Jill McMahon

Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase-increased expression with reduced muscle damage

The efficiency of plasmid gene transfer to skeletal muscle can be significantly improved by the application of an electrical field to the muscle following injection of plasmid DNA. However, this electrotransfer is associated with significant muscle damage which may result in substantial loss of transfected muscle fibres. Reduction of the voltage used in the technique can result in a decrease in muscle damage, with a concomitant reduction in expression, but without a significant decrease in the number of transfected fibres. Pre-treatment of the muscle with a solution of bovine hyaluronidase greatly increases the efficiency of plasmid gene transfer when used in conjunction with electrotransfer, but not when used alone. This combination treatment results in greatly enhanced levels of transfected muscle fibres without the increases in muscle damage associated with the electrotransfer process.

A non-apoptotic role for caspase-9 in muscle differentiation

Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells. Reduction of caspase-9 levels, using an shRNA construct, prevented caspase-3 activation and inhibited myoblast fusion. Myosin-heavy-chain expression, which accompanies myoblastic differentiation, was not caspase-dependent. Overexpression of Bcl-xL, a protein that inhibits caspase-9 activation, had the same effect on muscle differentiation as knockdown of caspase-9. These data suggest that the mitochondrial pathway is required for differentiation; however, the release of cytochrome c or Smac (Diablo) could not be detected, raising the possibility of a novel mechanism of caspase-9 activation during muscle differentiation.

The significance of measles virus antigen and genome distribution in the CNS in SSPE for mechanisms of viral spread and demyelination.

The distribution of measles virus (MV) antigen and genomic RNA in the central nervous system (CNS) in 10 cases of subacute sclerosing panencephalitis (SSPE) was determined using optimized immunocytochemistry and in situ hybridization techniques. As in previous reports neurons and oligodendrocytes were found to be the most frequently infected cells. It was confirmed that MV infection in neuronal processes was predominantly dendritic but there was also some evidence suggestive of occasional axonal involvement, a finding confirmed by electron microscopy. In addition MV genomic RNA was detected in neuronal processes, in some cases in the absence of demonstrable MV antigen. The relationship between myelin destruction and oligodendrocytic infection suggested that the demyelination may be solely the result of virus infection. A possible correlation between viral distribution and form and the clinical duration of disease was examined. Viral antigen and genome were equally abundant in the cerebral cortex in most short duration cases (lt; 6 months). However, in two of these cases viral RNA but not antigen was detected in the spinal cord. In long duration cases (gt;36 months) viral RNA was abundant in all areas of the CNS examined, frequently in the absence of demonstrable antigen. These findings may suggest viral spread in a cephalo-caudal direction, probably by transneuronal spread

High-efficiency plasmid gene transfer into dystrophic muscle

The efficiency of plasmid gene transfer in skeletal muscle is significantly enhanced by pretreatment with hyaluronidase and the application of an electrical field to the muscle following the injection of plasmid DNA, a process referred to as electrotransfer. However, the presence of increased levels of connective tissue in muscular dystrophies, such as Duchenne muscular dystrophy (DMD), may affect the efficiency of this process. Here we demonstrate that the efficiency of electrotransfer is not affected by increased levels of connective tissue in the mdx mouse model of DMD and that any damage induced by the electrotransfer process is not exacerbated in the dystrophic phenotype. However, increasing the concentration of hyaluronidase does not improve transfection efficiencies further. Unlike direct injection of plasmid DNA, the efficiency of electrotransfer is not dependent upon the sex and age of mice used. The combined treatment of hyaluronidase and electrotransfer results in highly efficient gene transfer in dystrophic muscle with limited muscle damage.

Gene-eluting Stents: Adenovirus-mediated Delivery of eNOS to the Blood Vessel Wall Accelerates Re-endothelialization and Inhibits Restenosis

Drug-eluting stents for coronary artery disease results in inhibition of smooth muscle cell (SMC) and endothelial cells which may increase the risk of stent thrombosis. In this study, we attempted to enhance re-endothelialization of deployed stents while simultaneously inhibiting intimal hyperplasia by overexpression of endothelial nitric oxide synthase (eNOS) delivery in the vasculature using an adenovirus gene-eluting stent. Re-endothelialization was significantly greater in vessels obtained from normocholesterolemic animals at day 14 (85.34% +/- 7.38 versus 62.66% +/- 10.49; P < 0.05) and day 28 (91.1% +/- 10 versus 63.1% +/- 22; P < 0.05) and hypercholesterolemic animals (96.97% +/- 3.2 versus 28.33% +/- 38.76; P < 0.05) at day 28 with AdeNOS-eluting stents. At day 28, there was a significant increase in the lumen size [AdeNOS 2.73 mm(2) +/- 1.18, AdbetaGal 0.98 mm(2) +/- 0.98, phosphorylcholine (PC) 1.87 mm(2) +/- 1.18; P < 0.05], and a significant reduction in neointimal formation (AdeNOS 2.32 mm(2) +/- 1.13, AdbetaGal 3.73 mm(2) +/- 0.95, PC 3.2 mm(2) +/- 0.94; P < 0.05), and percent restenosis (AdeNOS 45.23 +/- 20.81, AdbetaGal 79.6 +/- 20.31, PC 70.16 +/- 22.2; P < 0.05) in AdeNOS-stented vessels in comparison with controls from hypercholesterolemic animals, assessed by morphometry and quantitative coronary angiography (AdeNOS 15.95% +/- 7.63, AdbetaGal 56.9% +/- 38.6, PC 58 +/- 34.6; P < 0.05). Stent-based delivery of AdeNOS results in enhanced endothelial regeneration and reduction in neointimal formation as compared with controls. This seems to be a promising treatment strategy for preventing in-stent restenosis (ISR) while simultaneously reducing the risk of stent thrombosis.

Increased expression of endoplasmic reticulum stress-related signaling pathway molecules in multiple sclerosis lesions

Activation of endoplasmic reticulum (ER) stress-related cell signals has been reported in several neurologic disorders and may contribute to neurodegeneration. Endoplasmic reticulum stress is also linked to ischemic injury. However, activation of an ER stress response has not been investigated in multiple sclerosis (MS) lesions. We detected increased expression of ER stress-associated C/EBP homologous protein, immunoglobulin heavy chain-binding protein, and X-box-binding protein 1 in multiple cell types, including oligodendrocytes, astrocytes, T cells, and microglia in active MS lesions. Semiquantitative analysis of expression in active, chronic active, and chronic inactive lesions indicated that levels of immunoglobulin heavy chain-binding protein were significantly higher in acute lesions than in non-MS controls or MS normal-appearing white matter, and that ER stress-associated C/EBP homologous protein was upregulated to the greatest extent at the edges of chronic active lesions. Because demyelination may be triggered by a tissue response to ischemia-like conditions, changes in the hypoxia-related antigen D-110 were also investigated, and it was found that increased ER stress-associated C/EBP homologous protein expression can occur in either the presence or absence of D-110. A possible link between a perturbed ER and lesion development in MS suggests a signaling pathway that may represent a new therapeutic target in MS.

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

IntroductionA combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.MethodsTransduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.ResultsThe second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.ConclusionsCollectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.

Immune responses, not promoter inactivation, are responsible for decreased long-term expression following plasmid gene transfer into skeletal muscle

Long‐term high‐level in vivo gene expression appears to depend on the promoter chosen to drive the gene of choice. In many cases the promoter appears to ‘switch off’ some time after in vivo gene transfer. We demonstrate that, following intramuscular injection of β‐galactosidase reporter plasmids, promoter ‘switch off’ is due to elimination of fibres expressing the transferred reporter gene by activation of a Th1 (cytotoxic) immune response. This finding, in the absence of stimulation of the immune system by viral vector proteins, has implications not only for gene transfer experiments but for the future of muscle‐directed gene therapy.

The effects of blood–brain barrier disruption on glial cell function in multiple sclerosis

Dysfunction of the BBB (blood-brain barrier) is a major hallmark of MS (multiple sclerosis). Studies in our laboratories over the last decade have shown that increased BBB permeability is associated with decreased expression of TJ (tight junction) proteins in brain capillary endothelial cells. Results have revealed that TJ abnormalities were most common in active lesions (42% of vessels affected), but were also present in inactive lesions (23%) and in MS normal-appearing white matter (13%). Importantly, TJ abnormality was also positively associated with leakage of the serum protein fibrinogen which has recently been shown to be an activator of microglia. TJ abnormality and the resultant vascular permeability in both lesional and non-lesional white matter may impair tissue homoeostasis, which may have effects on disease progression, repair mechanisms and drug delivery.

Apoptosis in measles virus-infected human central nervous system tissues

The extent of apoptotic cell death was examined in central nervous system (CNS) tissues from three cases of subacute sclerosing panencephalitis (SSPE). Apoptosis was demonstrated by in situ end‐labelling of DNA in formalin‐fixed, paraffin‐embedded tissue sections. Measles virus and cell types were labelled by immunohistochemistry and/or in situ hybridization. Furthermore, bcl‐2 expression in SSPE was examined by immunohistochemistry. All three cases exhibited varying degrees of apoptosis in all CNS areas studied. Brain tissue from a non‐neurological control case did not show any significant apoptosis. Characterization of cell types demonstrated neurons, oligodendrocytes, lymphocytes and microglia undergoing apoptosis. A linear relationship could not be established between virus burden and the extent of apoptosis in any particular area. Virus‐negative cells were observed which were undergoing apoptosis. Bcl‐2 immunoreactivity in SSPE was confined to the infiltrating cell population. These results suggest that apoptosis of various cell types may contribute to the neuropathogenesis of measles virus infection in the human CNS, either as a direct effect of viral infection or by cytokine‐mediated responses.