RESUMEN
With an increasing number of gene therapy clinical trials and drugs reaching the market, it becomes important to standardize the methods that evaluate the efficacy and safety of gene therapy. We herein report the generation of lentiviral standards which are stable, cloned human cells prepared from the diploid HCT116 cell line and which carry a known number of lentiviral vector copies in their genome. These clones can be used as reference cellular materials for the calibration or qualification of analytical methods that quantify vector copy numbers in cells (VCN) or lentiviral vector genomic integration sites (IS). Cellular standards were used to show the superior precision of digital droplet PCR (ddPCR) over quantitative PCR (qPCR) for VCN determination. This enabled us to develop a new sensitive and specific VCN ddPCR method specific for the integrated provirus and not recognizing the transfer plasmid. The cellular standards, were also useful to assess the sensitivity and limits of a ligation-mediated PCR (LM-PCR) method to measure IS showing that at least 1% abundance of a single IS can be detected in a polyclonal population but that not all IS can be amplified with similar efficiency. Thus, lentiviral standards should be systematically used in all assays that assess lentiviral gene therapy efficacy and safety.
Asunto(s)
Variaciones en el Número de Copia de ADN , Terapia Genética , Genómica , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Lymph node stromal cells play a role in self-tolerance by presenting tissue antigens to T cells. Yet, immunomodulatory properties of lymphoid tissue stroma, particularly toward CD4+ T cells, remain insufficiently characterized by lack of tools to target antigens for presentation by stromal cells. A lentiviral vector was therefore designed for antigen delivery to MHC class II+ cells of nonhematopoietic origin. Following intravenous vector delivery, the transgene was detected in lymph node gp38+ stromal cells which were CD45- MHCII+ and partly positive for CD86 and CTLA4 or B7-H4. The transgene was not detected in classical dendritic cells of lymph nodes or spleen. Transgene-specific CD4+ and CD8+ T cell responses were primed and regulatory T cells were also induced but effector T cell response did not develop, even after a peptide boost. Antigen-specific CD8+ T cells were not cytolytic in vivo. Thus, expressing a neo-antigen in MHC-II+ lymph node stroma seems to trigger blunt CD4 T cell responses leading to antigen-specific CD8+ T cell anergy. These results open up new perspectives to further characterize lymph node stromal cell functional properties and to develop gene transfer protocols targeting lymph node stroma to induce peripheral tolerance.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Ganglios Linfáticos/inmunología , Células del Estroma/metabolismo , Animales , Presentación de Antígeno , Linfocitos T CD4-Positivos/inmunología , Anergia Clonal , Femenino , Genes MHC Clase II , Lentivirus/genética , Masculino , Ratones , Especificidad de ÓrganosRESUMEN
The immunogenic properties of recombinant adeno-associated virus (rAAV) gene transfer vectors remain incompletely characterized in spite of their usage as gene therapy vectors or as vaccines. Molecular interactions between rAAV and various types of antigen-presenting cells (APCs), as well as the impact of these interactions on transgene or capsid-specific immunization remain unclear. We herein show that binding motifs recognized by the capsid and which determine the vector tissue tropism are also critical for key immune activation processes. Using rAAV capsid serotype 1 (rAAV1) vectors which primary receptors on target cells are α2,3 and α2,6 N-linked sialic acids, we show that sialic acid-dependent binding of rAAV1 on APCs is essential to trigger CD4(+) T-cell responses by increasing rAAV1 uptake and contributing to antigenic presentation of both the capsid and transgene product although this involves different APCs. In addition, the nanoparticulate structure of the vector in itself appears to be sufficient to trigger mobilization and activation of some APCs. Therefore, combinations of structural and of serotype-specific cell-targeting properties of rAAV1 determine its complex immunogenicity. These findings may be useful to guide a selection of rAAV variants depending on the intended level of immunogenicity for either gene therapy or vaccination applications.
Asunto(s)
Dependovirus/genética , Nanopartículas/química , Serogrupo , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Cápside/inmunología , Dependovirus/inmunología , Femenino , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Inyecciones Intramusculares , Lectinas/inmunología , Ratones , Ratones Endogámicos C57BL , Nanosferas/química , Nanosferas/virología , Análisis de Componente Principal , Transgenes , Vacunas Sintéticas/virologíaRESUMEN
The salting process for meat transformation is a crucial step in conventional industry. Recent developments in label-free spectrometry techniques combined with machine learning hold great promise for high-precision salt processing. In this study, we applied UV fluorescence to characterize salting treatments in pig's Teres major muscle and predict NaCl concentrations. t-SNE analyses based on spectral measurements revealed clear differences between NaCl-free and salted treatments. However, salt treatments were not clearly identified. We then highlighted and exploited a variability seen in the emission spectra at the wavelengths 300, 318, and 360 nm, which reflected structural or compositional changes. Using this information, predictive models could accurately identify the five salted treatments with a high specificity and sensitivity or predict salt concentrations. This study paves the way toward the possibility for industrials to precisely adjust NaCl concentrations with precision during processing.
Asunto(s)
Manipulación de Alimentos , Cloruro de Sodio , Animales , Porcinos , Manipulación de Alimentos/métodos , Cloruro de Sodio/análisis , Carne/análisis , Músculos/química , Cloruro de Sodio Dietético , Aprendizaje AutomáticoRESUMEN
Salted and tumbled pork teres major muscle samples, with varying sodium chloride content (1.1 % to 1.9 %), were examined by UV fluorescence spectroscopy. Results indicated that muscle fluorescence varies with salt level as a consequence of the protein denaturation state. The 1.5 % NaCl level was the threshold beyond which the fluorescence properties no longer changed markedly. Changes in muscle fluorescence do not appear to be linearly related to salt levels. Hence, we explored whether the change in fluorescence relies on other factors relating to the variability of carcass characteristics and on muscle physicochemical changes that are partly dependent on stress response and on postmortem metabolism evolution.
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Carne , Cloruro de Sodio , Cloruro de Sodio/química , Carne/análisis , Músculo Esquelético/química , Cloruro de Sodio Dietético/análisis , Fluorescencia , Manipulación de Alimentos/métodosRESUMEN
Gene transfer efficacy is limited by unwanted immunization against transgene products. In some models, immunization may be avoided by regulating transgene expression with mir142.3p target sequences. Yet, it is unclear if such a strategy controls T-cell responses following recombinant adeno-associated viral vector (rAAV)-mediated gene transfer, particularly in muscle. In mice, intramuscular rAAV1 gene delivery of a tagged human sarcoglycan muscle protein is robustly immunogenic and leads to muscle destruction. In this model, the simple insertion of mir142.3p-target sequences in the transgene expression cassette modifies the outcome of gene transfer, providing high and persistent levels of muscle transduction in C57Bl/6 mice. Such regulated vector fails to prime specific CD4 and CD8 T cells; although, transgene tolerance seems to result from ignorance and could be broken by a robust antigenic challenge. While effective in normal mice, the mir142.3p-regulated transgene remains immunogenic in sarcoglycan-deficient dystrophic mice. In these mice, transgene expression is only prolonged but does not persist as effector CD4 and CD8 T-cell responses develop. Thus, using a mir142.3p-regulated transgene can improve rAAV muscle gene transfer results, but the level of efficacy depends on the context of application. In normal muscle, this strategy is sufficient to prevent immunization and functions even more effectively than tissue-specific promoters. In dystrophic models, additional strategies are required to fully control T-cell responses.