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1.
Int J Mol Sci ; 23(1)2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-35008999

RESUMO

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy.

2.
Mol Ther Nucleic Acids ; 25: 237-250, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34458008

RESUMO

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

3.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33804860

RESUMO

The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.


Assuntos
Redes Reguladoras de Genes , Doenças Neurodegenerativas/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Linhagem Celular Tumoral , Éxons , Células HEK293 , Humanos , Neurônios/metabolismo , Motivos de Nucleotídeos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transcriptoma
4.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805154

RESUMO

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.


Assuntos
Autoantígenos/metabolismo , Epidermólise Bolhosa Juncional/genética , Colágenos não Fibrilares/metabolismo , Oligonucleotídeos Antissenso/genética , Splicing de RNA , Processamento Alternativo , Biópsia , Linhagem Celular , Sobrevivência Celular , Epidermólise Bolhosa Juncional/metabolismo , Epidermólise Bolhosa Juncional/terapia , Éxons , Genótipo , Homozigoto , Humanos , Queratinócitos/citologia , Lipossomos/química , Mutação , Técnicas de Cultura de Órgãos , RNA Mensageiro/metabolismo
5.
J Invest Dermatol ; 141(4): 883-893.e6, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32946877

RESUMO

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide-based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.


Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/terapia , Terapia Genética/métodos , Oligonucleotídeos Antissenso/administração & dosagem , Cicatrização/genética , Animais , Biópsia , Linhagem Celular , Modelos Animais de Doenças , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Éxons/genética , Fibroblastos , Fibrose , Humanos , Queratinócitos , Camundongos , Camundongos Transgênicos , Mutação , Oligonucleotídeos Antissenso/genética , Cultura Primária de Células , Pele/efeitos dos fármacos
6.
Cell Commun Signal ; 18(1): 61, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32276641

RESUMO

BACKGROUND: Cutaneous squamous cell carcinomas (cSCC) are the primary cause of premature deaths in patients suffering from the rare skin-fragility disorder recessive dystrophic epidermolysis bullosa (RDEB), which is in marked contrast to the rarely metastasizing nature of these carcinomas in the general population. This remarkable difference is attributed to the frequent development of chronic wounds caused by impaired skin integrity. However, the specific molecular and cellular changes to malignancy, and whether there are common players in different types of aggressive cSCCs, remain relatively undefined. METHODS: MiRNA expression profiling was performed across various cell types isolated from skin and cSCCs. Microarray results were confirmed by qPCR and by an optimized in situ hybridization protocol. Functional impact of overexpression or knock-out of a dysregulated miRNA was assessed in migration and 3D-spheroid assays. Sample-matched transcriptome data was generated to support the identification of disease relevant miRNA targets. RESULTS: Several miRNAs were identified as dysregulated in cSCCs compared to control skin. These included the metastasis-linked miR-10b, which was significantly upregulated in primary cell cultures and in archival biopsies. At the functional level, overexpression of miR-10b conferred the stem cell-characteristic of 3D-spheroid formation capacity to keratinocytes. Analysis of miR-10b downstream effects identified a novel putative target of miR-10b, the actin- and tubulin cytoskeleton-associated protein DIAPH2. CONCLUSION: The discovery that miR-10b mediates an aspect of cancer stemness - that of enhanced tumor cell adhesion, known to facilitate metastatic colonization - provides an important avenue for future development of novel therapies targeting this metastasis-linked miRNA.


Assuntos
Carcinoma de Células Escamosas , Epidermólise Bolhosa Distrófica/patologia , MicroRNAs/fisiologia , Células-Tronco Neoplásicas , Neoplasias Cutâneas , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Invasividade Neoplásica , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Cultura Primária de Células , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
7.
J Invest Dermatol ; 140(10): 1985-1993.e5, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32142798

RESUMO

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.


Assuntos
Sistemas CRISPR-Cas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Edição de Genes , Células Cultivadas , Reparo do DNA por Junção de Extremidades , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Queratinócitos/metabolismo , Mutação , Ribonucleoproteínas/farmacologia
8.
Mol Ther Nucleic Acids ; 18: 496-507, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31670199

RESUMO

Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.

9.
J Invest Dermatol ; 139(8): 1699-1710.e6, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30998984

RESUMO

Epidermolytic ichthyosis is a skin fragility disorder caused by dominant-negative mutations in KRT1 or KRT10. No definitive restorative therapies exist that target these genetic faults. Gene editing can be used to efficiently introduce frameshift mutations to inactivate mutant genes. This can be applied to counter the effect of dominantly inherited diseases such as epidermolytic ichthyosis. In this study, we used transcription activator-like effector nuclease technology, to disrupt disease-causing mutant KRT10 alleles in an ex vivo cellular approach, with the intent of developing a therapy for patients with epidermolytic ichthyosis. A transcription activator-like effector nuclease was designed to specifically target a region of KRT10, upstream of a premature termination codon known to induce a genetic knockout. This proved highly efficient at gene disruption in a patient-derived keratinocyte cell line. In addition, analysis for off-target effects indicated no promiscuous gene editing-mediated disruption. Reversion of the keratin intermediate filament fragility phenotype associated with epidermolytic ichthyosis was observed by the immunofluorescence analysis of correctly gene-edited single-cell clones. This was in concurrence with immunofluorescence and ultrastructure analysis of murine xenograft models. The efficiency of this approach was subsequently confirmed in primary patient keratinocytes. Our data demonstrate the feasibility of an ex vivo gene-editing therapy for more than 95.6% of dominant KRT10 mutations.


Assuntos
Edição de Genes/métodos , Hiperceratose Epidermolítica/terapia , Filamentos Intermediários/metabolismo , Queratina-10/genética , Pele/patologia , Alelos , Animais , Biópsia , Linhagem Celular , Modelos Animais de Doenças , Éxons/genética , Estudos de Viabilidade , Feminino , Terapia Genética/métodos , Humanos , Hiperceratose Epidermolítica/genética , Hiperceratose Epidermolítica/patologia , Queratina-10/metabolismo , Queratinócitos/patologia , Queratinócitos/transplante , Masculino , Camundongos , Mutação , Cultura Primária de Células , Estabilidade Proteica , Pele/citologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética
10.
Matrix Biol Plus ; 4: 100017, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33543014

RESUMO

High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) - a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies for genetic diseases a realistic possibility. DEB is one disorder for which substantial progress has been made toward curative therapies and improved management of the disease. However, to increase their efficacy further preclinical studies are needed. The early neonatal lethality of complete collagen VII deficient mice, have led researches to resort to using models maintaining residual collagen VII expression or grafting of DEB model skin on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage. Here, we describe a novel resource toward this end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII in situ.

11.
Int J Mol Sci ; 19(3)2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29518954

RESUMO

In recent years, RNA trans-splicing has emerged as a suitable RNA editing tool for the specific replacement of mutated gene regions at the pre-mRNA level. Although the technology has been successfully applied for the restoration of protein function in various genetic diseases, a higher trans-splicing efficiency is still desired to facilitate its clinical application. Here, we describe a modified, easily applicable, fluorescence-based screening system for the generation and analysis of antisense molecules specifically capable of improving the RNA reprogramming efficiency of a selected KRT14-specific RNA trans-splicing molecule. Using this screening procedure, we identified several antisense RNAs and short rationally designed oligonucleotides, which are able to increase the trans-splicing efficiency. Thus, we assume that besides the RNA trans-splicing molecule, short antisense molecules can act as splicing modulators, thereby increasing the trans-splicing efficiency to a level that may be sufficient to overcome the effects of certain genetic predispositions, particularly those associated with dominantly inherited diseases.


Assuntos
Regulação da Expressão Gênica , Oligonucleotídeos Antissenso , Interferência de RNA , Splicing de RNA , Trans-Splicing , Linhagem Celular , Edição de Genes , Genes Reporter , Humanos , Sítios de Splice de RNA
12.
PLoS One ; 12(12): e0189324, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29228025

RESUMO

Primordial growth failure has been linked to defects in the biology of cell division and replication. The complex processes involved in microtubule spindle formation, organization and function have emerged as a dominant patho-mechanism in these conditions. The majority of reported disease genes encode for centrosome and centriole proteins, leaving kinetochore proteins by which the spindle apparatus interacts with the chromosomes largely unaccounted for. We report a novel disease gene encoding the constitutive inner kinetochore member CENPT, which is involved in kinetochore targeting and assembly, resulting in severe growth failure in two siblings of a consanguineous family. We herein present studies on the molecular and cellular mechanisms that explain how genetic mutations in this gene lead to primordial growth failure. In both, affected human cell lines and a zebrafish knock-down model of Cenpt, we observed aberrations in cell division with abnormal accumulation of micronuclei and of nuclei with increased DNA content arising from incomplete and/or irregular chromosomal segregation. Our studies underscore the critical importance of kinetochore function for overall body growth and provide new insight into the cellular mechanisms implicated in the spectrum of these severe growth disorders.


Assuntos
Proteínas Cromossômicas não Histona/genética , Transtornos do Crescimento/genética , Animais , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Modelos Animais , Peixe-Zebra/genética
13.
Mol Ther ; 25(11): 2585-2598, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28888469

RESUMO

With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.


Assuntos
Sistemas CRISPR-Cas , Epidermólise Bolhosa Simples/terapia , Edição de Genes/métodos , Queratina-14/genética , Queratinócitos/metabolismo , Reparo de DNA por Recombinação , Sequência de Bases , Células Cultivadas , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Epidermólise Bolhosa Simples/genética , Epidermólise Bolhosa Simples/metabolismo , Epidermólise Bolhosa Simples/patologia , Éxons , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons , Queratina-14/metabolismo , Queratinócitos/patologia , Queratinócitos/transplante , Terapia de Alvo Molecular , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/genética
14.
Mol Ther ; 25(11): 2573-2584, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28800953

RESUMO

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.


Assuntos
Sistemas CRISPR-Cas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/terapia , Edição de Genes/métodos , Queratinócitos/metabolismo , Terapia de Alvo Molecular , Animais , Sequência de Bases , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/metabolismo , Epidermólise Bolhosa Distrófica/patologia , Éxons , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Queratinócitos/patologia , Queratinócitos/transplante , Camundongos , Camundongos Nus , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Transplante Heterólogo , Resultado do Tratamento
15.
Int J Mol Sci ; 17(10)2016 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-27669223

RESUMO

RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3' and 5' RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders.


Assuntos
Colágeno Tipo VII/genética , RNA de Cadeia Dupla/metabolismo , Trans-Splicing , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Bases , Células Cultivadas , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Éxons , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Dados de Sequência Molecular , Mutação , RNA Mensageiro/química , RNA Mensageiro/metabolismo
16.
Mol Ther Nucleic Acids ; 5: e287, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26928235

RESUMO

RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5' trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5' RNA trans-splicing molecule, capable of replacing Col7a1 exons 1-15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5' RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

17.
Exp Neurol ; 269: 75-89, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25819103

RESUMO

The presence of neural stem/progenitor cells (NSPCs) in specific areas of the central nervous system (CNS) supports tissue maintenance as well as regeneration. The subependymal zone (SEZ), located at the lateral ventricle's wall, represents a niche for NSPCs and in response to stroke or demyelination becomes activated with progenitors migrating towards the lesion and differentiating into neurons and glia. The mechanisms that underlie this phenomenon remain largely unknown. The vascular niche and in particular blood-derived elements such as platelets, has been shown to contribute to CNS regeneration in different pathological conditions. Indeed, intracerebroventricularly administrated platelet lysate (PL) stimulates angiogenesis, neurogenesis and neuroprotection in the damaged CNS. Here, we explored the presence of platelets in the activated SEZ after a focal demyelinating lesion in the corpus callosum of mice and we studied the effects of PL on proliferating SEZ-derived NSPCs in vitro. We showed that the lesion-induced increase in the size of the SEZ and in the number of proliferating SEZ-resident NSPCs correlates with the accumulation of platelets specifically along the activated SEZ vasculature. Expanding on this finding, we demonstrated that exposure of NSPCs to PL in vitro led to increased numbers of cells by enhanced cell survival and reduced apoptosis without differences in proliferation and in the differentiation potential of NSPCs. Finally, we demonstrate that the accumulation of platelets within the SEZ is spatially correlated with reduced numbers of apoptotic cells when compared to other periventricular areas. In conclusion, our results show that platelet-derived compounds specifically promote SEZ-derived NSPC survival and suggest that platelets might contribute to the enlargement of the pool of SEZ NSPCs that are available for CNS repair in response to injury.


Assuntos
Plaquetas/citologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Células-Tronco Adultas/citologia , Animais , Lesões Encefálicas/patologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos Endogâmicos C57BL , Neurônios/citologia
18.
Int J Mol Sci ; 16(1): 1179-91, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25569093

RESUMO

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.


Assuntos
Oligonucleotídeos Antissenso/metabolismo , Trans-Splicing , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Íntrons , Mutação , Transfecção
19.
Eur J Immunol ; 44(12): 3747-57, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25179679

RESUMO

The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sµ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.


Assuntos
Antígeno B7-2/imunologia , Proliferação de Células , Citidina Desaminase/imunologia , Dano ao DNA/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/imunologia , Feminino , Regulação Leucêmica da Expressão Gênica/imunologia , Genes de Cadeia Pesada de Imunoglobulina/imunologia , Histonas/imunologia , Humanos , Região de Troca de Imunoglobulinas/imunologia , Cadeias mu de Imunoglobulina/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino
20.
Mol Oncol ; 7(6): 1056-68, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23998959

RESUMO

Targeting tumor marker genes by RNA trans-splicing is a promising means to induce tumor cell-specific death. Using a screening system we designed RNA trans-splicing molecules (RTM) specifically binding the pre-mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC). Specific trans-splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3-specific RTMs containing HSV-tk were analyzed regarding their trans-splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3-MG). Expression of the chimeric SLCO1B3-tk was detected by semi-quantitative RT-PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene-mediated apoptosis in SLCO1B3-MG expressing cells. The lead RTM also showed its potential to facilitate a trans-splicing reaction into the endogenous SLCO1B3 pre-mRNA in EB-SCC cells resulting in tk-mediated apoptosis. We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.


Assuntos
Biomarcadores Tumorais/genética , Terapia Genética/métodos , Proteínas de Neoplasias/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , RNA Neoplásico/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , Trans-Splicing/genética , Apoptose/genética , Biomarcadores Tumorais/biossíntese , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/biossíntese , Transportadores de Ânions Orgânicos Sódio-Independentes/biossíntese , RNA Neoplásico/metabolismo , Neoplasias Cutâneas/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto
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