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Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-offunction effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the [...]

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a deficiency of survival motor neuron (SMN) due to mutations in the SMN1 gene. In this study, an adeno-associated virus (AAV) vector expressing human SMN (AAV8-hSMN) was injected at birth into the CNS of mice modeling SMA. Western blot analysis showed that these injections resulted in widespread expression of SMN throughout the spinal cord, and this translated into robust improvement in skeletal muscle physiology, including increased myofiber size and improved neuromuscular junction architecture. Treated mice also displayed substantial improvements on behavioral tests of muscle strength, coordination, and locomotion, indicating that the neuromuscular junction was functional. Treatment with AAV8-hSMN increased the median life span of mice with SMA-like disease to 50 days compared with 15 days for untreated controls. Moreover, injecting mice with SMA-like disease with a human SMN-expressing self-complementary AAV vector--a vector that leads to earlier onset of gene expression compared with standard AAV vectors--led to improved efficacy of gene therapy, including a substantial extension in median survival to 157 days. These data indicate that CNS-directed, AAV-mediated SMN augmentation is highly efficacious in addressing both neuronal and muscular pathologies in a severe mouse model of SMA., Introduction Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene and loss of encoded SMN protein (1). The [...]

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease. axonal transport | deep cerebellar nuclei | gene therapy | lysosomal storage diseases | adeno-associated virus

Preconditioning in cultured cardiomyocytes elevates the expression of several protective genes including Glut-4 and heat shock protein (HSP)70. Hypoxiainducible factor-1 (HIF-1) is known to mediate the transcriptional activation of hypoxia-responsive genes. In this study, we examined the effect of adenovirus-mediated expression of constitutively stable hybrid forms of HIF-let on cardiomyocyte viability and gene expression. Cultured neonatal rat cardiomyocytes were subjected to simulated ischemia-reperfusion with or without preinfection with recombinant adenoviral vectors [Ad2/H1F-1[alpha]/herpes simplex virus protein VP 16 and Ad2/HIF-1[alpha]/nuclear factor-[kappa]B (NF-[kappa]B)]. Cellular viability and mRNA levels of several cardioprotective genes were measured. We demonstrated that infection with Ad2/HIF-1[alpha]VP16 and Ad2/ HIF-1[alpha]/NF-[kappa]B mimicked the upregulation of the mRNA levels of vascular endothelial growth factor (VEGF), Glut-1, Glut-4, HSP70, and inducible NO synthase (iNOS) and the protection of cultured neonatal rat cardiomyocytes by late-phase preconditioning against simulated ischemia-reperfusion. The same dose of a control viral vector expressing no transgene had no effect. Preconditioning also elevated HIF-1[alpha] protein levels. These results suggest that adenovirus-mediated expression of HIF-1[alpha]/VP16 or HIF-1[alpha]/NF-[kappa]B, a constitutively stable hybrid transcriptional factor, protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes. ischemic preconditioning: myocardial protection

Expression of angiopoietins in renal epithelial and clear cell carcinoma cells: regulation by hypoxia and participation in angiogenesis. Am J Physiol Renal Physiol 287: F649-F657, 2004. First published June 15, 2004; 10.1152/ajprenal.00028.2004.--The hereditary von Hippel-Lindau (VHL) syndrome predisposes sufferers to highly vascularized tumors such as renal clear cell carcinoma (RCC) and central nervous system hemangioblastoma. In RCC4 and RCC786-0 VH[L.sup.-] ceils with VHL mutations, the protein of hypoxia-inducible factor-1[alpha] (HIF-1[alpha] is constitutively stabilized and the mRNA levels of HIF target genes, including vascular endothelial growth factor (VEGF), are elevated. However, the expression of angiopoietins in these cells and their involvement in angiogenesis are not well known. In this study, we compared the mRNA levels of angiopoietins in human kidney proximal tubule epithelial (RPTE) and RCC4 and RCC786-0 VH[L.sup.-] cells. In RPTE cells, angiopoietin-4 (Ang-4) expression was selectively induced by hypoxia or by expression of a hybrid form of HIF-1[alpha]. Under normoxic conditions, the mRNA levels of Ang-4 were higher in RCC4 and RCC786-0 VH[L.sup.+] than RPTE cells. Angiopoietin-1 expression was detectable in RCC4 and RCC786-0 VH[L.sup.-] cells but not RPTE cells. In RCC786-0 VH[L.sup.+] cells, which were stably transfected with a wild-type copy of VHL, the mRNA levels of VEGF and Ang-4 were suppressed and the hypoxic response was restored. We also demonstrated that stimulation of endothelial tube formation by conditioned medium harvested from RCC4 cells was inhibited by a soluble Tie-2 receptor. These results suggest that the angiopoietin/Tie-2 system may participate in the angiogenic response to hypoxia in renal tissues and in tumor angiogenesis in renal carcinoma. hypoxia-inducible factor-[alpha] ; von Hippel-Lindau; vascular endothelial growth factor; Tie-2 receptor

The role of exogenous .nitric oxide (.NO) in the modulation of heterologous gene expression in polarized kidney epithelial cells that were stably transduced with a cDNA encoding human wild-type cystic fibrosis membrane conductance regulator (CFTR) was assessed. The results showed that .NO impaired the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms. Furthermore, the evidence indicated the potential presence of large amounts of .NO in the lung.

A study was conducted to examine the ability of deoxyspergualin (DSG) to influence the trafficking of Delta-F508-cystic fibrosis transmembrane conductance regulatory (CFTR) to the cell membrane. Chloride channel activity was analyzed using the halide-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)-quinolinium. Results indicated that the DSG treatment of human CF cells restored cAMP-stimulated CFTR Cl- channel activity at the plasma membrane.

A series of recombinant replication-defective adenovirus gene transfer vectors with different polyadenylation signal sequences were constructed with cystic fibrosis transmembrane conductance regulators (CTRF). The recombinant adenovirus gene vectors with built-in Ad2E4ORF6 backbones exhibited efficient gene transduction capabilities during cystic fibrosis gene therapy. The recombinant adenovirus gene vectors also corrected cyclic adenosine monophosphate-mediated Cl- secretion and fluid transport through gene transduction.

Overexpression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR), a chloride (Cl-) channel, arrests cell growth in the G(sub 2)/M phase and increases the cell volume of monkey kidney cells. CFTR overexpression in the G551D-CFTR mutant inhibits growth slightly, and there is an increase in the number of cells in the G(sub 2)/M phase. Decrease in the threshold of Cl- channel activity in the mutant produces growth defects. CFTR is capable of downregulating its activity.

Potent derivatives of butyrates may be effective in the cure of delta F508 or cystic fibrosis (CF) mutation-induced Chlorine ion channel defects because overexpression of mutant protein causes transfers of fractions of delta F508-cystic fibrosis transmembrane regulators (CFTR) into the plasma membrane. Incubation of delta F508-CFTR expressing cells at low temperature enhances transfer and maturation of mutant CFTR proteins.

Most lysosomal storage diseases (LSDs) have a significant neurological component, including types 2 and 3 Gaucher disease (neuronal forms of Gaucher disease; nGD). No therapies are currently available for nGD since the recombinant enzymes used in the systemic form of Gaucher disease do not cross the blood–brain barrier (BBB). However, a number of promising approaches are currently being tested, including substrate reduction therapy (SRT), in which partial inhibition of the synthesis of the glycosphingolipids (GSLs) that accumulate in nGD lowers their accumulation. We now induce nGD in mice by injection with conduritol B‐epoxide (CBE), an irreversible inhibitor of acid beta‐glucosidase (GCase), the enzyme defective in nGD, with or without co‐injection with Genz‐667161, a prototype for SRT which crosses the BBB. Significant neuropathology, and a reduction in lifespan, was observed upon CBE injection, and this was largely reversed by co‐injection with Genz‐667161, along with a reduction in glucosylceramide and glucosylsphingosine levels. Analysis of gene expression by RNAseq revealed that Genz‐667161 largely reversed the changes in genes and pathways that were differentially expressed upon CBE injection, specifically pathways of GSL metabolism, lipoproteins and other lipid metabolic pathways, lipid droplets, astrocyte activation, neuronal function, and to some extent, neuroinflammation. Together, this demonstrates the efficacy of SRT to reverse the effects of substrate accumulation on pathological components and pathways in nGD brain. [ABSTRACT FROM AUTHOR]