Istvan Danko

The efficient expression of intravascularly delivered DNA in rat muscle

Previous studies have demonstrated that muscle can take up and express naked DNA or RNA. This study demonstrates that the pDNA can be delivered to and expressed within skeletal muscle when injected rapidly, in a large volume and when all blood vessels leading into and out of the hindlimb are occluded. The additional use of collagenase, papaverine and ischemia raised expression moderately but was not critical. These results demonstrate that a nonviral method can lead to high levels of expression in the muscles of adult animals larger than mice.

The Frequency of Revertants in mdx Mouse Genetic Models for Duchenne Muscular Dystrophy

ABSTRACT: The mdx mouse has been used for the development of cellular and gene therapies for Duchenne muscular dystrophy. The relatively frequent occurrence of dystrophin-positive muscle cells called revertants has hampered these efforts by interfering with data interpretation. The mdx4cv and mdx5cv dystrophin mouse mutants have approximately 10-fold fewer revertants than the mdx mutant at both 2 and 6 mo. The mdx3cv dystrophin mouse mutant may be a useful model for some types of human dystrophin deficiencies in which the levels of dystrophin are low but not completely absent.

Direct gene transfer into muscle.

Gene therapy has great promise for the treatment and the prevention of a broad range of inherited and acquired diseases. Gene transfer methods currently explored include the use of viral vectors and physical-chemical methods. Plasmid DNA can be taken up by skeletal muscle cells in vivo without any special delivery mechanism and persist long-term in an extrachromosomal, non-replicative circular form. Thus, foreign genes can be expressed permanently in skeletal muscle. At present the efficiency of gene transfer is not high enough to treat genetic muscle diseases. However, even at the relatively low efficiency of expression we are able to achieve at present, plasmid DNA transfer seems to be a very promising way of programming cells in vivo to secrete proteins for immunization purposes.

Expression of Deletion-Containing Dystrophins in mdx Muscle: Implications for Gene Therapy and Dystrophin Function

ABSTRACT: The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane. Only those constructs containing the carboxyl terminus were able to stabilize dystrophin-associated proteins (DAP) at the membrane, consistent with other studies that suggest that this domain is critical to DAP binding. Colocalization with DAP was not necessary for membrane localization of the various dystrophin molecules. However, stabilization and co-localization of the DAP did seem to be a prerequisite for expression and/or stabilization of mutant dystrophins beyond 1 wk and these same criteria seemed important for mitigating the histopathological consequences of dystrophin deficiency.

765. Intravascular Delivery of Naked Plasmid DNA into Muscle for Long-Term Correction of Hyperbilirubinemia in the Gunn Rat

Background: We have shown previously that intra-arterial delivery of naked plasmid (pDNA) expressing human bilirubin glucuronosyl transferase (hUGT1A1) leads to bilirubin glucuronidation in muscle and significant decrease of total serum bilirubin (TBi) for at least 2 weeks in the UGT1A1 deficient Gunn rat (GR), animal model of Crigler-Najjar Syndrome Type I. This study was undertaken to evaluate the feasibility of repeat intravenous and intra-arterial pDNA delivery into skeletal muscle for long-term metabolic correction in GR.

187. Strategies of Intravascular Delivery of Naked DNA into Rat Skeletal Muscle

Background: Due to its large mass and ability to express naked plasmid DNA (pDNA), skeletal muscle is an attractive target for non-viral gene transfer. Further development of this approach requires improved stability of gene expression and identification of the minimal muscle mass required to maintain metabolic correction. In this study we evaluated the efficiency of various vascular access routes in different muscle groups of the rat hind-limb and comparation of viral and muscle-specific promoters for reporter gene expression.

1041. Treatment of Hyperbilirubinemia in the Gunn Rat by Intravascular Gene Transfer into Muscle with Naked Plasmid DNA (pDNA)

Background: Use of pDNA vectors to express hepatic enzymes in muscle would enable gene therapy for inborn errors of metabolism without the risks inherent in viral gene transfer into the liver. We tested the feasibility of this approach in the bilirubin uridine glucuronosyl transferase (UGT1A1) deficient Gunn rat, animal model of Crigler-Najjar syndrome type I. UGT1A1 deficiency results from mutations of the UGT gene and is characterized by hyperbiliriubinemia due to inefficient biliary excretion of bilirubin. Goal of treatment is prevention of the major complication, encephalopathy, by decreasing serum bilirubin levels.