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1.
Mol Ther ; 32(8): 2489-2504, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-38894541

RESUMO

Spinal muscular atrophy is a rare and progressive neuromuscular disease that, without treatment, leads to progressive weakness and often death. A plethora of studies have led to the approval of three high-cost and effective treatments since 2016. These treatments, nusinersen, onasemnogene abeparvovec, and risdiplam, have not been directly compared and have varying challenges in administration. In this review, we discuss the evidence supporting the use of these medications, the process of treatment selection, monitoring after treatment, the limited data comparing treatments, as well as future directions for investigation and therapy.


Assuntos
Atrofia Muscular Espinal , Oligonucleotídeos , Humanos , Atrofia Muscular Espinal/terapia , Oligonucleotídeos/uso terapêutico , Terapia Genética/métodos , Pirimidinas/uso terapêutico , Resultado do Tratamento , Compostos Azo
2.
Appl Microbiol Biotechnol ; 108(1): 154, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38240803

RESUMO

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.


Assuntos
Edição de Genes , Monascus , Monascus/genética , Monascus/metabolismo , Sistemas CRISPR-Cas , Marcação de Genes/métodos , Lovastatina/metabolismo , Agrobacterium tumefaciens/genética , DNA/metabolismo
3.
BMC Biol ; 21(1): 226, 2023 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-37864194

RESUMO

BACKGROUND: Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals. RESULTS: Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene. CONCLUSIONS: In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Feminino , Animais , Bovinos/genética , Masculino , Humanos , Edição de Genes/métodos , Marcação de Genes/métodos , Técnicas de Inativação de Genes , Mutação Puntual
4.
Alzheimers Dement ; 20(4): 3080-3087, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38343132

RESUMO

INTRODUCTION: Genetic studies conducted over the past four decades have provided us with a detailed catalog of genes that play critical roles in the etiology of Alzheimer's disease (AD) and related dementias (ADRDs). Despite this progress, as a field we have had only limited success in incorporating this rich complexity of human AD/ADRD genetics findings into our animal models of these diseases. Our primary goal for the gene replacement (GR)-AD project is to develop mouse lines that model the genetics of AD/ADRD as closely as possible. METHODS: To do this, we are generating mouse lines in which the genes of interest are precisely and completely replaced in the mouse genome by their full human orthologs. RESULTS: Each model set consists of a control line with a wild-type human allele and variant lines that precisely match the human genomic sequence in the control line except for a high-impact pathogenic mutation or risk variant.


Assuntos
Doença de Alzheimer , Humanos , Animais , Camundongos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Proteínas tau/genética , Mutação , Presenilina-1/genética , Precursor de Proteína beta-Amiloide/genética
5.
Int J Mol Sci ; 25(4)2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38396920

RESUMO

The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient's skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin's basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB.


Assuntos
Epidermólise Bolhosa , Humanos , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/terapia , Epidermólise Bolhosa/patologia , Pele/metabolismo , Epiderme/metabolismo , Vesícula , Mutação
6.
Plant Biotechnol J ; 21(12): 2641-2653, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37610064

RESUMO

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.


Assuntos
Sistemas CRISPR-Cas , Nicotiana , Nicotiana/genética , Sistemas CRISPR-Cas/genética , Protoplastos , Melhoramento Vegetal , Edição de Genes/métodos , Plantas/genética , Genoma de Planta
7.
FEMS Yeast Res ; 232023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-37500280

RESUMO

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.


Assuntos
Hanseniaspora , Hanseniaspora/genética , Saccharomyces cerevisiae/genética , Reação em Cadeia da Polimerase , Marcação de Genes
8.
Biochemistry (Mosc) ; 88(12): 2157-2178, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38462459

RESUMO

Throughout the years, several hundred million people with rare genetic disorders have been receiving only symptom management therapy. However, research and development efforts worldwide have led to the development of long-lasting, highly efficient, and safe gene therapy for a wide range of hereditary diseases. Improved viral vectors are now able to evade the preexisting immunity and more efficiently target and transduce therapeutically relevant cells, ensuring genome maintenance and expression of transgenes at the relevant levels. Hematological, ophthalmological, neurodegenerative, and metabolic therapeutic areas have witnessed successful treatment of hemophilia and muscular dystrophy, restoration of immune system in children with immunodeficiencies, and restoration of vision. This review focuses on three leading vector platforms of the past two decades: adeno-associated viruses (AAVs), adenoviruses (AdVs), and lentiviruses (LVs). Special attention is given to successful preclinical and clinical studies that have led to the approval of gene therapies: six AAV-based (Glybera® for lipoprotein lipase deficiency, Luxturna® for retinal dystrophy, Zolgensma® for spinal muscular atrophy, Upstaza® for AADC, Roctavian® for hemophilia A, and Hemgenix® for hemophilia B) and three LV-based (Libmeldy® for infantile metachromatic leukodystrophy, Zynteglo® for ß-thalassemia, and Skysona® for ALD). The review also discusses the problems that arise in the development of gene therapy treatments, which, nevertheless, do not overshadow the successes of already developed gene therapies and the hope these treatments give to long-suffering patients and their families.


Assuntos
Terapia Genética , Hemofilia A , Criança , Humanos , Vetores Genéticos , Hemofilia A/genética , Hemofilia A/terapia , Transgenes
9.
New Microbiol ; 46(2): 186-195, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37247239

RESUMO

Currently, the infection of hypervirulent Klebsiella pneumoniae (hvKp) is becoming increasingly serious and the virulent mechanisms of hvKp are still not very clear. An effective gene-editing method for genes on hvKp virulence plasmid can help us reveal related virulent mechanisms. There are a few reports focusing on the methods mentioned above, however with certain limitations. In this work, we first constructed the pRE112-basing recombinant suicide plasmid to knock out or replace the genes in the hvKp virulence plasmid based on the principle of homology recombination. Results showed that the target virulent genes iucA, iucB, iroB, and rmpA2 on the hvKp virulence plasmid were scarlessly knocked out or replaced by marker genes, and mutant hvKp strains with the expected phenotypes were obtained. These indicated that we established an efficient gene-editing method for genes on hvKp virulence plasmid, which could help us explore the functions of these genes and reveal the virulent mechanisms of hvKp.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Humanos , Virulência/genética , Klebsiella pneumoniae/genética , Plasmídeos/genética , Infecções por Klebsiella/tratamento farmacológico , Antibacterianos/uso terapêutico
10.
Int J Mol Sci ; 23(18)2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36142294

RESUMO

Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8-9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.


Assuntos
Edição de Genes , Oryza , Sistemas CRISPR-Cas , Carotenoides , DNA , Edição de Genes/métodos , Liases Intramoleculares , Oryza/genética , Espécies Reativas de Oxigênio , Replicon/genética
11.
Nervenarzt ; 93(6): 537-548, 2022 Jun.
Artigo em Alemão | MEDLINE | ID: mdl-35384490

RESUMO

BACKGROUND: In recent years the theoretical hope has become reality and the first hereditary neuromuscular diseases have become causally treatable. Neuromuscular diseases have thus become the pacemaker of this form of therapy for the whole of neurology. AIMS: This article describes the principles of precision gene therapy for neurogenetic diseases using examples of neuromuscular diseases. DISCUSSION: Various strategies of gene therapy have become established and are being tested in preclinical and clinical trials and evaluated as approved forms for long-term efficacy. The aim of every gene therapy is the modification or introduction of the target gene with initiation of a degradation of dysfunctional proteins. Various techniques, such as gene transfer, gene substitution or gene editing in vivo and ex vivo are now usable. For example, a modification of the pre-mRNA using antisense oligonucleotides or RNA interference (siRNA) can be used for exon skipping. An initiation of gene expression to produce the target protein can be based on a modification of the DNA by means of gene replacement, cell-based therapy (iPS cells), regulation by compensatory proteins or pharmacological treatment with so-called small molecules. Each method has advantages and complex disadvantages that must be individually evaluated. Phenotypic peculiarities of a rare disease often only become apparent through specific translational therapy. It is already becoming obvious that a very early point in timing of gene therapy is probably the most effective. Newborn screening is therefore gaining additional importance as early diagnosis can achieve the best possible success of therapies, possibly even preventively.


Assuntos
Distrofia Muscular de Duchenne , Doenças Neuromusculares , Terapia Genética , Humanos , Recém-Nascido , Distrofia Muscular de Duchenne/terapia , Doenças Neuromusculares/diagnóstico , Doenças Neuromusculares/genética , Doenças Neuromusculares/terapia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Splicing de RNA
12.
J Mol Cell Cardiol ; 150: 101-108, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33049255

RESUMO

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited myocardial disease characterized by unexplained left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. Clinical heterogeneity is wide, ranging from asymptomatic individuals to heart failure, arrhythmias and sudden death. HCM is often caused by mutations in genes encoding components of the sarcomere. Among them, MYBPC3, encoding cardiac myosin-myosin binding protein C is the most frequently mutated gene. Three quarter of pathogenic or likely pathogenic mutations in MYBPC3 are truncating and the resulting protein was not detected in HCM myectomy samples. The overall prognosis of the patients is excellent if managed with contemporary therapy, but still remains a significant disease-related health burden, and carriers with double heterozygous, compound heterozygous and homozygous mutations often display a more severe clinical phenotype than single heterozygotes. We propose these individuals as a good target population for MYBPC3 gene therapy.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/uso terapêutico , Terapia Genética , Fatores Etários , Animais , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/terapia , Proteínas de Transporte/química , Transplante de Coração , Humanos , Mutação/genética
13.
Appl Environ Microbiol ; 87(23): e0151021, 2021 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-34524900

RESUMO

Targeted gene insertion or replacement is a promising genome-editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum, targeted gene insertion and replacement remain a serious challenge due to the low efficiency of homologous recombination (HR) in this species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the nonhomologous end joining (NHEJ) pathway, at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma, we first inactivated the NHEJ pathway via disruption of the Ku70 gene (ku70) using a dual single guide RNA (sgRNA)-directed gene deletion method. Disruption of the ku70 gene significantly decreased NHEJ activity in G. lucidum. Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of targeted gene insertion and replacement, respectively, when target DNA with the orotidine 5'-monophosphate decarboxylase (ura3) gene and 1.5-kb homologous 5'- and 3'-flanking sequences was used as a donor template, compared to 3.3% and 0%, respectively, at these targeted sites for a control strain (Cas9 strain). Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum. IMPORTANCE Functional genomic studies in Ganoderma have been hindered by the absence of adequate genome-engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum, targeted gene insertion and replacement have remained a serious challenge due to the low efficiency of HR in these species, although such precise genome modifications, including site mutations, site-specific integrations, and allele or promoter replacements, would be incredibly valuable. In this work, we inactivated the NHEJ repair mechanism in G. lucidum by disrupting the ku70 gene using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70-disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.


Assuntos
Sistemas CRISPR-Cas , Mutagênese Insercional , Reishi , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Genômica , Reishi/genética
14.
J Exp Bot ; 72(13): 4796-4808, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33872346

RESUMO

Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yield and quality. Farmers can benefit from cultivars tolerant to herbicides such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimize the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L., we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa, for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS, resulting in amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring these substitutions were glyphosate-tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape.


Assuntos
Brassica napus , RNA Guia de Cinetoplastídeos , Brassica napus/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA , Glicina/análogos & derivados , Processamento Pós-Transcricional do RNA , Replicon , Glifosato
15.
Muscle Nerve ; 64(2): 153-155, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33959970

RESUMO

Implementation of newborn screening for spinal muscular atrophy (SMA) in 33 US states and increased genetic carrier screening have led to an increase in early, presymptomatic diagnosis of SMA. Early treatment is critically important and is recommended for presymptomatic infants with two to four copies of survival motor neuron 2. Currently, no specific treatment recommendations exist for preterm infants with SMA. The US Food and Drug Administration does not recommend using onasemnogene abeparvovec-xioi in preterm infants. Some insurance companies interpret "preterm" to be less than 40 weeks gestational age (GA) instead of the commonly accepted 37 weeks GA, which can be a barrier to treatment access. Given the risk of rapid decline in some infants, we recommend treatment of preterm infants when they reach 37 weeks GA, based on the definitions of term GA from the World Health Organization and Centers for Disease Control and Prevention, assuming all other treatment criteria are met.


Assuntos
Produtos Biológicos/uso terapêutico , Atrofia Muscular Espinal/tratamento farmacológico , Atrofias Musculares Espinais da Infância/tratamento farmacológico , Fatores de Tempo , Terapia Genética/métodos , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Neurônios Motores , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Triagem Neonatal , Atrofias Musculares Espinais da Infância/diagnóstico
16.
Proc Natl Acad Sci U S A ; 115(39): E9182-E9191, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30181272

RESUMO

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is "foreign," and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTRG2 mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.


Assuntos
DNA , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Força Muscular/genética , Distrofia Muscular de Duchenne/terapia , Plasmídeos , Animais , DNA/genética , DNA/farmacocinética , Modelos Animais de Doenças , Distrofina/genética , Distrofina/imunologia , Distrofina/metabolismo , Vetores Genéticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Força Muscular/imunologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/metabolismo , Plasmídeos/genética , Plasmídeos/farmacologia
17.
Hautarzt ; 71(2): 85-90, 2020 Feb.
Artigo em Alemão | MEDLINE | ID: mdl-31965203

RESUMO

BACKGROUND: Use of ex vivo stem cell gene therapy enables the correction of the genetic cause of a monogenetic skin disease. OBJECTIVES: The procedure and choice of gene therapy method in the course of ex vivo gene therapy of the skin are presented. MATERIALS AND METHODS: Current gene therapeutic applications focus on the addition or targeted correction of the respective gene within the genome. RESULTS: So far, gene replacement therapy has been successfully used in patients suffering from the blistering skin disease epidermolysis bullosa. Designer nuclease-based gene therapy approaches are at the preclinical stage. CONCLUSIONS: The selection of the gene therapy method depends on its safety profile, the target genodermatoses and the genetic mutation to correct.


Assuntos
Epidermólise Bolhosa , Dermatopatias , Transplante de Células-Tronco , Epidermólise Bolhosa/terapia , Terapia Genética , Humanos , Células-Tronco
18.
Nervenarzt ; 91(4): 294-302, 2020 Apr.
Artigo em Alemão | MEDLINE | ID: mdl-32076758

RESUMO

5­q-associated spinal muscular atrophy (SMA) has so far been a causally untreatable disease, which leads to severe, progressive physical restrictions due to the loss of spinal motor neurons. However, the monogenetic cause of the relatively short coding "survival motor neuron" (SMN) 1 gene sequence and the presence of almost identical gene copies, the SMN2 genes, offer favorable conditions for the development of new therapeutic approaches. While previously only supportive and palliative therapies could be used, new disease-modifying drugs are now available for the first time. Nusinersen, an antisense oligonucleotide (ASO), is the first drug that has received approval in Germany to treat SMA. Further therapeutic approaches such as the so-called "small molecules" or the gene replacement therapy are currently still being tested in clinical studies or are already waiting for approval by the European Medicines Agency (EMA). In this article, the most important disease-modifying drugs of SMA, the associated studies and their challenges are presented.


Assuntos
Terapia Genética , Atrofia Muscular Espinal , Oligonucleotídeos Antissenso , Terapia Genética/tendências , Alemanha , Humanos , Neurônios Motores , Atrofia Muscular Espinal/terapia , Oligonucleotídeos Antissenso/uso terapêutico
19.
Plant J ; 95(1): 5-16, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29668111

RESUMO

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Geminiviridae/genética , Marcação de Genes/métodos , Plantas Geneticamente Modificadas/genética , Replicon/genética , Solanum lycopersicum/genética , Alelos , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Genes de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala
20.
Pflugers Arch ; 471(5): 807-815, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-29971600

RESUMO

Hypertrophic cardiomyopathy (HCM) is an inherited myocardial disease with an estimated prevalence of 1:200 caused by mutations in sarcomeric proteins. It is associated with hypertrophy of the left ventricle, increased interstitial fibrosis, and diastolic dysfunction for heterozygous mutation carriers. Carriers of double heterozygous, compound heterozygous, and homozygous mutations often display more severe forms of cardiomyopathies, ultimately leading to premature death. So far, there is no curative treatment against HCM, as current therapies are focused on symptoms relief by pharmacological intervention and not on the cause of HCM. In the last decade, several strategies have been developed to remove genetic defects, including genome editing, exon skipping, allele-specific silencing, spliceosome-mediated RNA trans-splicing, and gene replacement. Most of these technologies have already been tested for efficacy and efficiency in animal- or human-induced pluripotent stem cell models of HCM with promising results. We will summarize recent technological advances and their implication as gene therapy options in HCM with a special focus on treating MYBPC3 mutations and its potential for being a successful bench to bedside example.


Assuntos
Cardiomiopatia Hipertrófica/genética , Terapia Genética/métodos , Animais , Cardiomiopatia Hipertrófica/terapia , Edição de Genes/métodos , Humanos
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