RESUMEN
Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional.
Asunto(s)
Células Madre Embrionarias/metabolismo , Células Epiteliales/metabolismo , Expresión Génica , ARN Mensajero/genética , Transfección/métodos , Transporte Activo de Núcleo Celular , Western Blotting , Diferenciación Celular/genética , Línea Celular , Núcleo Celular/metabolismo , Células Madre Embrionarias/citología , Células Epiteliales/ultraestructura , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , ARN Mensajero/metabolismo , Epitelio Pigmentado de la Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/metabolismoRESUMEN
We generated a knockout mouse model for guanidinoacetate N-methyltransferase (GAMT) deficiency (MIM 601240), the first discovered human creatine deficiency syndrome, by gene targeting in embryonic stem cells. Disruption of the open reading frame of the murine GAMT gene in the first exon resulted in the elimination of 210 of the 237 amino acids present in mGAMT. The creation of an mGAMT null allele was verified at the genetic, RNA and protein levels. GAMT knockout mice have markedly increased guanidinoacetate (GAA) and reduced creatine and creatinine levels in brain, serum and urine, which are key findings in human GAMT patients. In vivo (31)P magnetic resonance spectroscopy showed high levels of PGAA and reduced levels of creatine phosphate in heart, skeletal muscle and brain. These biochemical alterations were comparable to those found in human GAMT patients and can be attributed to the very similar GAMT expression patterns found by us in human and mouse tissues. We provide evidence that GAMT deficiency in mice causes biochemical adaptations in brain and skeletal muscle. It is associated with increased neonatal mortality, muscular hypotonia, decreased male fertility and a non-leptin-mediated life-long reduction in body weight due to reduced body fat mass. Therefore, GAMT knockout mice are a valuable creatine deficiency model for studying the effects of high-energy phosphate depletion in brain, heart, skeletal muscle and other organs.