scholarly journals Impeding transcription of expanded microsatellite repeats by deactivated Cas9

2017 ◽  
Author(s):  
Belinda S. Pinto ◽  
Tanvi Saxena ◽  
Ruan Oliveira ◽  
Héctor R. Méndez-Gómez ◽  
John D. Cleary ◽  
...  
Keyword(s):  
Corneal Dystrophy ◽  
Therapeutic Benefit ◽  
Systemic Delivery ◽  
Microsatellite Repeat ◽  
Aberrant Splicing ◽  
Adeno Associated Virus ◽  
Microsatellite Repeats ◽  
Repeat Sequences ◽  
Rna Foci ◽  
Splicing Patterns

SummaryTranscription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs’ endothelial corneal dystrophy, andC9orf72-ALS/FTD. Eliminating or reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that a deactivated form of the Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction in the abundance of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, andC9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

scholarly journals Aberrant Bcl-x splicing in cancer: from molecular mechanism to therapeutic modulation

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zhihui Dou ◽  
Dapeng Zhao ◽  
Xiaohua Chen ◽  
Caipeng Xu ◽  
Xiaodong Jin ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Human Cancer ◽  
Structural Characteristics ◽  
Therapy Resistance ◽  
Regulatory Elements ◽  
Aberrant Splicing ◽  
Clinical Role ◽  
Splicing Isoforms ◽  
The Impact ◽  
Splicing Patterns

AbstractBcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy.

scholarly journals Characterization of Duck Microsatellite Repeat Sequences

2001 ◽  
Vol 35 (4) ◽  
pp. 217-219 ◽  
Author(s):  
Hideaki TAKAHASHI ◽  
Masahiro SATOH ◽  
Mitsuru MINEZAWA ◽  
Tresnawati PURWADARIA ◽  
Hardi PRASETYO
Keyword(s):  
Microsatellite Repeat ◽  
Repeat Sequences

scholarly journals Duplex RNAs and ss-siRNAs Block RNA Foci Associated with Fuchs' Endothelial Corneal Dystrophy

2019 ◽  
Vol 29 (2) ◽  
pp. 73-81 ◽  
Author(s):  
Jiaxin Hu ◽  
Xiulong Shen ◽  
Frank Rigo ◽  
Thahza P. Prakash ◽  
V. Vinod Mootha ◽  
...  
Keyword(s):  
Corneal Dystrophy ◽  
Rna Foci

scholarly journals Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes

1999 ◽  
Vol 73 (11) ◽  
pp. 9314-9324 ◽  
Author(s):  
André Lieber ◽  
Dirk S. Steinwaerder ◽  
Cheryl A. Carlson ◽  
Mark A. Kay
Keyword(s):  
First Generation ◽  
Cultured Cells ◽  
Inverted Terminal Repeat ◽  
Stable Gene ◽  
Adeno Associated Virus ◽  
Transgene Integration ◽  
Repeat Sequences ◽  
Hybrid Vectors ◽  
Viral Genes

ABSTRACT Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus–adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (ΔAd.AAV) and stimulate transgene integration. We demonstrate here that ΔAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. ΔAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. ΔAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The ΔAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with ΔAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that ΔAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. ΔAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.

Adeno-Associated Virus-Mediated Rhodopsin Replacement Provides Therapeutic Benefit in Mice with a Targeted Disruption of the Rhodopsin Gene

2010 ◽  
Vol 21 (3) ◽  
pp. 311-323 ◽  
Author(s):  
Arpad Palfi ◽  
Sophia Millington-Ward ◽  
Naomi Chadderton ◽  
Mary O'Reilly ◽  
Tobias Goldmann ◽  
...  
Keyword(s):  
Therapeutic Benefit ◽  
Targeted Disruption ◽  
Adeno Associated Virus ◽  
Rhodopsin Gene

scholarly journals Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria

2020 ◽  
Vol 48 (9) ◽  
pp. 4658-4671 ◽  
Author(s):  
François Halloy ◽  
Pavithra S Iyer ◽  
Paulina Ćwiek ◽  
Alice Ghidini ◽  
Jasmin Barman-Aksözen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Bone Marrow Cells ◽  
Therapeutic Benefit ◽  
Loss Of Function ◽  
Erythropoietic Protoporphyria ◽  
Aberrant Splicing ◽  
In Trans ◽  
Patients Experience ◽  
Distinct Distribution

Abstract Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

Isolation and characterisation of microsatellite loci in the bush stone-curlew (Burhinus grallarius), a declining Australian bird

2013 ◽  
Vol 61 (6) ◽  
pp. 421
Author(s):  
Robert A. B. Mason ◽  
Catherine Price ◽  
Walter E. Boles ◽  
Karen-Anne Gray ◽  
Edwina Rickard ◽  
...  
Keyword(s):  
Microsatellite Loci ◽  
Allelic Diversity ◽  
Pcr Amplification ◽  
Habitat Modification ◽  
Microsatellite Repeat ◽  
Gene Encoding ◽  
Repeat Sequences ◽  
Introduced Predators ◽  
Ground Nesting

The bush stone-curlew (Burhinus grallarius Latham), a ground-nesting nocturnal bird, is endangered in southern Australia due to habitat modification and introduced predators. To provide tools for conservation, ecological and behavioural studies, we isolated variable microsatellite repeat sequences and designed primers for PCR amplification in this species. Primer pairs were developed and levels of diversity were assessed for eight microsatellite loci, including one locus linked to the gene encoding Microtubule-Associated Protein 2, a protein important for behavioural imprinting in birds, and one sex-linked locus. Isolated loci contained allelic diversity of between 5 and 17 alleles.

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences

1995 ◽  
Vol 6 (10) ◽  
pp. 714-724 ◽  
Author(s):  
R. T. Stone ◽  
J. C. Pulido ◽  
G. M. Duyk ◽  
S. M. Kappes ◽  
J. W. Keele ◽  
...  
Keyword(s):  
Genomic Library ◽  
Microsatellite Repeat ◽  
Repeat Sequences

scholarly journals Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation

2001 ◽  
Vol 75 (16) ◽  
pp. 7662-7671 ◽  
Author(s):  
Dongsheng Duan ◽  
Ziying Yan ◽  
Yongping Yue ◽  
Wei Ding ◽  
John F. Engelhardt
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Gene Delivery ◽  
Muscle Cells ◽  
Therapeutic Benefit ◽  
Transduction Efficiency ◽  
Great Promise ◽  
Adeno Associated Virus ◽  
Muscle Gene ◽  
Duchenne's Muscular Dystrophy

ABSTRACT Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improving the safety and therapeutic benefit in clinical trials. In the present study, we compared the efficiency of gene delivery to mouse muscle cells for recombinant AAV type 2 (rAAV-2) and rAAV-2cap5 (AAV-2 genomes pseudo-packaged into AAV-5 capsids). Despite similar levels of transduction by these two vectors in undifferentiated myoblasts, pseudotyped rAAV-2cap5 demonstrated dramatically enhanced transduction in differentiated myocytes in vitro (>500-fold) and in skeletal muscle in vivo (>200-fold) compared to rAAV-2. Serotype-specific differences in transduction efficiency did not directly correlate with viral binding to muscle cells but rather appeared to involve endocytic or intracellular barriers to infection. Furthermore, application of this pseudotyped virus in a mouse model of Duchenne's muscular dystrophy also demonstrated significantly improved transduction efficiency. These findings should have a significant impact on improving rAAV-mediated gene therapy in muscle.

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