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G Dickson

Publications and source records attributed to G Dickson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

3T3 Cells↗
PubMed1998

Effective restoration of dystrophin-associated proteins in vivo by adenovirus-mediated transfer of truncated dystrophin cDNAs.

A series of truncated dystrophin cDNAs (3.1-4.2 kbp) containing only three, three, two or one rod repeats with hinge 1 and 4 (named deltaDysAX2, AX11, AH3, M3, respectively) or no rod repeat retaining either hinge 1 or 4 (named deltaDysH1, H4, respectively) were constructed. These cDNAs were introduced into skeletal muscle of adult mdx mice using the adenovirus vector with a strong CAG promoter. deltaDysAX2, AX11, AH3 and deltaDysM3 expressed themselves successfully and recovered dystrophin-associated proteins effectively. Especially 3.7 kbp cDNA for deltaDysM3 offers the possibility of an approach utilizing newly developed virus vectors, such as an adeno-associated virus vector, toward gene therapy of Duchenne muscular dystrophy.

Adenoviridae↗
PubMed1998

Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle fibers.

In normal skeletal muscle fibers, dystrophin accumulates at the cytoplasmic face of the sarcolemma where it associates with dystrophin-associated proteins (DAPs). Several studies have recently shown that the neuronal isoform of nitric oxide synthase (nNOS) is also located at the sarcolemma, and that this membrane localization is mediated through interactions of nNOS with one of the DAPs, namely alpha 1-syntrophin. Since the lack of dystrophin in muscle fibers from Duchenne muscular dystrophy patients and mdx mice is accompanied by an absence of sarcolemmal nNOS, we examined in the present study, whether dystrophin gene replacement would lead to the restoration of nNOS at its appropriate subcellular location. To this end, tibialis anterior muscles from mdx4cv mice were directly injected with plasmid DNA encoding either full-length (pRSV-dys) or mini-(pRSV-dyB; lacking exons 17-48) dystrophin. For these experiments, we chose to study 10-week-old mdx4cv mice since at this developmental stage, muscles from these mice have already undergone several cycles of degeneration-regeneration. Immunofluorescence experiments performed on serial cross-sections revealed that approximately 50% of the dystrophin-positive fibers also exhibited significant levels of nNOS at their sarcolemma 2 weeks following gene transfer with pRSV-dys. Similar results were obtained with pRSV-dyB indicating that exons 17-48 of the dystrophin gene are not essential for the correct localization of nNOS in skeletal muscle fibers. Taken together with the recent demonstration that dystrophin gene transfer leads to significant physiological benefits our results suggest that dystrophin gene therapy using full-length or truncated dystrophin, also induces a rapid recovery of biochemical functions.

Animals↗
PubMed1998

Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER.

Cationic lipid-DNA complexes (lipoplexes) have been widely used as gene transfer vectors which avoid the adverse immunogenicity and potential for viraemia of viral vectors. With the long-term aim of gene transfer into skeletal muscle in vivo, we describe a direct in vitro comparison of two commercially available cationic lipid formulations, Lipofectamine and DOSPER. Optimisation of transfection was performed in the C2C12 mouse muscle cell line, before further studies in primary mouse myoblasts and C2C12 myotubes. Reporter gene constructs expressing either E. coli beta-galactosidase or green fluorescent protein (GFP) were used in order to evaluate transfection efficiency by histochemical staining or FACS analysis, respectively. Both lipid formulations were able to promote efficient, reproducible gene transfer in C2C12 cells, and to transfect primary mouse myoblast cultures successfully. However, DOSPER exhibited the important advantage of being able to transfect cells in the presence of serum of both bovine and murine origin. This feature allowed increased cell survival during in vitro transfections, and may be advantageous for direct in vivo gene transfer efficacy.

Animals↗
PubMed1998

Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors.

Plasma apolipoprotein AI (apoAI) and lecithin-cholesterol acyltransferase (LCAT) play important roles in reverse cholesterol transport, promoting the removal of excess cholesterol from peripheral cells and reducing formation of atherosclerotic lesions. Gene augmentation of either apoAI or LCAT, or both, are thus attractive targets for prevention or treatment of atherosclerosis. With the eventual aim of safe and efficient gene delivery to skeletal muscle, our chosen secretory platform for systemic delivery of anti-atherogenic proteins, we have constructed conventional and AAV-based plasmid vectors containing human apoAI or LCAT cDNAs; their efficacy was tested by lipoplex transfection of mouse C2C12 muscle cells or human 293 cells. The secretion of apoAI or LCAT by transduced cultures was two- to five-fold higher using AAV-based plasmid vectors than conventional plasmid vectors. Additionally, cells transfected with a bicistronic AAV-based vector containing an internal ribosome entry site (IRES) efficiently expressed both apoAI and LCAT simultaneously. Furthermore, AAV-based vector sequences were retained by host cells, whereas those of conventional plasmid vectors were lost. These studies indicate that ectopic overexpression of apoAI and LCAT in muscle tissue using AAV-based plasmid vectors might provide a feasible anti-atherogenic strategy in vivo.

Animals↗
PubMed1998

Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides.

Deletions and point mutations in the gene encoding the cytoskeletal protein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively. Frameshift mutations tend to result in a lack of dystrophin at the sarcolemma, destabilization of the membrane and degeneration of skeletal muscle. The mdx mouse is a valuable animal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy. This report represents a novel approach to correct dystrophin deficiency at the post-transcriptional level by transfection of muscle cells with antisense RNA. Essentially, 2'- O -methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after transfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22. Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relatively mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.

Animals↗
PubMed1998

[Expression of the human dystrophin gene in mdx mouse muscle fibers after transfection using liposomes and synthetic oligopeptides].

The number of dysrophin-positive fibers appearing in the femoral quadriceps muscle of mdx mice after injection of the full-length human dystrophin cDNA within the pHSADy plasmid was examined by means of immunohystochemical techniques. Transfection was carried out using lipofectamine (LFA), or synthetic oligopeptide complexes that provided the condensation of plasmid DNA (K8) and its release from endosomes gopeptide complexes that provided the condensation of plasmid DNA (K8) and its release from endosomes (JTS1). The LFA + pHSADy at a dose of 10 micrograms DNA did not affect the number of dystrophin-positive fibers at the site of injection (0.6-0.8%), whereas it caused a statistically significant increase in the number of these fibers in the same muscle of the contralateral leg (up to 2.3%). Injection of the SO + pHSADy complex resulted in the occurrence of dystrophin-positive muscle fibers characterized by a heterogeneous content and the distribution of dystrophin. The greatest number of dystrophin-positive fibers (about 16%) was observed under a ratio of pHSADy to K8 of 1:3 or 1:4. The observed maximal number of dystrophin-positive fibers after a single injection of SO + pHSADy was 3.8%, and it was 17.7% after three injections. These values were statistically significantly higher compared to intact mice (0.6%), the injection of pure plasmid (2.2%), or the intramuscular injection of sucrose (from 0.7 to 1.3%). A relatively high level of transfection (about 5%) was observed after an intracardiac injection of a large dose of the pHSADy (70 micrograms DNA). The perspectives of the targeted delivery of the dystrophin gene into muscles under conditions of parenteral administration are discussed.

Amino Acid Sequence↗
PubMed1997

Bacterial beta-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection.

Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial beta-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven by mouse leukaemia virus promotor (pMLVDy) were used throughout the studies. Musculus glutaeus superficialis of C57BL/6J and quadriceps femoris of mdx male mice were opened surgically under anesthesia and bombarded by means of the gene-gun technique originally developed by us. Different mixtures of gold and tungsten particles at ratios of 4:1, 1:1, 1:4 were applied. X-gal assay revealed marked beta-gal activity, both in total muscles and whole muscle fibers on histological sections, up to three months after transfection. The most intensive staining was observed after SV40-lacZ delivery. No staining was detected with LTR-lacZneo DNA as well as in untreated muscles. The higher tungsten particle concentration in the bombardment mixture correlated with more intense X-gal staining. At the gold/tungsten ratio of 1:4 the microparticles penetrated the musculus glutaeus superficialis and transfected the underlying musculus glutaeus medius as well. Immuno-cytochemical assay for human dystrophin revealed dystrophin positive myofibers (DPM) in the bombarded area up to two months after transfection. The proportion of DMP varied from 2.5% on day 17 up two 5% on day 60 after bombardment compared to only 0.5% in the control mdx mice. These results suggest the applicability of particle bombardment for gene delivery into muscle fibers.

Animals↗
PubMed1997

Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.

Animals↗
PubMed1997

Mini-dystrophin gene transfer in mdx4cv diaphragm muscle fibers increases sarcolemmal stability.

To date, all dystrophin gene transfer studies have been performed on mdx hindlimb skeletal muscles which in comparison to the severe deficits seen in muscles from patients afflicted with Duchenne muscular dystrophy (DMD), exhibit only modest morphological and functional changes. Since the mdx diaphragm muscle presents the same pathophysiological alterations characteristic of DMD muscles, we therefore injected recombinant plasmid DNA encoding the dystrophin mini-gene (pRSVdy-B) into diaphragm muscles of 10-week-old mdx4cv mice and examined the physiological consequences of dystrophin expression in a muscle that has undergone a phase of massive degeneration and regeneration. Immunoperoxidase and immunofluorescence experiments revealed that 1 and 3 weeks following gene transfer, approximately 17% of the fibers in a bundle of diaphragm muscle expressed dystrophin at the sarcolemma. Most importantly, this level of dystrophin expression was sufficient to protect all fibers present within these diaphragm muscle bundles from the damaging effects of repetitive lengthening contractions. In addition, dystrophin expression partially restored the ability of transduced mdx4cv muscle bundles to generate isometric tetanic tension following lengthening contractions. These results show that mini-dystrophin expression leads to rapid and significant functional improvements in diaphragm muscles of mdx4cv mice. Although these data provide encouraging results for future therapeutic strategies aimed at curing DMD, additional work will none the less be necessary to determine the full impact of dystrophin gene replacement. In this context, it is clear from the data presented here that the diaphragm muscle of the mdx mouse is an invaluable model system to address this critical issue.

Animals↗
PubMed1996

Transplantation of retroviral producer cells for in vivo gene transfer into mouse skeletal muscle.

We describe a new strategy for efficient in vivo gene transfer into skeletal muscle using retroviral vectors. Recombinant retroviral producer cells, previously treated with the cytostatic drug mitomycin C, were injected into regenerating muscle of adult nude, nude/mdx, and C57BL/10 mice. Using LacZ reporter gene activity, we detected efficient transduction in all mouse strains (Nude, mean 11%, range 4.2-21%; C57BL/10, mean 12%, range 3.4-20%; Nude mdx, mean 4.3%, range 2.1-7% at 4 weeks post-injection and 6.6%, range 1.3-12% at 12 weeks post-injection). Foreign gene expression was sustained at high levels for at least 3 months. This strategy allows muscle satellite cells to be transfected in vivo, forming a reservoir of the transgene for incorporation into new myofibers in subsequent rounds of degeneration and regeneration. Because of its efficiency and potentially broad application, this procedure represents a new strategy for in vivo genetic transfer in skeletal muscle and potentially in other tissues.

Animals↗
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↗