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1.
Nature ; 585(7823): 107-112, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32728218

RESUMO

Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours1,2. We are testing melanoma FixVac (BNT111)-an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma-in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination.


Assuntos
Antineoplásicos/uso terapêutico , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Melanoma/imunologia , Melanoma/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA Neoplásico/genética , Linfócitos T/imunologia , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Terapia Combinada , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Estadiamento de Neoplasias , Linfócitos T/citologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Vacinação
2.
Nature ; 547(7662): 222-226, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28678784

RESUMO

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of ß2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.


Assuntos
Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Melanoma/imunologia , Melanoma/terapia , Mutação/genética , Medicina de Precisão/métodos , RNA/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/imunologia , Antígenos CD8/imunologia , Vacinas Anticâncer/uso terapêutico , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoterapia/métodos , Melanoma/genética , Metástase Neoplásica , Recidiva Local de Neoplasia/prevenção & controle , Nivolumabe , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/imunologia , Vacinação , Microglobulina beta-2/deficiência
3.
Nature ; 534(7607): 396-401, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-27281205

RESUMO

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Imunoterapia/métodos , Melanoma/imunologia , Melanoma/terapia , RNA/administração & dosagem , Administração Intravenosa , Animais , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/genética , Antígenos Virais/genética , Autoantígenos/genética , Autoantígenos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Ensaios Clínicos Fase I como Assunto , Células Dendríticas/citologia , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Feminino , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Ativação Linfocitária/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , RNA/genética , Eletricidade Estática , Linfócitos T/citologia , Linfócitos T/imunologia , Receptor 7 Toll-Like/imunologia
4.
Mol Ther ; 27(4): 824-836, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30638957

RESUMO

Synthetic mRNA has emerged as a powerful tool for the transfer of genetic information, and it is being explored for a variety of therapeutic applications. Many of these applications require prolonged intracellular persistence of mRNA to improve bioavailability of the encoded protein. mRNA molecules are intrinsically unstable and their intracellular kinetics depend on the UTRs embracing the coding sequence, in particular the 3' UTR elements. We describe here a novel and generally applicable cell-based selection process for the identification of 3' UTRs that augment the expression of proteins encoded by synthetic mRNA. Moreover, we show, for two applications of mRNA therapeutics, namely, (1) the delivery of vaccine antigens in order to mount T cell immune responses and (2) the introduction of reprogramming factors into differentiated cells in order to induce pluripotency, that mRNAs tagged with the 3' UTR elements discovered in this study outperform those with commonly used 3' UTRs. This approach further leverages the utility of mRNA as a gene therapy drug format.


Assuntos
Regiões 3' não Traduzidas/genética , Biblioteca Gênica , Terapia Genética/métodos , Estabilidade de RNA , RNA Mensageiro/genética , Animais , Doadores de Sangue , Vacinas Anticâncer , Células Cultivadas , Reprogramação Celular/genética , Feminino , Fibroblastos , Técnicas de Transferência de Genes , Meia-Vida , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Vacinação
5.
Nucleic Acids Res ; 44(20): 9578-9590, 2016 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-27903882

RESUMO

Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5' end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization.


Assuntos
Difosfatos/química , Biossíntese de Proteínas , Análogos de Capuz de RNA , Capuzes de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas , Humanos , Estrutura Molecular , Ligação Proteica , Análogos de Capuz de RNA/síntese química , Fatores de Transcrição/metabolismo
6.
J Immunol ; 194(12): 6177-89, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25957167

RESUMO

The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients' immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simple, stable, robust, and scalable technology to generate reference samples that contain defined numbers of functional Ag-specific T cells. First, we show that RNA-engineered lymphocytes, equipped with selected TCRs, can repetitively deliver functional readouts of a controlled size across multiple assay platforms. We further describe a concept for the application of TCR-engineered reference samples to keep assay performance within or across institutions under tight control. Finally, we provide evidence that these novel control reagents can sensitively detect assay variation resulting from typical sources of error, such as low cell quality, loss of reagent stability, suboptimal hardware settings, or inaccurate gating.


Assuntos
Testes Imunológicos/métodos , Testes Imunológicos/normas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Expressão Gênica , Engenharia Genética , Antígenos HLA/química , Antígenos HLA/imunologia , Humanos , Imunoterapia/métodos , Peptídeos/química , Peptídeos/imunologia , Multimerização Proteica , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia
7.
Nucleic Acids Res ; 42(16): 10245-64, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25150148

RESUMO

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, ß- or γ-position of the 5',5'-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m2 (7,3'-O)GpppG. Higher expression of cancer antigens would make mRNAs containing m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2 favorable for anticancer immunization.


Assuntos
Boranos/química , Fosfatos/química , Inibidores da Síntese de Proteínas/química , Análogos de Capuz de RNA/química , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Células Dendríticas/metabolismo , Endorribonucleases/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Pirofosfatases/metabolismo , Análogos de Capuz de RNA/síntese química , Análogos de Capuz de RNA/metabolismo , Análogos de Capuz de RNA/farmacologia , Estereoisomerismo
8.
Bioorg Med Chem Lett ; 23(13): 3753-8, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23726029

RESUMO

Synthetic mRNA cap analogs are valuable tools in the preparation of modified mRNA transcripts with improved translational activity and increased cellular stability, and have recently attracted more attention because of their great potential in therapeutic applications. We have synthesized and tested isopropylidene dinucleotide cap analogs bearing a phosphorothioate group at the ß position of the 5',5'-triphosphate bridge (two diastereomers of 2',3'-iPr-m(7)GppSpG), as synthetically simpler alternatives to previously obtained phosphorothioate cap analogs. To evaluate the utility of the new compounds in biological systems we determined their affinity to translation initiation factor 4E (eIF4E), and tested their translational properties in rabbit reticulocyte lysates (RRL) and in human immature dendritic cells (hiDCs). In order to explain the properties of isopropylidene analogs we performed (1)H NMR conformational analysis and correlated the absolute configuration at the ß-phosphorous atom with previously synthesized m(7)GppSpG.


Assuntos
Alcenos/química , Fosfatos/química , Capuzes de RNA/farmacologia , RNA Mensageiro/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Capuzes de RNA/síntese química , Capuzes de RNA/química , RNA Mensageiro/genética , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Ativação Transcricional/genética
9.
Front Mol Biosci ; 10: 1291045, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38146535

RESUMO

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription. Materials and methods: In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed. Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs. Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.

10.
J Pharm Sci ; 112(5): 1364-1371, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36642376

RESUMO

mRNA vaccines have been established as a safe and effective modality, thanks in large part to the expedited development and approval of COVID-19 vaccines. In addition to the active, full-length mRNA transcript, mRNA fragment species can be present as a byproduct of the cell-free transcription manufacturing process or due to mRNA hydrolysis. In the current study, mRNA fragment species from BNT162b2 mRNA were isolated and characterized. The translational viability of intact and fragmented mRNA species was further explored using orthogonal expression systems to understand the risk of truncated spike protein or off-target antigen translation. The study demonstrates that mRNA fragments are primarily derived from premature transcriptional termination during manufacturing, and only full-length mRNA transcripts are viable for expression of the SARS-CoV-2 spike protein antigen.


Assuntos
Vacina BNT162 , COVID-19 , Humanos , Vacinas contra COVID-19 , SARS-CoV-2/genética , RNA Mensageiro/genética , Anticorpos Antivirais
11.
RNA ; 15(1): 153-75, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19029308

RESUMO

The removal of intervening sequences from a primary RNA transcript is catalyzed by the spliceosome, a large complex consisting of five small nuclear (sn) RNAs and more than 150 proteins. At the start of the splicing cycle, the spliceosome assembles anew onto each pre-mRNA intron in an ordered process. Here, we show that several small-molecule inhibitors of protein acetylation/deacetylation block the splicing cycle: by testing a small number of bioactive compounds, we found that three small-molecule inhibitors of histone acetyltransferases (HATs), as well as three small-molecule inhibitors of histone deacetylases (HDACs), block pre-mRNA splicing in vitro. By purifying and characterizing the stalled spliceosomes, we found that the splicing cycle is blocked at distinct stages by different inhibitors: two inhibitors allow only the formation of A-like spliceosomes (as determined by the size of the stalled complexes and their snRNA composition), while the other compounds inhibit activation for catalysis after incorporation of all U snRNPs into the spliceosome. Mass-spectrometric analysis of affinity-purified stalled spliceosomes indicated that the intermediates differ in protein composition both from each other and from previously characterized native A and B splicing complexes. This suggests that the stalled complexes represent hitherto unobserved intermediates of spliceosome assembly.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Inibidores de Histona Desacetilases , Splicing de RNA/efeitos dos fármacos , Spliceossomos/metabolismo , Acetilação , Catálise , Células HeLa , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Humanos , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteínas Nucleares Pequenas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/efeitos dos fármacos
12.
RNA Biol ; 8(1): 35-43, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21289486

RESUMO

RNAs with optimized properties are increasingly investigated as a tool to deliver the genetic information of complete antigens into professional antigen-presenting dendritic cells for HLA haplotype-independent antigen-specific vaccination against cancer. As the dose of the antigen and duration of its presentation are critical factors for generating strong and sustained antigen-specific immune responses, improvement of the immunobioavailability of RNA-based vaccines has been a recurrent subject of research. Substantial increase of the amount of antigen produced from RNA can be achieved by optimizing RNA stability and translational efficiency. Both features are determined by cis-acting elements in the RNA, namely the 5' cap, the poly(A) tail, and the sequence of the coding and non-coding regions, which interact with corresponding trans-acting factors. This article summarizes recent developments in identifying optimized RNA for expression of foreign proteins in dendritic cells, as well as their implications for immunotherapy based on antigen-encoding RNA.


Assuntos
Vacinas Anticâncer/farmacocinética , Imunoterapia/métodos , Capuzes de RNA/metabolismo , RNA/farmacocinética , Regiões 3' não Traduzidas , Apresentação de Antígeno/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Neoplasias/terapia , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Poliadenilação , RNA/imunologia , RNA/uso terapêutico , Capuzes de RNA/imunologia , Estabilidade de RNA
13.
Sci Transl Med ; 13(610): eabc7804, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34516826

RESUMO

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.


Assuntos
Citocinas , Neoplasias , Citocinas/genética , Humanos , Neoplasias/genética , Neoplasias/terapia , RNA Mensageiro
14.
Mol Biol Cell ; 17(7): 3221-31, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16687569

RESUMO

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs.


Assuntos
Corpos Enovelados/metabolismo , Corpos Enovelados/ultraestrutura , Proteínas Nucleares/análise , Ribonucleoproteínas Nucleares Pequenas/análise , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Corpos Enovelados/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células HeLa , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Mutação , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/antagonistas & inibidores , Proteínas de Transporte Nucleocitoplasmático/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/análise , Proteínas do Complexo SMN , Spliceossomos/metabolismo , Spliceossomos/ultraestrutura
15.
Wiley Interdiscip Rev RNA ; 6(5): 471-99, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26061157

RESUMO

In recent years, the interest in using messenger RNA (mRNA) as a therapeutic means to tackle different diseases has enormously increased. This holds true not only for numerous preclinical studies, but mRNA has also entered the clinic to fight cancer. The advantages of using mRNA compared to DNA were recognized very early on, e.g., the lack of risk for genomic integration, or the expression of the encoded protein in the cytoplasm without the need to cross the nuclear membrane. However, it was generally assumed that mRNA is just not stable enough to give rise to sufficient expression of the encoded protein. Yet, an initially small group of mRNA aficionados could demonstrate that the stability of mRNA and the efficiency, by which the encoded protein is translated, can be significantly increased by selecting the right set of cis-acting structural elements (including the 5'-cap, 5'- and 3'-untranslated regions, poly(A)-tail, and modified building blocks). In parallel, significant advances in RNA packaging and delivery have been made, extending the potential for this molecule. This paved the way for further work to prove mRNA as a promising therapeutic for multiple diseases. Here, we review the developments to optimize mRNA regarding stability, translational efficiency, and immune-modulating properties to enhance its functionality and efficacy as a therapeutic. Furthermore, we summarize the current status of preclinical and clinical studies that use mRNA for cancer immunotherapy, for the expression of functional proteins as so-called transcript (or protein) replacement therapy, as well as for induction of pluripotent stem cells.


Assuntos
Imunoterapia , Células-Tronco Pluripotentes Induzidas/imunologia , Neoplasias , RNA Mensageiro , RNA , Animais , Humanos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , RNA/genética , RNA/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia
16.
Methods Mol Biol ; 969: 55-72, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296927

RESUMO

The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5'-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2'- or 3'-positions of m(7)Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen production and presentation by dendritic cells. Protocols are presented for synthesis of modified mRNAs, their introduction into cells and whole animals, and measurement of their translational efficiency and stability.


Assuntos
Biossíntese de Proteínas , Análogos de Capuz de RNA/química , Estabilidade de RNA , RNA Mensageiro , Transfecção/métodos , Animais , Humanos , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/genética
17.
Curr Gene Ther ; 12(5): 347-61, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22827224

RESUMO

Several viral and non-viral vectors have been developed for exogenous protein expression in specific cells. Conventionally, this purpose is achieved through the use of recombinant DNA. But mainly due to the risks associated with permanent genetic alteration of cells, safety and ethical concerns have been raised for the use of DNA-based vectors in human clinical therapy. In the last years, synthetic messenger RNA has emerged as powerful tool to deliver genetic information. RNA vectors exhibit several advantages compared to DNA and are particularly interesting for applications that require transient gene expression. RNA stability and translation efficiency can be increased by cis-acting structural elements in the RNA such as the 5'-cap, the poly(A)-tail, untranslated regions and the sequence of the coding region. Here we review recent developments in the optimization of messenger RNA as vector for modulation of protein expression emphasizing on stability, transfection and immunogenicity. In addition, we summarize current pre-clinical and clinical studies using RNA-based vectors for immunotherapy, T cell, stem cell as well as gene therapy.


Assuntos
Técnicas de Transferência de Genes , RNA Mensageiro/genética , Animais , Expressão Gênica , Terapia Genética , Vetores Genéticos , Humanos , Imunidade Inata , Processamento de Proteína Pós-Traducional , Estabilidade de RNA , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo
18.
Cancer Res ; 72(5): 1081-91, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22237626

RESUMO

Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients.


Assuntos
Vacinas Anticâncer/uso terapêutico , Exoma , Melanoma Experimental/terapia , Mutação Puntual/imunologia , Animais , Vacinas Anticâncer/classificação , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Epitopos/genética , Feminino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de DNA , Vacinação , Vacinas de Subunidades Antigênicas/uso terapêutico
19.
Curr Genet ; 42(5): 241-51, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12589463

RESUMO

Most primary messenger RNA transcripts (pre-mRNAs) in eukaryotes contain intervening sequences that must be precisely removed to generate a functional mRNA. The excision of the intervening sequences, the introns, from a pre-mRNA and the concomitant joining of the flanking sequences, the exons, is called pre-mRNA splicing. Pre-mRNA splicing takes place in large ribonucleoprotein machinery, the spliceosome. Although the function and components of this machinery appear to be highly conserved between organisms, many distinct differences between budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe, have been found, emphasizing their evolutionary distance. Most interestingly, fission yeast appears to reflect the more conservative evolutionary development regarding pre-mRNA splicing. Many spliceosomal components, including the five small nuclear RNAs, which most likely form the catalytic core of the spliceosome, show a higher degree of similarity with the components of the splicing machinery found in mammals. In addition, several regulatory components of the spliceosome detected in mammals are absent in Sac. cerevisiae, but present in Sch. pombe. Here, we review recent progress made in our understanding of the control of pre-mRNA splicing in Sch. pombe. The focus is on Prp4p kinase, first discovered in fission yeast and also present in mammals, but absent in Sac. cerevisiae. Results from both mammals and Sch. pombe suggest that Prp4p plays a key role in regulating pre-mRNA splicing and in connecting this process with the cell cycle.


Assuntos
Precursores de RNA/genética , Splicing de RNA/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Schizosaccharomyces/genética , Animais , Sequência de Bases , Sequência Conservada , Mamíferos , Conformação de Ácido Nucleico , Transcrição Gênica
20.
Proc Natl Acad Sci U S A ; 99(14): 9145-9, 2002 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-12087126

RESUMO

Prp8 is the largest and most highly conserved protein in the spliceosome yet its mechanism of function is poorly understood. Our previous studies implicate Prp8 in control of spliceosome activation for the first catalytic step of splicing, because substitutions in five distinct regions (a-e) of Prp8 suppress a cold-sensitive block to activation caused by a mutation in U4 RNA. Catalytic activation of the spliceosome is thought to require unwinding of the U1 RNA/5' splice site and U4/U6 RNA helices by the Prp28 and Prp44/Brr2 DExD/H-box helicases, respectively. Here we show that mutations in regions a, d, and e of Prp8 exhibit allele-specific genetic interactions with mutations in Prp28, Prp44/Brr2, and U6 RNA, respectively. These results indicate that Prp8 coordinates multiple processes in spliceosome activation and enable an initial correlation of Prp8 structure and function. Furthermore, additional genetic interactions with U4-cs1 support a two-state model for this RNA conformational switch and implicate another splicing factor, Prp31, in Prp8-mediated spliceosome activation.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Temperatura Baixa , RNA Helicases DEAD-box , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , RNA Helicases , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Splicing de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
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