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1.
Mol Ther ; 26(1): 304-319, 2018 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28988711

RESUMO

Directed evolution continues to expand the capabilities of complex biomolecules for a range of applications, such as adeno-associated virus vectors for gene therapy; however, advances in library design and selection strategies are key to develop variants that overcome barriers to clinical translation. To address this need, we applied structure-guided SCHEMA recombination of the multimeric adeno-associated virus (AAV) capsid to generate a highly diversified chimeric library with minimal structural disruption. A stringent in vivo Cre-dependent selection strategy was implemented to identify variants that transduce adult neural stem cells (NSCs) in the subventricular zone. A novel variant, SCH9, infected 60% of NSCs and mediated 24-fold higher GFP expression and a 12-fold greater transduction volume than AAV9. SCH9 utilizes both galactose and heparan sulfate as cell surface receptors and exhibits increased resistance to neutralizing antibodies. These results establish the SCHEMA library as a valuable tool for directed evolution and SCH9 as an effective gene delivery vector to investigate subventricular NSCs.


Assuntos
Dependovirus/genética , Engenharia Genética , Vetores Genéticos/genética , Ventrículos Laterais/citologia , Células-Tronco Neurais/metabolismo , Transdução Genética , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Dependovirus/classificação , Dependovirus/ultraestrutura , Galactose/metabolismo , Biblioteca Gênica , Técnicas de Transferência de Genes , Terapia Genética/métodos , Genoma Viral , Heparitina Sulfato/metabolismo , Humanos , Imageamento Tridimensional , Camundongos , Modelos Moleculares , Mutação , Conformação Proteica
2.
Cancer Immunol Immunother ; 60(1): 99-109, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20960189

RESUMO

Survivin is overexpressed by 70-80% of pancreatic cancers, and is associated with resistance to chemotherapy and a poor prognosis. Gemcitabine has been a standard treatment for patients with advanced pancreatic cancer for a decade. Recent reports have demonstrated that gemcitabine treatment attenuates the tumor-suppressive environment by eliminating CD11b(+)/Gr-1(+) myeloid-derived suppressor cells (MDSCs). We hypothesize that a cancer vaccine targeting survivin can achieve enhanced efficacy when combined with gemcitabine. In this study, we tested this hypothesis using modified vaccinia Ankara (MVA) expressing full-length murine survivin. The poorly immunogenic mouse pancreas adenocarcinoma cell line, Pan02, which expresses murine survivin and is syngeneic to C57BL/6, was used for this study. Immunization with MVA-survivin resulted in a modest therapeutic antitumor effect on established Pan02 tumors. When administered with gemcitabine, MVA-survivin immunization resulted in significant tumor regression and prolonged survival. The enhanced vaccine efficacy was associated with decreased CD11b(+)/Gr-1(+) MDSCs. To analyze the survivin-specific immune response to MVA-survivin immunization, we utilized a peptide library of 15mers with 11 residues overlapping from full-length murine survivin. Splenocytes from mice immunized with MVA-survivin produced intracellular γ-interferon in response to in vitro stimulation with the overlapping peptide library. Increased survivin-specific CD8(+) T cells that specifically recognized the Pan02 tumor line were seen in mice treated with MVA-survivin and gemcitabine. These data suggest that vaccination with MVA-survivin in combination with gemcitabine represents an attractive strategy to overcome tumor-induced peripheral immune tolerance, and this effect has potential for clinical benefit in pancreatic cancer.


Assuntos
Adenocarcinoma/terapia , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Pancreáticas/terapia , Fragmentos de Peptídeos/metabolismo , Proteínas Repressoras/metabolismo , Vaccinia virus/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígeno CD11b/biossíntese , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Vetores Genéticos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/imunologia , Interferon gama/genética , Interferon gama/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Receptores de Superfície Celular/biossíntese , Indução de Remissão , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Survivina , Vacinação , Vaccinia virus/imunologia , Gencitabina
3.
Cell Rep ; 27(13): 3780-3789.e4, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242412

RESUMO

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dependovirus , Eletroporação , Técnicas de Introdução de Genes , Infecções por Parvoviridae , Ribonucleoproteínas , Animais , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Camundongos , Camundongos Transgênicos , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
4.
Curr Opin Neurobiol ; 50: 163-170, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29614429

RESUMO

Optimization of traditional replication-competent viral tracers has granted access to immediate synaptic partners of target neuronal populations, enabling the dissection of complex brain circuits into functional neural pathways. The excessive virulence of most conventional tracers, however, impedes their utility in revealing and genetically perturbing cellular function on long time scales. As a promising alternative, the natural capacity of adeno-associated viral (AAV) vectors to safely mediate persistent and robust gene expression has stimulated strong interest in adapting them for sparse neuronal labeling and physiological studies. Furthermore, increasingly refined engineering strategies have yielded novel AAV variants with enhanced target specificity, transduction, and retrograde trafficking in the CNS. These potent vectors offer new opportunities for characterizing the identity and connectivity of single neurons within immense networks and modulating their activity via robust delivery of functional genetic tools.


Assuntos
Vetores Genéticos/genética , Vias Neurais/fisiologia , Engenharia de Proteínas/métodos , Animais , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/metabolismo , Humanos , Transporte Proteico/genética
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