Co-delivery of indoleamine 2,3-dioxygenase prevents loss of expression of an antigenic transgene in dystrophic mouse muscles.

A significant problem affecting gene therapy approaches aiming at achieving long-term transgene expression is the immune response against the protein product of the therapeutic gene, which can reduce or eliminate the therapeutic effect. The problem is further exacerbated when therapy involves targeting an immunogenic tissue and/or one with a pre-existing inflammatory phenotype, such as dystrophic muscles. In this proof-of-principle study, we co-expressed a model antigen, bacterial ß-galactosidase, with an immunosuppressive factor, indoleamine 2,3-dioxygenase 1 (IDO1), in muscles of the mdx mouse model of Duchenne muscular dystrophy. This treatment prevented loss of expression of the transgene concomitant with significantly elevated expression of T-regulatory (Treg) markers in the IDO1-expressing muscles. Moreover, co-expression of IDO1 resulted in reduced serum levels of anti-ß-gal antibodies. These data indicate that co-expression of genes encoding immunomodulatory enzymes controlling kynurenine pathways provide a viable strategy for preventing loss of transgenes targeted into dystrophic muscles with pre-existing inflammation.

Impact of P2RX7 ablation on the morphological, mechanical and tissue properties of bones in a murine model of duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an inherited, lethal disorder characterised by progressive muscle degeneration and associated bone abnormalities. We have previously demonstrated that P2RX7 purinergic receptors contribute to the pathogenesis of DMD, and found that P2RX7 ablation alleviated the severity of the disease. In this work we have used a dystrophic mdx mouse crossed with the global P2RX7 receptor to generate a knockout mouse (mdx/P2X7-/-), and compared its morphometric, mechanical and tissue properties against those of mdx, as well as the wild type (WT) and the P2RX7 knockout (P2X7-/-). Micro-computed tomography (µCT), three-point bending testing, scanning electron microscopy (SEM) and nano-indentation were utilised in the study. The bones were analysed at approximately 4 weeks of age to examine the impact of P2RX7 ablation on the bone properties during the acute disease phase, before muscle wasting is fully developed. The results show that P2RX7 purinoceptor ablation has produced improvement or significant improvement in some of the morphological, the mechanical and the tissue properties of the dystrophic bones examined. Specifically, although the ablation produced smaller bones with significantly lower total cross-section area (Tt.Ar) and Second Moment of Area (SMA), significantly higher cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar) and trabecular bone volume fraction (BV/TV) are found in the mdx/P2X7-/- mice than in any other types. Further, the mdx/P2X7-/- bones have relatively higher average flexural strength, work-to-fracture and significantly higher strain to failure compared with those of mdx, suggesting greater resistance to fracture. Indentation modulus, elasticity and creep are also significantly improved in the knockout cortical bones over those of mdx. These findings seem to suggest that specific pharmacological blockade of P2RX7 may improve dystrophic bones, with a potential for therapeutic application in the treatment of the disease.

Changes in immunolocalisation of beta-dystroglycan and specific degradative enzymes in the osteoarthritic synovium.

OBJECTIVE: To investigate the immunolocalisation of beta-dystroglycan (beta-DG) and specific matrix metalloproteinases (MMPs)-3, -9, -13 and a disintegrin like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) within the joint tissues of patients with osteoarthritis (OA) and unaffected controls. DESIGN: Cartilage, synovium and synovial fluid were obtained from the hip joints of five osteoarthritic (patients undergoing total hip replacement) and five control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for beta-DG protein using Western blot technique and by immunohistochemistry for tissue distribution of beta-DG, MMP-3, -9, -13, and ADAMTS-4. RESULTS: beta-DG was detected in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Importantly, beta-DG was detected in endothelium of blood vessels of OA synovium, but not in the control endothelium. In the endothelium of osteoarthritic synovial blood vessels, beta-DG co-localised with MMP-3 and -9. MMP-13 and ADAMTS-4 showed no endothelial staining, and only weak staining of the vascular smooth muscle was found. In contrast, we did not detect beta-DG protein in cartilage or synovial fluid. CONCLUSIONS: beta-DG has been shown to have a role in angiogenesis, and our results demonstrate for the first time that there are clear differences in beta-DG staining between OA and control synovial blood vessels. The specific immunolocalisation of beta-DG within endothelium of inflamed OA blood vessels and its co-localisation with MMP-3 and -9, reported to have pro-angiogenic roles and believed to be involved in beta-DG cleavage, may also suggest that beta-DG plays a role in angiogenesis accompanying OA.

Intramuscular plasmid DNA injection can accelerate autoimmune responses.

We have investigated if the administration of plasmid vectors engineered for gene delivery into mammalian muscle induced the production of anti-double stranded (ds) DNA and anti-nuclear autoantibodies in normal and autoimmunity-prone mouse models. In normal mice, repeated injection of plasmid DNA did not trigger an anti-DNA response. The presence of eukaryotic transcription factor binding sites in plasmid vectors did not increase autoantibody formation in these animals. In contrast, repeated injection of such plasmids in autoimmunity-prone MRL/MpJ mice caused a significant increase in both anti-dsDNA and anti-nuclear antibody levels. Thus the repeated administration of bacterial plasmids containing eukaryotic promoter elements may induce immune responses with generation of antibodies cross-reacting not only with the mammalian DNA, but also with nuclear antigens. The potential for iatrogenic autoimmunity in susceptible individuals should be considered.

Structural diversity despite strong evolutionary conservation in the 5'-untranslated region of the P-type dystrophin transcript.

Analysis of the 5'-flanking regions of the Purkinje (P-) dystrophin genes and mRNAs in different species revealed strong sequence conservation but functional diversity. Multiple transcription initiation sites were identified in cerebella and muscles, tissues expressing P-dystrophin. The predominant initiation site was conserved, with another muscle-specific site located upstream. Despite sequence homology, significant tissue- and species-specific structural diversity in the P-type 5'-ends exists, including alternative splicing within the 5'-untranslated region combined with alternative splicing of intron 1. One amino terminus is conserved in mammals and, to a lesser extent, in chicken. However, alternative usage of ATG codons may result in a choice of N-termini or translation of short upstream ORFs in different species. Promoter activity of a fragment upstream of the cap site was shown by transient expression in myoblasts and in vivo following intramuscular injection. It is tissue- and developmentally regulated. Analysis of promoter deletions suggests the existence of negative regulatory elements in the proximal region.

Prospects and problems of gene therapy: an update.

The gene therapy approach can vary from delivering extra copies of a gene, through modifications of a genome using the properties of ribozymes or chimeraplasts, to injection of modified cells. For the treatment of genetic deficits the ultimate goal would be the repair of the mutated gene in the target tissue(s). The techniques required for such an approach are emerging, albeit slowly. Therefore, delivery of an extra copy of a normal gene in a specific vector remains the predominant approach. Moreover, this method finds wider applications in gene therapy relating to disorders other than heritable defects, e.g., malignancies, cardiovascular diseases and infections. The major and most intensive areas of research are: i) vectors and delivery methods, ii) regulation of transgene expression and iii) stability of expression. Targeting of the therapeutic gene is being accomplished by using viral vectors or non-viral delivery systems, either ex vivo or in vivo. The choice of vectors and delivery routes depends on the nature of the target cells and the required levels and stability of expression. Although there have been the first positive clinical results and significant technical achievements over the past 2 years, there are still obstacles to the development of effective clinical products and these remain largely unchanged. The most important barriers are the low levels and stability of expression and immune responses to vectors and/or gene products. The safety aspects of gene therapy have become painfully evident with the first death conclusively linked to gene therapy. The progress in AAV and lentiviral vectors, improved regulation of transgene expression and advances in stem cell technology are among the recent most exciting developments.

Kainate-evoked changes in dystrophin messenger RNA levels in the rat hippocampus.

Dystrophin and dystroglycan messenger RNAs are expressed in specific brain areas, including regions of the cortex and the hippocampus, and in such neurons dystrophin has been localized to postsynaptic densities. In the present study we examined by in situ hybridization the effect of neuronal activation and neurotoxicity induced by kainate and pentylenetetrazole administered in vivo on dystrophin and dystroglycan expression in the rat brain. Kainate injection resulted in a transient but dramatic decrease in dystrophin transcript levels in the dentate gyrus granule cells, neurons not affected by kainate neurotoxicity, 6 h after injection. There was also a strong, concomitant increase in dystrophin messenger RNA levels in the CA3 subfield. At 24-72 h after kainate injection, the dystrophin transcript in the dentate granule cells returned to control levels, while it decreased gradually in the CA subfields, coinciding with the neurodegeneration observed in these areas. Comparable results were obtained with pan-dystrophin probes and probes specific to the short, G-dystrophin (Dp71) isoform that predominates in the dentate gyrus. This indicates that any dystrophin transcript that might be expressed in these areas responds to kainate in the same manner. In contrast, kainate insult had no significant effect on the dystroglycan messenger RNA levels in these hippocampal areas at 6 h post-injection. At later times. however, there was a gradual decrease in the dystroglycan messenger RNA in those areas which respond to the kainate insult with extensive neuronal death. For comparison, seizures which are not associated with progressive neurodegeneration were induced by pentylenetetrazole: in this situation the dystrophin and dystroglycan messenger RNA levels remained unchanged in all areas of the hippocampal formation. Since activation of glutamate receptors is thought to be involved in some forms of synaptic plasticity in the adult hippocampus, our data indicate that the dystrophin gene behaves as a candidate plasticity-related gene responding to glutamate.

beta-dystrobrevin, a member of the dystrophin-related protein family.

The importance of dystrophin and its associated proteins in normal muscle function is now well established. Many of these proteins are expressed in nonmuscle tissues, particularly the brain. Here we describe the characterization of beta-dystrobrevin, a dystrophin-related protein that is abundantly expressed in brain and other tissues, but is not found in muscle. beta-dystrobrevin is encoded by a 2.5-kb alternatively spliced transcript that is found throughout the brain. In common with dystrophin, beta-dystrobrevin is found in neurons of the cortex and hippocampal formation but is not found in the brain microvasculature. In the brain, beta-dystrobrevin coimmunoprecipitates with the dystrophin isoforms Dp71 and Dp140. These data provide evidence that the composition of the dystrophin-associated protein complex in the brain differs from that in muscle. This finding may be relevant to the cognitive dysfunction affecting many patients with Duchenne muscular dystrophy.

Differential expression of syntrophins and analysis of alternatively spliced dystrophin transcripts in the mouse brain.

Expression of syntrophin genes, encoding members of the dystrophin-associated protein complex, was studied in the mouse brain. In the hippocampal formation there is distinctive co-localization of specific syntrophins with certain dystrophin isoforms in neurons, e.g. alpha1-syntrophin with the C-dystrophin in CA regions and beta2-syntrophin with the G-dystrophin in the dentate gyrus. Expression of the alpha1-syntrophin is predominant in CA regions and the olfactory bulb and it is also present in the cerebral cortex and the dentate gyrus. The beta2-syntrophin mRNA is most abundant in the dentate gyrus and is also evident in the pituitary, the cerebral cortex and in Ammon's horn and in traces in the caudate putamen. The choroid plexus was labelled by both alpha1- and beta2-syntrophin-specific probes. The expression of syntrophins in the brain correlates with expression of dystrophins and dystroglycan. There are brain areas such as the cerebral cortex where several different syntrophins and dystrophins are expressed together. Syntrophin expression co-localizes with utrophin in the choroid plexus and caudate putamen. Finally, no syntrophin was detected in the cerebellar Purkinje cells where the specific dystrophin isoform (P-type) is present. This specific distribution of syntrophins in the brain is particularly interesting, as muscle syntrophin interacts with neuronal nitric oxide synthase. This may suggest that the dystrophin-associated protein complex may be involved in synaptic organisation and signal transduction machinery in both muscle and neurons. The dystrophin isoform, with exons 71-74 spliced out and hence lacking syntrophin binding sites, had been believed to be predominant in the brain, but our analyses using in situ hybridization, S1 nuclease protection and the semi-quantitative polymerase chain reaction revealed that this alternatively spliced mRNA is a minor, low abundance form in the brain.

Gene transfer and expression of human alpha-galactosidase from mouse muscle in vitro and in vivo.

Lysosomal storage disorders are amenable to treatment by enzyme replacement. Genetic modification of muscle via direct injection of expression vectors might represent an alternative method of providing the defective enzymes, if adequate and long-lasting expression levels can be achieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme alpha-galactosidase (alpha-gal). In differentiated myotubes, a construct containing the myosin light chain 1/3 enhancer in combination with the human cytomegalovirus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit beta-myosin heavy chain promoter, or containing the CMV promoter only. Increased enzymatic activity was detectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in alpha-gal were able to take up the enzyme from medium conditioned by transfected myoblasts. This did not occur in the presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the first report in which a correctly processed form of human alpha-gal was expressed and secreted from differentiated muscle cells. Direct injection of a plasmid expression vector into mouse tibialis anterior muscle showed significantly increased levels of alpha-gal 7 days after injection.

Kainate-evoked modulation of gene expression in rat brain.

Kainate is a glutamate analog that produces neuronal excitation resulting in seizures within hours following its intraperitoneal injection into adult rats. Then, at 2-3 days after the treatment, neurodegeneration of apoptotic character can be observed in limbic system. As a consequence, plastic reorganization and glial reactivation phenomena occur. These physiological and pathological responses are reflected by specific changes in gene expression, that can be dissected according to their spatio-temporal patterns. The early phase of gene expression observed in all hippocampal subfields appears to reflect a sudden burst of spiking activity. Changes in mRNA levels restricted to dentate gyrus are suggestive of a link to neuronal plasticity. The late gene expression response implies its correlation either to neuronal cell death or glial reactivation, depending on cellular localization of gene products. Thus analysis of the temporal and spatial gene expression pattern in the hippocampus after kainate treatment may provide clues revealing specific phenomena to which gene expression could be attributed.

Gene therapy: panacea or placebo? I. Strategies and limitations of gene therapy.

Progress in genetics and molecular medicine has led to characterization of many disorders at the level of specific genes. Techniques for reliable diagnosis of these disorders have been developed in parallel. Attempts are currently being made to develop DNA-based therapeutic procedures to correct genetic diseases. These procedures are known under a common name of gene therapy. The initial step in gene therapy is the delivery of the gene of interest into target cells. There are several conceptually different approaches to achieve this, e.g. targeting can be accomplished by using viral vectors (transduction) or DNA-mediated routes (transformation) either ex vivo or in vivo. The selection of vector systems and the choice of delivering genes either into isolated cells or directly in the organism depend on factors like: the nature of target cell or organ, the levels of expression required, stability and/or regulation of expression, safety, etc. Many viruses have been adapted for use as vectors for gene therapy, according to their specific properties. Viral genomes have been modified to remove their ability to replicate and to increase the cloning capacity. Non-viral gene delivery systems rely on cellular mechanisms to import DNA into the cell nucleus and different methods to enhance DNA uptake have been attempted. Despire many significant achievements there are still obstacles to the development of effective clinical products. Most significant are the low levels and stability of expression of introduced genes and immune responses to vectors and/or gene products.

Gene therapy: panacea or placebo? II. Main applications of gene therapy.

For most disorders the ideal goal of gene therapy is the repair of the mutated gene in the target tissue. However, the techniques required for such an approach are still at an early stage of development. Most current research is directed towards delivery of normal gene sequences in order to generate active protein and compensate for the lack of endogenous production. This approach may be suitable for the treatment of recessive monogenic disorders, but is inappropriate for dominantly inherited disorders. Therefore, gene therapy was originally intended as a most promising approach for the treatment of inborn errors of metabolism. However, apart from the correction of heritable genetic disorders, gene delivery has many potential applications including treatment of malignancies, atherosclerosis and vascular proliferative disorders, rheumatoid arthritis and viral infections. The authors discuss significant examples marking progress in the development of gene therapy for specific diseases, present some hopes and hurdles that have arisen from some recent preclinical and early clinical trials.

Dystroglycan mRNA expression during normal and mdx mouse embryogenesis: a comparison with utrophin and the apo-dystrophins.

Alpha dystroglycan (156 kDa DAG) and beta dystroglycan (43 kDa DAG) are encoded by the same gene and are components of the dystrophin-associated membrane glycoprotein complex. The dystroglycans together with dystrophin form a link between the extracellular matrix and the intracellular cytoskeleton of the muscle fibre. Using in situ hybridisation to mRNA in embryo sections we have examined the expression of the mouse dystroglycan gene. Dystroglycan transcripts are ubiquitously expressed throughout development but are most abundant in cardiac, skeletal and smooth muscle and in ependymal cells lining the developing neural tube and brain. The expression patterns of dystroglycan and dystrophin overlap in major muscle systems during development, suggesting that the dystrophin-dystroglycan complex plays an important role during myogenesis. In contrast, the major sites of utrophin expression do not co-localize with those of dystroglycan suggesting that utrophin may interact with a distinct membrane-associated complex in these non-muscle sites. In mdx embryos the pattern of distribution of dystroglycan mRNA remains unchanged, as do those of utrophin and apo-dystrophin mRNAs. This observation implies that the observed changes in the relative abundance of DAGs and utrophin in dystrophin-deficient muscle occur post-transcriptionally.

Editing of human alpha-galactosidase RNA resulting in a pyrimidine to purine conversion.

During a study of the gene coding for alpha-galactosidase (EC 3.2.1.22), the lysosomal enzyme deficient in Fabry's disease, RT-PCR amplification of alpha-galactosidase mRNAs obtained from three different tissues isolated from males revealed a substantial number of clones with a U to A conversion at the nucleotide position 1187. Such a modification of the coding sequence would result in an amino acid substitution in the C-terminal region (Phe396Tyr) of the enzyme. Neither PCR analysis of the genomic sequence nor the RT-PCR amplification of RNA obtained by in vitro transcription of the wild-type cDNA showed this change in the sequence. Multiple genes, pseudogenes are allelic variants were excluded. Hence, we propose RNA editing as a mechanism responsible for this base change in the alpha-galactosidase RNA.

G-utrophin, the autosomal homologue of dystrophin Dp116, is expressed in sensory ganglia and brain.

The utrophin gene is closely related to the dystrophin gene in both sequence and genomic structure. The Duchenne muscular dystrophy (DMD) locus encodes three 14-kb dystrophin transcripts in addition to several smaller isoforms, one of which, Dp116, is specific to peripheral nerve. We describe here the corresponding 5.5-kb mRNA from the utrophin locus. This transcript, designated G-utrophin, is of particular interest because it is specifically expressed in the adult mouse brain and appears to be the predominant utrophin transcript in this tissue. G-utrophin is expressed in brain sites generally different from the regions expressing beta-dystroglycan. During mouse embryogenesis G-utrophin is also seen in the developing sensory ganglia. Our data confirm the close evolutionary relationships between the DMD and utrophin loci; however, the functions for the corresponding proteins probably differ.

Specific expression of G-dystrophin (Dp71) in the brain.

Duchenne muscular dystrophy is associated with mental retardation and several dystrophin transcripts are differentially expressed in specific brain areas. G-dystrophin (Dp71) is known to be the predominant isoform in the brain. We have localized its mRNA to be present predominantly in the dentate gyrus and in the olfactory bulb. This distribution is specific and significantly different from that for the full-size dystrophin transcripts, present mainly in CA regions of the hippocampus, in the cerebral cortex and in cerebellar Purkinje cells. Furthermore, our data show that the various dystrophins co-localize with the dystroglycan in the brain.