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1.
Proc Natl Acad Sci U S A ; 115(46): E10907-E10914, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30381459

RESUMO

The translational potential of cell-based therapies is often limited by complications related to effectively engineering and manufacturing functional cells. While the use of electroporation is widespread, the impact of electroporation on cell state and function has yet to be fully characterized. Here, we use a genome-wide approach to study optimized electroporation treatment and identify striking disruptions in the expression profiles of key functional transcripts of human T cells. These genetic disruptions result in concomitant perturbation of cytokine secretion including a 648-fold increase in IL-2 secretion (P < 0.01) and a 30-fold increase in IFN-γ secretion (P < 0.05). Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vivo, with electroporated T cells failing to demonstrate sustained antigen-specific effector responses when subjected to immunological challenge. In contrast, cells subjected to a mechanical membrane disruption-based delivery mechanism, cell squeezing, had minimal aberrant transcriptional responses [0% of filtered genes misregulated, false discovery rate (FDR) q < 0.1] relative to electroporation (17% of genes misregulated, FDR q < 0.1) and showed undiminished effector responses, homing capabilities, and therapeutic potential in vivo. In a direct comparison of functionality, T cells edited for PD-1 via electroporation failed to distinguish from untreated controls in a therapeutic tumor model, while T cells edited with similar efficiency via cell squeezing demonstrated the expected tumor-killing advantage. This work demonstrates that the delivery mechanism used to insert biomolecules affects functionality and warrants further study.


Assuntos
Engenharia Celular/métodos , Microfluídica/métodos , Células Dendríticas/imunologia , Eletroporação/métodos , Humanos , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Transcriptoma
2.
Curr Gene Ther ; 9(1): 9-19, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19275567

RESUMO

Numerous studies have investigated the potential use of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic since its discovery in 1995--because TRAIL is a potent inducer of apoptosis in tumor cells but not in normal cells and tissues. Consequently, a great deal is known about TRAIL/TRAIL receptor expression, the molecular components of TRAIL receptor signaling, and methods of altering tumor cell sensitivity to TRAIL-induced apoptosis. Our laboratory was the first to report the possibility of TRAIL gene transfer therapy as an alternative method of using TRAIL as an antitumor therapy. As with recombinant proteins administered systemically, intratumoral TRAIL gene delivery also has limitations that can restrict its full potential. Translating the preclinical TRAIL studies into the clinic has started, with the hope that TRAIL will exhibit robust tumoricidal activity against human primary tumors in situ with minimal toxic side effects.


Assuntos
Terapia Genética , Neoplasias/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Ensaios Clínicos como Assunto , Humanos , Neoplasias/genética , Neoplasias/patologia , Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
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