RESUMEN
The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.
Asunto(s)
Ingeniería Celular , Biología Sintética , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Procesamiento Proteico-Postraduccional , Ingeniería Metabólica , Mamíferos/metabolismoRESUMEN
Progress in synthetic biology has enabled the construction of designer cells that sense biological inputs, and, in response, activate user-defined biomolecular programs. Such engineered cells provide unique opportunities for treating a wide variety of diseases. Current strategies mostly rely on cell-surface receptor systems engineered to convert binding interactions into activation of a transcriptional program. Genetic control systems are emerging as an appealing alternative to receptor-based sensors as they overcome the need for receptor engineering and result in cellular behaviors that operate over therapeutically relevant timescales. Genetic control systems include synthetic gene networks, RNA-based sensors, and post-translational tools. These technologies present fundamental challenges, including the requirement for precise integration with innate pathways, the need for parts orthogonal to existing circuitries, and the metabolic burden induced by such complex cell engineering endeavors. This review discusses the challenges in the design of genetic control systems for cellular therapies and their translational applications.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Biología Sintética , Ingeniería Celular , Redes Reguladoras de Genes/genética , Procesamiento Proteico-Postraduccional , Ingeniería GenéticaRESUMEN
BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are two highly aggressive and generally incurable gliomas with little therapeutic advancements made in the past several decades. Despite immense initial success of chimeric antigen receptor (CAR) T cells for the treatment of leukemia and lymphoma, significant headway into the application of CAR-T cells against solid tumors, including gliomas, is still forthcoming. The integrin complex alphav beta3 (αvß3) is present on multiple and diverse solid tumor types and tumor vasculature with limited expression throughout most normal tissues, qualifying it as an appealing target for CAR-T cell-mediated immunotherapy. METHODS: Patient-derived DIPG and GBM cell lines were evaluated by flow cytometry for surface expression of αvß3. Second-generation CAR-T cells expressing an anti-αvß3 single-chain variable fragment were generated by retroviral transduction containing either a CD28 or 4-1BB costimulatory domain and CD3zeta. CAR-T cells were evaluated by flow cytometry for CAR expression, memory phenotype distribution, and inhibitory receptor profile. DIPG and GBM cell lines were orthotopically implanted into NSG mice via stereotactic injection and monitored with bioluminescent imaging to evaluate αvß3 CAR-T cell-mediated antitumor responses. RESULTS: We found that patient-derived DIPG cells and GBM cell lines express high levels of surface αvß3 by flow cytometry, while αvß3 is minimally expressed on normal tissues by RNA sequencing and protein microarray. The manufactured CAR-T cells consisted of a substantial frequency of favorable early memory cells and a low inhibitory receptor profile. αvß3 CAR-T cells demonstrated efficient, antigen-specific tumor cell killing in both cytotoxicity assays and in in vivo models of orthotopically and stereotactically implanted DIPG and GBM tumors into relevant locations in the brain of NSG mice. Tumor responses were rapid and robust with systemic CAR-T cell proliferation and long-lived persistence associated with long-term survival. Following tumor clearance, TCF-1+αvß3 CAR-T cells were detectable, underscoring their ability to persist and undergo self-renewal. CONCLUSIONS: These results highlight the potential of αvß3 CAR-T cells for immunotherapeutic treatment of aggressive brain tumors with reduced risk of on-target, off-tumor mediated toxicity due to the restricted nature of αvß3 expression in normal tissues.