Dominic J. Wells

Zebrafish melanopsin: Isolation, tissue localisation and phylogenetic position

Photoreception is best understood in retinal rods and cones, but it is not confined to these cells. In non-mammals, intrinsically photosensitive cells have been identified within several structures including the pineal, hypothalamus and skin. More recently novel light sensitive cells have been identified in the inner/basal retina of both teleosts and rodents. Melanopsin has been proposed as the photopigment mediating many of these non-rod, non-cone responses to light. However, much about the melanopsin gene family remains to be clarified including their potential role as photopigments, and taxonomic distribution. We have isolated the first orthologue of melanopsin from a teleost fish and show expression of this gene in a sub-set of retinal horizontal cells (type B). Zebrafish melanopsin, and orthologues of this gene, differ markedly from the vertebrate photopigment opsins. The putative counterion is not a glutamate but a tyrosine, the putative G-protein binding domain in the third cytoplasmic loop is not conserved, and they show low levels of amino acid identity (approximately 27%) to both the known photopigment opsins and to other members of the melanopsin family. Mouse melanopsin is only 58% identical to Xenopus, and 68% identical to zebrafish. By contrast, the photosensory opsin families show approximately 75% conservation. On the basis of their structure, genomic organisation, discrete evolutionary lineage, and their co-expression with other opsins, the melanopins are not obvious photosensory opsins. They might represent a separate branch of photopigment evolution in the vertebrates or they may have a non-direct photosensory function, perhaps as a photoisomerase, in non-rod, non-cone light detection.

Immune responses to dystrophin: implications for gene therapy of Duchenne muscular dystrophy

Introduction of dystrophin by gene transfer into the dystrophic muscles of Duchenne muscular dystrophy (DMD) patients has the possibility of triggering an immune response as many patients will not have been exposed to some (or all) of the epitopes of dystrophin. This could in turn lead to cytotoxic destruction of transfected muscle fibres. We assessed such concerns in the dystrophin-deficient mdx mouse using plasmid DNA as the gene transfer system. This avoids complications associated with the administration of viral proteins. Gene transfer of cDNAs encoding mouse full-length or a truncated minidystrophin did not evoke either a humoral or cytotoxic immune response. Mdx mice may be tolerant due to the presence of rare ‘revertant’ dystrophin-positive fibres in their skeletal muscles. In contrast, gene transfer of human full-length or minidystrophin provoked both humoral and cytotoxic responses leading to destruction of the transfected fibres. These experiments demonstrate the potential risk of deleterious effects following gene therapy in DMD patients and lead us to suggest that patients enrolled in gene therapy trials should ideally have small, preferably point, mutations and evidence of ‘revertant’ dystrophin-positive muscle fibres.

Relocalization of neuronal nitric oxide synthase (nNOS) as a marker for complete restoration of the dystrophin associated protein complex in skeletal muscle

A lack of effective treatments for Duchenne muscular dystrophy, a fatal X-linked myopathy, has focused attention on the possibility of gene therapy. The aim of the gene therapy approach is the restoration of the dystrophin associated complex of proteins, one member of which is neuronal nitric oxide synthase, an important enzyme in signal transduction. Transgenic mdx mice and plasmid gene transfer of both human and murine recombinant dystrophins was used to assess whether nNOS could be restored to the sarcolemma following dystrophin gene transfer at a variety of levels of expression. Murine revertant fibres and human patients with different dystrophin deletions were used to assess the relationship between exon deletion and loss of neuronal nitric oxide synthase localization to the sarcolemma. We demonstrate that the domain encoded by exons 45-48 is required for localization of neuronal nitric oxide synthase to the sarcolemma. On the basis of these observations we suggest that neuronal nitric oxide synthase is a useful marker for complete restoration of the dystrophin associated complex and should be used as one of the criteria for selecting the recombinant molecule to be used for gene therapy in Duchenne muscular dystrophy.

An IL-7 fusion protein that shows increased thymopoietic ability

The role of IL-7 during thymopoiesis has led to it being the focus of a number of therapeutic interventions. However, its small size and pleiotropic nature present problems for thymus-directed therapies. We have created a fusion molecule between the extracellular N-terminal domain of CCR9 and IL-7, which has the potential to overcome these difficulties. This novel fusion protein retains the thymopoietic activity of IL-7 and the ligand-binding ability of CCR9. As a thymopoietic agent, compared with IL-7, it shows an enhanced retention in the thymus, increased de novo T cell production, and increased thymic output. Old mice receiving the fusion protein show improved CD8 T cell responses and reduced viral load after infection with influenza virus compared with those receiving IL-7. This chimeric molecule offers a novel therapeutic strategy that may result in the production of an effective immunorestorative agent.

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins

One of the possible therapies for Duchenne muscular dystrophy (DMD) is the introduction of a functional copy of the dystrophin gene into the patient. For this approach to be effective, therapeutic levels and long-term expression of the protein need to be achieved. However, immune responses to the newly expressed dystrophin have been predicted, particularly in DMD patients who express no dystrophin or only very truncated versions. In a previous study, we demonstrated a strong humoral and cytotoxic immune response to human dystrophin in the mdx mouse. However, the mdx mouse was tolerant to murine dystrophin, possibly due to the endogenous expression of dystrophin in revertant fibres or the other nonmuscle dystrophin isoforms. In the present study, we delivered human and murine dystrophin plasmids by electrotransfer after hyaluronidase pretreatment to increase gene transfer efficiencies. Tolerance to murine dystrophin was still seen with this improved gene delivery. Tolerance to exogenous recombinant full-length human dystrophin was seen in mdx transgenic lines expressing internally deleted versions of human dystrophin. These results suggest that the presence of revertant fibres may prevent the development of serious immune responses in patients undergoing dystrophin gene therapy.

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.

Ectopic Expression of NCAM in Skeletal Muscle of Transgenic Mice Results in Terminal Sprouting at the Neuromuscular Junction and Altered Structure But Not Function

The neuromuscular system provides an excellent model for the analysis of molecular interactions involved in the development and plasticity of synaptic contacts. The neural cell adhesion molecule (NCAM) is believed to be involved in the development and plasticity of the neuromuscular junction, in particular the axonal sprouting response observed in paralyzed and denervated muscle. In order to explore the role of myofiber NCAM in modulating the differentiation of motor neurons, we generated transgenic mice expressing a GPI-anchored NCAM isoform that is normally found in developing and denervated muscle, under the control of a skeletal muscle-specific promoter. This results in the constitutive expression of NCAM at postnatal ages, a time when the endogenous mouse NCAM is absent from the myofiber. We found that a significant number of neuromuscular junctions in adult transgenic animals displayed terminal sprouting (>20%) reminiscent of that elicited in response to cessation of neuromuscular activity. Additionally, a significant increase in the size and complexity of neuromuscular synapses as a result of extensive intraterminal sprouting was detected. Electrophysiological studies, however, revealed no significant alterations of neuromuscular transmission at this highly efficient synapse. Sprouting in response to paralysis or following nerve crush was also significantly enhanced in transgenic animals. These results suggest that in this ectopic expression model NCAM can directly modulate synaptic structure and motor neuron-muscle interactions. The results contrast with knockout experiments of the NCAM gene, where very limited changes in the neuromuscular system were observed.

Therapeutic restoration of dystrophin expression in Duchenne muscular dystrophy.

It is 20xa0years since the discovery of the genetic defect causing Duchenne muscular dystrophy (DMD). This X-linked progressive and fatal myopathy is due to the absence of a functional version of a critical sub-sarcolemmal protein called dystrophin that appears to act both as a structural and as a signalling molecule in the muscle fibre. A number of molecular approaches have been developed to restore the expression of dystrophin in DMD patients. Pre-clinical experiments have demonstrated the potential of delivery of recombinant versions of the DMD gene using viral or non-viral vectors and importantly several of these systems are compatible with vascular delivery, an essential feature as all muscles are affected in this condition. Other studies have shown that antisense oligonucleotides can modify the splicing of the primary transcript to provide an internally truncated but still functional protein. Alternatively, in approximately 10–20% of cases it is possible to chemically persuade the translational machinery to read-through a pre-mature stop codon. The pre-clinical results of the last 4xa0years have encouraged the development of clinical trials for all of the above.

Recombinant micro-genes and dystrophin viral vectors

An effective gene therapy for Duchenne muscular dystrophy ideally relies on the ability to provide long-term expression to muscle tissue of the missing protein, dystrophin. Early work in the mdx mouse using a 6.3 kb mini-dystrophin cDNA, carried out in either adenoviral or retroviral vectors was generally successful, however, expression was only transient. In an attempt to remedy this problem, two approaches are being investigated. The first of these is a hybrid vector system that combines the efficacy of gene transfer into skeletal muscle of adenoviral vectors with the long-term stability of retroviral vectors. The second utilises the inherently efficient transducing properties and stability of the adeno-associated viral delivery system. Using highly truncated micro-dystrophin cDNAs we have shown that both vector systems were able to restore dystrophin and dystrophin-associated protein expression at the plasma membrane of mdx mice for prolonged periods of time. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. These studies suggest that hybrid adenoviral-retroviral and adeno-associated viral vectors are capable of ameliorating dystrophic pathology at the cellular level and as such are useful tools in the development of a gene therapy for Duchenne muscular dystrophy.