Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT), which catalyzes the conversion of glyoxylate to glycine. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only therapeutic strategy available to prevent kidney failure. Gene therapy is an attractive option to provide a definitive cure for PH1. Towards this goal, we are investigating helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. We have injected PH1 mice with an HDAd encoding the AGT under the control of a liver-specific promoter and observed a sustained reduction of oxalate urinary excretion and reduction in kidney stone formation. Recently, we have developed a minimally invasive method to improve the therapeutic index of HDAd (Brunetti-Pierri et al., 2009 ). This approach based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver, results in higher efficiency of hepatocyte transduction using clinically relevant low vector doses. Therefore, this method may permit correction of PH1 using clinically relevant doses of HDAd and may thus pave the way to clinical application of HDAd for PH1 gene therapy. The use of antisense genetic therapy for RNA mis-splicing diseases has gained increased attention as the splicing changes account for up to 15% of all mutations and with massive parallel genomic sequencing of individual patients the number of splicing mutations will be increased. Although the number of patients who can be potentially treated is low, it represents an excellent therapeutical option representing a type of personalized molecular medicine which is especially relevant for diseases for which there is to date no efficient treatment. In this work we summarize the splicing modulations explored to date especially targeted to deep intronic changes and the potential use to reprogram the splicing process using antisense therapy against intronic and exonic new or cryptic splice sites. In addition, we present our recent data in the investigation of new transporter structures that are thought to provide effective in vivo delivery. We are working on an octa-guanidine dendrimer covalently linked to specific morpholinos and also a new approach using locked nucleic acids monomers (LNA) bound to carbosilane dendrimers. We have successfully recovered the splicing process in MUT, PTPS, PCCA and PCCB disease cellular models suggesting that we are closer to applying the antisense therapy in animal models. A recently discovered chemical entity, PTC124 (Ataluren) was shown to selectively induce ribosomal readthrough of premature but not normal termination codons. In vitro experiments and subsequent treatment of mdx mice showed a promising improvement in the form of functional recovery. This led us to investigate the effect of PTC124 on nonsense mediated premature termination in two of the three cerebral creatine deficiency syndrome genes, namely the creatine transporter gene (SLC6A8) and the guanidinoacetate methyl-transferase gene (GAMT). We obtained PTC124 from Selleck Chemicals (Texas, USA) and incubated patient's cell-lines containing nonsense mutations with 17 μM of PTC124 for 46 days (SLC6A8, n=2) and 14 days (GAMT, n=3) and collected cells every 7 days. We measured intra-cellular creatine content and formation of 2H3-13 C2-creatine using a two-step derivatization procedure, followed by quantification with GC-MS. With all cell-lines we found no increase of function, indicated by low intra-cellular creatine content (SLC6A8) and no formed 2H3-13 C2-creatine (GAMT) as compared to the wild-type celllines. This led us to conclude that PTC124 (Selleck Chemicals) cannot induce ribosomal readthrough of the five tested nonsense mediated termination codons in the SLC6A8 and GAMT gene. However, it should be taken into account that we were unable to validate the obtained compound. Background: Batten disease (Juvenile Neuronal Ceroid-Lipofuscinosis, JNCL), is a recessively inherited untreatable neurodegenerative disorder caused by mutations in the CLN3 gene. The mechanism by which the abnormal or missing protein leads to neuronal cell death remains unclear. It has been shown that a downstream effect is abnormal intracellular calcium accumulation which may lead to apoptosis. Previously, we demonstrated reversal of the calcium effect in a neuroblastoma cell line using amlodipine and other calcium channel antagonists. Objectives: The aim of this study is to investigate the neuroprotective role of amlodipine in a primary neuronal model of CLN3. We studied calcium changes and indicators of apoptosis in amlodipine-treated primary rat neurons with siRNA CLN3 knock down. Results: We demonstrated that intracellular calcium is elevated in CLN3 siRNA-silenced primary neurons. We also demonstrate that amlodipine; an L-type calcium channel antagonist can reverse the aberrant calcium elevations at pharmacological levels. Furthermore, we demonstrated that amlodipine can protect from etoposide-induced apoptosis in the siRNAsilenced neurons. Conclusion: This study indicates that amlodipine, in addition to its wellestablished role as a calcium channel antagonist, can also function as a neuroprotective drug. It suggests that currently available calcium channel antagonists may be potential therapeutic agents for Batten disease. Background: Fabry disease (FD) is a lysosomal storage disease resulting from a deficiency of the enzyme alpha-galactosidase A. Defects in this enzyme lead to accumulation of ceramide trihexoside (CTH) in the endothelium and with patients becoming susceptible to early onset stroke, cardiovascular and kidney disease. Objectives: The aetiology of the kidney damage associated with FD and CTH accumulation is poorly understood. We describe the development of novel glycolipid arrays by immobilising glycolipids to a chip surface and using them to study proteins which interact with CTH in the kidney. Methods: CTH and the ganglioside GM1 (positive control) were immobilised on RS100 arrays and incubated with adolescent rat kidney homogenates. Those proteins interacting with each glycolipid were then identified using QTOF mass spectrometry. Results: A number of proteins were identified that bound specifically to CTH including hamartin, chloride channel protein 1 and tubulin alpha 3 chain; which have been implicated previously in kidney disease. Interestingly, several other proteins were found to bind specifically to GM1. Conclusions: Glycosphingolipid arrays show much potential for studying protein: lipid interactions. Using this technology we have identified several proteins that could play a role in the complex pathogenesis of kidney disease in FD. Clinical molecular testing in IMD is currently PCR-based precluding the identification of deletions which account for a variable fraction (1-25%) of mutant alleles depending on the gene involved. We have developed a highresolution comparative genomic hybridization array (Metabolarray®) for the detection of exonic copy number changes in 205 genes involved in IMD which are currently diagnosed in the laboratory. The array consists of 62,979 oligos spread genome wide, with 40,555 hybridizing to target genes with an average spacing of about 250 bp and 26,678 covering the rest of the genome. For validation, we have retrospectively analyzed a series of IMD patients carriers of exonic deletions previously genotyped by different methods (MLPA, SNP-arrays). All the heterozygous and homozygous deletions even of a single exon were detected using the Metabolarray®. In a series of prospectively evaluated patients, we have identified a novel 2 Kb deletion in the PCCB gene encompassing exons 4 and 5. Our results show that this new molecular tool which can be easily modified to analyze additional genes may be highly efficient in screening for exon copy number changes in IMD patients with incomplete genotype. Background: Degradation of cytoplasmic tRNAs and of mitochondrial mRNAs are part of a cellular response to environmental stress, such as oxidative stress and may regulate protein synthesis or lead to apoptosis. PTCD1 is a novel human protein that was recently shown to decrease the levels of mitochondrial Leucyl-tRNAs. Objectives: To investigate, whether targeted degradation of mitochondrial tRNAs is part of the cellular response to oxidative stress, and whether PTCD1 might be involved in this process. Methods: We compared the effect of oxidative stress (induced by 500 μM tert-butyl hydroperoxide for 1 h followed by 0-3.5 h recovery) on PTCD1 knockdown , on PTCD1 over-expressing and on the respective control HepG2 cells. RNA levels were quantified by quantitative real-time PCR. Results and Discussion: Oxidative stress treatment rapidly decreased the steady state levels of the mitochondrial Leucyl-tRNAs (tRNALeuUUR, tRNALeuCUN), but increased the mRNA steady state levels of the mitochondrial superoxide dismutase (SOD2) and of the transcription factor ATF4 in mock-transfected control cells. PTCD1 knockdown could not prevent the decrease of tRNALeuUUR and tRNALeuCUN in response to oxidative stress. These results suggest, that PTCD1 is not involved in a targeted degradation of mitochondrial Leucyl-tRNAs in response to oxidative stress. Objective: To investigate the globle expression profiling of microRNAs in brain tissues of fetuses with anencephaly and normal control by using microRNA microarray, and predict the target genes of some specific miRNAs with bioinformatic method. Methods: The profiling of miRNAs from brain tissues of 3 fetuses with anencephaly and normal brains was detected using an Affymetrix GeneChip® miRNA microarray. Then some of the significantly misexpressed miRNAs were selected to validate the microarray assay results by real-time RT-PCR analysis. Furthermore, the target genes of these miRNAs were predicted with some online databases. Results: 1. Compared to the normal group, anencephaly group has a specific miRNA expression profile. There were 73 up-regulated miRNAs and 14 down-regulated miRNAs. 2. The microarray findings were extended using Real-time RT-PCR for 10 miRNAs. Of these miRNAs validated, 8 miRNAs were up-regulated, whereas one was down-regulated. In addition, mir-125a was found to be higher in anencephaly group than in the normal group, which contradicted the result detected by the microarray. Conclusion: This study shows that brain tissues of anencephaly group have a significantly misexpressed miRNA expression profile compared to that of normal group. And these misexpressed miRNAs may involve in the pathogenesis of NTDs. Conflict of Interest declared. PDHA2 gene encodes the testis-specific isoform of E1α, the target subunit for most pyruvate dehydrogenase complex deficiencies, and it was postulated that its activation in somatic tissues would represent a conceptual therapy. Accordingly, we aimed to unveil the mechanisms regulating PDHA2 tissue-specific expression. Basal transcriptional activity of different PDHA2 promoter constructs was assayed in three different somatic cell lines, and in vitro methylation experiments were also performed. A cell line (SH-SY5Y) was treated with 5'-aza-2'-deoxycytidine(DAC). PDHA2 methylation status was evaluate by MS-PCR, and mRNA levels analyzed by qRT-PCR; ChIP assays were performed and the recovered DNA analyzed by qRT-PCR. We found that PDHA2 promoter-directed transcription occurred in cultured somatic cells, with no obvious differences between cell lines, and that in vitro methylation clearly reduced transcriptional activity of the constructs. We observed that DAC treatment induced a significant demethylation of an exonic CpG island, while the core promoter remained fully methylated. Moreover, this demethylation was concomitant with PDHA2 gene derepression. Finally, ChIP assays confirmed the involvement of DNA methylation in PDHA2 gene expression.