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1.
Gene Ther ; 31(3-4): 128-143, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37833563

RESUMO

Adeno-associated virus (AAV) vector gene therapy is a promising approach to treat rare genetic diseases; however, an ongoing challenge is how to best modulate host immunity to improve transduction efficiency and therapeutic outcomes. This report presents two studies characterizing multiple prophylactic immunosuppression regimens in male cynomolgus macaques receiving an AAVrh10 gene therapy vector expressing human coagulation factor VIII (hFVIII). In study 1, no immunosuppression was compared with prednisolone, rapamycin (or sirolimus), rapamycin and cyclosporin A in combination, and cyclosporin A and azathioprine in combination. Prednisolone alone demonstrated higher mean peripheral blood hFVIII expression; however, this was not sustained upon taper. Anti-capsid and anti-hFVIII antibody responses were robust, and vector genomes and transgene mRNA levels were similar to no immunosuppression at necropsy. Study 2 compared no immunosuppression with prednisolone alone or in combination with rapamycin or methotrexate. The prednisolone/rapamycin group demonstrated an increase in mean hFVIII expression and a mean delay in anti-capsid IgG development until after rapamycin taper. Additionally, a significant reduction in the plasma cell gene signature was observed with prednisolone/rapamycin, suggesting that rapamycin's tolerogenic effects may include plasma cell differentiation blockade. Immunosuppression with prednisolone and rapamycin in combination could improve therapeutic outcomes in AAV vector gene therapy.


Assuntos
Ciclosporina , Sirolimo , Masculino , Humanos , Animais , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Sirolimo/metabolismo , Ciclosporina/metabolismo , Plasmócitos , Prednisolona/farmacologia , Prednisolona/uso terapêutico , Prednisolona/metabolismo , Terapia Genética , Vetores Genéticos/genética , Macaca/genética , Dependovirus
2.
Phys Biol ; 15(6): 065006, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30124431

RESUMO

Peptide amphiphile micelles (PAMs) are attractive vehicles for the delivery of a variety of therapeutic and prophylactic peptides. However, a key limitation of PAMs is their lack of preferential targeting ability. In this paper, we describe our design of a PAM system that incorporates a DNA oligonucleotide amphiphile (antitail amphiphile-AA) to form A/PAMs. A cell-targeting DNA aptamer with a 3' extension sequence (tail) complementary to the AA is annealed to the surface to form aptamer-displaying PAMs (Aptamer~A/PAMs). Aptamer~A/PAMs are small, anionic, stable nanoparticles capable of delivering a large mass percentage peptide amphiphile (PA) compared to targeting DNA components. Aptamer~A/PAMs are stable for over 4 h in the presence of biological fluids. Additionally, the aptamer retains its cell-targeting properties when annealed to the A/PAM, thus leading to enhanced delivery to a specifically-targeted B-cell leukemia cell line. This exciting modular technology can be readily used with a library of different targeting aptamers and PAs, capable of improving the bioavailability and potency of the peptide cargo.


Assuntos
Aptâmeros de Nucleotídeos/química , Sistemas de Liberação de Medicamentos , Micelas , Peptídeos/química , Peptídeos/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Nanopartículas/química , Nanopartículas/ultraestrutura
3.
J Immunol ; 196(10): 4003-13, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27084103

RESUMO

The scaffold molecule POSH is crucial for the regulation of proliferation and effector function in CD8(+) T cells. However, its role in CD4(+) T cells is not known. In this study, we found that disruption of the POSH scaffold complex established a transcriptional profile that strongly skewed differentiation toward Th2, led to decreased survival, and had no effect on cell cycle entry. This is in stark contrast to CD8(+) T cells in which POSH regulates cell cycle and does not affect survival. Disruption of POSH in CD4(+) T cells resulted in the loss of Tak1-dependent activation of JNK1/2 and Tak1-mediated survival. However, in CD8(+) T cells, POSH regulates only JNK1. Remarkably, each type of T cell had a unique composition of the POSH scaffold complex and distinct posttranslational modifications of POSH. These data indicate that the mechanism that regulates POSH function in CD4(+) T cells is different from CD8(+) T cells. All together, these data strongly suggest that POSH is essential for the integration of cell-type-specific signals that regulate the differentiation, survival, and function of T cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Sobrevivência Celular , Proteínas do Citoesqueleto/metabolismo , Equilíbrio Th1-Th2 , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Proteínas do Citoesqueleto/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Especificidade de Órgãos , Processamento de Proteína Pós-Traducional , Transdução de Sinais
4.
Nat Commun ; 9(1): 2283, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29891903

RESUMO

Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19-21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50-80 kDa, 175-250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs.


Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , Nanoestruturas/administração & dosagem , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , Cães , Sistemas de Liberação de Medicamentos , Endocitose , Endossomos/metabolismo , Corantes Fluorescentes/química , Humanos , Leucemia de Células B/genética , Leucemia de Células B/metabolismo , Leucemia de Células B/terapia , Microscopia Confocal , Nanoestruturas/química , Nanotecnologia , Conformação de Ácido Nucleico
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