Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free "GeneSwitch" vector.

Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3-4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - "Gene Switch" vector

Identification of Domains and Amino Acids Essential to the Collagen Galactosyltransferase Activity of GLT25D1

Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166–168) and second motif (amino acids 461–463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein

Natural swimming speed of Dascyllus reticulatus increases with water temperature

Recent research on the relationship between coral reef water temperature and fish swimming activity has stated that swimming speed is inversely correlated with temperature above a species’ optimum temperature (Johansen, J. L., and Jones, G. P. 2011. Increasing ocean temperature reduces the metabolic performance and swimming ability of coral reef damselfishes. Global Change Biology, 17: 2971–2979; Johansen, J. L.,Messmer,V., Coker, D. J., Hoey, A. S., and Pratchett, M. S. 2014. Increasing ocean temperatures reduce activity patterns of a large commercially important coral reef fish. Global Change Biology, 20: 1067–1074). For tropical coral reefs, one anticipated consequence of global warming is an increase of ≥38C in average water temperature in addition to greater thermal fluctuations [IPCC (Intergovernmental Panel on Climate Change). 2007. Summary for policymakers. In Climate Change 2007: The Physical Science Basis. Contribution of Working, Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Ed. by S. Solomon, D. Qin, and M. Manning et al. Cambridge University Press, Cambridge, UK; Lough, J. 2007. Climate and climate change on the Great Barrier Reef. In Climate Change and the Great Barrier Reef. Ed. by J. Johnson and P. A. Marshall, pp. 15–50. Great Barrier Reef Marine Park Authority and Australian Greenhouse Office, Townsville, Qld, Australia; Johansen and Jones, 2011]. Evaluating the behaviour of coral reef associated fish species at different temperatures can help to assess their sensitivity to climate change. In this study, the speed of freely swimming fish in a natural setting is investigated as a function of seasonal changes in water temperature, as contrasted with systematic temperature increases in a fish tank.Weshowthat Dascyllus reticulatusswim faster as a function of increased water temperature over the range 20.9–30.38C. The experiments were carried out using 3.6 million fish trajectories observed at the Kenting National Park in Taiwan. Fish speed was computed by detecting and tracking the fish through consecutive video frames, then converting image speeds to scene speeds. Temperatureswere grouped into 10 intervals. The data reveal an2 mms21 increase in average speed per additional temperature degree over the range of 20.9–30.38C. The Mann–Kendall test using the mean and median speed for each interval revealed that there is a speed increase trend as temperature increases at the 0.05 significance level, rather than a random increase. Our results complement previous studies that investigated the effect of temperature on the swimming performance of different fish species in the laboratory (Johansen and Jones, 2011; Myrick, C. A. and Cech, J. J. 2000. Swimming performance of four California stream fishes: temperature effects. Environmental Biology of Fishes, 58: 289–295; Ojanguren, A. F. and Bran ̃a, F. 2000. Thermal dependence of swimming endurance in juvenile brown trout. Journal of Fish Biology, 56: 1342–1347; Lough 2007; Johansen et al., 2014)

Hampered Foraging and Migratory Performance in Swans Infected with Low-Pathogenic Avian Influenza A Virus

It is increasingly acknowledged that migratory birds, notably waterfowl, play a critical role in the maintenance and spread of influenza A viruses. In order to elucidate the epidemiology of influenza A viruses in their natural hosts, a better understanding of the pathological effects in these hosts is required. Here we report on the feeding and migratory performance of wild migratory Bewick's swans (Cygnus columbianus bewickii Yarrell) naturally infected with low-pathogenic avian influenza (LPAI) A viruses of subtypes H6N2 and H6N8. Using information on geolocation data collected from Global Positioning Systems fitted to neck-collars, we show that infected swans experienced delayed migration, leaving their wintering site more than a month after uninfected animals. This was correlated with infected birds travelling shorter distances and fuelling and feeding at reduced rates. The data suggest that LPAI virus infections in wild migratory birds may have higher clinical and ecological impacts than previously recognised

Repair and Polyadenylation of a Naturally Occurring Hepatitis C Virus 3′ Nontranslated Region-Shorter Variant in Selectable Replicon Cell Lines

The 3′ nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific cis-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3′ end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3′ NTR may also be essential in host defense against exoribonucleases. During 3′-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3′ end of the genome. These shorter 3′ ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3′ ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3′ terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3′ ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3′ end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed

Targeting RNA-mediated toxicity in C9orf72 ALS and/or FTD by RNAi-based gene therapy

A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts

Targeting RNA-Mediated Toxicity in C9orf72 ALS and/or FTD by RNAi-Based Gene Therapy

Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients

The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases.RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G4C2)44-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients. Keywords: C9orf72, ALS, FTD, gene therapy, microRN

Targeting RNA-mediated toxicity in C9orf72 ALS and/or FTD by RNAi-based gene therapy

A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts