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1.
Sci Rep ; 7: 46740, 2017 04 24.
Article in English | MEDLINE | ID: mdl-28436437

ABSTRACT

Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington's disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection.


Subject(s)
Huntingtin Protein/genetics , Huntington Disease/genetics , Mutant Proteins/genetics , Polymorphism, Single Nucleotide , Adult , Aged , Alleles , Cells, Cultured , Cohort Studies , Genotype , Humans , Huntington Disease/blood , Middle Aged , Myeloid Cells/metabolism , RNA Interference
2.
J Huntingtons Dis ; 5(1): 33-8, 2016.
Article in English | MEDLINE | ID: mdl-27003665

ABSTRACT

BACKGROUND: Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington's disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments. OBJECTIVES: We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA. METHODS: Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection. RESULTS: Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites. CONCLUSIONS: Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage.


Subject(s)
Huntingtin Protein/genetics , Huntington Disease/genetics , Mutation/genetics , RNA Interference , RNA, Messenger/genetics , Trinucleotide Repeats/genetics , Animals , Corpus Striatum/metabolism , Disease Models, Animal , Exons/genetics , Gene Knockdown Techniques , Huntingtin Protein/analysis , Huntingtin Protein/metabolism , Liver/metabolism , Mice , RNA, Messenger/analysis , RNA, Messenger/metabolism
3.
J Huntingtons Dis ; 2(4): 491-500, 2013.
Article in English | MEDLINE | ID: mdl-25062733

ABSTRACT

BACKGROUND: Huntington's disease is caused by expansion of CAG trinucleotide repeats in the first exon of the huntingtin gene, which is essential for both development and neurogenesis. Huntington's disease is autosomal dominant. The normal allele contains 6 to 35 CAG triplets (average, 18) and the mutant, disease-causing allele contains >36 CAG triplets (average, 42). OBJECTIVE: We examined 279 postmortem brain samples, including 148 HD and 131 non-HD controls. A total of 108 samples from 87 HD patients that are heterozygous at SNP rs362307, with a normal allele (18 to 27 CAG repeats) and a mutant allele (39 to 73 CAG repeats) were used to measure relative abundance of mutant and wild-type huntingtin mRNA. METHODS: We used allele-specific, quantitative RT-PCR based on SNP heterozygosity to estimate the relative amount of mutant versus normal huntingtin mRNA in postmortem brain samples from patients with Huntington's disease. RESULTS: In the cortex and striatum, the amount of mRNA from the mutant allele exceeds that from the normal allele in 75% of patients. In the cerebellum, no significant difference between the two alleles was evident. Brain tissues from non-HD controls show no significant difference between two alleles of huntingtin mRNAs. Allelic differences were more pronounced at early neuropathological grades (grades 1 and 2) than at late grades (grades 3 and 4). CONCLUSION: More mutant HTT than normal could arise from increased transcription of mutant HTT allele, or decreased clearance of mutant HTT mRNA, or both. An implication is that equimolar silencing of both alleles would increase the mutant HTT to normal HTT ratio.


Subject(s)
Brain/metabolism , Huntington Disease/genetics , Nerve Tissue Proteins/genetics , RNA, Messenger/genetics , Adult , Aged , Aged, 80 and over , Allelic Imbalance , Female , Humans , Huntingtin Protein , Huntington Disease/metabolism , Male , Middle Aged , Mutant Proteins/genetics , Mutant Proteins/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Trinucleotide Repeat Expansion
4.
Mol Phylogenet Evol ; 54(2): 561-70, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19679193

ABSTRACT

We investigated the phylogenetic relationships and estimated the history of species diversification and biogeography in the bufonid genus Ansonia from Southeast Asia, a unique organism with tadpoles adapted to life in strong currents chiefly in montane regions and also in lowland rainforests. We estimated phylogenetic relationships among 32 named and unnamed taxa using 2461bp sequences of the mitochondrial 12S rRNA, tRNA(val), and 16S rRNA genes with equally-weighted parsimony, maximum likelihood, and Bayesian methods of inference. Monophyletic clades of Southeast Asian members of the genus Ansonia are well-supported, allowing for the interpretation of general biogeographic conclusions. The genus is divided into two major clades. One of these contains two reciprocally monophyletic subclades, one from the Malay Peninsula and Thailand and the other from Borneo. The other major clade primarily consists of Bornean taxa but also includes a monophyletic group of two Philippine species and a single peninsular Malaysian species. We estimated absolute divergence times using Bayesian methods with external calibration points to reconstruct the relative timing of faunal exchange between the major landmasses of Southeast Asia.


Subject(s)
Bufonidae/genetics , Evolution, Molecular , Phylogeny , Animals , Asia, Southeastern , Bayes Theorem , Bufonidae/classification , DNA, Mitochondrial/genetics , Geography , Likelihood Functions , Sequence Alignment , Sequence Analysis, DNA
5.
Nat Methods ; 5(11): 951-3, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18931668

ABSTRACT

Allele-specific silencing using small interfering RNAs targeting heterozygous single-nucleotide polymorphisms (SNPs) is a promising therapy for human trinucleotide repeat diseases such as Huntington's disease. Linking SNP identities to the two HTT alleles, normal and disease-causing, is a prerequisite for allele-specific RNA interference. Here we describe a method, SNP linkage by circularization (SLiC), to identify linkage between CAG repeat length and nucleotide identity of heterozygous SNPs using Huntington's disease patient peripheral blood samples.


Subject(s)
Huntington Disease/genetics , Polymorphism, Single Nucleotide/genetics , Trinucleotide Repeats/genetics , Humans , Molecular Sequence Data
6.
Mol Phylogenet Evol ; 49(1): 318-26, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18723097

ABSTRACT

The phylogenetic relationships among 46 samples from 27 populations of the Japanese giant salamander, Andriasjaponicus and its congener, A. davidianus from China was investigated, using 3664 bp sequences of the mitochondrial genes NADH1, NADH3, cyt b and CR, partial NADH6 and intervening genes. In phylogenetic trees constructed by MP, ML, and Bayesian methods, the family Cryptobranchidae and the genus Andrias both form monophyletic groups. Japanese A. japonicus and Chinese A. davidianus are sister taxa and can be regarded as separate species despite a small degree of genetic differentiation. Andriasjaponicus is divided into central and western clades, but the phylogenetic relationships within the latter clade are unresolved. As previously reported from allozyme analyses, A. japonicus exhibits little genetic differentiation, in strong contrast to salamanders of the genus Hynobius with which their distributions overlap. This reduced genetic variability in A. japonicus is attributable to a unique mating system of polygyny, delayed sexual maturity, notable longevity, life in a stable aquatic environment, and gigantism, as well as bottleneck effects following habitat fragmentation and extinction of local populations during Quaternary glaciations. The species is thus susceptible to extinction by potential environmental fluctuations, and requires extensive conservation measures.


Subject(s)
Evolution, Molecular , Genetic Variation , Urodela/classification , Urodela/genetics , Animals , Bayes Theorem , Conservation of Natural Resources , DNA, Mitochondrial/genetics , Ecosystem , Genes, Mitochondrial , Haplotypes , Likelihood Functions , Mitochondria/genetics , Phylogeny , Sequence Analysis, DNA , Species Specificity
7.
Mol Phylogenet Evol ; 38(3): 659-66, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16403658

ABSTRACT

We investigated the phylogenetic relationships among 20 species of Oriental torrent frogs in the genus Amolops and its allies from China and Southeast Asia based on 1346-bp sequences of the mitochondrial 12S and 16S rRNA genes. Oriental species of the tribe Ranini form a monophyletic group containing 11 clades (Rana temporaria + Pseudoamolops, R. chalconota, four clades of Amolops, Meristogenys, three clades of Huia species, and Staurois) for which the phylogenetic relationships are unresolved. The genus Amolops consists of southern Chinese, southwestern Chinese, Thai, and Vietnamese-Malaysian lineages, but their relationships are also unresolved. The separation of southern and southwestern lineages within China conforms to previous morphological and karyological results. Species of Huia do not form a monophyletic group, whereas those of Meristogenys are monophyletic. Because P. sauteri is a sister species of R. temporaria, distinct generic status of Pseudoamolops is unwarranted.


Subject(s)
Ranidae/classification , Animals , Polymerase Chain Reaction , RNA, Ribosomal, 16S/genetics , Ranidae/genetics , Sequence Analysis, DNA , Species Specificity
8.
Mol Reprod Dev ; 73(1): 40-9, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16177983

ABSTRACT

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.


Subject(s)
Spermatogenesis/physiology , Ubiquitin Thiolesterase/biosynthesis , Ubiquitin Thiolesterase/genetics , Animals , Apoptosis/genetics , Caspase 3 , Caspases/metabolism , Down-Regulation/genetics , Immunohistochemistry , Infertility, Male/enzymology , Infertility, Male/genetics , Male , Mice , Mice, Transgenic , Phenotype , Polyubiquitin/metabolism , Proliferating Cell Nuclear Antigen/biosynthesis , Proliferating Cell Nuclear Antigen/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Sertoli Cells/metabolism , Spermatocytes/cytology , Spermatocytes/enzymology , Ubiquitin/metabolism , Ubiquitin Thiolesterase/physiology , Up-Regulation/genetics , Vimentin/biosynthesis , Vimentin/genetics
9.
Neurosci Res ; 53(3): 241-9, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16095740

ABSTRACT

Huntington's disease (HD) is an autosomal dominant inheritable neurodegenerative disorder currently without effective treatment. It is caused by an expanded polyglutamine (poly Q) tract in the corresponding protein, huntingtin (htt), and therefore suppressing the huntingtin expression in brain neurons is expected to delay the onset and mitigate the severity of the disease. Here, we have used small interfering RNAs (siRNAs) directed against the huntingtin gene to repress the transgenic mutant huntingtin expression in an HD mouse model, R6/2. Results showed that intraventricular injection of siRNAs at an early postnatal period inhibited transgenic huntingtin expression in brain neurons and induced a decrease in the numbers and sizes of intranuclear inclusions in striatal neurons. Treatments using this siRNA significantly prolonged model mice longevity, improved motor function and slowed down the loss of body weight. This work suggests that siRNA-based therapy is promising as a future treatment for HD.


Subject(s)
Genetic Therapy/methods , Huntington Disease/genetics , Huntington Disease/therapy , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , RNA, Small Interfering/therapeutic use , Animals , Body Weight/genetics , Corpus Striatum/metabolism , Corpus Striatum/pathology , Corpus Striatum/physiopathology , Disease Models, Animal , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Humans , Huntingtin Protein , Huntington Disease/physiopathology , Injections, Intraventricular , Intranuclear Inclusion Bodies/genetics , Intranuclear Inclusion Bodies/metabolism , Intranuclear Inclusion Bodies/pathology , Male , Mice , Mice, Transgenic , Motor Activity/genetics , Nerve Tissue Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Peptides/genetics , Peptides/metabolism , RNA, Small Interfering/genetics , Survival Rate , Transgenes/genetics , Treatment Outcome , Trinucleotide Repeat Expansion/genetics
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