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1.
EMBO Rep ; 23(6): e53955, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35393769

RESUMO

Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame-disrupting mutations in the DMD gene. Although exon-skipping antisense oligonucleotides (AOs) are clinically approved and can correct DMD, insufficient muscle delivery limits efficacy. If AO activity can be enhanced by safe dietary supplements, clinical trials for efficacy can be undertaken rapidly to benefit patients. We showed previously that intravenous glycine enhanced phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscles in mdx mice. Here, we demonstrate that the combination of oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body-wide function and phenotypic rescue of dystrophin /utrophin double knock-out (DKO) mice without any overt adverse effects. The DKO mice treated with the combination without altering the approved administration protocol of PMO show improved cardio-respiratory and behavioral functions. Metformin and glycine individually are ineffective in DMD patients, but the combination of PMO with clinically-approved oral glycine and metformin might improve the efficacy of the treatment also in DMD patients. Our data suggest that this combination therapy might be an attractive therapy for DMD and potentially other muscle diseases requiring systemic treatment with AOs.


Assuntos
Distrofina , Metformina , Animais , Distrofina/genética , Terapia Genética/métodos , Glicina/uso terapêutico , Humanos , Metformina/uso terapêutico , Camundongos , Camundongos Endogâmicos mdx , Morfolinos/genética , Morfolinos/uso terapêutico , Músculo Esquelético , Utrofina/genética
2.
Mol Ther ; 31(12): 3579-3593, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37919900

RESUMO

Neoantigen-based cancer vaccines are emerging as promising tumor therapies, but enhancement of immunogenicity can further improve therapeutic outcomes. Here, we demonstrate that anchoring different peptide neoantigens on subcutaneously administered serum exosomes promote lymph node homing and dendritic cell uptake, resulting in significantly enhanced antigenicity in vitro and in vivo. Exosomes anchoring of melanoma peptide neoantigens augmented the magnitude and breadth of T cell response in vitro and in vivo, to a greater extent with CD8+ T cell responses. Simultaneous decoration of different peptide neoantigens on serum exosomes induced potent tumor suppression and neoantigen-specific immune responses in mice with melanoma and colon cancer. Complete tumor eradication and sustainable immunological memory were achieved with neoantigen-painted serum exosome vaccines in combination with programmed cell death protein 1 (PD-1) antibodies in mice with colon cancer. Importantly, human serum exosomes loaded with peptide neoantigens elicited significant tumor growth retardation and immune responses in human colon cancer 3-dimensional (3D) multicellular spheroids. Our study demonstrates that serum exosomes direct in vivo localization, increase dendritic cell uptake, and enhance the immunogenicity of antigenic peptides and thus provides a general delivery tool for peptide antigen-based personalized immunotherapy.


Assuntos
Vacinas Anticâncer , Neoplasias do Colo , Exossomos , Melanoma , Humanos , Animais , Camundongos , Antígenos de Neoplasias , Exossomos/metabolismo , Peptídeos , Imunoterapia/métodos , Neoplasias do Colo/terapia , Neoplasias do Colo/tratamento farmacológico
3.
J Med Virol ; 93(7): 4603-4607, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33719033

RESUMO

We compared the performance of five assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on nasopharyngeal swab samples: Roche "cobas," Luminex "ARIES," MiRXES "Fortitude," Altona "RealStar," and Thermo Fisher Scientific "TaqPath." A total of 94 nasopharyngeal swab samples were obtained from 80 confirmed coronavirus disease 2019 cases in the first 2 weeks of illness (median, 7 days; range, 2-14 days) and 14 healthy controls. After collection, all samples were transported to the hospital clinical laboratory within 24 h. These samples were tested on all five assays within 3 days of sample receipt. Of the 94 samples, 69 yielded the same result on all platforms, resulting in an agreement of 73.4% (69 of 94). Of these, 14 were the healthy control swabs which all tested negative, demonstrating good specificity across all platforms. The ARIES assay had the lowest detection rate (68.8%), followed by Fortitude (85.0%), RealStar (86.3%), cobas (95.0%), and TaqPath (100%). Statistically significant differences were observed for ARIES, Fortitude, and RealStar when compared against the best performing TaqPath using McNemar's χ2 test. A consensus result was established based on the results obtained by the cobas, Fortitude, RealStar, and TaqPath. Six discrepancies had failed to reach a consensus and were adjudicated using the Cepheid Xpert Xpress SARS-CoV-2. Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS-CoV-2 gene targets. On the other hand, the ARIES assay was the least sensitive, thus warranting the need for assay re-optimization before go-live at the testing laboratory.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Genes Virais/genética , Humanos , Nasofaringe/virologia , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade
4.
Mol Ther ; 28(5): 1339-1358, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32209436

RESUMO

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.


Assuntos
Proliferação de Células/efeitos dos fármacos , Transplante de Células/métodos , Glicinérgicos/administração & dosagem , Glicina/administração & dosagem , Morfolinos/administração & dosagem , Distrofia Muscular de Duchenne/tratamento farmacológico , Mioblastos/transplante , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/transplante , Animais , Modelos Animais de Doenças , Sinergismo Farmacológico , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/fisiologia , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
5.
Mol Ther ; 27(3): 571-583, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30711446

RESUMO

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-ß, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-ß3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-ß pathway proteins.


Assuntos
Exossomos/metabolismo , Fibrose/terapia , Nefropatias/terapia , MicroRNAs/fisiologia , Atrofia Muscular/terapia , Animais , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Exossomos/genética , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose/genética , Nefropatias/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Atrofia Muscular/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo
6.
Mol Ther ; 27(12): 2111-2122, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501034

RESUMO

The development of new therapies to slow down or halt the progression of Parkinson's disease is a health care priority. A key pathological feature is the presence of alpha-synuclein aggregates, and there is increasing evidence that alpha-synuclein propagation plays a central role in disease progression. Consequently, the downregulation of alpha-synuclein is a potential therapeutic target. As a chronic disease, the ideal treatment will be minimally invasive and effective in the long-term. Knockdown of gene expression has clear potential, and siRNAs specific to alpha-synuclein have been designed; however, the efficacy of siRNA treatment is limited by its short-term efficacy. To combat this, we designed shRNA minicircles (shRNA-MCs), with the potential for prolonged effectiveness, and used RVG-exosomes as the vehicle for specific delivery into the brain. We optimized this system using transgenic mice expressing GFP and demonstrated its ability to downregulate GFP protein expression in the brain for up to 6 weeks. RVG-exosomes were used to deliver anti-alpha-synuclein shRNA-MC therapy to the alpha-synuclein preformed-fibril-induced model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved the clinical symptoms. Our results confirm the therapeutic potential of shRNA-MCs delivered by RVG-exosomes for long-term treatment of neurodegenerative diseases.


Assuntos
Encéfalo/metabolismo , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Exossomos/genética , Doença de Parkinson/terapia , RNA Interferente Pequeno/genética , alfa-Sinucleína/administração & dosagem , Animais , Regulação da Expressão Gênica , Terapia Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença de Parkinson/genética , Doença de Parkinson/patologia , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/genética
7.
Nucleic Acids Res ; 45(13): 7870-7885, 2017 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-28575281

RESUMO

We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach.


Assuntos
Terapia Genética/métodos , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/terapia , Ataxina-7/antagonistas & inibidores , Ataxina-7/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Íntrons , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Genéticos , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Splicing de RNA , Ataxias Espinocerebelares/metabolismo , Transfecção
8.
Nucleic Acids Res ; 43(13): 6568-78, 2015 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-26089392

RESUMO

Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs, and will be especially useful for co-delivery of coding genes and RNAi. A specific family of mirtrons - 3'-tailed mirtrons - has hairpins precisely defined on the 5' end by the 5' splice site and 3' end by the branch point. Here, we present design principles for artificial 3'-tailed mirtrons and demonstrate, for the first time, efficient gene knockdown with tailed mirtrons within eGFP coding region. These artificial tailed mirtrons, unlike canonical mirtrons, have very few sequence design restrictions. Tailed mirtrons targeted against VEGFA mRNA, the misregulation of which is causative of several disorders including cancer, achieved significant levels of gene knockdown. Tailed mirtron-mediated knockdown was further shown to be splicing-dependent, and at least as effective as equivalent artificial intronic miRNAs, with the added advantage of very defined cleavage sites for generation of mature miRNA guide strands. Further development and exploitation of this unique mirtron biogenesis pathway for therapeutic RNAi coupled into protein-expressing genes can potentially enable the development of precisely controlled combinatorial gene therapy.


Assuntos
Técnicas de Silenciamento de Genes , Íntrons , Interferência de RNA , Sítios de Splice de RNA , Fator A de Crescimento do Endotélio Vascular/genética , Células HEK293 , Humanos , MicroRNAs/química , Splicing de RNA , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
Nanomedicine ; 11(4): 879-83, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25659648

RESUMO

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. FROM THE CLINICAL EDITOR: Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.


Assuntos
Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/ultraestrutura , Cromatografia em Gel , Células HEK293 , Humanos , Ultrafiltração
12.
RNA ; 18(7): 1328-37, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22647847

RESUMO

Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs. Here we present a design algorithm for artificial mirtrons and demonstrate, for the first time, efficient gene knockdown of myotonic dystrophy protein kinase (DMPK) target sequences in Renilla luciferase 3' UTR and subsequently pathogenic DMPK mRNA, causative of Type I myotonic dystrophy, using artificial mirtrons cloned as eGFP introns. Deep sequencing of artificial mirtrons suggests that functional mature transcripts corresponding to the designed sequence were produced in high abundance. They were further shown to be splicing-dependent, Drosha-independent, and partially dependent on exportin-5, resulting in the precise generation of pre-miRNAs. In a murine myoblast line containing a pathogenic copy of human DMPK with more than 500 CUG repeats, the DMPK artificial mirtron corrected DM1-associated splicing abnormalities of the Serca-1 mRNA, demonstrating the therapeutic potential of mirtron-mediated RNAi. Thus, further development and exploitation of the unique properties of mirtrons will benefit future research and therapeutic RNAi applications as an alternative to conventional RNAi strategies.


Assuntos
Técnicas de Silenciamento de Genes , Íntrons , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Processamento Alternativo , Animais , Sequência de Bases , Linhagem Celular , Inativação Gênica , Humanos , Carioferinas/metabolismo , Camundongos , Dados de Sequência Molecular , Mioblastos/metabolismo , Miotonina Proteína Quinase , Proteínas Serina-Treonina Quinases/metabolismo , Ribonuclease III/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transcrição Gênica
13.
Nucleic Acids Res ; 40(19): 9863-75, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22848108

RESUMO

Mirtrons are a recently described category of microRNA (miRNA) relying on splicing rather than processing by the microprocessor complex to generate pre-miRNA precursors of the RNA interference (RNAi) pathway. Their discovery and subsequent verification provides important information about a distinct class of miRNA and inherent advantages that could be exploited to silence genes of interest. These include micro-processor-independent biogenesis, pol-II-dependent transcription, accurate species generation and the delivery of multiple artificial mirtrons as introns within a single host transcript. Here we determined the sequence motifs required for correct processing of the mmu-miR-1224 mirtron and incorporated these into artificial mirtrons targeting Parkinson's disease-associated LRRK2 and α-synuclein genes. By incorporating these rules associated with processing and splicing, artificial mirtrons could be designed and made to silence complementary targets either at the mRNA or protein level. We further demonstrate with a LRRK2 targeting artificial mirtron that neuronal-specific silencing can be directed under the control of the human synapsin promoter. Finally, multiple mirtrons were co-delivered within a single host transcript, an eGFP reporter, to allow simultaneous targeting of two or more targets in a combinatorial approach. Thus, the unique characteristics of artificial mirtrons make this an attractive approach for future RNAi applications.


Assuntos
MicroRNAs/química , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , alfa-Sinucleína/genética , Linhagem Celular , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mimetismo Molecular , Neurônios/metabolismo , Doença de Parkinson/genética , Regiões Promotoras Genéticas , Splicing de RNA , RNA Mensageiro/química
14.
Nucleic Acids Res ; 40(1): 438-48, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21914725

RESUMO

Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT-PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.


Assuntos
MicroRNAs/metabolismo , Interferência de RNA , Precursores de RNA/metabolismo , Animais , Linhagem Celular , Humanos , Íntrons , Camundongos , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , Splicing de RNA , Ribonuclease III/fisiologia
15.
EMBO Mol Med ; 16(4): 1027-1045, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38448545

RESUMO

Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXOEAA-PMO). EXOEAA-PMO elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXOEAA-PMO elicited therapeutic levels of dystrophin restoration and functional improvements in mdx mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.


Assuntos
Exossomos , Distrofia Muscular de Duchenne , Animais , Camundongos , Distrofina/genética , Camundongos Endogâmicos mdx , Exossomos/metabolismo , Morfolinos/metabolismo , Morfolinos/farmacologia , Morfolinos/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos/metabolismo , Oligonucleotídeos/uso terapêutico
16.
Hum Mol Genet ; 20(3): 413-21, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21062902

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kDa cytoskeletal protein, dystrophin. There is no effective treatment, and affected individuals die from respiratory failure and cardiomyopathy by age 30. Here, we investigated whether cardiomyopathy could be prevented in animal models of DMD by increasing diaphragm utrophin or dystrophin expression and thereby restoring diaphragm function. In a transgenic mdx mouse, where utrophin was over expressed in the skeletal muscle and the diaphragm, but not in the heart, we found cardiac function, specifically right and left ventricular ejection fraction as measured using in vivo magnetic resonance imaging, was restored to wild-type levels. In mdx mice treated with a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that resulted in high levels of dystrophin restoration in the skeletal muscle and the diaphragm only, cardiac function was also restored to wild-type levels. In dystrophin/utrophin-deficient double-knockout (dKO) mice, a more severely affected animal model of DMD, treatment with a PPMO again produced high levels of dystrophin only in the skeletal muscle and the diaphragm, and once more restored cardiac function to wild-type levels. In the dKO mouse, there was no difference in heart function between treatment of the diaphragm plus the heart and treatment of the diaphragm alone. Restoration of diaphragm and other respiratory muscle function, irrespective of the method used, was sufficient to prevent cardiomyopathy in dystrophic mice. This novel mechanism of treating respiratory muscles to prevent cardiomyopathy in dystrophic mice warrants further investigation for its implications on the need to directly treat the heart in DMD.


Assuntos
Cardiomiopatias/prevenção & controle , Diafragma/fisiopatologia , Distrofina/metabolismo , Morfolinas/farmacologia , Distrofia Muscular Animal/tratamento farmacológico , Utrofina/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Diafragma/efeitos dos fármacos , Diafragma/metabolismo , Distrofina/genética , Coração/fisiopatologia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Camundongos Transgênicos , Morfolinos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/fisiopatologia , Volume Sistólico , Utrofina/genética
17.
Front Bioeng Biotechnol ; 11: 1271297, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38026864

RESUMO

Introduction: Aberrant microRNA (miRNA) expressions are often discovered in many life threatening diseases such as cancer. In particular, recent studies show combinations of miRNA levels have greater diagnostic accuracy as opposed to single miRNA levels. For point-of-care applications, rapid and sensitive isothermal amplification with loop-mediated isothermal amplification (LAMP) has gained significant interest. Method: We developed a cost-effective point-of-care testing (POCT) device for multiple miRNAs that can integrate miRNA signals into a single output. Results and Discussion: We demonstrate that the loop primers for LAMP can be broken and be used for miRNA detection. This split-LAMP approach provides a logic AND-gate output for two distinct miRNA inputs. We then show that this is potentially useable in point-of-care testing using pH-sensitive dye to give a rapid, colorimetric endpoint readout within 30 min. This novel logic gate approach can potentially be extended to multiple miRNAs such that there can be a powerful diagnostic concept for multiple short RNAs in a point-of-care rapid test.

18.
Front Genet ; 14: 1086865, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36911398

RESUMO

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.

19.
Bioengineering (Basel) ; 10(5)2023 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-37237590

RESUMO

Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.

20.
Mol Ther ; 19(7): 1295-303, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21505427

RESUMO

Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here, we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to >50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-phosphorodiamidate morpholino (PMO) is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics.


Assuntos
Éxons/genética , Miocárdio/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Animais , Western Blotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Transdução Genética
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