Search

Background Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death. Methodology/Principal Findings RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-β (Ifnb1), Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division. Conclusions/Significance Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection., Alfred P. Sloan Foundation, David & Lucile Packard Foundation

Variants in the IMP2 (insulin-like growth factor 2 [IGF2] mRNA-binding protein 2) gene are implicated in susceptibility to type 2 diabetes. We describe the ability of mammalian target of rapamycin (mTOR) to regulate the cap-independent translation of IGF2 mRNA through phosphorylation of IMP2, an oncofetal RNA-binding protein. IMP2 is doubly phosphorylated in a rapamycin-inhibitable, amino acid-dependent manner in cells and by mTOR in vitro. Double phosphorylation promotes IMP2 binding to the IGF2 leader 3 mRNA 5′ untranslated region, and the translational initiation of this mRNA through eIF-4E- and 5′ cap-independent internal ribosomal entry. Unexpectedly, the interaction of IMP2 with mTOR complex 1 occurs through mTOR itself rather than through raptor. Whereas depletion of mTOR strongly inhibits IMP2 phosphorylation in cells, comparable depletion of raptor has no effect; moreover, the ability of mTOR to phosphorylate IMP2 in vitro is unaffected by the elimination of raptor. Dual phosphorylation of IMP2 at the mTOR sites is evident in the mouse embryo, likely coupling nutrient sufficiency to IGF2 expression and fetal growth. Doubly phosphorylated IMP2 is also widely expressed in adult tissues, including islets of Langerhans., National Institutes of Health (U.S.) (NIH grant CA73818), National Institutes of Health (U.S.) (NIH grant DK17776), National Institutes of Health (U.S.) (NIH grant DK057521), United States. American Recovery and Reinvestment Act of 2009

Influenza A virus is a highly infectious agent that cause seasonal epidemics affecting 5-15% of the world population with mild to severe illness and possibly death. While this pathogen represents a significant disease burden to the human population, it can also infect a wide range of animals including swine and land-based poultry, which are thought to serve as intermediate hosts between the human and natural wild aquatic bird reservoir. Here, two viruses, a swine-origin pandemic H1N1 and a seasonal human H3N2 are examined for segment fitness during co-infection of in vivo animal models. In three independent co-infections, reassortment between seasonal and pandemic viruses resulted in the selection of an H1N2 virus with a seasonal PB1 with an otherwise pandemic internal gene constellation. Selection for the seasonal PB1 and NA as well as the pandemic M segment was observed to occur rapidly during segment resolution. As pandemic M gene reassortant strains are being consistently identified in the field, studies were performed to identify the genetic determinants in pandemic M gene selection. Research here shows that both the M1 capsid protein and M2 ion channel from the pandemic virus are sufficient to drive the selection of the entire M segment. As swine represent an important intermediate host for the adaptation of potentially pandemic viruses, including pandemic M gene reassortant strains, alternative DNA and recombinant baculovirus-based platforms are investigated for their ability to generate influenza viruses from porcine polymerase I promoters and serve as potential vaccine candidates. Research here shows that influenza A virus can be rescued de novo using the porcine polymerase I promoter in an eight plasmid system. Furthermore, a single bacmid can be constructed that rescues influenza virus or baculovirus encoding the influenza reverse genetic system in mammalian tissue culture or Sf9 cells, respectively. These represent a new generation of species-tailored va

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2012., Cataloged from PDF version of thesis., Includes bibliographical references (p. 83-89)., Somatic cells can be reprogrammed to a pluripotent stem-cell state by ectopic expression of defined proteins. However, existing reprogramming methods take several weeks, suffer from low efficiencies, and most use DNA-based vectors, which carry mutagenesis risks. Here, we describe efficient and rapid reprogramming of human cells using RNA. Within two weeks, fibroblasts from 7 adult patients, including 5 Parkinson's patients aged 53 to 85, formed colonies that exhibited gene expression consistent with pluripotent stem cells. Established lines generated teratomas in vivo, and differentiated into tyrosine hydroxylase-positive neurons in vitro. Genetic analysis using array comparative genomic hybridization with an 8.9kb median probe spacing demonstrated that RNA reprogramming can yield lines free of copy number variations. The very high efficiency of this technique allowed us to reprogram single adult fibroblasts to pluripotency with a 44% success rate (n = 9). Our results suggest that the efficiency and kinetics of reprogramming methods need not be limited by a fundamental stochastic element as has been suggested. Due to the high efficiency, speed, reliability, and integration-free nature of RNA reprogramming, this technique will likely become the method of choice for generating disease and patient-specific pluripotent stem cells., by Matthew Angel., Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008., Includes bibliographical references (p. 53-56)., An experimental study was performed to evaluate a novel method of controlling protein expression by repeatedly transfecting cells with in vitro-transcribed RNA. Transcripts encoding six factors, each known to play an important role in cell-type specification and maintenance, were designed and synthesized. Aspects of the design were optimized, and the intracellular stability and translation efficiency of the ivT RNA were quantified. Transcripts were delivered to cells by electroporation, and a method of increasing the rate at which cells recover from ivT-RNA transfection by combined knockdown of innate-immune-related genes was developed. Using this technique a high, approximately steady-state level of protein expression was transduced in MRC-5 human fetal-lung fibroblasts by repeated transfection with capped, poly(A)+ ivT RNA encoding a protein with an intracellular half-life of approximately three days. Transfection at 24-hour intervals with ivT RNA encoding a less stable protein resulted in protein expression that peaked twelve hours after each transfection, and diminished before the next transfection. In both cases, cells sustained a high rate of proliferation. In this study, extended transient transfection by repeated delivery of ivT RNA was shown to transduce expression of defined factors in cultured cells without genetic modification or the extensive screening required in small-molecule approaches and with significant control over the level of transduced protein. This technique may become a powerful tool in the development of new directed-differentiation and cell-type-conversion protocols., by Matthew Angel., S.M.