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Illkirch-Graffenstaden, France

Wooddell C.I.,Roche Holding AG | Hegge J.O.,Roche Holding AG | Zhang G.,University of Wisconsin - Madison | Sebestyen M.G.,Roche Holding AG | And 8 more authors.
Human Gene Therapy

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 μg/g and maximal expression at approximately 400 μg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus. © 2011, Mary Ann Liebert, Inc. Source

Wooddell C.I.,Roche Holding AG | Subbotin V.M.,Roche Holding AG | Sebestyen M.G.,Roche Holding AG | Griffin J.B.,Roche Holding AG | And 7 more authors.
Human Gene Therapy

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA. © 2011, Mary Ann Liebert, Inc. Source

Wooddell C.I.,Roche Holding AG | Zhang G.,University of Wisconsin - Madison | Griffin J.B.,Roche Holding AG | Hegge J.O.,Roche Holding AG | And 2 more authors.
Muscle and Nerve

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD-positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD+ muscle fibers in 12-19-month-old mdx mice increased 3-fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD. In contrast, the intra- and interlimb amounts of EBD in 2-7-month-old mdx mice were much too variable. A therapeutic effect can more readily be measured in old mdx mice. These results will be useful in the design of therapy protocols using the mdx mouse. © 2009 Wiley Periodicals, Inc. Source

Zhang G.,University of Wisconsin - Madison | Zhang G.,Roche Holding AG | Wooddell C.I.,Roche Holding AG | Hegge J.O.,Roche Holding AG | And 5 more authors.
Human Gene Therapy

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight. The HLV gene delivery procedure was tolerated well by the dystrophic mice and repeat injections could be performed over an extended period of time. Multiple gene deliveries additively increased the amount of dystrophin protein and also increased the percentages of dystrophin-expressing myofibers. Plasmids expressing dystrophin from a cytomegalovirus (CMV) promoter construct containing the HMG1 intron provided stable dystrophin expression for the life of the mouse and provided significant benefit to the limbs. EBD staining showed that dystrophin gene delivery preserved myofibers in the CMV-HMGi-mDys-injected leg by 2.5-to 5-fold in large groups of muscles and by 2.5-fold throughout the injected legs, compared with the contralateral control legs injected with a nonexpressing plasmid. A similar degree of protection was measured in young adult mice evaluated soon after the last gene delivery and in aged mice injected over an extended period of time. This degree of protection resulted from 18 to 20% of the normal level of dystrophin protein, with 11-16% dystrophin-expressing myofibers. These studies show promise for the use of HLV injections to deliver therapeutic doses of full-length dystrophin-expressing plasmids for long-lasting protection of skeletal muscles in patients with DMD. © Copyright 2010, Mary Ann Liebert, Inc. Source

Transgene | Date: 2013-04-29

Chemicals for use in research in the field of chemistry bacteriology, virology, immunology, molecular biology, genetic engineering, gene therapy and human and veterinary medicine, namely, chemical reagents, lytic reagents, bacteriostatic reagents, fungicidal reagents, enzyme reagents, rinsing reagents and cleaning reagents all for use with hematology, cytological, particulate, virological and chemical analytical automated and semiautomated study and analysis instruments, machines and systems used in diagnostic, industrial, biological, and cytology laboratories all for scientific or research use; monoclonal antibodies and antigens for diagnostic, scientific, or research use; reagents, diluents and buffers as packaged in ready-to-use dispensing packages for scientific or research use; cell markers used as reagents for diagnostic, scientific, or research use; dyes and stains for microscopic specimen slides and flow cytometry used in diagnostic or scientific research; microspheres, fluoroscopes and monodispersed particles for use as standards, calibrators and carrier receptor sites employed with cytology and particle study and analysis instruments and systems used in the biological, industrial, and academic fields for scientific or research use; whole blood hematology reference control solutions for calibration in electronic particle laboratory analysis for diagnostic, scientific or research use; diluents, standards, stabilizers, buffers, reference control compositions, immersion oil, sheath fluids and wetting fluids for scientific and research use; diagnostic laboratory reagents for hemoglobin testing for scientific and research use; reagents for hemoglobin testing for scientific and research use; reagents for use with automatic chemical analysis instruments in biological, chemical, industrial and clinical laboratories for scientific or research use; and cell markers for diagnostic and research use. Pharmaceutical preparations for use in treatment of cancer, central nervous system diseases, multiple sclerosis, autoimmune diseases and hypersensitivities, infectious diseases and hematological conditions. Research and scientific studies in the fields of chemistry, bacteriology, genetics, human and veterinary medicine.

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