-on system displays a high background activity. To overcome this unregulated expression we used the tetracycline-dependent transcriptional silencer (tTS), which binds the tetO inducible promoter in the absence of Dox. Controlled gene expression was analyzed in vivo by delivering combinations of Dox-regulated luciferase reporter construct, rtTA, and tTS expression plasmids into mouse muscle, using electrotransfer. Elevated luciferase expression levels were observed in the absence of doxycycline, and a 10-fold induction was obtained after drug administration. In contrast, when tTS was added, background expression was dramatically lowered by three to four orders of magnitude, and induction was maintained. The tTS system was then used to control expression of a therapeutic gene in experimental arthritis. DBA/1 mice were coinjected with plasmids encoding the antiinflammatory interleukin-10 cytokine under the control of the tetO promoter, the rtTA, and the tTS. Electrotransfer resulted in a dose-dependent increase in IL-10 expression, maintained over a 3-month period, and significant inhibitory effects on collagen-induced arthritis. We conclude that the use of tTS significantly improves the utility of the rtTA system for somatic gene transfer by reducing background activity.

Mannosidase I inhibition rescues the human α-sarcoglycan R77C recurrent mutation

Limb girdle muscular dystrophy type 2D (LGMD2D, OMIM600119) is a genetic progressive myopathy that is caused by mutations in the human alpha-sarcoglycan gene (SGCA). Here, we have introduced in mice the most prevalent LGMD2D mutation, R77C. It should be noted that the natural murine residue at this position is a histidine. The model is, therefore, referred as Sgca(H77C/H77C). Unexpectedly, we observed an absence of LGMD2D-like phenotype at histological or physiological level. Using a heterologous cellular model of the sarcoglycan complex formation, we showed that the R77C allele encodes a protein that fails to be delivered to its proper cellular localization in the plasma membrane, and consequently to the disappearance of a positively charged residue. Subsequently, we transferred an AAV vector coding for the human R77C protein in the muscle of Sgca-null mice and were able to pharmacologically rescue the R77C protein from endoplasmic reticulum-retention using proteasome or mannosidase I inhibitors. This suggests a therapeutic approach for LGMD2D patients carrying mutations that impair alpha-sarcoglycan trafficking.

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.

Femoral intra-arterial injection: a tool to deliver and assess recombinant AAV constructs in rodents whole hind limb.

With the aim of simplifying recombinant‐adeno‐associated virus (rAAV) delivery in muscle, a new femoral intra‐arterial technique was designed and tested in rodents (rats and mice). Two serotypes, several promoters and transgenes (reporter or therapeutic) were tested using this administration route. The new route is both easy to perform and efficient. Its usefulness as a tool to assess gene delivery constructs in the muscle was established in the context of recombinant AAV serotypes 1 and 2, and with the ubiquitous CMV and two muscle‐specific (C5‐12 and CK6) promoters. Both serum monitoring of a secreted protein (murine alkaline phosphatase: muSEAP) and slide staining were used to compare the different constructs. Significantly different patterns of expression in kinetics of expression (muSEAP) and homogeneity of fiber transduction (staining) were evidenced with the different promoters tested, and compared with intra‐muscular expression patterns. Detailed studies of differential transduction in leg and thigh muscles showed equivalent efficacy, except in rectus femoris, and to a lesser extent in soleus. In light of these results and prior data, intra‐arterially mediated gene transfer mechanism is discussed. Copyright

261. Recombinant Adeno-Associated Viral (rAAV) Microdystrophin Vectors as Therapeutic Tools for Duchenne Muscular Dystrophy (DMD)

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder affecting 1:3500 male individuals, caused by recessive mutations in the dystrophin gene. The size of the gene (2.4Mb) and mRNA (14kb) in addition to immunogenicity problems and inefficient transduction of mature myofibres by currently available vector systems (that could incorporate the full dystrophin cDNA cassette) are formidable obstacles to the development of gene therapy. AAV vectors overcome many of the problems associated with other vector systems but accommodate limited transgene capacity (<5kb). More than 8 AAV vector serotypes have been identified to date with certain serotypes (1, 5, 6, 7) displaying more favourable tropism in transducing muscle fibers compared to the traditionally used AAV2.

Non viral gene therapy in arthritis by in vivo intramuscular IL-10 DNA electrotransfer

Intramuscular electroporation of DNA is an attractive technique for nonviral gene transfer of therapeutic genes in inflammatory/autoimmune disease such as rheumatoid arthritis (RA). We have developed in vivo electroporation for efficient cytokine gene transfer in collagen induced arthritis.