Loss of HtrA2/Omi activity in non-neuronal tissues of adult mice causes premature aging.

mnd2 mice die prematurely as a result of neurodegeneration 30-40 days after birth due to loss of the enzymatic activity of the mitochondrial quality control protease HtrA2/Omi. Here, we show that transgenic expression of human HtrA2/Omi in the central nervous system of mnd2 mice rescues them from neurodegeneration and prevents their premature death. Interestingly, adult transgenic mnd2 mice develop accelerated aging phenotypes, such as premature weight loss, hair loss, reduced fertility, curvature of the spine, heart enlargement, increased autophagy, and death by 12-17 months of age. These mice also have elevated levels of clonally expanded mitochondrial DNA (mtDNA) deletions in their tissues. Our results provide direct genetic evidence linking mitochondrial protein quality control to mtDNA deletions and aging in mammals.

Gene delivery of antioxidant enzymes inhibits human immunodeficiency virus type 1 gp120-induced expression of caspases.

Caspases are implicated in neuronal death in neurodegenerative and other central nervous system (CNS) diseases. In a rat model of human immunodeficiency virus type 1 (HIV-1) associated neurocognitive disorders (HAND), we previously characterized HIV-1 envelope gp120-induced neuronal apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. In this model, neuronal apoptosis occurred probably via gp120-induced reactive oxygen species (ROS). Antioxidant gene delivery blunted gp120-related apoptosis. Here, we studied the effect of gp120 on different caspases (3, 6, 8, 9) expression. Caspases production increased in the rat caudate-putamen (CP) 6h after gp120 injection into the same structure. The expression of caspases peaked by 24h. Caspases colocalized mainly with neurons. Prior gene delivery of the antioxidant enzymes Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) into the CP before injecting gp120 there reduced levels of gp120-induced caspases, recapitulating the effect of antioxidant enzymes on gp120-induced apoptosis observed by TUNEL. Thus, HIV-1 gp120 increased caspases expression in the CP. Prior antioxidant enzyme treatment mitigated production of these caspases, probably by reducing ROS levels.

Intracisternal rSV40 administration provides effective pan-CNS transgene expression.

Potential genetic treatments for many generalized central nervous system (CNS) diseases require transgene expression throughout the CNS. Using oxidant stress and apoptosis caused by HIV-1 envelope gp120 as a model, we studied pan-CNS neuroprotective gene delivery into the cisterna magna (CM). Recombinant SV40 vectors carrying Cu/Zn superoxide dismutase or glutathione peroxidase were injected into rat CMs following intraperitoneal administration of mannitol. Sustained transgene expression was seen in neurons throughout the CNS. On challenge, 8 weeks later with gp120 injected into the caudate putamen, significant neuroprotection was documented. Thus, intracisternal administration of antioxidant-carrying rSV40 vectors may be useful in treating widespread CNS diseases such as HIV-1-associated neurocognitive disorders characterized by oxidative stress.

Muscle-directed gene therapy for hemophilia B with more efficient and less immunogenic AAV vectors.

BACKGROUND: Adeno-associated viral vector (AAV)-mediated and muscle-directed gene therapy is a safe and non-invasive approach to treatment of hemophilia B and other genetic diseases. However, low efficiency of transduction, inhibitor formation and high prevalence of pre-existing immunity to the AAV capsid in humans remain as main challenges for AAV2-based vectors using this strategy. Vectors packaged with AAV7, 8 and 9 serotypes have improved gene transfer efficiencies and may provide potential alternatives to overcome these problems. OBJECTIVE: To compare the long-term expression of canine factor IX (cFIX) levels and anti-cFIX antibody responses following intramuscular injection of vectors packaged with AAV1, 2, 5, 7, 8 and 9 capsid in immunocompetent hemophilia B mice. RESULTS: Highest expression was detected in mice injected with AAV2/8 vector (28% of normal), followed by AAV2/9 (15%) and AAV2/7 (10%). cFIX expression by AAV2/1 only ranged from 0 to 5% of normal levels. High incidences of anti-cFIX inhibitor (IgG) were detected in mice injected with AAV2 and 2/5 vectors, followed by AAV2/1. None of the mice treated with AAV2/7, 2/8 and 2/9 developed inhibitors or capsid T cells. CONCLUSIONS: AAV7, 8 and 9 are more efficient and safer vectors for muscle-directed gene therapy with high levels of transgene expression and absence of inhibitor formation. The absence of antibody response to transgene by AAV7, 8 and 9 is independent of vector dose but may be due to the fact that these three serotypes are associated with high level distribution to, and transduction of, hepatocytes following i.m. injection.

Bone marrow-derived cells migrate to line the vessels of the CNS: opportunities for gene delivery to CNS vasculature.

Using bone marrow (BM)-directed gene transfer and permanent transduction via recombinant SV40-derived vectors, we previously reported that BM-derived cells may be progenitors of CNS cells, such as neurons in normal adult animals. In this study, we asked whether the same was true for the CNS blood vessels, that is, whether marrow-resident precursors can migrate to the vasculature of the CNS. SV40-derived gene delivery vectors, carrying marker epitopes (FLAG or AU1), appended to carrier proteins, were injected directly into the femoral BM of rats or rabbits. Controls received intramarrow SV(BUGT), a control vector. Transgene expression was then examined in the vasculature. Endothelial cells expressing the transgenes were observed in the vessels of the striatum, principally localized in laminin- or CD31-positive structures (markers of brain blood vessels). Results in both animal models and with both transgenes were similar. Thus, under physiologic conditions and in the absence of CNS or vascular injury, BM-derived cells can migrate to, and form an endothelial lining for, brain blood vessels. Intramarrow gene delivery may provide an avenue to deliver genes to the vascular endothelium of the CNS.

Gene transfer to the rhesus monkey brain using SV40-derived vectors is durable and safe.

Gene transfer to central nervous system (CNS) has been approached using various vectors. Recombinant SV40-derived vectors (rSV40s) transduce human neurons and microglia effectively in vitro and in rodent brains in vivo, so we tested rSV40s gene transfer to rhesus monkey CNS in vivo, to characterize the distribution, duration and safety of such gene delivery. We used rSV40s carrying HIV-1 RevM10 with a carboxyl-terminal AU1 epitope tag as a marker, and others with the antioxidant enzymes, Cu/Zn superoxide dismutase and glutathione peroxidase. Vectors were injected stereotaxically into the caudate nucleus. Transgene expression was studied at 1 and 6 months by immunostaining serial brain sections. After intraparenchymal administration, numerous transgene-expressing cells were seen, with a longitudinal extent of 20 mm. In neurons and, more rarely, microglial cells, transgene expression remained strong throughout the 6-month study period. Astrocytes and oligodendroglia were not transduced. No evidence of inflammation or tissue damage was observed. SV40-derived vectors may thus be useful for long-term gene expression in the monkey brain and, potentially, in the human brain.

In vitro and in vivo functional characterization of gutless recombinant SV40-derived CFTR vectors.

In cystic fibrosis (CF), respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa. Despite considerable improvement in patient survival, conventional therapies are mainly supportive. Recent progress toward gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy. As alternative gene therapy vectors for CF, we examined the feasibility of using recombinant SV40-derived vectors (rSV40s), which may circumvent some of these obstacles. To accommodate the large cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, we removed not only SV40 Tag genes, but also all capsid genes. We, therefore, tested whether 'gutless' rSV40s could be packaged and were able to express a functional human CFTR cDNA. The results from our in vitro analysis determined that rSV40-CFTR was able to successfully result in the expression of CFTR protein, which localized to the plasma membrane and restored channel function to CFTR-deficient cells. Similarly, in vivo experiments delivering rSV40-CFTR to the lungs of Cftr-/- mice resulted in a reduction of the pathology associated with intra-tracheal P. aeruginosa challenge. rSV40-CFTR-treated mice had less weight loss when compared with control-treated mice as well as demonstrably reduced lung inflammation as evidence by histology and reduced inflammatory cytokines in the broncho-alveolar lavage. The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways. These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted.

Protecting neurons from HIV-1 gp120-induced oxidant stress using both localized intracerebral and generalized intraventricular administration of antioxidant enzymes delivered by SV40-derived vectors.

Human immunodeficiency virus-1 (HIV-1) is the most frequent cause of dementia in adults under 40. We sought to use gene delivery to protect from HIV-1-related neuron loss. Because HIV-1 envelope (Env) gp120 elicits oxidant stress and apoptosis in cultured neurons, we established reproducible parameters of Env-mediated neurotoxicity in vivo, then tested neuroprotection using gene delivery of antioxidant enzymes. We injected 100-500 ng mul(-1)gp120 stereotaxically into rat caudate-putamens (CP) and assayed brains for apoptosis by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) 6-h to 14-day post-injection. Peak apoptosis occurred 1 day after injection of 250 and 500 ng microl(-1)gp120. TUNEL-positive cells mostly expressed neuronal markers (NeuroTrace), although some expressed CD68 and so were most likely microglial cells. Finally, we compared neuroprotection from gp120-induced apoptosis provided by localized and generalized intra-central nervous system (CNS) gene delivery. Recombinant SV40 vectors carrying Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) were injected into the CP, where gp120 was administered 4-24 weeks later. Alternatively, we inoculated the vector into the lateral ventricle (LV), with or without prior intraperitoneal (i.p.) administration of mannitol. Intracerebral injection of SV(SOD1) or SV(GPx1) significantly protected neurons from gp120-induced apoptosis throughout the 24-week study. Intraventricular vector administration protected from gp120 neurotoxicity comparably, particularly if preceded by mannitol i.p. Thus, HIV-1 gp120 is neurotoxic in vivo, and intracerebral or intra-ventricular administration of rSV40 vectors carrying antioxidant enzymes is neuroprotective. These findings suggest the potential utility of both localized and widespread gene delivery in treating neuroAIDS and other CNS diseases characterized by excessive oxidative stress.

Strategies for CNS-directed gene delivery: in vivo gene transfer to the brain using SV40-derived vectors.

Gene transfer to the central nervous system (CNS) has been approached using various vectors. Recombinant SV40-derived vectors (rSV40s) transduce neurons and microglia effectively in vitro, so we tested rSV40s gene transfer to the CNS in vivo, and characterized the distribution, duration and cell types transduced. We used rSV40s carrying Human Immunodeficiency Virus Type 1 Net protein (HIV-1 Nef) with a C-terminal FLAG epitope tag as a marker, and another with Cu/Zn superoxide dismutase (SOD1). Rats were given vectors stereotaxically, either intraparenchymally into the caudate-putamen (CP) or into the lateral ventricle (LV). FLAG expression was studied for 3 months by immunostaining serial brain sections. After intraparenchymal administration, numerous transgene-expressing cells were seen, many as far as 4 mm from the injection site. Transgene expression remained strong throughout the 3-month study period. Coimmunostaining for lineage markers showed that neurons and, more rarely, microglial cells were tranduced, except astrocytes and oligodendroglia. After injection into the LV, high levels of transgene expression were detected throughout the frontal cortex by Western analysis. Systemic mannitol-induced hyperosmolarity further augmented LV transgene delivery. SV40-derived vectors may, thus, be useful for long-term gene expression in the brain, whether locally by intraparenchymal administration or diffusely by intraventricular injection, with or without mannitol.

Antioxidant enzyme gene delivery to protect from HIV-1 gp120-induced neuronal apoptosis.

Human immunodeficiency virus-1 (HIV-1) infection in the central nervous system (CNS) may lead to neuronal loss and progressively deteriorating CNS function: HIV-1 gene products, especially gp120, induce free radical-mediated apoptosis. Reactive oxygen species (ROS), are among the potential mediators of these effects. Neurons readily form ROS after gp120 exposure, and so might be protected from ROS-mediated injury by antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD1) and/or glutathione peroxidase (GPx1). Both enzymes detoxify oxygen free radicals. As they are highly efficient gene delivery vehicles for neurons, recombinant SV40-derived vectors were used for these studies. Cultured mature neurons derived from NT2 cells and primary fetal neurons were transduced with rSV40 vectors carrying human SOD1 and/or GPx1 cDNAs, then exposed to gp120. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Transduction efficiency of both neuron populations was >95%, as assayed by immunostaining. Transgene expression was also ascertained by Western blotting and direct assays of enzyme activity. Gp120 induced apoptosis in a high percentage of unprotected NT2-N. Transduction with SV(SOD1) and SV(GPx1) before gp120 challenge reduced neuronal apoptosis by >90%. Even greater protection was seen in cells treated with both vectors in sequence. Given singly or in combination, they protect neuronal cells from HIV-1-gp120 induced apoptosis. We tested whether rSV40 s can deliver antioxidant enzymes to the CNS in vivo: intracerebral injection of SV(SOD1) or SV(GPx1) into the caudate putamen of rat brain yielded excellent transgene expression in neurons. In vivo transduction using SV(SOD1) also protected neurons from subsequent gp120-induced apoptosis after injection of both into the caudate putamen of rat brain. Thus, SOD1 and GPx1 can be delivered by SV40 vectors in vitro or in vivo. This approach may merit consideration for therapies in HIV-1-induced encephalopathy.

Phase I dose-finding and pharmacokinetic study of docetaxel and vinorelbine as first-line chemotherapy for metastatic breast cancer.

BACKGROUND AND PURPOSE: Anthracycline-containing regimens are widely used in advanced breast cancer. However, there is a need for new, non-anthracycline regimens that are active in patients for whom anthracyclines are contraindicated. The aim of this study was to determine the maximum tolerated dose (MTD), the dose-limiting toxicities (DLTs) and recommended doses of docetaxel and vinorelbine as first-line chemotherapy in patients with metastatic breast cancer. The pharmacokinetics of both drugs was also evaluated. PATIENTS AND METHODS: Thirty-four women with first-line metastatic breast cancer were treated with docetaxel, 60-100 mg/m2 (day 1), and vinorelbine, 20-22.5 mg/m2 (days 1 and 5), repeated every three weeks and administered on an outpatient basis. RESULTS: Two MTDs were determined: MTD1 was defined at the dose level using docetaxel 75 mg/m2, and vinorelbine 22.5 mg/m2 DLT being a grade 3 stomatitis that was more related to the dose of vinorelbine than that of docetaxel. Therefore, the study continued with a fixed dose of vinorelbine, 20 mg/m2, and docetaxel 85-100 mg/m2. MTD2 was defined at the dose level combining docetaxel, 100 mg/m2, and vinorelbine, 20 mg/m2; DLTs were grade 3 stomatitis and severe asthenia. Fluid retention was observed in 41% of patients but was never severe or a reason for patient discontinuation. In comparison with historical experience, Daflon 500 did not seem to increase the efficacy of the three-day corticosteroid premedication by further reducing the incidence or severity of fluid retention. No significant neurotoxicity was observed and no patient discontinued the study due to this site effect. Activity was observed at all dose levels and at all metastatic sites, with an overall response rate of 71% (95% CI: 52.0%-85.8%). The median time to progression was 31.4 weeks (95% CI: 12-48 weeks) and median survival was 15.6 months (95% CI: 2.6-26.6 months). The pharmacokinetics of docetaxel and vinorelbine were not modified between day 1 and day 3 when the two drugs were combined with the day 1 administration schedule used in this study. CONCLUSION: The recommended doses for phase II studies are docetaxel, 75 mg/m2 (day 1), plus vinorelbine, 20 mg/m2 (days 1 and 5), repeated every three weeks. At these doses, the combination was found to be active and well tolerated.

iNOS expression in dystrophinopathies can be reduced by somatic gene transfer of dystrophin or utrophin.

BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy.

X-linked vacuolated myopathy : TNF-alpha and IFN-gamma expression in muscle fibers with MHC class I on sarcolemma.

The presence and the distribution of tumor necrosis factor-alpha, interferon-gamma, and p65 subunit of nuclear factor-kappaB, molecules known to induce synergistically and to mediate major histocompatibility complex (MHC) class I expression, were determined in muscle sections from control and X-linked vacuolated myopathy patients. MHC class I colocalized with tumor necrosis factor-alpha and interferon-gamma, as well as with p65, in most of the membrane attack complex- and/or calcium-positive muscle fibers in X-linked vacuolated myopathy. These results suggest that the expression of MHC class I in X-linked vacuolated myopathy could be induced by tumor necrosis factor-alpha and interferon-gamma and partly mediated by nuclear factor-kappaB.

Linkage of X-linked myopathy with excessive autophagy (XMEA) to Xq28.

X-linked myopathy with excessive autophagy (XMEA, MIM 310440) is a rare inherited mild myopathy. We have used 32 polymorphic markers spanning the entire X chromosome to exclude most of the chromosome except the Xq28 region in a large XMEA family. Using three additional families for linkage analysis, we have obtained a significant two-point lod score with marker DXS1183 (Z = 2.69 at theta = 0). Multipoint linkage analysis confirmed the assignment of the disease locus with a maximal lod score of 2.74 obtained at recombination fraction zero. Linkage of XMEA to the Xq28 region is thus firmly established. In addition, we have ruled out the Emery-Dreifuss muscular dystrophy to be allelic with XMEA by direct sequencing of the emerin gene in three of our families.

Sodium withdrawal contractures in developing and regenerating rat extensor digitorum longus muscles.

During postnatal development of extensor digitorum longus (EDL) muscle, sodium withdrawal contractures were observed during the first 6 days after birth, and not after this time. In regenerating EDL muscles, zero-Na contractures were demonstrated: (1) 7 days after bupivacaine injection, but not 14 or 90 days after this injection; (2) 7, 14, and 90 days after autotransplantation; and (3) 7, 14, and 90 days after the intervention in sliced muscles. The present findings emphasize the role of the denervation in the development of zero-Na contractures in the regenerating muscles and suggest that a calcium-sodium exchange across the sarcolemma may appear in these muscles.

X-linked vacuolated myopathy: membrane attack complex deposition on the surface membrane of injured muscle fibers is not accompanied by S-protein.

We have studied the expression of S-protein on the muscle from patients with X-linked vacuolated myopathy [characterized by the deposition of the complement C5b-9 membrane attack complex (MAC) over abnormal muscle fibers] and controls by immunocytochemistry and immunoblotting. No expression was detected on muscle from controls and patients with X-linked vacuolated myopathy. These findings suggest that S-protein does not render the MAC inactive in X-linked vacuolated myopathy. This situation may be due to the fact that the pathways of MAC activation and the expression of S-protein in X-linked vacuolated myopathy are different from the ones observed in ischemic and/or necrotic, or immune diseases. These results emphasize the role of the membrane complement regulatory proteins (i.e., CD59) in X-linked vacuolated myopathy.

Abnormalities of cardiac repolarization in multiple sclerosis: relationship with a model of allergic encephalomyelitis in rat.

Ventricular repolarization was investigated for the first time in 48 multiple sclerosis (MS) patients using measurement of QTc interval on standard electrocardiographic recordings. The repolarization process was prolonged significantly in MS compared to control subjects (P = 0.0001). This result was confirmed with an animal model of MS, i.e., the experimental allergic encephalomyelitis in rat. The contribution of prolonged QT to syncopal attack or sudden cardiac death in MS patients need further investigation.

Long-term external calcium dependence of autotransplanted and sliced extensor digitorum longus muscle contractility.

Contractile responses were studied in autotransplanted and sliced extensor digitorum longus (EDL) muscles at different times after the surgical intervention (3 and 12 months). The contraction of autotransplanted and sliced EDL muscles remained strongly dependent upon the extracellular Ca2+ concentration ([Ca]o) 3 and even 12 months after the intervention. It could be suggested that in autotransplanted and sliced EDL muscles, neural control could be involved in the persistent dependence of contractility toward [Ca]o.

A randomised clinical trial comparing interferon-alpha and intravenous immunoglobulin in polyneuropathy associated with monoclonal IgM. The IgM-associated Polyneuropathy Study Group.

OBJECTIVES: The polyneuropathy associated with a monoclonal IgM directed to the myelin associated glycoprotein (MAG) is a specific entity with a putative causal link between the IgM and the neuropathy. The small benefit offered by alkylating agents or plasma exchanges in these patients justifies the search for alternative treatments. METHODS: A 12 month multicentre, prospective, randomised, open clinical trial was carried out comparing intravenous immunoglobulin (IVIg; 2g/kg and then 1 g/kg every three weeks) and recombinant interferon-alpha (IFN-alpha; 3 MU/m2 subcutaneously three times weekly). The main end point was a clinical neuropathy disability score (CNDS) after six months of treatment. Twenty patients were enrolled; 10 were assigned to IVIg and 10 to IFN-alpha. RESULTS: At six months, one out of 10 patients treated with IVIg had a CNDS improvement of more than 20% whereas eight out of 10 patients treated with IFN-alpha had such an improvement (P=0.005). The mean CNDS worsened by 2.3 (SD 7.6) (8%) in the IVIg group whereas it improved by 7.5 (SD 11.1) (31%) in the IFN-alpha group (P=0.02). This improvement persisted after 12 months and was mainly related to an improvement of the sensory component (P=0.02) whereas the motor component was unchanged (P=0.39). Electrophysiological data did not show improvement of motor nerve conduction velocities whereas sensory nerve conduction velocities improved in the upper limbs. A decrease in the level of the monoclonal IgM was seen in two patients treated with IFN-alpha. At the end of the treatment, antibody activity to MAG was still detected in the serum of all patients. CONCLUSION: IVIg, as used in this study, did not improve patients with polyneuropathy and monoclonal IgM. By contrast, although its mechanism of action remains to be fully elucidated, IFN-alpha was effective in eight out of 10 patients at six months.