DM1 CTG expansions affect insulin receptor isoforms expression in various tissues of transgenic mice.

Myotonic dystrophy (DM1) is a dominant autosomal multisystemic disorder caused by the expansion of an unstable CTG trinucleotide repeat in the 3' untranslated region of the DMPK gene. Nuclear accumulation of the enlarged CUG-containing DMPK transcripts has a deleterious effect on the regulation of alternative splicing of some RNAs and has a central role in causing the symptoms of DM1. In particular, Insulin Receptor (IR) mRNA splicing defects have been observed in the muscle of DM1 patients. In this study, we have investigated IR splicing in insulin-responsive tissues (i.e. skeletal muscles, adipose tissue, liver) and pancreas and we have studied glucose metabolism in mice carrying the human genomic DM1 region with expanded (>350 CTG) or normal (20 CTG) repeats and in wild-type mice. Mice carrying DM1 expansions displayed a tissue- and age-dependent abnormal regulation of IR mRNA splicing in all the tissues that we investigated. Furthermore, these mice showed a basal hyperglycemia and glucose intolerance which disappeared with age. Our findings show that deregulation of IR splicing due to the DM1 mutation can occur in different mouse tissues, suggesting that CTG repeat expansions might also result in IR misplicing not only in muscles but also in other tissues in DM1 patients.

Total vascular exclusion of the liver enhances the efficacy of retroviral-mediated associated thymidine kinase and interleukin-2 genes transfer against multiple hepatic tumors in rats.

BACKGROUND: Recent advances in gene transfer technology render gene therapy an attractive treatment of disseminated liver metastases for which other treatments remain disappointing. In this setting, total vascular exclusion of the liver could improve gene transfer to cancer cells and prevent extrahepatic vector spreading during portal infusion of therapeutic genes. We evaluate the efficiency of combined herpes simplex virus type-1 thymidine kinase (HSV1-TK) and interleukin-2 retrovirus-mediated gene transfer through the portal vein, under total vascular exclusion of the liver, in a model of macroscopic multiple liver metastases in rats. METHODS: Multifocal liver metastases were established in BDIX rats with intraportal injection of DHDK12 colon cancer cells. On randomization, rats received either vector-producing cells or saline solution under total vascular exclusion of the liver. Vector-producing cells released retroviral vectors encoding Lac-Z in marking studies or HSV1-TK or interleukin-2 in therapeutic studies. Rats were either killed for pathologic studies, or followed for survival. RESULTS: Total vascular exclusion of the liver markedly improved gene transfer efficacy in marking studies. In therapeutic studies we observed a significant reduction in tumor volume of treated rats compared with untreated controls (2170 +/- 310 mm(3)). Although singular HSV1-TK or interleukin-2 gene transfer showed significant efficacy, the greatest tumor volume regression was observed in rats treated with combined HSV1-TK + interleukin-2 gene therapy (145 +/- 60 mm(3); P =.0001 vs control). This translated into an increased median survival rate compared with either control rats (P =.006) or rats treated with single gene therapy. CONCLUSION: In a rat model, a significant antitumoral effect against macroscopic multifocal liver metastases can be observed after retrovirus-mediated HSV1-TK and interleukin-2 gene transfer through the portal vein under total vascular exclusion of the liver, followed by ganciclovir administration. We believe that this well-tolerated and efficient therapeutic approach deserves clinical evaluation in patients with disseminated colorectal liver metastases.

CTG repeat instability and size variation timing in DNA repair-deficient mice.

Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene. We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA-PKcs (non-homologous end-joining). Genes of the non-homologous end-joining and homologous recombination pathways did not seem to affect repeat instability. Only lack of Rad52 led to a slight decrease in expansion range. Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model. Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations. Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability. We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.