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Gene Replacement (gene + replacement)
Selected AbstractsLimb-girdle muscular dystrophy type 2D gene therapy restores ,-sarcoglycan and associated proteins,,ANNALS OF NEUROLOGY, Issue 3 2009Jerry R. Mendell MD Objective ,-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression. Methods A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis muscle was conducted. Control sides received saline. A 3-day course of methylprednisolone accompanied gene transfer without further immune suppression. Results No adverse events were encountered. SGCA gene expression increased 4,5-fold over control sides when examined at 6 weeks (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in the 3-month subject. Adeno-associated virus serotype 1 (AAV1)-neutralizing antibodies were seen as early as 2 weeks. Neither CD4+ nor CD8+ cells were increased over contralateral sides. Scattered foci of inflammation could be found, but showed features of programmed cell death. Enzyme-linked immunospot (ELISpot) showed no interferon-, response to ,-SG or AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 subjects successfully achieved study aims, precluding the need for additional enrollment. Interpretation The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy. Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design of vascular delivery gene therapy trials. Ann Neurol 2009;66:290,297 [source] From gene replacement to gene manipulationDEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 8 2007Peter Baxter No abstract is available for this article. [source] Allogeneic bone marrow transplantation with reduced conditioning (RC-BMT)EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2001Lars Vindeløv Abstract: Allogeneic bone marrow transplantation with conventional conditioning (CC-BMT) has the potential of curing various malignant and non-malignant diseases. The curative mechanisms encompass 1) stem cell support for myeloablative radio-chemotherapy, 2) the graft-versus-tumor (GVT) effect, 3) gene replacement for genetic diseases and 4) immunoablation for autoimmune diseases. CC-BMT is characterized by high intensity conditioning, the requirement of prolonged and expensive hospital treatment and a treatment related mortality (TRM) of 10,50% depending on diagnosis, disease stage, patient age and donor type. Recent preclinical and clinical progress has resulted in the emergence of new concepts and procedures that allow replacement of patient bone marrow and immune system with that of the donor by a transplant procedure with markedly reduced conditioning (RC-BMT). This type of transplant, sometimes referred to as mini-BMT, activates curative mechanisms 2,4, which for a number of diseases seems sufficient for cure. It avoids the severe organ toxicity of myeloablative radio-chemotherapy and the complications of profound neutropenia. Patients beyond the age limit of conventional BMT (50,60 yr) may therefore be candidates for this type of transplant as well as patients which because of other medical conditions or the type of disease for which the transplant is needed are poor candidates for CC-BMT. The procedure can be performed in an outpatient setting. The resulting cost reduction should contribute to making allogenic BMT more readily available. This review describes basic concepts and procedures involved in RC-BMT and summarizes preliminary results obtained with RC-BMT in different transplant centers. [source] Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategiesINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2003C.A. Collins Summary., Duchenne's muscular dystrophy (DMD) is a lethal childhood disease caused by mutations of the dystrophin gene, the protein product of which, dystrophin, has a vital role in maintaining muscle structure and function. Homologues of DMD have been identified in several animals including dogs, cats, mice, fish and invertebrates. The most notable of these are the extensively studied mdx mouse, a genetic and biochemical model of the human disease, and the muscular dystrophic Golden Retriever dog, which is the nearest pathological counterpart of DMD. These models have been used to explore potential therapeutic approaches along a number of avenues including gene replacement and cell transplantation strategies. High-throughput screening of pharmacological and genetic therapies could potentially be carried out in recently available smaller models such as zebrafish and Caenorhabditis elegans. It is possible that a successful treatment will eventually be identified through the integration of studies in multiple species differentially suited to addressing particular questions. [source] Age-dependent and tissue-specific CAG repeat instability occurs in mouse knock-in for a mutant Huntington's disease geneJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2001Hiroshi Ishiguro Abstract Huntington's disease (HD) is a neurodegenerative disorder characterized by the expansion of CAG repeats in exon 1 of the HD gene. To clarify the instability of expanded CAG repeats in HD patients, an HD model mouse has been generated by gene replacement with human exon 1 of the HD gene with expansion to 77 CAG repeats. Chimeric proteins composed of human mutated exon 1 and mouse huntingtin are expressed ubiquitously in brain and peripheral tissues. One or two CAG repeat expansion was found in litters from paternal transmission, whereas contraction of CAG repeat in litters was observed through maternal transmission. Elderly mice show greater CAG repeat instability than younger mice, and a unique case was observed of an expanded 97 CAG repeat mouse. Somatic CAG repeat instability is particularly pronounced in the liver, kidney, stomach, and brain but not in the cerebellum of 100-week-old mice. The same results of expanded CAG repeat instability as observed in this HD model mouse were confirmed in the human brain of HD patients. Glial fibrillary acidic protein (GFAP)-positive cells have been found to be increased in the substantia nigra (SN), globus pallidus (GP), and striatum (St) in the brains of 40-week-old affected mice, although without neuronal cell death. The CAG repeat instability and increase in GFAP-positive cells in this mouse model appear to mirror the abnormalities in HD patients. The HD model mouse may therefore have advantages for investigations of molecular mechanisms underlying instability of CAG repeats. J. Neurosci. Res. 65:289,297, 2001. © 2001 Wiley-Liss, Inc. [source] A highly efficient gene-targeting system for Aspergillus parasiticusLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2008P.-K. Chang Abstract Aims:, To establish a system that greatly increases the gene-targeting frequency in Aspergillus parasiticus. Methods and Results:, The ku70 gene, a gene of the nonhomologous end-joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O -methylsterigmatocystin (OMST). The NHEJ-deficient strain, RH,ku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA-topoisomerase I complex inhibitor, camptothecin, and the DNA-damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA -targeting frequency in RH,ku70 reached 96%. Conclusions:, The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ-deficient RH,ku70, easy creation of homologous ends for integration, and the ptr -based selection form a highly efficient gene-targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies. Significance and Impact of the Study:, The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus. [source] The tetraspanin BcPls1 is required for appressorium-mediated penetration of Botrytis cinerea into host plant leavesMOLECULAR MICROBIOLOGY, Issue 3 2004M. Gourgues Summary Animal tetraspanins are membrane proteins controlling cell adhesion, morphology and motility. In fungi, the tetraspanin MgPls1 controls an appressorial function required for the penetration of Magnaporthe grisea into host plants. An orthologue of MgPLS1, BcPLS1, was identified in the necrotrophic fungal plant pathogen Botrytis cinerea. We constructed a Bcpls1::bar null mutant by targeted gene replacement. Bcpls1::bar is not pathogenic on intact plant tissues of bean, tomato or rose, but it infects wounded plant tissues. Both wild type and Bcpls1::bar differentiate appressoria on plant and artificial surfaces, a process involving an arrest of polarized growth, apex swelling and its cell wall reinforcement. Although wild-type appressoria allowed the penetration of the fungus into the host plant within 6,12 h, no successful penetration events were observed with Bcpls1::bar, suggesting that its appressoria are not functional. An eGFP transcriptional fusion showed that BcPLS1 was specifically expressed in conidia, germ tubes and appressoria during host penetration. Our results indicate that BcPLS1 is required for the penetration of B. cinerea into intact host plants. The defect in pathogenicity of Bcpls1::bar also demonstrates that functional B. cinerea appressoria are required for a successful penetration process. As Bcpls1::bar and Mgpls1,::hph penetration defects are similar, fungal tetraspanins are likely to be required for an essential appressorial function widespread among fungi. [source] Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregationMOLECULAR MICROBIOLOGY, Issue 5 2002Vinod S. Dubey Summary Cell wall lipids of Mycobacterium tuberculosis containing multiple methylbranched fatty acids play critical roles in pathogenesis and thus offer targets for new antimycobacterial drugs. Mycocerosic acid synthase gene (mas) encodes the enzyme that produces one class of such acids. Seven mas -like genes (msls) were identified in the genome. One of them, msl3, originally annotated as two separate genes, pks 3 and pks 4, is now shown to constitute a single open reading frame, which encodes a 220.3 kDa protein. Msl3 was disrupted using a phage mediated delivery system and the gene replacement in the mutant was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Biochemical analysis showed that the msl3 mutant does not produce mycolipanoic acids and mycolipenic (phthienoic) acids, the major constituents of polyacyl trehaloses and thus lacks this cell wall lipid, but synthesizes all of the other classes of lipids. The absence of the major acyl chains that anchor the surface-exposed acyltrehaloses causes a novel growth morphology; the cells stick to each other, most probably via the intercellular interaction between the exposed hydrophobic cell surfaces, manifesting a bead-like growth morphology without affecting the overall growth rate. [source] Genome-wide transcriptomic and proteomic analysis of the primary response to phosphate limitation in Streptomyces coelicolor M145 and in a ,phoP mutantPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2007Antonio Rodríguez-García Abstract Phosphate limitation in Streptomyces and in other bacteria triggers expression changes of a large number of genes. This response is mediated by the two-component PhoR,PhoP system. A Streptomyces coelicolor ,phoP mutant (lacking phoP) has been obtained by gene replacement. A genome-wide analysis of the primary response to phosphate limitation using transcriptomic and proteomic studies has been made in the parental S. coelicolor M145 and in the ,phoP mutant strains. Statistical analysis of the contrasts between the four sets of data generated (two strains under two phosphate conditions) allowed the classification of all genes into 12 types of profiles. The primary response to phosphate limitation involves upregulation of genes encoding scavenging enzymes needed to obtain phosphate from different phosphorylated organic compounds and overexpression of the high-affinity phosphate transport system pstSCAB. Clear interactions have been found between phosphate metabolism and expression of nitrogen-regulated genes and between phosphate and nitrate respiration genes. PhoP-dependent repressions of antibiotic biosynthesis and of the morphological differentiation genes correlated with the observed ,phoP mutant phenotype. Bioinformatic analysis of the presence of PHO boxes (PhoP-binding sequences) in the upstream regions of PhoP-controlled genes were validated by binding of PhoP, as shown by electrophoretic mobility shift assays. [source] Dysferlin overexpression in skeletal muscle produces a progressive myopathyANNALS OF NEUROLOGY, Issue 3 2010Louise E. Glover Objective The dose,response effects of dysferlin transgenesis were analyzed to determine if the dysferlin-deficient myopathies are good candidates for gene replacement therapy. Methods We have generated 3 lines of transgenic mice, expressing low, mid, and high levels of full-length human dysferlin from a muscle-specific promoter. Transgenic skeletal muscle was analyzed and scored for morphological and functional deficits. Results Overexpression of dysferlin in mice resulted in a striking phenotype of kyphosis, irregular gait, and reduced muscle mass and strength. Moreover, protein dosage correlated with phenotype severity. In contrast to dysferlin-null skeletal muscle, no evidence of sarcolemmal impairment was revealed. Rather, increased levels of Ca2+ -regulated, dysferlin-binding proteins and endoplasmic reticulum stress chaperone proteins were observed in muscle lysates from transgenic mice as compared with controls. Interpretation Expression levels of dysferlin are important for appropriate function without deleterious or cytotoxic effects. As a corollary, we propose that future endeavors in gene replacement for correction of dysferlinopathy should be tailored to take account of this. ANN NEUROL 2010;67:384,393 [source] The molecular genetics of the genodermatoses: progress to date and future directionsBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2003A.D. Irvine Summary The Human Genome Mapping Project and allied rapid advances in genetic technology over the past decade have facilitated accurate association of allelic variations in several genes with specific skin phenotypes. Currently the genetic bases of the majority of the more common genodermatoses have been elucidated. In scientific terms this work has been extraordinarily successful and has yielded many new biological insights. These advances, although exciting, have yet to be translated into direct benefit for patients with these diseases. Genetic counselling has been greatly aided by gene identification, by the better understanding of genotype,phenotype correlation and by the disclosure of unexpected genetic mechanisms in some families. Knowledge of the molecular basis of these disorders has also been vital in enabling DNA-based prenatal diagnosis in several conditions and DNA-based preimplantation diagnosis has been used in a selected few. While this successful period of gene mapping is now nearing completion, progress towards the next goal, that of developing therapeutic strategies based on the knowledge of these underlying genetic mechanisms, has proven frustratingly slow. Despite the ready access to the skin compared with solid internal organs, the challenges of cutaneous gene therapy are legion and many technical issues need to be surmounted to enable gene replacement or modification of gene expression to have a useful role in these disorders. In this article we make a comprehensive review of progress to date in gene identification, genotype,phenotype correlation, prenatal diagnosis and cutaneous gene therapy, and we examine future directions for research in this field. [source] | |||||||||||||