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G Dickson

Publications and source records attributed to G Dickson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Expression of full-length human dystrophin cDNA in mdx mouse muscle by HVJ-liposome injection.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder which is caused by a defect of dystrophin, a 427-kDa muscle cell membrane protein. One of the possible means of DMD therapy is to express the dystrophin gene in patients' muscles. In this study, full length dystrophin cDNA was expressed in mdx (muscular dystrophy model) mouse muscle using the hemagglutinating virus of Japan (HVJ)-liposome method. With the HVJ-liposome method, the lacZ reporter genes were expressed in 50-80% of cultured mdx mouse myoblasts, which suggested its potential usefulness for an in vivo gene study. Three expression vectors containing human full length dystrophin cDNA driven by Rous sarcoma virus (RSV), mouse leukemia virus, or human dystrophin promoters, were used. HVJ-liposomes containing these plasmids were directly injected into mdx mouse quadriceps muscle. The highest efficiency of expression of dystrophin was in 26% of the muscle fibers at the injected site on day 3 after HVJ-liposome injection of the RSV-based vector. The expression was decreased on day 10. The study thus demonstrates the feasibility of full length human dystrophin cDNA transfer and dystrophin expression using HVJ-liposomes in vivo.

Animals↗
PubMed1995

Dystrophin expression in the hair cells of the cochlea.

Dystrophin is normally expressed in a number of tissues including muscle, brain and the outer plexiform layer of the retina. In Duchenne and Becker muscular dystrophy abnormal or deficient dystrophin expression leads to muscle degeneration and has been implicated in mental retardation and a form of night blindness. We have examined the expression of dystrophin immunoreactivity in cochlear tissues of normal guinea-pig and mouse, and whether expression is perturbed in the cochlea of the dystrophic MDX mouse. A single band of approximately 427 kDa, corresponding to a full-length isoform of dystrophin was detected in guinea-pig and normal mouse but was absent from the MDX mouse. Cochleae from guinea-pig, normal and MDX mouse also showed a second dystrophin isoform of 116 kDa molecular weight with the C-terminal specific antibody. Immunostained guinea pig cochlear half turns were examined by laser scanning confocal microscopy. Dystrophin was localized in both inner and outer hair cells with staining patterns which were qualitatively similar with both antibodies. In the outer hair cells labelling of the lateral wall was especially distinctive. The synaptic region of both hair cell types was also strongly labelled.

Animals↗
PubMed1995

Liposome-mediated gene transfer into normal and dystrophin-deficient mouse myoblasts.

A range of tissue types has now been targeted for development of gene therapeutic procedures both to correct genetic defects and to treat acquired disease. In particular, skeletal muscle holds great importance, not exclusively for the treatment of inherited muscle disorders but also as a platform for the expression of heterologous recombinant proteins, destined to immunise the host or to serve some systemic therapeutic goal. With respect to the X-linked myopathy Duchenne muscular dystrophy (DMD), several gene therapy protocols are being developed that focus on complementing primary genetic defects in the DMD gene by introducing copies of recombinant gene constructs into muscle cells both ex vivo and in vivo. In the present study the potential use of a range of polycationic liposomes as physical gene delivery systems for skeletal muscle has been examined. Using a LacZ reporter gene under optimised conditions up to 40% transfection efficiencies were obtained with the mouse myoblast cell line C2C12. With primary cultures of normal and dystrophin-deficient mdx mouse muscle, up to 10% transfection efficiency was obtained with reporter gene constructs, and high levels of recombinant human dystrophin expression were observed following transfer of dystrophin cDNA gene constructs. These in vitro studies indicate that cationic liposomes can be used to deliver recombinant genes to muscle cells at high efficiency and form a basis to expand investigations into in vivo expression of recombinant dystrophin protein either by direct intramuscular gene transfer or via implantation of transfected myoblasts.

Animals↗
PubMed1995

Efficiency of in vivo gene transfer using murine retroviral vectors is strain-dependent in mice.

Retroviral vectors can be used to transduce cultured cells at high frequencies, but efficient transduction of target cells in vivo has proved difficult and little is known about the factors that influence the efficiency of retroviral infection. Many commonly used mouse strains harbor endogenous C-type proviruses, some of which are expressed and have circulating antibodies against the viral envelope glycoproteins that cross-react with the Moloney strain of murine leukemia virus (MoMLV), from which most current retroviral vectors are derived. We have investigated the relative efficiency of retroviral-mediated gene transfer into regenerating skeletal muscle of a variety of mouse strains using a MoMLV-based vector. Humoral immune competence and interference between endogenous MLVs and exogenous recombinant MoMLV were observed to affect the efficiency of retroviral-mediated transfection in vivo. Our results indicate that the mouse genetic background and immune status need to be considered when choosing a preclinical model for in vivo retroviral-mediated gene transfer.

Animals↗
PubMed1995

Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder with a high spontaneous mutation rate and no effective treatment, hence development of genetic based therapies is an important goal. We report that expression of a recombinant human minidystrophin cDNA, compatible with current viral vectors, can significantly reduce the myopathic phenotype in transgenic mdx mice, even when expressed at only 20-30% of endogenous dystrophin levels at the sarcolemma. To the extent that data obtained in mouse studies are applicable to DMD, the virtual elimination of morphological and biochemical abnormalities in the mdx mouse supports the use of this cDNA in somatic gene therapy protocols for DMD.

Animals↗
PubMed1994

Adenovirus-mediated gene transfer to murine retinal cells in vitro and in vivo.

Adenovirus-mediated gene transfer to retinal cells was evaluated using the replication-defective recombinant adenovirus vector Ad2/CMVlacZ-1 (coding for beta-galactosidase) both in an in vitro murine culture model and in vivo in adult mice. In vitro, no difference in infectability of neuronal and glial cells was observed, and 50% of neurons expressed the exogenous gene at low viral concentration (10 pfu/cell). In vivo, intraocular injection of 3 x 10(6) pfu Ad2/CMVlacZ-1 resulted in expression of the transferred beta-galactosidase gene in retinal pigment epithelium and ganglion cells. These results demonstrate that Ad2/CMVlacZ-1 is an effective vector for gene transfer into retinal cells.

Adenoviridae↗
PubMed1994

Generalized vascular permeability and pulmonary function in patients following serious trauma.

It has been suggested that the adult respiratory distress syndrome (ARDS) is a manifestation of a generalized vascular permeability defect. Low-level urinary albumin excretion reflects changes in systemic vascular permeability in a variety of acute inflammatory conditions including trauma. To test the hypothesis that impaired pulmonary function is associated with increased systemic vascular permeability, 44 trauma patients with Injury Severity Scores (ISS) ranging from 9 to 75 were studied over 3 days. Urinary albumin was measured from admission and expressed as the albumin/creatinine ratio (ACR). In 24 mechanically ventilated patients mean inspired oxygen/fraction (FIO2) and mean arterial oxygen tension (PO2) were used to calculate the PO2/FIO2 ratio. For each study day patients were divided into group I, mean FIO2 > 0.5; group II, mean FIO2 < 0.5; and group III, those breathing spontaneously. During the first posttrauma period the log ACR and the PO2/FIO2 ratio were inversely related (r = -0.712; p < 0.001), and the log ACR predicted PO2/FIO2 independent of ISS (p = 0.001). The log mean ACR (SD) for groups I and III were 34.0 (5.6) and 8.7 (2.9) mg/mmol, respectively (Mann Whitney p = 0.013). Following trauma, pulmonary dysfunction is associated with increased vascular permeability in remote organs.

Adolescent↗
PubMed1994

Independent localization of dystrophin N- and C-terminal regions to the sarcolemma of mdx mouse myofibres in vivo.

Dystrophin has been proposed to associate with the skeletal muscle membrane by way of a glycoprotein complex that interacts with its C-terminal domains. Transfection of mdx mouse myotubes in culture or myofibres in vivo with recombinant genes encoding human dystrophin deletion mutants shows, however, that not only the C terminus of dystrophin but also its N-terminal actin-binding domain can locate independently to the muscle sarcolemma. This observation suggests that lack of sarcolemma-associated dystrophin in Duchenne muscular dystrophy (DMD) muscle may result from enhanced degradation of truncated mutation products rather than their inability per se to associate with the sarcolemma.

Animals↗
PubMed1993

Trans-activation of the murine dystrophin gene in human-mouse hybrid myotubes.

Myotube cultures of the myogenic cell line, C2, produce significantly lower levels of dystrophin than primary mouse cultures. We demonstrate that expression of the C2 dystrophin gene increases 10-fold in hybrid myotubes formed by fusion of C2 and dystrophin-deficient human myoblasts from a Duchenne muscular dystrophy patient. These results indicate that C2 cells are deficient in endogenous gene regulatory factors which enhance dystrophin expression, and that the C2 cell line may therefore be used to identify putative trans-acting factors involved in the regulation of dystrophin gene expression.

Animals↗
PubMed1993

Magnetic affinity cell sorting (MACS) separation and flow cytometric characterization of neural cell adhesion molecule-positive, cultured myogenic cells from normal and dystrophic dogs.

We developed a magnetic affinity cell sorting (MACS) assay based on differential expression of neural cell adhesion molecule (NCAM) isoforms in muscle cell cultures from normal and dystrophic dogs. NCAM is expressed during normal muscle differentiation, but has not been extensively examined within the context of muscle disease. A myogenic MACS assay could potentially maximize chances of obtaining normal nonsenescent, low-passage myogenic cells capable of proliferating in vitro and in vivo following transplantation. Myoblast-specific anti-NCAM polyclonal antibody directed against the NCAM isoform associated with muscle cell proliferation more effectively separated mixed canine cultures than did monoclonal antibodies directed against differentiated NCAM isoforms in the MACS assay. Flow cytometry using 5.1H11 anti-NCAM monoclonal antibody was then performed on normal and dystrophic fractionated cells and the results from these two groups were compared to each other and to nonfractionated cell populations. Normal canine cell cultures that had not been separated contained a larger percentage of FACscan-positive cells than did corresponding dystrophic canine cell cultures. Prior polyclonal anti-NCAM MACS separation of dystrophic cultures yielded higher numbers of adherent cells and higher gating percentages of 5.1H11-positive cell populations than did normal cultures. However, cells from dystrophic animals exhibited lower mean fluorescent expression of NCAM than normal cells.

Animals↗