RESUMEN
Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors. The fluorescence labelling procedure uses the (3-(2-furoyl) quinoline-2-carboxaldehyde dye, and the CE-SDS LIF method enables the evaluation of in-process besides final product samples. This method outperforms other analytical methods, such as SDS-polyacrylamide gel electrophoresis with Sypro Ruby staining, in terms of sensitivity, resolution, and high-throughput capability. Notably, this CE-SDS LIF method was also successfully implemented to characterize enveloped viruses such as Maraba virus and lentivirus, whose development as biopharmaceuticals is now restricted by the lack of suitable analytical tools. This method was also qualified for quantification of rAAV2 according to the International Council for Harmonisation guidelines. Overall, our work shows that CE-SDS LIF is a precise and sensitive analytical platform for in-process sample analysis and quantification of different virus-based targets, with a great potential for application in biomanufacturing.
Asunto(s)
Electroforesis Capilar , Virión , Electroforesis Capilar/métodos , Dodecil Sulfato de Sodio , Electroforesis en Gel de PoliacrilamidaRESUMEN
Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical breakthroughs. As of April 2021, there are two rAAV-based gene therapies approved and more than two-hundred active clinical trials (approximately thirty in Phase III). However, the expected increase in demand for rAAV vectors still poses several challenges. Discussed herein are key aspects related to R&D needs and Chemistry, Manufacturing and Control (CMC) efforts required to attend this growing demand. Authors provide their perspective on strategic topics for rAAV-based therapies success: scalability and productivity; improved safety; increased process understanding combined with development of orthogonal bioanalytics that are able to identify, monitor and control Critical Quality Attributes (CQAs) during bioprocessing.
Asunto(s)
Dependovirus , Vectores Genéticos , Dependovirus/genética , Terapia Genética , Vectores Genéticos/genéticaRESUMEN
Mammalian CST (CTC1-STN1-TEN1) complex fulfills numerous functions including rescue of the stalled replication forks and termination of telomerase action. In fission yeast lacking the CTC1 ortholog, the Stn1-Ten1 complex restricts telomerase action via its sumoylation-mediated interaction with Tpz1TPP1. We identify a small ubiquitin-like modifier (SUMO)-interacting motif (SIM) in the carboxyl-terminal part of Stn1 and show that this domain is crucial for SUMO and Tpz1-SUMO interactions. Point mutations in the SIM (Stn1-226) lead to telomere elongation, impair Stn1-Ten1 recruitment to telomeres, and enhance telomerase binding, revealing that Stn1 SIM domain contributes to the inhibition of telomerase activity at chromosome ends. Our results suggest that Stn1-Ten1 promotes DNA synthesis at telomeres to limit single-strand DNA accumulation. We further demonstrate that Stn1 functions in the replication of telomeric and subtelomeric regions in a Taz1-independent manner. Genetic analysis reveals that misregulation of origin firing and/or telomerase inhibition circumvents the replication defects of the stn1-226 mutant. Together, our results show that the Stn1-Ten1 complex has a dual function at telomeres by limiting telomerase action and promoting chromosome end replication.
Asunto(s)
Chaperonas Moleculares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/genética , Telómero/metabolismo , Replicación del ADN , ADN de Cadena Simple , Expresión Génica , Modelos Biológicos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutación , Unión Proteica , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genéticaRESUMEN
The tumor suppressor protein BRCA2 is a key component of the homologous recombination pathway of DNA repair, acting as the loader of RAD51 recombinase at sites of double-strand breaks. Here we show that BRCA2 associates with telomeres during the S and G2 phases of the cell cycle and facilitates the loading of RAD51 onto telomeres. Conditional deletion of Brca2 and inhibition of Rad51 in mouse embryonic fibroblasts (MEFs), but not inactivation of Brca1, led to shortening of telomeres and accumulation of fragmented telomeric signals--a hallmark of telomere fragility that is associated with replication defects. These findings suggest that BRCA2-mediated homologous recombination reactions contribute to the maintenance of telomere length by facilitating telomere replication and imply that BRCA2 has an essential role in maintaining telomere integrity during unchallenged cell proliferation. Mouse mammary tumors that lacked Brca2 accumulated telomere dysfunction-induced foci. Human breast tumors in which BRCA2 was mutated had shorter telomeres than those in which BRCA1 was mutated, suggesting that the genomic instability in BRCA2-deficient tumors was due in part to telomere dysfunction.
Asunto(s)
Proteína BRCA2/fisiología , Recombinasa Rad51/metabolismo , Telómero/metabolismo , Animales , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Reparación del ADN , Fase G2 , Eliminación de Gen , Inestabilidad Genómica , Ratones , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/genética , Fase S , Telómero/químicaRESUMEN
Germ-line mutations in breast cancer 1, early onset (BRCA1) result in predisposition to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify p53-binding protein 1 (53BP1) as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell-cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Notably, loss of 53BP1 partially restores the homologous-recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.
Asunto(s)
Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Genes BRCA1 , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Mutación , Animales , Proteínas Reguladoras de la Apoptosis , Proteína BRCA2/deficiencia , Proteína BRCA2/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Ciclo Celular , Proliferación Celular , Proteínas Cromosómicas no Histona , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Eliminación de Gen , Genes BRCA2 , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Mutagénesis Insercional , Células Tumorales Cultivadas , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Cucurbitacin R is known to exhibit an anti-inflammatory effect in different experimental models of inflammation. In this article, we outline the effect of cucurbitacin R on T lymphocyte proliferation, cytokine production, and nuclear factor activation, as well as its influence on various experimental models of delayed-type hypersensitivity (DTH) in mice. Cucurbitacin R reduced the proliferation of phytohemagglutinin A-stimulated human T lymphocytes (IC(50), 18 microM), modifying the cell cycle, as well as the production of cytokines [interleukin (IL)-2, IL-4, IL-10, and especially interferon-gamma] and the induction of the principal cyclins implicated in the cell cycle (A(1), B(1), D(2), and E). These effects are brought on by a novel, selective inhibition of nuclear factor AT (NFAT) by cucurbitacin R, with no concomitant effect on other transcription factors such as activator protein-1. In addition, we tested the in vivo effects of cucurbitacin R in three experimental models of DTH, as well as its effects on T lymphocyte proliferation, the cell cycle, cytokines, and cyclins. Although cucurbitacin R was found to reduce the inflammatory response brought on by both oxazolone and dinitrofluorobenzene, its activity was even more pronounced against sheep red blood cell-induced edema in mouse paws, with a clear reduction in the production of IL-1beta, IL-4, and tumor necrosis factor alpha in the inflamed paw. In conclusion, cucurbitacin R has the potential to be a new immunosuppressive agent with antiproliferative effects through the inhibition of the NFAT with anti-inflammatory activity in DTH reactions.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Hipersensibilidad a las Drogas/tratamiento farmacológico , Hipersensibilidad Tardía/prevención & control , Factores de Transcripción NFATC/antagonistas & inhibidores , Triterpenos/farmacología , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Ciclo Celular/efectos de los fármacos , Ciclinas/metabolismo , Dinitrofluorobenceno/farmacología , Oído/patología , Femenino , Pie/patología , Humanos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/patología , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Células Jurkat , Ratones , Factores de Transcripción NFATC/metabolismo , Oxazolona/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Triterpenos/uso terapéuticoRESUMEN
Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Proteínas Proto-Oncogénicas/metabolismo , Triterpenos/farmacología , Proteínas ras/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Proteínas Proto-Oncogénicas p21(ras) , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
We have studied the effects of dihydrocucurbitacin B, a triterpene isolated from Cayaponia tayuya roots, on different models of delayed type hypersensitivity (DTH) in mice, as well as on T-lymphocyte proliferation and the mediators involved. In experiments with mice, dihydrocucurbitacin B inhibited the inflammatory reactions induced by oxazolone, dinitrofluorobenzene, and sheep red blood cells, reducing both the edema and cell infiltration. Moreover, the analysis of inflamed tissues showed that dihydrocucurbitacin B reduced the presence of the most relevant cytokines implicated in these processes, including interleukin-1 beta, interleukin-4, and tumor necrosis factor-alpha. Dihydrocucurbitacin B was also found to inhibit the proliferation of phytohemagglutinin-stimulated human T lymphocytes (IC(50) = 1.48 microM), halting the cell cycle in the G(0) phase. In addition, the triterpene reduced the production of interleukin-2, interleukin-4, interleukin-10, and interferon-gamma in human T lymphocytes, and it hampered the induction of the principal cyclins involved in the cell cycle, including A(1), B(1), D(2), and E(1). Finally, dihydrocucurbitacin B was found to exert a selective inhibition on the nuclear factor of activated T cells (NFAT) in human lymphocytes without affecting the calcium influx. Taken together, these results suggest that dihydrocucurbitacin B curbs DTH reactions by inhibiting NFAT, which in turn suppresses the proliferation of the most relevant cells involved in DTH reactions, namely the T cells.
Asunto(s)
Hipersensibilidad Tardía/tratamiento farmacológico , Activación de Linfocitos/efectos de los fármacos , Triterpenos/farmacología , Animales , Ciclinas/efectos de los fármacos , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Modelos Animales de Enfermedad , Inflamación/prevención & control , Ratones , Factores de Transcripción NFATC/antagonistas & inhibidores , Fase de Descanso del Ciclo CelularRESUMEN
Scropolioside A, an iridoid isolated from Scrophularia auriculata ssp. pseudoauriculata, showed anti-inflammatory properties against different experimental models of delayed-type hypersensitivity. This iridoid reduced the oedema induced by oxazolone by 79% (72 h) at 0.5 mg/ear while reducing that induced by sheep red blood cells by 47% (18 h), 45% (24 h) and 36% (48 h) at 10 mg/kg. In vivo it reduced both oedema formation and cell infiltration whereas in vitro it reduced the proliferation of activated T-lymphocytes (IC50 of 67.74 microM). Treatment with scropolioside A (100 microM) 18 and 24 h after phytohemagglutinin stimulation increased the number of cells arrested in the subG(0) phase whereas treatment 3 h after stimulation clearly increased the number of cells that passed to the S phase. Scropolioside A also inhibited the production of prostaglandin E2, leukotriene B4, nitric oxide, interleukin-1beta, interleukin-2, interleukin-4, tumour necrosis factor-alpha and interferon-gamma, but had no effect on the production of interleukin-10. Moreover, it modified the expression of both nitric oxide synthase-2 and cyclooxygenase-2, as well as the activation of nuclear factor-kappaB in RAW 264.7 macrophages.
Asunto(s)
Antiinflamatorios/farmacología , Glucósidos/farmacología , Piranos/farmacología , Alérgenos , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Citocinas/biosíntesis , Dinoprostona/inmunología , Oído/patología , Edema/inducido químicamente , Eritrocitos/inmunología , Femenino , Humanos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/tratamiento farmacológico , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/patología , Leucotrieno B4/inmunología , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Oxazolona , Elastasa Pancreática/inmunología , Ovinos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
The aim of this study was to investigate the effects of cucurbitacin R on an experimental model of adjuvant-induced arthritis in rats. The treatment of arthritic rats with cucurbitacin R (1 mg/kg p.o. daily) modified the evolution of the clinical symptoms, whereas the histopathology of paws demonstrated a reduction in the signs of arthritis. Compared with the control group, radiography of the tibiotarsal joints of cucurbitacin R-treated rats showed a decrease in joint damage and soft tissue swelling of the footpad. The in vivo study of the expression of proinflammatory enzymes (nitric-oxide synthase-2 and cyclooxygenase-2) with the aid of the Western blot technique, and that of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) by means of enzyme-linked immunosorbent assays demonstrated a clear decrease in both the enzymes and the mediators in paw homogenates. The analysis for prostaglandin E(2), nitric oxide, and TNF-alpha production in RAW 264.7 macrophages, as well as that for TNF-alpha in human lymphocytes, indicated a reduction of all mediators. The expression of cyclooxygenase-2 was not modified in RAW 264.7 macrophages, whereas the expression of nitric-oxide synthase-2 was clearly diminished. Moreover, cucurbitacin R was found to inhibit signal transducer and activator of transcription 3 activation in the lymphocytes of both healthy and arthritic men. These experimental data on the chronic model, together with previously reported activity on acute and subchronic experimental models, justify the anti-inflammatory activity of cucurbitacin R and provide further evidence for the therapeutic potential of a group of natural products as anti-inflammatory agents.