RNA preparations containing purified modified RNA for reprogramming cells

Abstract

To provide compositions and methods for reprogramming somatic cells using purified RNA preparations comprising single-strand mRNA encoding an iPS cell inducing factor.SOLUTION: Provided is a method for inducing mammalian cells existing in vitro to produce a recombinant protein, the method including contacting the mammalian cells with a preparation of mRNA synthesized in vitro, containing an open reading frame encoding a recombinant protein, the mRNA being obtained by in vitro transcription and containing Ψ(5-β-D-ribofuranosyluracil), or Ψ(5-β-D-ribofuranosyluracil) containing a modified nucleobase, the mRNA preparation being purified using a purification step of removing RNA contaminant molecules that are immunogenic and toxic to the cells by inducing an innate immune response.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application Serial No. 61 / 267,312, filed December 7, 2009, the entire contents of which are incorporated herein by reference.

[0002] This application also claims priority to U.S. Application Serial No. 11 / 990,646, filed March 27, 2009, which is a U.S. national registration of PCT / US06 / 32372, filed August 21, 2006, which claims priority to U.S. Provisional Application No. 60 / 710,164, filed August 23, 2005, all of which are incorporated herein by reference in their entireties.

[0003] FIELD OF THE INVENTION The present invention relates to compositions and methods for altering or reprogramming the differentiation state of eukaryotic cells, including human or other animal cells, by contacting the eukaryotic cells with a purified RNA preparation comprising or consisting of one or more different single-stranded mRNA molecules, each encoding a reprogramming factor (e.g., an iPS cell inducing factor). The purified single-stranded mRNA molecules preferably contain at least one modified nucleoside (e.g., pseudouridine (abbreviated by the Greek letter "psi" or "Ψ"), 5-methylcytosine (m-methylcytosine), or 5-methylcytosine (m-methylcytosine) in place of at least some of the corresponding unmodified conventional nucleosides (e.g., in place of substantially all of the corresponding unmodified A, C, G, or T conventional nucleosides). 5 C), 5-methyluridine (m 5 U), 2'-O-methyluridine (Um or m 2’-O U), 2-thiouridine (s 2 U), and N 6 -methyladenosine (m 6A) selected from the group consisting of: A) and B). In addition, the single-stranded mRNA molecule is preferably purified to be substantially free of RNA contaminant molecules that would activate unintended responses, reduce the expression of the single-stranded mRNA, and / or activate RNA sensors in cells. In certain embodiments, the purified RNA preparation is substantially free of RNA contaminant molecules that are double-stranded and / or uncapped RNA that are shorter or longer than the full-length single-stranded mRNA molecule. [Background technology]

[0004] In 2006, it was reported that the introduction of genes encoding four protein factors (OCT4 (octamer-4; POU class 5 homeobox 1), SOX2 (SRY (sex determining region Y)-box 2), KLF4 (Krueppel-like factor 4), and c-MYC) into differentiated mouse somatic cells induces the cells to become pluripotent stem cells (referred to herein as "induced pluripotent stem cells," "iPS cells," or "iPSCs") (Takahashi and Yamanaka 2006). Following this original report, pluripotent stem cells were also derived by transforming human somatic cells with genes encoding similar human protein factors (OCT4, SOX2, KLF4, and c-MYC) (Takahashi et al. 2007) or with genes encoding human OCT4 and SOX2 and two other human factors, NANOG and LIN28 (Lin-28 homolog A) (Yu et al. 2007). All of these methods use retroviruses or lentiviruses to integrate genes encoding reprogramming factors into the genome of the transformed cells, and somatic cells were only reprogrammed into iPS cells over extended periods of time (e.g., more than a week).

[0005] The generation of iPS cells from differentiated somatic cells holds great promise as a possible means for treating diseases through cell transplantation. The possibility of generating iPS cells from somatic cells derived from individual patients may also enable the development of patient-specific therapies with reduced risk due to immune rejection. Furthermore, the generation of iPS cells from disease-specific somatic cells holds promise as a means for researching and developing drugs to treat specific disease states (Ebert et al. 2009, Lee et al. 2009, Maehr et al. 2009).

[0006] Viral delivery of genes encoding protein reprogramming factors (or "iPSC factors") provides a highly efficient method for generating iPS cells from somatic cells, but genomic integration of foreign DNA, whether random or nonrandom, can produce unpredictable results and ultimately lead to cancer (Nakagawa et al. 2008). Emerging reports indicate that iPS cells can be generated (at lower efficiency) using other methods that do not require genomic integration. For example, it has been shown that iPS cells can be generated by repeated transfection of mouse embryonic fibroblasts with expression plasmids containing genes for OCT4, SOX2, KLF4, and c-MYC (Okita et al. 2008). Induced pluripotent stem cells have also been generated from human somatic cells by introducing plasmids that expressed genes encoding human OCT4, SOX2, c-MYC, KLF4, NANOG, and LIN28 (Yu et al. 2009). Other successful approaches to generating iPS cells include treating somatic cells with recombinant protein reprogramming factors (Zhou et al. 2009), non-integrating adenovirus (Stadtfeld et al. 2008), or piggyback transposons (Woltjen et al. 2009) to deliver the reprogramming factors. Currently, generating iPS cells using these non-viral delivery techniques to deliver reprogramming factors is extremely inefficient. Methods for generating iPS cells for potential clinical use need to increase the speed and efficiency of iPS cell formation while maintaining genomic integrity. Summary of the Invention

[0007] The present invention provides compositions and methods for reprogramming the differentiation state of eukaryotic cells, including human or other animal cells, by contacting the cells with a purified RNA preparation comprising or consisting of one or more different single-stranded mRNA molecules each encoding a reprogramming factor (e.g., an iPS cell inducer). The purified single-stranded mRNA molecules preferably contain at least one of the following amino acids: (e.g., pseudouridine (Ψ), 5-methylcytosine (m ... 5 C), 5-methyluridine (m 5 U), 2'-O-methyluridine (Um or m 2’-O U), 2-thiouridine (s 2 U), and N 6 -methyladenosine (m 6 A) contains at least one modified nucleoside (selected from the group consisting of A) in place of the corresponding unmodified A, C, G, or T normal nucleoside. In addition, the single-stranded mRNA molecule is preferably purified to be substantially free of RNA contaminant molecules that would activate unintended responses, reduce expression of single-stranded mRNA, and / or activate RNA sensors (e.g., double-stranded RNA-dependent enzymes) in cells. In certain embodiments, the purified RNA preparation is substantially free of RNA contaminant molecules that are shorter or longer than full-length single-stranded mRNA molecules, double-stranded RNA, and / or uncapped RNA. In some preferred embodiments, the present invention provides compositions and methods for programming differentiated eukaryotic cells, including human or other animal somatic cells, by contacting them with a purified RNA preparation comprising or consisting of one or more different single-stranded mRNA molecules each encoding an iPS cell-inducing factor.

[0008] In some embodiments, the present invention provides a method for altering the differentiation state of a somatic cell, comprising introducing an mRNA encoding an iPS cell inducer into the somatic cell to generate a reprogrammed, dedifferentiated cell, wherein the mRNA comprises at least one 5-methylcytidine (or other modified base described herein).

[0009] In one embodiment, the present invention provides a method for reprogramming cells exhibiting a first differentiated state or phenotype into cells exhibiting a second differentiated state or phenotype, the method comprising introducing a purified RNA preparation containing a modified mRNA molecule encoding at least one reprogramming factor into cells exhibiting the first differentiated state and culturing the cells under conditions in which the cells exhibit the second differentiated state. In one embodiment, the modified mRNA molecule contains at least one modified nucleoside selected from the group consisting of pseudouridine and 5-methylcytidine. In one embodiment, the cells are derived from a human or animal. In a further embodiment, the purified RNA preparation comprises i) a first single-stranded mRNA encoding a first iPS cell inducer, wherein substantially all of the first single-stranded complete mRNA contains at least one pseudouridine residue and / or at least one 5-methylcytidine residue, and ii) is substantially free of RNA contaminant molecules capable of activating an RNA sensor in the somatic cell. In certain embodiments, the RNA contaminant molecule is selected from the group consisting of a partial mRNA encoding only a portion of the iPS cell induction factor, an RNA molecule smaller than the full-length mRNA, an RNA molecule larger than the full-length mRNA, a double-stranded mRNA molecule, and an uncapped mRNA molecule.

[0010] In some embodiments, the present invention provides a method for reprogramming (e.g., dedifferentiating or transdifferentiating) somatic cells, comprising contacting the somatic cells with a purified RNA preparation to produce reprogrammed cells, wherein the purified RNA preparation comprises: i) a first single-stranded mRNA encoding a first iPS cell inducer, wherein substantially all of the first single-stranded complete mRNA comprises at least one pseudouridine residue and / or at least one 5-methylcytidine residue; and ii) is substantially free of contaminant molecules (e.g., RNA contaminant molecules) that can activate an RNA sensor in the somatic cells. In certain embodiments, the RNA contaminant molecule comprises a partial mRNA encoding only a portion of the iPS cell inducer, a single-stranded run-on mRNA encoding the iPS cell inducer and at least one additional portion of the iPS cell inducer, a double-stranded mRNA molecule, and an uncapped mRNA molecule. In certain embodiments, the first single-stranded mRNA also does not encode an additional portion of the first iPS cell inducer.

[0011] In some embodiments, the reprogrammed cells are dedifferentiated cells (e.g., stem cells or stem cell-like cells). In other embodiments, the reprogrammed cells are transdifferentiated cells (e.g., skin cells are reprogrammed into neuronal cells, or other types of changes). In further embodiments, the first single-stranded mRNA encodes the complete first iPS-inducing factor (e.g., the mRNA encodes the complete coding sequence for a particular iPS-inducing factor). In other embodiments, the contacting further comprises (e.g., after a period of time) contacting the somatic cell with a growth factor and / or cytokine. In further embodiments, the contacting further comprises contacting the somatic cell with an immune response inhibitor.

[0012] In some embodiments, all or nearly all of the uridine nucleosides in the first single-stranded mRNA are replaced with pseudouridine nucleosides. In other embodiments, all or nearly all of the cytidine nucleosides in the first single-stranded mRNA are replaced with 5-methylcytidine nucleosides or other bases listed herein.

[0013] In certain embodiments, the invention provides a method for producing reprogrammed cells, comprising contacting somatic cells with a purified RNA preparation to produce reprogrammed cells that can survive in culture for at least 10 days (e.g., at least 10 days...at least 13 days...at least 16 days...at least 20 days...at least 40 days...or a number of days capable of forming a cell line), wherein the purified RNA preparation comprises a first single-stranded mRNA encoding an iPS cell inducer, and wherein a majority of the first single-stranded mRNA comprises at least one pseudouridine residue and / or at least one 5-methylcytidine residue.

[0014] In some embodiments, the purified RNA preparation is free of RNA contaminant molecules in an amount that would activate an immune response in somatic cells sufficient to prevent the reprogrammed cells from surviving in culture for at least 10 days (e.g., at least 10 days...at least 15 days...at least 20 days...at least 40 days, or longer). In other embodiments, the RNA contaminant molecules include partial mRNAs encoding only a portion of the iPS cell inducer, single-stranded mRNAs that fully encode the iPS cell inducer and encode at least one additional portion of the iPS cell inducer, double-stranded mRNA molecules, uncapped mRNA molecules, and mixtures thereof. In some embodiments, the reprogrammed cells generated can form a reprogrammed cell line. In other embodiments, the purified RNA preparation is free of RNA contaminant molecules in an amount that would activate an immune response in somatic cells sufficient to prevent the generation of a reprogrammed cell line.

[0015] In certain embodiments, the RNA contaminant molecule is selected from the group consisting of a partial mRNA encoding only a portion of the iPS cell inducer, a single-stranded mRNA encoding the iPS cell inducer and encoding at least one additional portion of the iPS cell inducer, a double-stranded mRNA molecule, an uncapped mRNA molecule, and a mixture thereof.

[0016] In some embodiments, the present invention provides a method for producing a reprogrammed cell line, comprising: a) contacting somatic cells with a purified RNA preparation to produce reprogrammed cells, wherein the purified RNA preparation contains mRNA encoding an iPS cell inducer, and wherein the majority of the mRNA contains at least one pseudouridine residue and / or at least one 5-methylcytidine residue; and b) culturing the dedifferentiated cells to produce a reprogrammed cell line. In other embodiments, the purified RNA preparation does not contain contaminant molecules in amounts that would activate an immune response in somatic cells sufficient to prevent the production of a reprogrammed cell line. In some embodiments, the immune response involves the activation of RNA sensors in somatic cells.

[0017] In some embodiments, the present invention provides a method for reprogramming somatic cells, comprising contacting the somatic cells with a purified RNA preparation to produce reprogrammed cells, wherein the purified RNA preparation comprises i) a first single-stranded mRNA encoding a first iPS cell inducer, wherein substantially all of the first single-stranded mRNA comprises at least one pseudouridine residue and / or at least one 5-methylcytidine residue, and ii) a) a partial mRNA encoding only a portion of the first iPS cell inducer, and b) substantially no double-stranded mRNA molecules. In further embodiments, the first single-stranded mRNA also does not encode an additional portion of the first iPS cell inducer. In certain embodiments, the first single-stranded mRNA completely encodes the first iPS cell inducer. In other embodiments, the purified RNA preparation also encodes the first iPS cell inducer and is substantially free (or essentially free, or virtually free, or no) of a single-stranded second mRNA encoding at least one additional portion of the first iPS cell inducer. In other embodiments, substantially all of the first single-stranded complete mRNA is 5'-capped. In other embodiments, the purified RNA preparation is also substantially free of uncapped mRNA molecules. In some embodiments, substantially all of the first single-stranded mRNA comprises at least one pseudouridine residue. In additional embodiments, substantially all of the first single-stranded mRNA comprises at least one 5-methylcytidine residue. In other embodiments, substantially all of the first single-stranded mRNA comprises at least one pseudouridine residue and at least one 5-methylcytidine residue.

[0018] In some embodiments, the purified RNA preparation comprises a transfection reagent. In other embodiments, the purified RNA preparation is obtained by HPLC purification of an RNA sample containing a significant amount of partial mRNA and double-stranded mRNA. In further embodiments, the purified RNA preparation is essentially free of partial mRNA and single-stranded mRNA. In some embodiments, the purified RNA preparation is essentially free, virtually free, or free of double-stranded mRNA molecules. In other embodiments, the purified RNA preparation is essentially free, virtually free, or free of uncapped mRNA molecules. In some embodiments, substantially all of the first single-stranded mRNA is polyadenylated. In other embodiments, the first single-stranded complete mRNA is capped with 7-methylguanosine.

[0019] In some embodiments, the first iPS cell inducer is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. In other embodiments, the purified RNA preparation further comprises i) a second single-stranded mRNA encoding a second iPS cell inducer, wherein the second single-stranded mRNA comprises at least one pseudouridine residue and / or at least one 5-methylcytidine residue, and ii) is further substantially free of a) a partial mRNA encoding only a portion of the second iPS cell inducer, and b) double-stranded mRNA. In other embodiments, the purified RNA preparation further encodes the second iPS cell inducer and is substantially free of single-stranded mRNA encoding at least one additional portion of the second iPS cell inducer. In some embodiments, the second iPS cell inducer is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. In certain embodiments, the somatic cells are fibroblasts. In other embodiments, the reprogrammed cells are pluripotent stem cells. In other embodiments, the dedifferentiated cells express NANOG and TRA-1-60. In some embodiments, the cells are in vitro. In further embodiments, the cells are in culture. In certain embodiments, the cells are in mouse embryonic fibroblast (MEF) conditioned medium.

[0020] In some embodiments, the present invention provides a composition comprising a purified RNA preparation, wherein the purified RNA preparation i) comprises a first single-stranded mRNA encoding a first iPS cell inducing factor, wherein the first single-stranded mRNA comprises at least one pseudouridine residue and / or at least one 5-methylcytidine residue, and ii) is substantially free of RNA contaminant molecules capable of activating an RNA sensor in somatic cells. In certain embodiments, the present invention provides a composition comprising a purified RNA preparation, wherein the purified RNA preparation i) comprises a first single-stranded mRNA encoding a first iPS cell inducing factor, wherein the first single-stranded complete mRNA comprises at least one pseudouridine residue and / or at least one 5-methylcytidine residue, and ii) is substantially free of a) a partial mRNA encoding only a portion of the first iPS cell inducing factor, and b) double-stranded RNA.

[0021] In certain embodiments, the purified RNA preparation also encodes the first iPS cell inducer and is substantially free of single-stranded, subsequent mRNA encoding at least one additional portion of the first iPS cell inducer. In some embodiments, the purified RNA preparation further comprises i) a second single-stranded mRNA encoding a second iPS cell inducer, wherein the second single-stranded complete mRNA contains at least one pseudouridine residue and / or at least one 5-methylcytidine residue, and ii) a) a second single-stranded mRNA encoding the first iPS cell inducer and is substantially free of single-stranded, subsequent mRNA encoding at least one additional portion of the second iPS cell inducer.

[0022] In some embodiments, the present invention provides a composition comprising in vitro synthesized mRNA encoding the MYC gene, wherein the in vitro synthesized mRNA comprises at least one pseudouridine residue and / or at least one 5-methylcytidine residue. In certain embodiments, the composition is substantially free of RNA contaminant molecules capable of activating RNA sensors in somatic cells.

[0023] In certain embodiments, the present invention provides a method for inducing mammalian cells to produce MYC protein, comprising contacting mammalian cells with in vitro synthesized mRNA encoding MYC gene, wherein the in vitro synthesized mRNA contains at least one pseudouridine residue and / or at least one 5-methylcytidine residue, thereby inducing the mammalian cells to produce MYC protein. In other embodiments, the mammalian cells are dendritic cells. In other embodiments, the mammalian cells are alveolar cells, astrocytes, microglia, or neurons.

[0024] In some embodiments, the present invention provides methods of treating a subject, comprising contacting the subject with a MYC protein-producing mammalian cell as described above and herein.

[0025] In a further embodiment, the present invention provides a method for synthesizing an in vitro transcribed RNA molecule encoding a MYC gene, the method comprising combining an isolated RNA polymerase, a template nucleic acid sequence encoding a MYC gene, unmodified nucleotides, and pseudouridine or 5-methylcytidine modified nucleotides under conditions such that an in vitro transcribed RNA molecule encoding a MYC gene containing at least one pseudouridine or 5-methylcytidine residue is produced.

[0026] Experiments conducted during the development of embodiments of the present invention have shown that mRNA molecules can be administered to cells to induce the dedifferentiation process and generate dedifferentiated cells, including pluripotent stem cells. Thus, the present invention provides compositions and methods for generating iPS cells. Surprisingly, administration of mRNA can provide highly efficient generation of iPS cells.

[0027] The present invention also provides RNA molecules, oligoribonucleotide molecules, and polyribonucleotide molecules that contain pseudouridine or modified nucleosides, gene therapy vectors that contain them, methods for synthesizing them, and methods for gene replacement, gene therapy, gene transcription silencing, and in vivo delivery of therapeutic proteins to tissues that contain said molecules.The present invention also provides a method for reducing the immunogenicity of RNA molecules, oligoribonucleotide molecules, and polyribonucleotide molecules.

[0028] In some embodiments, the present invention provides methods for dedifferentiating somatic cells, comprising introducing mRNA encoding one or more iPSC-inducing factors into the somatic cells to produce dedifferentiated cells.

[0029] In some embodiments, the present invention provides a method for dedifferentiating somatic cells, comprising introducing mRNA encoding one or more iPSC-inducing factors into somatic cells, and maintaining the cells under conditions that allow the cells to survive and that allow the mRNA introduced into the cells to be translated in sufficient amounts and for sufficient time to produce dedifferentiated cells. In some preferred embodiments, the dedifferentiated cells are induced pluripotent stem cells.

[0030] In some embodiments, the present invention provides methods for altering the differentiation state (or differentiated state) of a eukaryotic cell, comprising introducing into the cell mRNA encoding one or more reprogramming factors, and maintaining the cell under conditions in which the cell is viable and in which the mRNA introduced into the cell is translated in sufficient amounts and for a sufficient time to produce a cell that exhibits an altered state of differentiation compared to the cell into which the mRNA was introduced.

[0031] In some embodiments, the present invention provides a method for changing the differentiation state of eukaryotic cells, comprising introducing mRNA encoding one or more reprogramming factors into cells, and maintaining the cells under conditions in which the cells are viable and the mRNA introduced into the cells is translated in sufficient quantity and for sufficient time to produce cells that exhibit a changed differentiation state compared to the cells into which the mRNA is introduced.In some embodiments, the changed differentiation state is a dedifferentiated state compared to the cells into which the mRNA is introduced.For example, in some embodiments, the cells that exhibit a changed differentiation state are pluripotent stem cells that dedifferentiate compared to the somatic cells into which the mRNA is introduced (e.g., fibroblasts, cardiomyocytes, or cells that differentiate into another differentiated cell type).In some embodiments, the cells into which the mRNA is introduced are somatic cells of one cell lineage, phenotype, or function, and the cells that exhibit a changed differentiation state are somatic cells that exhibit a cell lineage, phenotype, or function that is different from that of the cells into which the mRNA is introduced; therefore, in these embodiments, the method results in transdifferentiation (Graf and Enver 2009).

[0032] The present invention is not limited with respect to the particular cell into which the mRNA is introduced. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is derived from any multicellular eukaryotic organism. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is selected from among human cells and other animal cells. While the work presented herein was performed using human or other animal cells, the applicant further asserts that the methods of the present invention also relate to the reprogramming of other eukaryotic cells (e.g., plant cells and fungal cells), comprising contacting human and animal cells with a purified RNA preparation consisting of one or more purified single-stranded mRNA molecules, each encoding a protein reprogramming factor (e.g., a transcription factor). In some embodiments of any of the above methods, the cell into which the mRNA is introduced is a normal cell derived from an organism without a known disease. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is a cell derived from an organism with a known disease. In some embodiments of any of the above methods, the cells into which the mRNA is introduced are cells without a known pathology. In some embodiments of any of the above methods, the cells into which the mRNA is introduced are cells that exhibit a disease state or a known pathology (cancer cells, or pancreatic beta cells that exhibit metabolic characteristics characteristic of diabetic cells).

[0033] The present invention is not limited to the use of specific cell types (e.g., specific somatic cell types) in embodiments of the present methods, which involve introducing mRNA encoding one or more iPSC cell inducers to generate dedifferentiated cells (e.g., iPS cells). Any cells that can be dedifferentiated using iPS cell inducers are contemplated. Such cells include, but are not limited to, fibroblasts, keratinocytes, adipocytes, lymphocytes, T cells, B cells, mononuclear cord blood cells, buccal mucosa cells, hepatocytes, HeLa, MCF-7, or other cancer cells. In some embodiments, the cells are present in vitro (e.g., in culture) or in vivo. In some embodiments, when developing in culture, cell-free conditioned medium (e.g., MEF-conditioned medium) is used. As shown below, such medium provided enhanced iPS cell development. The present invention, however, is not limited to the culture conditions used. Any culture conditions or media currently known or later identified as useful in the methods of the present invention (e.g., for generating iPS cells from somatic cells and maintaining the cells) are contemplated for use with the present invention. For example, although not preferred, in some embodiments of the methods, a feeder cell layer is used instead of conditioned medium to culture the cells treated using the methods.

[0034] In some embodiments of any of these methods, the step of introducing the mRNA comprises delivering the mRNA to a cell (e.g., a somatic cell of a human or other animal) using a transfection reagent (e.g., TRANSIT™ mRNA Transfection Reagent, MirusBio, Madison, WI).

[0035] However, the present invention is not limited by the nature of the transfection method used. Indeed, any known or future-identified transfection process capable of delivering mRNA molecules to cells in vitro or in vivo is contemplated, including methods of delivering mRNA to cells in culture or in a life-supporting medium, whether isolated cells or cells comprising eukaryotic tissues or organs, or methods of delivering mRNA to cells in vivo in organisms such as humans, animals, plants, or fungi. In some embodiments, the transfection reagent comprises a lipid (e.g., a liposome, a micelle, etc.). In some embodiments, the transfection reagent comprises a nanoparticle or nanotube. In some embodiments, the transfection reagent comprises a cationic compound (e.g., polyethyleneimine, or PEI). In some embodiments, the transfection method uses an electric current to deliver the mRNA to cells (e.g., by electroporation). In some embodiments, the transfection method uses biolistic techniques to deliver the mRNA to cells (eg, a "gene gun" or "biolistic particle delivery system").

[0036] The data presented herein show that, with respect to mRNA introduced into cells, a certain amount of mRNA used in the examples described herein resulted in a higher efficiency and more rapid induction of pluripotent stem cells from the specific somatic cells used than other amounts of mRNA.However, the method of the present invention is not limited to the use of a specific amount of mRNA introduced into cells.For example, in some embodiments, mRNA was introduced into approximately 3 x 10 cells in a 10 cm plate using a total of three doses, each containing 18 micrograms of six different mRNAs, each encoding a different human reprogramming factor. 5 of human fibroblasts, but in that embodiment, greater or lesser amounts of mRNA were used to transduce the cells.

[0037] The present invention is not limited to the particular chemical form of mRNA used, although certain forms of mRNA may yield more efficient results. However, in some preferred embodiments, the mRNA contains nucleotides containing nucleotides such as pseudouridine (Ψ), 5-methylcytosine (m ... 5 C), 5-methyluridine (m 5 U), 2'-O-methyluridine (Um or m 2’-O U), 2-thiouridine (s 2 U), and N 6 -methyladenosine (m 6A) at least one modified nucleoside (selected from the group consisting of A) in place of at least a portion of the corresponding unmodified normal nucleosides (in some preferred embodiments, at least one modified nucleoside replaces substantially all of the corresponding unmodified A, C, G, or T normal nucleosides). In some embodiments, the mRNA is polyadenylated. In some preferred embodiments, the mRNA is prepared by polyadenylation of in vitro transcribed (IVT) RNA, the method comprising contacting the IVT RNA with poly(A) polymerase (e.g., yeast RNA polymerase or E. coli poly(A) polymerase). In some embodiments, the mRNA is polyadenylated during IVT by using a DNA template encoding a poly(A) tail. Regardless of whether the RNA is polyadenylated using poly(A) polymerase or during IVT of a DNA template, in some preferred embodiments, the mRNA includes a poly(A) tail (e.g., a poly(A) tail having 50 to 200 nucleotides, e.g., preferably 100 to 200, 150 to 200 nucleotides, or more than 150 nucleotides), although in some embodiments, longer or shorter poly(A) tails are used. In some embodiments, the mRNA used in the present methods is capped. To maximize the efficiency of expression in cells, it is preferred that the majority of mRNA molecules contain a cap. In some preferred embodiments, the mRNA used in the present methods is synthesized in vitro by incubating uncapped primary RNA in the presence of a capping enzyme system. In some preferred embodiments, the primary RNA used in the capping enzyme reaction is synthesized by in vitro transcription (IVT) of a DNA molecule encoding the RNA to be synthesized. The DNA encoding the RNA to be synthesized contains an RNA polymerase promoter, to which RNA polymerase binds and initiates transcription.It is also known in the art that mRNA molecules often have regions of different sequences located before the translation initiation codon and after the untranslated translation termination codon.These regions, called 5' prime untranslated region (5'UTR) and 3' prime untranslated region (3'UTR), respectively, can affect mRNA stability, mRNA localization, and the translation efficiency of the mRNA they are linked to.Some 5'UTR and 3'UTR, such as those for alpha globin and beta globin, are known to improve mRNA stability and mRNA expression. Thus, in some preferred embodiments, the mRNA encoding the reprogramming factor (e.g., an iPSC induction factor) exhibits a 5'UTR and / or 3'UTR (e.g., an alpha globin or beta globin 5'UTR and / or 3'UTR, e.g., a Xenopus or human alpha globulin or beta globulin 5'UTR and / or 3'UTR, or, e.g., a tobacco etch virus (TEV) 5'UTR) that results in greater mRNA stability and higher mRNA expression in the cells.

[0038] IVT can be performed using any RNA polymerase, as long as mRNA synthesis from the RNA-encoding DNA template is specifically and efficiently initiated from the respective cognate RNA polymerase promoters and full-length mRNA is obtained. In some preferred embodiments, the RNA polymerase is selected from T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase. In some other embodiments, capped RNA is synthesized co-transcriptionally by using a dinucleotide cap analog in the IVT reaction (e.g., using the AMPLICAP™ T7 Kit or the MESSAGEMAX™ T7 ARCA-CAPPED MESSAGE Transcription Kit, EPICENTRE or CellScript, Madison, WI, USA). When capping is performed co-transcriptionally, preferably, the dinucleotide cap analog is an anti-reverse cap analog (ARCA). However, capping using a capping enzyme system following the use of a separate IVT reaction, which results in approximately 100% of the RNA being capped, is typically preferred over co-transcriptional capping, which results in only about 80% of the RNA being capped. Thus, in some preferred embodiments, a higher percentage of the mRNA molecules used in the methods of the invention are capped (greater than 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% of the population of mRNA molecules are capped). In some preferred embodiments, the mRNA used in the methods of the invention is capped with a Cap 1 structure, meaning that the 2' hydroxyl of the ribose at the penultimate nucleotide relative to the cap nucleotide is methylated. However, in some embodiments, the mRNA used in the methods is capped with a Cap 0 structure, meaning that the 2' hydroxyl of the ribose at the penultimate nucleotide relative to the cap nucleotide is not methylated.With some, but not all, transcripts, transfection of eukaryotic cells with mRNA capped with a Cap 1 structure results in higher levels or longer duration of protein expression in the transfected cells compared to transfection of the same mRNA from the same cells but with a Cap 0 structure. In some embodiments, the mRNA used in the methods of the invention has a modified cap nucleotide. In several experiments conducted prior to the experiments presented in the Examples herein, Applicant discovered that when 1079 or IMR90 human fibroblasts were transfected with OCT4 mRNA containing either uridine or pseudouridine instead of uridine, the pseudouridine-containing mRNA was translated at a higher level or for a longer duration than the uridine-containing mRNA. Therefore, in some preferred embodiments, one or more or all of the uridines contained in the mRNA(s) used in the methods of the present invention are replaced by pseudouridine (e.g., by substituting pseudouridine-5'-triphosphate in the IVT reaction to synthesize the RNA instead of uridine-5'-triphosphate). However, in some embodiments, the mRNA used in the methods of the present invention contains uridine and does not contain pseudouridine. In some preferred embodiments, the mRNA contains (pseudouridine (Ψ), 5-methylcytosine (m). 5 C), 5-methyluridine (m 5 U), 2'-O-methyluridine (Um or m 2’-O U), 2-thiouridine (s 2 U), and N 6 -methyladenosine (m 6 In some preferred embodiments, the mRNA comprises at least one modified nucleoside selected from the group consisting of pseudouridine (Ψ) and 5-methylcytosine (m), in place of at least some of the corresponding unmodified conventional nucleosides (e.g., in place of substantially all of the corresponding unmodified A, C, G, or T conventional nucleosides).5 In some preferred embodiments, the mRNA comprises at least one modified nucleoside selected from the group consisting of pseudouridine (Ψ) and 5-methylcytosine (mC). 5 In addition, to achieve a particular goal, the nucleobase, sugar moiety, or internucleotide linkage in one or more of the nucleotides of the mRNA introduced into a eukaryotic cell in any of the methods of the invention may comprise a modified nucleobase, sugar moiety, or internucleotide linkage.

[0039] The present invention is not limited by the source of mRNA delivered to eukaryotic cells in any of the methods of the present invention.In some embodiments, such as those described in the Examples, the mRNA is synthesized by in vitro transcription of a DNA template comprising a gene cloned in a linearized plasmid vector, or by in vitro transcription of a DNA template synthesized by PCR or RT-PCR (i.e., by IVT of PCR by-products), capping with a capping enzyme system, or by co-transcriptional capping by incorporating a dinucleotide cap analog (e.g., ARCA) during IVT and polyadenylation using poly(A) polymerase.In some preferred embodiments, the mRNA is synthesized by IVT of a DNA template comprising a gene cloned in a linearized plasmid vector or a PCR or RT-PCR amplification product, wherein the DNA template encodes a 3' poly(A) tail.In some other embodiments, the mRNA delivered to eukaryotic cells in any of the methods of the present invention is directly derived from a cell or biological sample. For example, in some embodiments, mRNA from a cell or biological sample is obtained by amplifying mRNA from the cell or biological sample using an RNA amplification reaction, and capping the amplified mRNA using a capping enzyme system or co-transcriptional capping by incorporation of a dinucleotide cap analog (e.g., ARCA) during IVT, and polyadenylation of the amplified mRNA using poly(A) polymerase if the amplified mRNA does not already contain a poly(A) tail encoded by the template from the RNA amplification reaction.

[0040] With respect to methods involving the introduction of mRNA encoding one or more iPS cell inducers to generate dedifferentiated cells (e.g., iPS cells), the present invention is not limited by the nature of the iPS cell inducer used. Any mRNA encoding one or more protein inducers now known or later discovered to find use in dedifferentiation is contemplated for use in the present invention. In some embodiments, one or more mRNAs encoding KLF4, LIN28, c-MYC, NANOG, OCT4, or SOX2 are employed. Oct-3 / 4 and certain members of the Sox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified as transcriptional regulators involved in the induction process. However, additional genes, including certain members of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (C-myc, L-myc, and N-myc), Nanog, and LIN28, have been confirmed to enhance induction efficiency. Any one or more of such factors may be used as desired.

[0041] Although the compositions and methods of the present invention can be used to generate iPS cells, the present invention is not limited to the generation of such cells.For example, in some embodiments, the mRNA encoding one or more reprogramming factors is introduced into cells to generate cells with a different differentiation state compared with the cells into which the mRNA is introduced.For example, in some embodiments, the mRNA encoding one or more iPS cell inducers is used to generate dedifferentiated cells that are not iPS cells.Such cells can be used in research, drug screening and other applications.

[0042] In some embodiments, the present invention further provides a method for employing the dedifferentiated cells produced by the above method. For example, such cells are found to be used in research, drug screening, and therapeutic applications in humans or other animals. For example, in some embodiments, the produced cells are found to be used in identifying and characterizing iPS cell-inducing factors and other factors associated with differentiation or dedifferentiation. In some embodiments, the produced dedifferentiated cells are transplanted into an organism or into tissues existing in vitro or in vivo. In some embodiments, the organism, tissue, or culture system that is the host of the produced cells is exposed to a test compound, and the effect of the test compound on the cells or on the organism, tissue, or culture system is observed or measured.

[0043] In some embodiments, dedifferentiated cells (e.g., iPS cells) generated using the above methods are further processed to generate differentiated cells having the same differentiation state or cell type as the somatic cells from which the dedifferentiated cells were derived. In other embodiments, dedifferentiated cells (e.g., iPS cells) generated using the above methods are further processed to generate differentiated cells having a different differentiation state or cell type than the somatic cells from which the dedifferentiated cells were derived. In some embodiments, differentiated cells are generated from dedifferentiated cells (e.g., iPS cells) generated by introducing, during one or more treatments, mRNA encoding one or more reprogramming factors into the generated iPS cells and maintaining the mRNA-introduced cells under conditions that allow the cells to survive and differentiate into cells with an altered differentiation state or cell type compared to the generated dedifferentiated cells (e.g., iPS cells) into which the mRNA encoding one or more reprogramming factors was introduced. In some of these embodiments, the generated differentiated cells with an altered differentiation state are used for research, drug screening, and therapeutic applications (e.g., in humans or other animals). For example, the differentiated cells generated find use in identifying and characterizing reprogramming factors associated with differentiation. In some embodiments, the differentiated cells generated are transplanted into an organism or into an existing tissue in vitro or in vivo. In some embodiments, the organism, tissue, or culture system hosting the differentiated cells generated is exposed to a test compound, and the effect of the test compound on the organism, tissue, or culture system is observed or measured.

[0044] In some preferred embodiments of the method, which includes introducing mRNA encoding one or more iPSC-inducing factors into somatic cells and maintaining the cells under conditions in which the cells are viable and the introduced mRNA is expressed in sufficient amounts and for sufficient time to generate dedifferentiated cells (e.g., wherein the dedifferentiated cells are induced pluripotent stem cells), the sufficient time to generate dedifferentiated cells is less than one week. In some preferred embodiments of the method, the reprogramming efficiency for generating dedifferentiated cells is less than 1 week for 3×10 cells into which the mRNA is introduced. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 3 x 10 cells per 3 x 10 cells transfected with mRNA. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 3 x 10 cells transfected with mRNA, and the reprogramming efficiency is 100 or more dedifferentiated cells (e.g., iPSCs) per 3 x 10 cells transfected with mRNA. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 150 or more dedifferentiated cells (e.g., iPSCs) per 3×10 transfected cells. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells (e.g., iPSCs) is greater than 200 dedifferentiated cells per 3 x 10 transfected cells. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 300 or more dedifferentiated cells (e.g., iPSCs) per 3×10 transfected cells. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 3×10 to 400 dedifferentiated cells (e.g., iPSCs) per 3×10 input cells. 5In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 3 x 10 cells transfected with mRNA, and the reprogramming efficiency is 500 or more dedifferentiated cells (e.g., iPSCs) per 3 x 10 cells transfected. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 3 x 10 cells transfected with mRNA, and is 600 or more dedifferentiated cells (e.g., iPSCs) per 3 x 10 cells transfected. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 700 or more dedifferentiated cells (e.g., iPSCs) per 3×10 transfected cells. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 800 or more dedifferentiated cells (e.g., iPSCs) per 3×10 transfected cells. 5 In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is 3×10 to 900 dedifferentiated cells (e.g., iPSCs) per 3×10 transfected cells. 51000 or more dedifferentiated cells (e.g., iPSCs) per transfected cell. Thus, in some preferred embodiments, this method was more than two-fold more efficient than published protocols involving delivery of reprogramming factors using viral vectors (e.g., lentiviral vectors). In some preferred embodiments, this method was more than five-fold more efficient than published protocols involving delivery of reprogramming factors using viral vectors (e.g., lentiviral vectors). In some preferred embodiments, this method was more than ten-fold more efficient than published protocols involving delivery of reprogramming factors using viral vectors (e.g., lentiviral vectors). In some preferred embodiments, this method was more than twenty-fold more efficient than published protocols involving delivery of reprogramming factors using viral vectors (e.g., lentiviral vectors). In some preferred embodiments, this method was more than twenty-five-fold more efficient than published protocols involving delivery of reprogramming factors using viral vectors (e.g., lentiviral vectors). In some preferred embodiments, this method was more than 30 times more efficient than published protocols involving delivery of reprogramming factors using viral vectors (e.g., lentiviral vectors). In some preferred embodiments, this method was more than 35 times more efficient than published protocols involving delivery of reprogramming factors using viral vectors (e.g., lentiviral vectors). In some preferred embodiments, this method was more than 40 times more efficient than published protocols involving delivery of reprogramming factors using viral vectors (e.g., lentiviral vectors).

[0045] The present invention further provides compositions (systems, kits, reaction mixtures, cells, mRNA) used or useful in the methods and / or produced by the methods described herein. For example, in some embodiments, the present invention provides mRNA encoding an iPS cell inducer, wherein the mRNA has pseudouridine instead of uridine.

[0046] The present invention further provides a composition comprising a transfection reagent and mRNAs encoding iPS cell inducers (e.g., a mixture of a transfection reagent and mRNAs). In some preferred embodiments, the composition comprises mRNAs encoding multiple iPS cell inducers (e.g., two or more, three or more, four or more, five or more, or six), including, but not limited to, KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.

[0047] The compositions may further include any other reagents or components sufficient, necessary, or useful for carrying out any of the methods described herein, including, but not limited to, transfection reagents, medium (e.g., MEF-conditioned medium), cells (e.g., somatic cells, iPS cells), containers, boxes, buffers, inhibitors (e.g., RNase inhibitors), labels (e.g., fluorescent, luminescent, radioactive, etc.), positive and / or negative control molecules, reagents for generating capped mRNA, dry ice or other ice packs, instructions for use, cell culture equipment, detection / analysis equipment, and the like.

[0048] The present invention provides RNA molecules, oligoribonucleotide molecules, and polyribonucleotide molecules containing pseudouridine or modified nucleosides, gene therapy vectors containing them, gene therapy and gene transcription silencing methods containing them, methods for reducing their immunogenicity, and methods for synthesizing them.

[0049] In one embodiment, the present invention provides messenger RNAs containing pseudouridine residues. In another embodiment, the present invention provides RNA molecules encoding proteins of interest, wherein the RNA molecules contain pseudouridine residues. In another embodiment, the present invention provides in vitro transcribed RNA molecules containing pseudouridine or modified nucleosides. In another embodiment, the present invention provides in vitro synthesized oligoribonucleotides containing pseudouridine or modified nucleosides, wherein the modified nucleosides are m 5 Cm 5 U, m 6 A, s 2 U, Ψ, or 2'-O-methyl-U. In another embodiment, the present invention provides a gene therapy vector comprising an in vitro synthesized polyribonucleotide molecule, wherein the polyribonucleotide molecule comprises a pseudouridine or modified nucleoside.

[0050] In another embodiment, the present invention provides double-stranded RNA (dsRNA), which comprises pseudouridine or modified nucleoside as part of its sequence, and further comprises siRNA or shRNA.In another embodiment, the dsRNA molecule is more than 50 nucleotides in length.Each possibility represents a separate embodiment of the present invention.

[0051] In another embodiment, the invention provides a method for inducing a mammalian cell to produce a recombinant protein, comprising contacting the mammalian cell with an in vitro synthesized RNA molecule encoding the recombinant protein, wherein the in vitro synthesized RNA molecule comprises a pseudouridine or a modified nucleoside, thereby inducing the mammalian cell to produce the recombinant protein.

[0052] In another embodiment, the invention provides a method of treating anemia in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule, wherein the in vitro synthesized RNA molecule encodes erythropoietin, thereby treating the anemia in the subject.

[0053] In another embodiment, the invention provides a method for treating vasospasm in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule, wherein the in vitro synthesized RNA molecule encodes inducible nitric oxide synthase (iNOS), thereby treating vasospasm in the subject.

[0054] In another embodiment, the invention provides a method for improving the viability of a cell in a subject, comprising contacting the cell with an in vitro synthesized RNA molecule, wherein the in vitro synthesized RNA molecule encodes a heat shock protein, thereby improving the viability of the cell in the subject.

[0055] In another embodiment, the invention provides a method for reducing the incidence of restenosis of a blood vessel following a vasodilation procedure, comprising contacting cells of the blood vessel with an in vitro synthesized RNA molecule, wherein the in vitro synthesized RNA molecule encodes a heat shock protein, thereby reducing the incidence of restenosis in the subject.

[0056] In another embodiment, the present invention provides a method for enhancing hair growth from follicles in the scalp of a subject, comprising contacting cells of the scalp with an in vitro synthesized molecule, wherein the in vitro synthesized RNA molecule encodes telomerase or an immunosuppressive protein, thereby enhancing hair growth from the hair follicles.

[0057] In another embodiment, the present invention provides a method for inducing expression of an enzyme having antioxidant activity in a cell, comprising contacting the cell with an in vitro synthesized RNA molecule, wherein the in vitro synthesized RNA molecule encodes an enzyme having antioxidant activity, thereby inducing expression of the enzyme in the cell.

[0058] In another embodiment, the invention provides a method for treating cystic fibrosis in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule, wherein the in vitro synthesized RNA molecule encodes the cystic fibrosis transmembrane conductance regulator (CFTR), thereby treating cystic fibrosis in the subject.

[0059] In another embodiment, the invention provides a method for treating X-linked agammaglobulinemia in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule, wherein the in vitro synthesized RNA molecule encodes Bruton's tyrosine kinase, thereby treating the X-linked agammaglobulinemia.

[0060] In another embodiment, the invention provides a method for treating adenosine deaminase severe combined immunodeficiency (ADA SCID) in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule, wherein the in vitro synthesized RNA molecule encodes ADA, thereby treating ADA SCID.

[0061] In another embodiment, the invention provides a method for producing a recombinant protein, comprising contacting an in vitro translation apparatus with an in vitro synthesized polyribonucleotide, wherein the in vitro synthesized polyribonucleotide comprises a pseudouridine or modified nucleoside, thereby producing the recombinant protein.

[0062] In another embodiment, the invention provides a method for synthesizing in vitro transcribed RNA molecules containing modified nucleotides having pseudouridine-modified nucleosides, comprising contacting an isolated polymerase with a mixture of unmodified and modified nucleotides.

[0063] In another embodiment, the present invention provides an in vitro transcription apparatus comprising unmodified nucleotides, nucleotides containing pseudouridine or modified nucleosides, and a polymerase. In another embodiment, the present invention provides an in vitro transcription kit comprising unmodified nucleotides, nucleotides containing pseudouridine or modified nucleosides, and a polymerase. Each possibility represents a separate embodiment of the present invention. [Brief explanation of the drawings]

[0064] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. [Figure 1]Figure 1 shows that mRNA encoding each of six human reprogramming factors, prepared as described in the Examples, is translated and localized to the predicted subcellular locations after transfection into human neonatal 1079 fibroblasts. Untreated human 1079 fibroblasts: Photographs A, E, I, M, Q, and U show phase-contrast images of untreated human 1079 fibroblasts that had not been transformed with mRNA encoding a reprogramming factor, while photographs B, F, J, N, R, and V show fluorescence images of the same fields after the cells were stained with antibodies specific for each reprogramming factor. These results indicate that there was little or no endogenous reprogramming factor protein in untreated human 1079 fibroblasts. Treated human 1079 fibroblasts: Photographs C, G, K, O, S, and W show phase-contrast images of human 1079 fibroblasts transfected with mRNA encoding the indicated reprogramming factors, while photographs D, H, L, P, T, and X show fluorescent images of the same fields after the cells were stained with antibodies specific for each reprogramming factor 24 hours after transfection. These results demonstrate that each of the reprogramming factor proteins was expressed in human 1079 fibroblasts 24 hours after transfection with mRNA encoding the individual reprogramming factor, and that the reprogramming factor proteins localized to their predicted subcellular locations. A-T are 20x magnifications. U-X are 10x magnifications. [Figure 2] Figure 2 shows that mRNAs encoding human reprogramming factors (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) generate iPS cells in human somatic cells. Figure 2 shows brightfield (A, C) and immunofluorescence (B, D) images of iPS cells 12 days after final transfection with mRNAs encoding the reprogramming factors. NANOG staining is observed in colony #1 (B, D). Images A and B are at 10x magnification. C and D are at 20x magnification. [Figure 3]Figure 3 shows that iPS colonies derived from human 1079 and IMR90 somatic cells are positive for NANOG and TRA-1-60. Figure 3 shows phase-contrast (A, D, G) and immunofluorescence (B, C, E, F, H, I) images of iPS colonies derived from 1079 cells (A, D) and IMR90 cells (G). The same iPS colony shown in (A) is positive for both NANOG (B) and TRA-1-60 (C). The iPS colony shown in (D) is NANOG-positive (E) and TRA-1-60-positive (F). An iPS colony generated from IMR90 fibroblasts (G) is also positive for NANOG (H) and TRA-1-60 (I). All images are at 20x magnification. [Figure 4] Figure 4 shows that rapid and efficient iPSC colony formation was achieved by transfecting cells with mRNAs encoding reprogramming factors in MEF-conditioned medium. More than 200 colonies were detected 3 days after the final transfection. IMR90 cells were transfected in triplicate with 36 μg of each reprogramming mRNA (i.e., encoding KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) in 10 cm dishes. Representative iPSC colonies are shown at 4x (A, B), 10x (C-E), and 20x magnification (F). Eight days after the final mRNA transfection with mRNAs encoding the six reprogramming factors, more than 1,000 iPSC colonies were counted in IMR90 cells transfected with 18 μg (G, I) or 36 μg (H) of each of the six mRNAs. Representative colonies are shown at 4x magnification (G-H) and 10x magnification (I). [Figure 5]Figure 5 shows that 1079- and IMR90-derived iPSC colonies are positive for NANOG and TRA-1-60. Eight days after final mRNA transfection with 36 μg of mRNA for each of the six reprogramming factors, 1079-derived iPSC colonies (shown in A, D, and G) are positive for NANOG (B, E, and H) and TRA-1-60 (C, F, and I). Eight days after final mRNA transfection with 18 μg (J-L) or 36 μg (M-O) of mRNA for each of the six reprogramming factors, IMR90-derived iPS colonies are also positive for NANOG (K, N) and TRA-1-60 (L, O). [Figure 6] TNF-α production by MDDCs transfected with natural RNA shows that unmodified in vitro synthesized RNA, bacterial RNA, and mammalian mitochondrial RNA are highly immunogenic, whereas other mammalian RNAs are weakly immunogenic. Human MDDCs were incubated with Lipofectin® alone or complexed with R-848 (1 μg / mL) or 293 cells (total RNA, nuclear RNA, and cytoplasmic RNA), mouse heart (polyA+ mRNA), human platelet mitochondrial RNA, bovine tRNA, bacterial tRNA, and total RNA (E. coli) with or without RNase digestion. After 8 hours, TNF-α was measured in the supernatant by ELISA. Mean values ​​± standard error are shown. Results are representative of three independent experiments. [Figure 7]TLR-dependent activation by RNA shows that m6A and s2U modifications block TLR3 signaling, whereas all modifications block TLR7 and TLR8 signaling, and that less-modified bacterial RNA and unmodified in vitro-transcribed RNA activate all three TLRs. (A) Aliquots (1 μg) of in vitro-transcribed RNA-1571 with or without m5C, m6A, Ψ, m5U, or S2U nucleoside modifications were analyzed on a denaturing agarose gel followed by ethidium bromide staining and UV illumination. (B) 293 cells expressing human TLR3, TLR7, TLR8, and a control vector were treated with Lipofectin® alone, Lipofectin®-R-848 (1 μg / mL), or RNA (5 μg / mL). Note the modified nucleosides present in RNA-730 and RNA-1571. (C) CpG ODN-2006 (5 μg / mL), LPS (1.0 μg / mL), and RNA isolates were obtained from rat liver, a mouse cell line (TUBO), and human spleen (whole), human platelet mitochondrial RNA, or two different E. coli sources. 293-hTLR9 cells served as controls. After 8 hours, IL-8 was measured in the supernatant by ELISA. Mean values ​​± standard error are shown. Cell lines containing siRNA targeting hTLR3 are indicated with an asterisk. Results are representative of four independent experiments. [Figure 8]Cytokine production by RNA-transfected dendritic cells shows that all modifications block activation of cytokine-producing dendritic cells, whereas only the uridine modification blocks blood-derived dendritic cell activation. MDDCs generated using GM-CSF / IL-4 (A, C) or GM-CSF / IFN-α MDDCs (B), and primary dendritic cells 1 and 2 (D) were treated with Lipofectin® alone, Lipofectin®-R-848 (1 μg / mL), or RNA (5 μg / mL) for 8–16 hours. Note the modified nucleosides present in RNA-1571. TNF-α, IL-12 (p70), and IFN-α were measured in the supernatant by ELISA. Mean values ​​± standard error are shown. Results are representative of 10 (A and C), 4 (B), and 6 (D) independent experiments. E. Activation of dendritic cells by RNA. MDDCs were treated for 20 hours with Lipofectin® alone or complexed with 1 μg / mL poly(I):(C) or R-848 as a positive control (top panel), or with Lipofectin® complexed with the indicated RNA (5 μg / mL, bottom panel). Note the modified nucleoside present in RNA-1886. Expression of CD83, CD80, and HLA-DR was determined by flow cytometry. [Figure 8E] E. Activation of dendritic cells with RNA. MDDCs were treated for 20 hours with Lipofectin® alone or complexed with 1 μg / mL poly(I):(C) or R-848 as a positive control (top panel), or with Lipofectin® complexed with the indicated RNA (5 μg / mL, bottom panel). Note the modified nucleoside present in RNA-1886. Expression of CD83, CD80, and HLA-DR was determined by flow cytometry. [Figure 9]Activation of dendritic cells with RNA demonstrates that all nucleoside modifications inhibit RNA-mediated dendritic cell activation. MDDCs were treated for 20 hours with Lipofectin® alone, Lipofectin®-R-848 (1 μg / mL), or RNA-1571 (5 μg / mL) modified as indicated. (A) CD83 and HLA-DR staining. (B) TNF-α levels and mean fluorescence of CD80 and CD86 in the supernatant in response to incubation with RNA. The volume of medium was increased 30-fold for flow cytometry, as indicated by the asterisk. Data are representative of four independent experiments. [Figure 10]We transcribed capped RNA-1571 containing different amounts (0, 1, 10, 50, 90, 99, and 100% modified nucleosides relative to the corresponding unmodified NTP) and found that modifying only a few nucleosides resulted in inhibition of dendritic cell activation. (A) All transcripts were digested to monophosphate and analyzed by reverse-phase HPLC to determine the relative amount of modified nucleoside incorporation. Representative absorbance profiles obtained at the indicated (Ψ:U) ratios are shown. Elution times are noted for pseudouridine (Ψ), cytidine (C), guanosine (G), uridine (U), 7-methylguanosine ("m7G"), and adenosine ("A"). (B) Modified nucleoside content of RNA-1571. The expected percentages of m6A, Ψ (pseudouridine), or m5C in RNA-1571 were calculated based on the relative amounts of modified NTPs in the transcription reaction and nucleoside composition of RNA-1571 (A: 505, U: 451, C: 273, G: 342). Values ​​for the measured modified nucleoside content were determined based on quantification of HPLC chromatograms. Note: A: Values ​​(%) for m6ATP, ΨTP, or m5CTP relative to ATP, UTP, and CTP, respectively. B: Values ​​for m6A, Ψ, and m5C monophosphate relative to all NMPs. (C) MDDCs were transfected with capped RNA-1571 (5 μg / mL) complexed with Lipofectin® containing the indicated amounts of m6A, Ψ, or m5C. After 8 hours, TNF-α was measured in the supernatant. Data are expressed as relative inhibition of TNF-α. Mean values ​​± standard error obtained in three independent experiments are shown. [Figure 11]Expression of TNF-α by dendritic cells transfected with oligoribonucleotides demonstrates that as few as one modified nucleoside can reduce dendritic cell activation. (A) The sequences of chemically synthesized oligoribonucleotides (ORN1-4) (SEQ ID NOS: 6-9) or in vitro transcribed oligoribonucleotides (ORN5-6) (SEQ ID NOS: 10-11) are shown. The positions of the modified nucleosides Um (2'-O-methyluridine), mC, and Ψ are highlighted. Human MDDCs were transfected with Lipofectin® alone (medium), R-848 (1 μg / mL), or Lipofectin® complexed with RNA (5 μg / mL). Where indicated, cells were treated with 2.5 μg / mL cycloheximide (CHX). After 8 hours of incubation, TNF-α was measured in the supernatant. (C) RNA from cells was analyzed by Northern blotting. Representative mean values ​​± standard error of three independent experiments are shown. [Figure 12] A. Ψ-modified mRNA does not stimulate proinflammatory cytokine production in vivo. Serum samples (6 h postinjection) were analyzed by ELISA, revealing that 3 μg of unmodified mRNA induced higher levels of IFN-α than 3 μg of Ψ-modified mRNA (P<0.001). The level of IFN-α induced by 3 μg of Ψ-modified mRNA was similar to that obtained when animals were injected with uncomplexed lipofectin. Values ​​are expressed as mean ± standard error (n=3 or 5 animals / group). B. Similar results were observed for TNF-α. [Figure 13] mRNA containing pseudouridine (Ψ) does not activate PKR. Ψ: pseudouridine. Control: unmodified RNA. m5C: mRNA with m5C modification. [Figure 14] Increased expression of luciferase from pseudouridine-containing mRNA in rabbit reticulocyte lysates. Luc-Y: mRNA with pseudouridine modification, luc-C: unmodified RNA. Data are presented by normalizing luciferase activity to unmodified luciferase RNA. [Figure 15] Increased expression of Renilla from pseudouridine-containing mRNA in cultured cells. A. 293 cells. B. Mouse primary bovine bone marrow-derived mouse dendritic cells. Renilla-Y: mRNA with pseudouridine modification, renilla-C: unmodified RNA. RNA was modified with m5C, m6A, and m5U as indicated. [Figure 16] A. Additive effect of 3' and 5' elements on the translation efficiency of Ψ-modified mRNA. 293 cells were transfected with conventional firefly luciferase and mRNA with a Ψ-modified, 5'-cap (capLuc), 50-nucleotide-long 3' polyA tail (TEVlucA50), either both or neither of these elements (capTEVlucA50 and Luc, respectively). Cells were lysed after 4 hours, and luciferase activity was measured in 1 / 20 aliquots of the total lysate. B. Ψ-modified mRNA is more stable than unmodified mRNA. 293 cells transfected with capTEVlucAn containing unmodified or Ψ-modified nucleosides were lysed at the indicated times after transfection. Aliquots (1 / 20) of the lysates were assayed for luciferase. Standard errors are too small to visualize with error bars. C. Enhanced β-galactosidase expression using Ψ-modified mRNA compared to conventional mRNA. 293 cells seeded in 96-well plates were transfected with mRNA (0.25 μg / well) encoding bacterial β-galactosidase (lacZ) complexed with lipofectin. Transcripts had a cap and 3' polyA tail that was either 30 nucleotides long (caplacZ) or up to 200 nucleotides long (caplacZ-An). Constructs made with conventional U or Ψ nucleosides were tested. 24 hours after transfection, cells were fixed and stained with X-gal. Images were taken from representative wells by inverted microscopy (40x and 100x magnification). [Figure 17]A. Renilla expression after intracerebral injection of modified or unmodified encoding mRNA. Eight injections were performed in the rat cerebral cortex. One hemisphere was injected with capped Renilla-encoding RNA with pseudouridine modifications (capRenilla-Y), while the corresponding hemisphere was injected with capped RNA without nucleoside modifications (capRenilla-C). Data from two animals (6 injection sites) are shown. BG: Lower levels of detection in this assay. B. Intravenously delivered Ψ-modified mRNA is expressed in the spleen. Ψ mRNA (0.3 μg capTEVlucAn / mouse) complexed with lipofectin was administered via tail vein injection. Animals were sacrificed 2 and 4 hours post-injection, and luciferase activity was measured in aliquots (1 / 10) of organs homogenized in lysis buffer. Values ​​represent luciferase activity in all organs. C. Ψ-modified mRNA exhibits greater stability and translation in vivo. Mice were intravenously delivered with capTEVlucAn (0.3 μg / 60 μL / individual) complexed with lipofectin, with or without the Ψ-modification. Animals were sacrificed 1, 4, and 24 hours after injection, and half of their spleens were processed for luciferase enzyme measurement (left panel), and the other half for RNA analysis (right panel). Luciferase activity was measured in an aliquot (1 / 5) of homogenate prepared from half the spleen. Plotted values ​​represent luciferase activity in the entire spleen and are expressed as the mean ± standard error (n = 3 or 4 / time point). D. Expression of firefly luciferase after intratracheal injection of mRNA. capTEVluc-Y: pseudouridine-modified capped RNA encoding firefly luciferase. CapTEVluc-C: capped RNA without nucleoside modifications. [Figure 18]Protein production depends on the amount of mRNA delivered intravenously in mice.In a volume of 60 μL / individual, the indicated amount of lipofectin-complexed nucleic acid, capTEVlucAn mRNA, and pCMVluc plasmid DNA were delivered intravenously to mice.The animals injected with mRNA or plasmid DNA were sacrificed 6 hours or 24 hours after injection, respectively, and luciferase activity was measured in an aliquot (1 / 10) of the animal's spleen homogenized in lysis buffer.The values ​​from each animal are shown, with the short horizontal line indicating the mean and ND indicating non-detectable. [Figure 19] Expression of firefly luciferase after intratracheal delivery of the encoding mRNA. The mRNA was complexed with lipofectin (or PEI, as noted), and animals were injected with 0.3 μg of Ψ-modified or unmodified mRNA encoding firefly luciferase before sacrifice 3 hours later. Lungs were harvested and homogenized, and luciferase activity was measured in lysed organ aliquots. [Figure 20] Ψ-modified mRNA does not induce inflammatory mediators after pulmonary delivery. Induction of TNF-α and IFN-α in serum after intratracheal delivery of unmodified or Ψ-modified luciferase-encoding mRNA. Serum levels of TNF-α and IFN-α were determined by ELISA 24 hours after mRNA delivery. [Figure 21] Results from Example 35 are shown: mRNA encoding firefly or Renilla luciferase with the indicated modifications was complexed with lipofectin and delivered to mouse dendritic cells (A) and HEK293T cells (B). Human dendritic cells were transfected with mRNA encoding firefly or Renilla luciferase complexed with TransIT with the indicated modifications (C). Data are expressed as fold change compared to unmodified mRNA. [Figure 22]Results from Example 36 are shown. The T7 polymerase transcription reaction used to generate mRNA results in a large amount of RNA of the correct size, but also contains contaminants. This is visualized by applying the RNA to a reverse-phase HPLC column, which separates RNA based on size under denaturing conditions. Ψ-modified TEV-luciferase-A51 RNA was applied to the HPLC column at 38% Buffer B and subjected to a linear gradient of increasing Buffer B up to 55%. The profile showed both smaller than expected and larger than expected contaminants. [Figure 23] Results from Example 37 are shown: (A) EPO-encoding mRNA with the indicated modifications and with or without HPLC purification was delivered to mouse dendritic cells, and EPO levels in the supernatant were measured 24 hours later. M5C / Ψ-modified mRNA had the highest level of translation before HPLC purification, whereas Ψ-modified mRNA had the highest translation after HPLC purification. (B) Human dendritic cells were transfected with Renilla-encoding mRNA with the indicated modifications, with or without HPLC purification. [Figure 24] Results from Example 38 are shown. (A) Human dendritic cells were transfected with RNA complexed with TransIT containing the indicated modifications, with or without HPLC purification. IFN-α levels were measured 24 hours later. HPLC purification increased the immunogenicity of unmodified RNA, depending on the sequence, as other unmodified RNAs had similar levels of IFN-α or reduced levels after HPLC purification. Ψ-modified RNA had unmeasurable levels of IFN-α, similar to control-treated dendritic cells. (B) Ψ-modified RNA before (-) and after HPLC purification (P1 and P2) was analyzed for double-stranded RNA using a dot blotting method with a monoclonal antibody specific for double-stranded RNA (J2). RNA purification removed double-stranded RNA contaminants. (C) Ψ-modified RNA encoding iPS factors is immunogenic, and the RNA is removed by HPLC of the RNA. [Figure 25]mRNA encoding sequences for KLF4 (SEQ ID NO: 12) and LIN28 (SEQ ID NO: 13) are provided. [Figure 26] mRNA encoding sequences for cMYC (SEQ ID NO: 14) and NANOG (SEQ ID NO: 15) are provided. [Figure 27] mRNA encoding sequences for OCT4 (SEQ ID NO: 16) and SOX2 (SEQ ID NO: 17) are provided. [Figure 28] Figure 28 shows that mRNAs encoding human reprogramming factors (KLF4, c-MYC, OCT4, and SOX2) generate iPS cells in primary human keratinocyte cells. Figure 28 shows phase-contrast images of iPS colony formation in HEKn cells 2 days (A) and 11 days (B) and 20 days (C) after final transfection with mRNAs encoding the four reprogramming factors. Images are at 10x magnification. [Figure 29] Figure 29 shows that mRNA encoding human reprogramming factors (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) generates iPS cells in human keratinocytes that are positive for known iPS cell markers. Figure 29 shows phase-contrast images of colonies derived from HEKn cells (A, D, and G). The same iPS colony shown in (A) is positive for KLF4 (B) and LIN28 (C). The iPS colony shown in (D) is SSEA4-positive (E) and TRA-1-60-positive (F). The iPS colony shown in (G) is NANOG-positive (H). All images are at 20x magnification. [Figure 30] Figure 30 shows the increased expression of three iPS-related messages in HEKn cells transfected with four reprogramming mRNAs (KLF4, c-MYC, OCT4, and SOX2) that do not contain the reprogramming factor NANOG. The increased expression of the messages is detected by quantitative PCR and normalized to GAPDH expression. The expression level of each message is shown relative to the level observed in the original cell line. DETAILED DESCRIPTION OF THE INVENTION

[0065] (definition) The present invention will be understood and interpreted in accordance with the terms defined below.

[0066] As used herein, "substantially all" in reference to single-stranded intact mRNA containing pseudouridine or 5-methylcytidine residues means that at least 95% of the single-stranded intact mRNA present in a sample has either a pseudouridine or a 5-methylcytidine residue.

[0067] As used herein, "essentially all" in reference to single-stranded intact mRNA containing pseudouridine or 5-methylcytidine residues means that at least 99% of all single-stranded intact mRNA present in a sample has either a pseudouridine or 5-methylcytidine residue.

[0068] As used herein, an "RNA contaminant molecule" is a molecule that contains RNA residues and that, when transfected into a cell, is capable of at least partially activating an immune response (e.g., by activating RNA sensors such as RNA-dependent protein kinase (PKR), retinoic acid-inducible gene I (RIG-I), Toll-like receptor (TLR) 3, TLR7, TLR8, and oligoadenylate synthetase (OAS)), or an RNA molecule that is capable of at least partially activating an RNA interference (RNAi) response in the cell (e.g., including responses to large double-stranded RNA molecules or to small double-stranded RNA molecules (siRNAs)). Exemplary RNA contaminant molecules include, but are not limited to, partial or non-full-length mRNAs that encode only a portion of a reprogramming molecule (e.g., a non-full-length iPS cell inducer), for example, without being bound by theory, single-stranded mRNAs that are larger than the full-length mRNA that encodes a reprogramming factor (e.g., an iPS cell inducer) by "follow-up IVT" or other mechanisms, double-stranded large or small mRNA molecules, and uncapped mRNA molecules.

[0069] As used herein, a purified RNA preparation is "substantially free" of an RNA contaminant molecule (or a particular recited RNA contaminant) if less than 0.5% of the total RNA in the purified RNA preparation consists of the RNA contaminant molecule (or the particular recited RNA contaminant). The amount and relative amounts of non-contaminant mRNA molecules and RNA contaminant molecules (or the particular RNA contaminant) can be determined by HPLC or other methods used in the art to separate and quantify RNA molecules.

[0070] As used herein, a purified RNA preparation is "essentially free" of an RNA contaminant molecule (or a particular recited RNA contaminant) if less than 1.0% of the total RNA in the purified RNA preparation consists of the RNA contaminant molecule (or the particular recited RNA contaminant). The amount and relative amounts of non-contaminant mRNA molecules and RNA contaminant molecules (or the particular RNA contaminant) can be determined by HPLC or other methods used in the art to separate and quantify RNA molecules.

[0071] As used herein, a purified RNA preparation is "substantially free" of an RNA contaminant molecule (or a particular recited RNA contaminant) if less than 0.1% of the total RNA in the purified RNA preparation consists of the RNA contaminant molecule (or the particular recited RNA contaminant). The amount and relative amounts of non-contaminant mRNA molecules and the RNA contaminant molecule (or the particular RNA contaminant) can be determined by HPLC or other methods used in the art to separate and quantify RNA molecules.

[0072] As used herein, a purified RNA preparation is "free" of an RNA contaminant molecule (or a particular recited RNA contaminant) if less than 0.01% of the total RNA in the purified RNA preparation consists of the RNA contaminant molecule (or the particular recited RNA contaminant). The amount and relative amounts of non-contaminant mRNA molecules and RNA contaminant molecules (or the particular RNA contaminant) can be determined by HPLC or other methods used in the art to separate and quantify RNA molecules.

[0073] The terms "comprises," "containing," "having," "including," and "including" should be interpreted as "including, but not limited to," unless otherwise stated. The terms "a," "an," and "the" and similar referents in the context of describing the invention, particularly in the context of the appended claims, should be interpreted as covering both the singular and the plural, unless otherwise stated. The use of any and all examples or exemplary language (such as, for example, or such as) is intended merely to describe aspects or embodiments of the invention and should not be interpreted as limiting its scope unless otherwise asserted.

[0074] With respect to the use of the word "derived," such as RNA (including mRNA) or polypeptide "derived" from a sample, biological sample, cell, tumor, or the like, it means that either the RNA or polypeptide is present in the sample, biological sample, cell, tumor, or the like, or was generated using RNA in the sample, biological sample, cell, tumor, or the like, by a process such as an in vitro transcription or RNA amplification reaction, in which the RNA or polypeptide is encoded by or is a copy of all or part of the RNA or polypeptide molecule in the original sample, biological sample, cell, tumor, or the like. By way of example, such RNA can be derived from an in vitro transcription or RNA amplification reaction, with or without cDNA cloning, rather than obtained directly from the sample, biological sample, cell, tumor, or the like, so long as the original RNA used in the in vitro transcription or RNA amplification reaction was derived from the sample, biological sample, cell, tumor, or the like. The terms "sample" and "biological sample" are used in their broadest sense and include samples or specimens obtained from any source that contains or may contain eukaryotic cells, including biological and environmental sources. As used herein, the term "sample," when used to refer to a biological sample obtained from an organism, includes bodily fluids (e.g., blood or saliva), feces, biopsies, swabs (e.g., buccal swabs), isolated cells, exudates, and the like. Organisms include fungi, plants, animals, and humans. However, these examples should not be construed as limiting the types of samples or organisms that find use in the present invention. In addition, for conducting research related to or studying results related to the use of the methods or compositions of the present invention, in some embodiments, "sample" or "biological sample" includes fixed cells, processed cells, cell lysates, and the like.In some embodiments, such as those in which mRNA is delivered to cells from organisms with a known disease or to cells exhibiting a disease state or known pathology, "sample" or "biological sample" also includes bacteria or viruses.

[0075] As used herein, the term "incubating" and variations thereof means contacting one or more components of a reaction with another component or components under conditions and for a time sufficient for the desired reaction product to form.

[0076] As used herein, a "nucleoside" refers to a nucleobase (e.g., the conventional nucleobases: guanine (G), adenine (A), thymine (T), uracil (U), and cytosine (C)) or a modified nucleobase (e.g., 5-methylcytosine (mC)) covalently linked to a pentose sugar (e.g., ribose or 2'-deoxyribose). 5 C)), whereas a "nucleotide" or "mononucleotide" consists of a nucleoside phosphorylated at one of the hydroxyl groups of the pentose sugar. Linear nucleic acid molecules are said to have a "5' end" (5'-end) and a "3' end" (3'-end) because, except for capping or adenylation (e.g., adenylation by ligase), mononucleotides are linked unidirectionally via phosphodiester bonds to produce oligonucleotides or polynucleotides such that the phosphate on the 5' carbon of one mononucleotide sugar moiety is linked to the oxygen on the 3' carbon of the sugar moiety of the adjacent mononucleotide. Thus, the terminus of a linear single-stranded oligonucleotide or polynucleotide or the terminus of one strand of a linear double-stranded nucleic acid (RNA or DNA) is called the "5' end" if its 5' phosphate is bound or not bound to the oxygen on the 3' carbon of the mononucleotide sugar moiety, or the "3' end" if its 3' oxygen is not bound to the 5' phosphate attached to the sugar of another mononucleotide. A terminal nucleotide, as used herein, is the nucleotide at the end position of the 3' or 5' terminus.

[0077] To achieve a particular goal, the nucleobase, sugar moiety, or internucleoside (or internucleotide) linkage in one or more of the nucleotides of the mRNA introduced into a eukaryotic cell in any of the methods of the invention may comprise a modified base, sugar moiety, or internucleoside linkage. For example, in addition to the other modified nucleotides discussed elsewhere herein for practicing the methods of the invention, one or more of the nucleotides of the mRNA may also comprise a xanthine, aliamino-uracil, aliamino-thymidine, hypoxanthine, 2-aminoadenine, 5-propynyluracil, 5-propynylcytosine, 4-thiouracil, 6-thioguanine, aza- or deazauracil, aza- or deazathymidine, aza- or deazacytosine, aza- or deazaadenine, or aza- or deazaguanine, or a biotin moiety, a digoxigenin moiety, a fluorescent or chemiluminescent moiety, a quenching moiety, or a nucleotide to achieve one or more other specific goals. and / or one or more of the nucleotides of the mRNA can have a sugar moiety such as, but not limited to, 2'-fluoro-2'-deoxyribose or 2'-O-methyl-ribose, which provide resistance to some nucleases, or 2'-amino-2'-deoxyribose or 2'-azido-2'-deoxyribose, which can be labeled by reaction with a visible, fluorescent, infrared fluorescent, or other detectable dye or chemical having an electrolyte moiety, a photolabile moiety, an alkynyl moiety, or other reactive chemical moiety.

[0078] In some embodiments of the present invention, one or more of the nucleotides of the mRNA contain modified internucleoside linkages, such as phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages that are resistant to some nucleases, including in dinucleotide cap analogs used in IVT reactions for co-transcriptional capping of RNA (Grudzien-Nogalska et al. 2007) or at poly(A) termini (by incorporation of nucleotides with modified phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages during IVT of RNA, or by incorporation of ATP containing modified phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages into the poly(A) termini of RNA by polyadenylation using poly(A) polymerase). The present invention is not limited to the listed modified nucleobases, sugar moieties, or internucleoside linkages, which are presented to illustrate examples that may be used for specific purposes in the methods.

[0079] As used herein, a "nucleic acid" or "polynucleotide" or "oligonucleotide" is a covalently linked sequence of nucleotides in which the 3' position of the sugar moiety of one nucleotide is linked by a phosphodiester bond to the 5' position of the sugar moiety of the next nucleotide, and the nucleotides are linked in a specific sequence, i.e., the linear order of the nucleotides. In some embodiments, the nucleic acid or polynucleotide or oligonucleotide consists of or comprises 2'-deoxyribonucleotides (DNA). In some embodiments, the oligonucleotide consists of or comprises ribonucleotides (RNA).

[0080] The terms "isolated" or "purified," when used in reference to a polynucleotide or nucleic acid, as in "isolated RNA" or "purified RNA," refer to a nucleic acid that has been identified and separated from at least one contaminant that normally accompanies it in its source. Thus, isolated or purified nucleic acids (e.g., DNA and RNA) exist in a form or setting that is different from that found in nature or that existed before being subjected to a treatment or purification method. For example, a given DNA sequence (e.g., a gene) is found in a host cell chromosome, along with other genes and structural and functional proteins, and a specific RNA (e.g., a specific mRNA encoding a specific protein), and is found in the cell as a mixture with numerous other RNAs and other cellular components. Isolated or purified polynucleotides or nucleic acids can exist in single-stranded or double-stranded form.

[0081] "Cap" or "capped nucleotide" refers to a nucleoside-5'-triphosphate that is used as a substrate by a capping enzyme system under suitable reaction conditions and thereby attached to the 5'-end of a primary RNA transcript or an uncapped RNA, including RNA with a 5'-diphosphate. A nucleotide so attached to the RNA is also referred to herein as a "capped nucleotide." A "capped nucleotide" is a guanine nucleotide attached through its 5' end to the 5' end of a primary RNA transcript. An RNA having a capped nucleotide attached to its 5' end is referred to as a "capped RNA" or "capped RNA transcript" or "capped transcript." Common capped nucleosides are 7-methylguanosine or N-methylguanosine. 7 -methylguanosine (sometimes called the "standard cap"), 7 G), in which case it is capped RNA or "m 7 G-capped RNA" is m 7 G(5')ppp(5')N1(pN) xas -OH(3') or more simply m 7 GpppN1(pN) x Or m 7 G[5']ppp[5']N, wherein m 7 G represents a 7-methylguanosine cap nucleoside, ppp represents a triphosphate bridge between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, and N1(pN) x -OH(3') represents the primary RNA transcript, of which N1 is the 5'-most nucleotide, "p" represents a phosphate group, "G" represents a guanosine nucleoside, and "m 7 " represents the methyl group at the 7-position of guanine, and "[5']" indicates the position where "p" is attached to the ribose of the cap nucleotide and the first nucleoside ("N") of the mRNA transcript. In addition to this "standard cap," various other natural and synthetic cap analogs are known in the art. RNA having any cap nucleotide is called "capped RNA." Capped RNA can occur naturally from a biological sample or can be obtained by in vitro capping of RNA having a 5' triphosphate group or RNA having a 5' diphosphate group using a capping enzyme system (e.g., the vaccinia capping enzyme system or the Saccharomyces cerevisiae capping enzyme system). Alternatively, capped RNA can be obtained by in vitro transcription (IVT) of a DNA template containing an RNA polymerase promoter, in which, in addition to GTP, the IVT reaction can include a dinucleotide cap analog (e.g., m) using methods known in the art (e.g., using the AMPLICAP™ T7 Capping Kit or the MESSAGEMAX™ T7 ARCA-CAPPED MESSAGE Transcription Kit, EPICENTRE, or CellScript). 7 GpppG Cap Analog, or N 7 -methyl, 2'-O-methyl-GpppG ARCA cap analog, or N 7-methyl, 3'-O-methyl-GpppG (ARCA cap analog).

[0082] Capping of 5′-triphosphorylated primary mRNA transcripts in vivo (or using capping enzyme systems in vitro) occurs through several enzymatic steps ( Higman et al. 1992 , Martin et al. 1975 , Myette and Niles 1996 ).

[0083] The following enzymatic reactions are involved in capping eukaryotic mRNA: (1) RNA triphosphatase cleaves the 5'-triphosphate of mRNA to a diphosphate. pppN1(p)N x -OH(3') → ppN1(pN) x -OH(3') + Pi , then (2) RNA guanyltransferase catalyzes the attachment of GTP to the 5'-diphosphate of the 5'-most nucleotide (N1) of mRNA, ppN1(pN) x -OH(3') + GTP → G(5')ppp(5')N1(pN) x -OH(3') + PPi ,lastly, (3) Guanine-7-methyltransferase, which uses S-adenosyl-methionine (AdoMet) as a cofactor, catalyzes the methylation of the 7-nitrogen of guanine in the cap nucleotide.

[0084] G(5')ppp(5')N1(pN) x -OH(3') + AdoMet → m 7 G(5')ppp(5')N1(pN) x -OH(3') + AdoHyc RNA resulting from the action of the enzymatic activities of RNA triphosphatase and RNA guanyltransferase, as well as RNA that is additionally methylated by guanine-7-methyltransferase enzymatic activity, is referred to herein as "5'-capped RNA" or "capped RNA," and "capping enzyme system" or, more simply, "capping enzyme" herein refers to any combination of one or more polypeptides having enzymatic activity that results in "capped RNA." Capping enzyme systems, including cloned forms of such enzymes, have been identified and purified from many sources and are well known in the art (Banerjee 1980, Higman et al. 1992, Higman et al. 1994, Myette and Niles 1996, Shuman 1995, Shuman 2001, Shuman et al. 1980, Wang et al. 1997). Any capping enzyme system capable of converting uncapped RNA having a 5' polyphosphate into capped RNA can be used to provide the capped RNA for any of the embodiments of the present invention. In some embodiments, the capping enzyme system is a poxvirus capping enzyme system. In some preferred embodiments, the capping enzyme system is a vaccinia virus capping enzyme. In some embodiments, the capping enzyme system is a Saccharomyces cerevisiae capping enzyme. Also, in view of the fact that genes encoding RNA triphosphatase, RNA guanyltransferase, and guanine-7-methyltransferase from one source can complement deletions in one or all of these genes from another source, the capping enzyme system can be derived from one source, or one or more of the RNA triphosphatase, RNA guanyltransferase, and / or guanine-7-methyltransferase activities can comprise polypeptides from different sources.

[0085] A "modified capped nucleotide" of the present invention refers to a capped nucleotide in which the sugar, nucleobase, or internucleoside linkage is chemically modified compared to the corresponding normal 7-methylguanosine capped nucleotide. Examples of modified capped nucleotides include (i) modified 2'- or 3'-deoxyguanosine-5'-triphosphate (or guanine 2'- or 3'-deoxyribonucleic acid-5'-triphosphate) in which the 2'- or 3'-deoxy position of the deoxyribose sugar moiety is replaced with a group containing an amino group, an azido group, a fluorine group, a methoxy group, a thiol (or mercapto) group, or a methylthio (or methylmercapto) group; or (ii) modified 2'- or 3'-deoxyguanosine-5'-triphosphate (or guanine 2'- or 3'-deoxyribonucleic acid-5'-triphosphate) in which the O of the guanine base is replaced with a group containing an amino group, an azido group, a fluorine group, a methoxy group, a thiol (or mercapto) group, or a methylthio (or methylmercapto) group. 6 (iii) a cap nucleotide containing a modified guanosine-5'-triphosphate in which the oxygen is methylated, or (iv) 3'-deoxyguanosine. For clarity, it will be understood herein that "alkoxy-substituted deoxyguanosine-5'-triphosphate" can also be referred to as "O-alkyl-substituted guanosine-5'-triphosphate," and by way of example, but not limitation, 2'-methoxy-2'-deoxyguanosine-5'-triphosphate (2'-methoxy-2'-dGTP) and 3'-methoxy-3'-deoxyguanosine-5'-triphosphate (3'-methoxy-3'-dGTP) can be referred to herein as 2'-O-methylguanosine-5'-triphosphate (2'-OMe-GTP) and 3'-O-methylguanosine-5'-triphosphate (3'-OMe-GTP), respectively. After a modified cap nucleotide is attached to the 5' end of an uncapped RNA (or an RNA having a 5'-diphosphate) comprising a primary RNA transcript, the portion of the modified cap nucleotide attached to the uncapped RNA (or an RNA having a 5'-diphosphate) comprising a primary RNA transcript may be referred to herein as a "modified cap nucleoside" (i.e., without reference to the attached phosphate group), but is sometimes referred to as a "modified cap nucleotide."

[0086] "Modified nucleotide-capped RNA" refers to a capped RNA molecule synthesized using a capping enzyme system and a modified cap nucleotide, including capped RNA synthesized co-transcriptionally in an in vitro transcription reaction in which the cap nucleotide at its 5' end contains a modified cap nucleotide, or a modified dinucleotide cap analog containing a chemical modification at the cap nucleotide. In some embodiments, the modified dinucleotide cap analog is an anti-inverted cap analog or ARCA (Grudzien et al. 2004, Jemielity et al. 2003, Grudzien-Nogalska et al. 2007, Peng et al. 2002, Stepinski et al. 2001).

[0087] "Primary RNA" or "primary RNA transcript" means an RNA molecule synthesized in vivo or in vitro by an RNA polymerase and in which the RNA molecule has a triphosphate at the 5'-carbon of its 5'-most nucleotide.

[0088] "RNA amplification reaction" or "RNA amplification method" refers to a method for increasing the amount of RNA corresponding to one or more desired RNA sequences in a sample.For example, in some embodiments, the RNA amplification method includes: (a) synthesizing a first-strand cDNA complementary to one or more desired RNA molecules by RNA-dependent DNA polymerase extension of one or more primers annealed to the desired RNA molecules; (b) synthesizing a double-stranded cDNA from the first-strand cDNA using a process in which a functional RNA polymerase promoter binds; and (c) contacting the double-stranded DNA with an RNA polymerase bound to the promoter under transcription conditions to obtain RNA corresponding to one or more desired RNA molecules.Unless otherwise specified in connection with a specific embodiment of the present invention, the RNA amplification reaction according to the present invention refers to a sense RNA amplification reaction, which refers to an RNA amplification reaction that synthesizes sense RNA (e.g., RNA that has the same sequence as mRNA or other primary RNA transcripts, rather than the complement of mRNA or other primary RNA transcripts). Sense RNA amplification reactions known in the art that are encompassed within this definition include, but are not limited to, the methods for synthesizing sense RNA described in Ozawa et al. (Ozawa et al. 2006) and in U.S. Patent Applications Nos. 20090053775, 20050153333, 20030186237, 20040197802, and 20040171041. The RNA amplification method described in U.S. Patent Application No. 20090053775 is a preferred method for obtaining amplified RNA from one or more cells, which is then used to generate mRNA for use in the methods of the present invention.

[0089] "Poly-A polymerase" ("PAP") refers to a template-independent RNA polymerase found in most eukaryotes, prokaryotes, and eukaryotic viruses that preferentially uses ATP to incorporate AMP residues into the 3' hydroxylated ends of RNA. Because PAP enzymes studied from plants, animals, bacteria, and viruses all catalyze the same overall reaction (Edmonds 1990), are highly structurally conserved (Gershon 2000), and PAP loses essential specificity for particular sequences or sizes of RNA molecules when separated from proteins that recognize the AAUAAA polyadenylation signal (Wilusz and Shenk 1988), purified wild-type and recombinant PAP enzymes from any of a variety of sources can be used for the present invention. In some embodiments, the PAP enzyme from Saccharomyces (e.g., from Saccharomyces cerevisiae) is used for polyadenylation to produce a purified RNA preparation that comprises or consists of one or more modified mRNAs, each of which encodes a reprogramming factor (e.g., an iPS cell induction factor). In some embodiments, the PAP enzyme from Escherichia coli is used for polyadenylation to produce a purified RNA preparation that comprises or consists of one or more modified mRNAs, each of which encodes a reprogramming factor (e.g., an iPS cell induction factor).

[0090] "Reprogramming factor," when used alone or in combination with other factors or conditions, refers to a protein, polypeptide, or other biomolecule that results in a change in the differentiation state of a cell into which the reprogramming factor is introduced or expressed. In some preferred embodiments of the methods of the invention, the reprogramming factor is a protein or polypeptide encoded by an mRNA introduced into a cell, thereby generating a cell that exhibits an altered state of differentiation compared to the cell into which the mRNA was introduced. In some preferred embodiments of the methods of the invention, the reprogramming factor is a transcription factor. One embodiment of a reprogramming factor used in the methods of the invention is an "iPS cell induction factor."

[0091] An "iPS cell inducer" or "iPSC inducer" is a protein, polypeptide, or other biomolecule that can be used alone or in combination with other reprogramming factors to generate iPS cells from somatic cells. Examples of iPS cell inducers include OCT4, SOX2, c-MYC, KLF4, NANOG, and LIN28. iPS cell inducers include full-length polypeptide sequences or biologically active fragments thereof. Similarly, mRNAs encoding iPS cell inducers can encode full-length polypeptides or biologically active fragments thereof. mRNA encoding sequences for exemplary iPS inducers are shown in Figure 25 (KLF4 and LIN28), Figure 26 (cMYC and NANOG), and Figure 27 (OCT4 and SOX2). In certain embodiments, the present invention employs the sequences shown in these figures or similar sequences, including mRNA molecules that additionally comprise oligoribonucleotides attached to these mRNA sequences that exhibit any of the 5' and 3' UTR sequences, Kozak sequences, IRES sequences, cap nucleotides, and / or poly(A) sequences used in the experiments described herein, or oligoribonucleotides that are generally known in the art and can be used in place of the sequences used herein by attaching them to mRNA sequences encoding these proteins for the purposes of optimizing translation of individual mRNA molecules in cells and improving the stability of the molecules in cells to achieve the methods described herein.

[0092] "Differentiation" or "intracellular differentiation" refers to the natural biological process by which cells or cell types (e.g., fertilized egg cells, cells in embryos, or cells in eukaryotes) that exhibit less specialized differentiation become more specialized cells or cell types. Scientists, including biologists, cell biologists, immunologists, and embryologists, use various methods and criteria to define, describe, or classify different cells according to their "cell type," "differentiation state," or "state of differentiation." Generally, cells are defined, described, or classified based on one or more phenotypes exhibited by the cells, which may include shape, biochemical or metabolic activity or function, or the presence of certain biomolecules in the cell (e.g., based on staining that reacts with specific biomolecules) or on the cell (e.g., based on the binding of one or more antibodies that react with specific biomolecules on the cell surface). For example, in some embodiments, different cell types are identified and sorted using a cell sorter or a fluorescence-activated cell sorter (FACS) machine. "Differentiation" or "cell differentiation" can also occur in cells in culture.

[0093] The term "reprogramming," as used herein, refers to differentiation or intracellular differentiation that occurs in response to the delivery of one or more reprogramming factors to a cell, either directly (e.g., by delivery of a protein or polypeptide reprogramming factor to the cell) or indirectly (e.g., by delivery of a purified RNA preparation of the invention comprising one or more mRNA molecules each encoding a reprogramming factor), and maintaining the cell under conditions conducive to differentiation (e.g., medium, temperature, oxygen and CO2 levels, matrix, and other environmental conditions). The term "reprogramming," as used herein, is not intended to mean or refer to a particular direction or pathway of differentiation, and does not exclude the process of proceeding in a direction or pathway of differentiation beyond that normally observed in nature. Thus, in different embodiments of the present invention, "reprogramming" means and includes any and all of the following: (1) "Dedifferentiation," which refers to the process of a cell or cell type (e.g., mammalian fibroblasts, keratinocytes, muscle cells, or nerve cells) exhibiting a more specialized state of differentiation proceeding to a cell or cell type (e.g., iPS cells) exhibiting a less specialized state of differentiation; (2) "Transdifferentiation," which refers to the process of a cell or cell type (e.g., a mammalian fibroblast, keratinocyte, or neuron) exhibiting a more specialized state of differentiation that proceeds to another more specialized state of differentiation or cell type (e.g., a fibroblast or keratinocyte to a muscle cell), and (3) "Redifferentiation" or "expected differentiation" or "spontaneous differentiation" means the process of a cell exhibiting any particular state of differentiation or cell type, whether occurring in vivo in an organism or culture (e.g., in response to one or more reprogramming factors), proceeding to another state of differentiation or cell type that would naturally be expected if the cell were present in its natural location and environment (e.g., in an embryo or organ).

[0094] Description of the Invention The present invention provides compositions and methods for reprogramming the differentiation state of eukaryotic cells, including human or other animal cells, by contacting the cells with a purified RNA preparation comprising or consisting of one or more different single-stranded mRNA molecules each encoding a reprogramming factor (e.g., an iPS cell inducer). The purified single-stranded mRNA molecules preferably contain one or more of the following amino acids: pseudouridine (Ψ), 5-methylcytosine (m ... 5 C), 5-methyluridine (m 5 U), 2'-O-methyluridine (Um or m 2’-O U), 2-thiouridine (s 2 U), and N 6The single-stranded mRNA molecule preferably contains -methyladenosine in place of at least some (e.g., including substantially all) of the corresponding unmodified A, C, G, or T conventional nucleosides. In addition, the single-stranded mRNA molecule is preferably purified to be substantially free of RNA contaminant molecules that would activate unintended responses, reduce expression of the single-stranded mRNA, and / or activate RNA sensors in the cell. In certain embodiments, the purified RNA preparation is substantially free of RNA contaminant molecules that are shorter or longer than full-length single-stranded mRNA molecules, double-stranded RNA, and / or uncapped RNA. In some preferred embodiments, the present invention provides compositions and methods for reprogramming differentiated eukaryotic cells, including somatic cells of humans or other animals, by contacting the cells with a purified RNA preparation comprising or consisting of one or more different single-stranded mRNA molecules each encoding an iPS cell-inducing factor.

[0095] In some embodiments, the mRNA used in purified RNA preparation is purified to remove substantially, essentially, or virtually all contaminants, including substantially all RNA contaminants.The present invention is not limited to the purification method used to purify mRNA, and the present invention includes the use of any method known in the art or developed in the future to purify mRNA and remove contaminants, including RNA contaminants that interfere with the intended use of mRNA.For example, in a preferred embodiment, mRNA purification removes contaminants that are toxic to cells (for example, by inducing innate immune response in cells, or in the case of RNA contaminants that comprise double-stranded RNA, by inducing RNA interference (RNAi), for example, through siRNA or long RNAi molecules) and contaminants that directly or indirectly reduce the translation of mRNA in cells.In some embodiments, mRNA is purified by HPLC using the method described herein, including in examples. In some embodiments, mRNA is purified using a polymer resin matrix comprising a styrene-divinylbenzene copolymer derivatized with C18, and a triethylamine acetate (TEAA) ion-pairing agent is used in the column along with an acetonitrile gradient to elute the mRNA and separate it from RNA contaminants in a size-dependent manner. In some embodiments, mRNA purification is performed using HPLC, while in some other embodiments, a gravity-flow column is used for purification. In some embodiments, mRNA is purified using the method described in the book "RNA Purification and Analysis" by Douglas T. Gjerde, Lee Hoang, and David Hornby, published by Wiley-VCH, 2009, which is incorporated herein by reference. In some embodiments, mRNA purification is performed in a non-denaturing mode (e.g., at a temperature below about 50 ° C, e.g., at ambient temperature). In some embodiments, mRNA purification is performed in a denaturing mode (e.g., at a temperature above about 72 ° C).Of course, those skilled in the art know that the denaturation temperature depends on the melting temperature (Tm) of the mRNA being purified and the melting temperature of any RNA, DNA, or RNA / DNA hybrids contaminating the mRNA. In some other embodiments, mRNA is purified as described by Mellits KH et al. (Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNA1 from a T7 vector. Nucleic Acids Research 18: 5401-5406, 1990, the entire contents of which are incorporated herein by reference). These authors used a three-step purification method to remove contaminants, which can be used in embodiments of the present invention. Step 1 was 8% polyacrylamide gel electrophoresis in 7 M urea (denaturing conditions). The major RNA band was excised from the gel slice and subjected to 8% polyacrylamide gel electrophoresis under non-denaturing conditions (urea-free), and the major band was recovered from the gel slice. Further purification was performed on a cellulose CF-11 column using an ethanol-salt buffer mobile phase to separate double-stranded RNA from single-stranded RNA (Franklin RM. 1966. Proc. Natl. Acad. Sci. USA 55: 1504-1511; Barber R. 1966. Biochem. Biophys. Acta114:422; and Zelcer A et al. 1982. J. Gen. Virol. 59: 139-148, all of which are incorporated herein by reference), and the final purification step was cellulose chromatography.In some other embodiments, mRNA is purified using a hydroxylapatite (HAP) column under non-denaturing conditions or at higher temperatures (e.g., as described by Pays E. 1977. Biochem. J. 165: 237-245; Lewandowski LJ et al. 1971. J. Virol. 8: 809-812; Clawson GA and Smuckler EA. 1982. Cancer Research 42: 3228-3231; and / or Andrews-Pfannkoch C et al. 2010. Applied and Environmental Microbiology 76: 5039-5045, all of which are incorporated herein by reference). In some other embodiments, mRNA is purified by weak anion exchange liquid chromatography under non-denaturing conditions (e.g., as described by Easton LE et al. 2010. RNA 16: 647-653, which is incorporated herein by reference, for removing in vitro transcription reactions). In some embodiments, mRNA is purified using any combination of the foregoing methods or other methods known in the art or developed in the future. In yet another embodiment, mRNA used in the compositions and methods of the present invention is purified using a process comprising treating the mRNA with an enzyme that specifically acts on (e.g., digests) one or more contaminating RNAs or nucleic acids (including, e.g., DNA) but does not act on (e.g., does not digest) the desired mRNA. For example, in some embodiments, mRNA used in the compositions and methods of the present invention is purified using a process comprising treating with a ribonuclease III (RNase III) enzyme (e.g., E. coli RNase III), and then the mRNA is purified separately from RNase III digestion products. By ribonuclease III (RNase III) enzyme herein is meant an enzyme that digests double-stranded RNA larger than about 12 base pairs, leaving double-stranded RNA fragments.In some embodiments, the mRNA used in the compositions and methods of the invention is purified using a process that involves treating the mRNA with one or more other enzymes that specifically digest one or more contaminating RNAs or nucleic acids (including, for example, DNA).

[0096] The present invention provides the RNA molecules, oligoribonucleotide molecules and polyribonucleotide molecules that contain pseudouridine or modified nucleosides, the gene therapy vectors that contain them, the gene therapy and gene transcription silencing methods that contain them, the method for reducing their immunogenicity, and the method for synthesizing them.These modified sequences are preferably present in the purified RNA preparation described herein.

[0097] In one embodiment, the present invention provides messenger RNAs comprising pseudouridine residues. In another embodiment, the messenger RNA encodes a protein of interest. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides RNA molecules encoding a protein of interest, wherein the RNA molecules comprise pseudouridine residues. In another embodiment, the present invention provides in vitro transcribed RNA molecules comprising pseudouridine. In another embodiment, the present invention provides in vitro transcribed RNA molecules comprising modified nucleosides.

[0098] As provided herein, the present invention provides methods for synthesizing in vitro transcribed RNA molecules containing pseudouridine and / or modified nucleosides. In another embodiment, the present invention provides messenger RNA molecules containing pseudouridine residues.

[0099] In another embodiment, the in vitro transcribed RNA molecule of the methods and compositions of the present invention is synthesized by T7 phage RNA polymerase. In another embodiment, the molecule is synthesized by SP6 phage RNA polymerase. In another embodiment, the molecule is synthesized by T3 phage RNA polymerase. In another embodiment, the molecule is synthesized by a polymerase selected from the above polymerases. In another embodiment, the in vitro transcribed RNA molecule is an oligoribonucleotide. In another embodiment, the in vitro transcribed RNA molecule is a polyribonucleotide. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides in vitro synthesized oligoribonucleotides comprising pseudouridine or modified nucleosides, wherein the modified nucleosides are m 5 Cm 5 U, m 6 A, s 2 In another embodiment, the present invention provides an in vitro synthesized polyribonucleotide comprising pseudouridine or a modified nucleoside, wherein the modified nucleoside is m 5 Cm 5 U, m 6 A, s 2 U, Ψ, or 2'-O-methyl-U.

[0100] In another embodiment, the in vitro synthesized oligoribonucleotide or polyribonucleotide is a short hairpin (sh)RNA. In another embodiment, the in vitro synthesized oligoribonucleotide is a short interfering RNA (siRNA). In another embodiment, the in vitro synthesized oligoribonucleotide is any other type of oligoribonucleotide known in the art. Each possibility represents a separate embodiment of the present invention.

[0101] In another embodiment, the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule of the methods and compositions of the present invention further comprises an open reading frame encoding a functional protein.In another embodiment, the RNA molecule or oligoribonucleotide molecule functions without encoding a functional protein (e.g., in transcriptional silencing), such as RNzymes.Each possibility represents a separate embodiment of the present invention.

[0102] In another embodiment, the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule further comprises a poly-A tail. In another embodiment, the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule does not comprise a poly-A tail. Each possibility represents a separate embodiment of the present invention.

[0103] In another embodiment, the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule further comprises an m7GpppG cap. In another embodiment, the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule does not comprise an m7GpppG cap. Each possibility represents a separate embodiment of the present invention.

[0104] In another embodiment, the RNA, oligoribonucleotide molecule, or polyribonucleotide molecule further comprises a cap-independent translation enhancer. In another embodiment, the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule does not comprise a cap-independent translation enhancer. In another embodiment, the cap-independent translation enhancer is the tobacco etch virus (TEV) cap-independent translation enhancer. In another embodiment, the cap-independent translation enhancer is any other cap-independent translation enhancer known in the art. Each possibility represents a separate embodiment of the present invention.

[0105] In another embodiment, the present invention provides a gene therapy vector comprising an in vitro synthesized polyribonucleotide molecule, wherein the polyribonucleotide molecule comprises pseudouridine or a modified nucleoside.

[0106] In another embodiment, the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule of the methods and compositions of the present invention comprises pseudouridine. In another embodiment, the RNA molecule or oligoribonucleotide molecule comprises a modified nucleoside. In another embodiment, the RNA molecule or oligoribonucleotide molecule is an RNA molecule or oligoribonucleotide synthesized in vitro. Each possibility represents a separate embodiment of the present invention.

[0107] "Pseudouridine" refers, in another embodiment, to m 1 acp 3 (1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine). In another embodiment, the term refers to m 1 In another embodiment, the term refers to Ψm (2'-O-methylpseudouridine). In another embodiment, the term refers to m 5 In another embodiment, the term refers to m 3 The term refers to Ψ (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that has not been further modified. In another embodiment, the term refers to the monophosphate, diphosphate, or triphosphate of any of the preceding pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

[0108] In another embodiment, the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule of methods and compositions of the present invention is a therapeutic oligoribonucleotide.

[0109] In another embodiment, the present invention provides a method for delivering a recombinant protein to a subject, the method comprising the step of contacting the subject with an RNA molecule, oligoribonucleotide molecule, polyribonucleotide molecule, or gene therapy vector of the present invention, thereby delivering the recombinant protein to the subject.

[0110] In another embodiment, the present invention provides a double-stranded RNA (dsRNA) molecule that comprises pseudouridine or modified nucleosides and further comprises siRNA or short hairpin RNA (shRNA).In another embodiment, the dsRNA molecule is more than 50 nucleotides in length.Each possibility represents a separate embodiment of the present invention.

[0111] In another embodiment, pseudouridine or modified nucleoside is present within the siRNA sequence.In another embodiment, pseudouridine or modified nucleoside is present outside the siRNA sequence.In another embodiment, one or more pseudouridine and / or modified nucleoside residues are present both inside and outside the siRNA sequence.Each possibility represents a separate embodiment of the present invention.

[0112] In another embodiment, siRNA or shRNA is contained internally in dsRNA molecule.In another embodiment, siRNA or shRNA is contained at one end of dsRNA molecule.In another embodiment, one or more siRNA or shRNA is contained at one end of dsRNA molecule, while another one or more is contained internally.Each possibility represents a separate embodiment of the present invention.

[0113] In another embodiment, the length of an RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule (e.g., a single-stranded RNA (ssRNA) molecule or a dsRNA molecule) of methods and compositions of the present invention is greater than 30 nucleotides in length. In another embodiment, the RNA molecule or oligoribonucleotide is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides.In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides. Each possibility represents a separate embodiment of the present invention.

[0114] In another embodiment, the dsRNA molecule of the method and composition of the present invention is produced by in vitro transcription.In another embodiment, the in vitro transcription step utilizes T7 phage RNA polymerase.In another embodiment, the in vitro transcription utilizes SP6 phage RNA polymerase.In another embodiment, the in vitro transcription utilizes an RNA polymerase selected from the above polymerases.In another embodiment, the in vitro transcription utilizes any other RNA polymerase known in the art.Each possibility represents a separate embodiment of the present invention.

[0115] In another embodiment, dsRNA molecules can be processed by cellular enzymes to produce siRNA or shRNA. In another embodiment, the cellular enzyme is an endonuclease. In another embodiment, the cellular enzyme is Dicer. Dicer is an RNaseIII-family nuclease that initiates RNA interference (RNAi) and related phenomena by producing small RNAs that determine the specificity of these gene silencing pathways (Bernstein E, Caudy AA et al., Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409(6818): 363-6). In another embodiment, the cellular enzyme is any other cellular enzyme known in the art that can cleave dsRNA molecules. Each possibility represents a separate embodiment of the present invention.

[0116] In another embodiment, the dsRNA molecule contains two siRNAs or shRNAs. In another embodiment, the dsRNA molecule contains three siRNAs or shRNAs. In another embodiment, the dsRNA molecule contains four or more siRNAs or shRNAs. In another embodiment, the siRNAs and / or shRNAs are released from the dsRNA molecule by cellular enzymes. Each possibility represents a separate embodiment of the present invention.

[0117] In another embodiment, the present invention provides a method for administering siRNA or shRNA to a cell, comprising administering a dsRNA molecule of the present invention, wherein the cell processes the dsRNA molecule to produce the siRNA or shRNA, thereby administering the siRNA or shRNA to the cell.

[0118] In another embodiment, the modified nucleoside in the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule of the methods and compositions of the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G). Each possibility represents a separate embodiment of the present invention.

[0119] In another embodiment, the modified nucleoside of methods and compositions of the present invention is m 5 In another embodiment, the modified nucleoside is m 5 U (5-methyluridine). In another embodiment, the modified nucleoside is m 6 A(N 6 In another embodiment, the modified nucleoside is s-methyladenosine. 2 In another embodiment, the modified nucleoside is U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[0120] In other embodiments, the modified nucleoside is m l A (l-methyladenosine), m 2 A (2-methyladenosine), Am (2'-O-methyladenosine), ms 2 m G A(2-methylthio-N 6 -methyladenosine), i G A (~isopentenyladenosine), ms z i6A(2-methylthio-N 6 Isopentenyladenosine), io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine), ms 2 io 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine), g6 A(N 6 -glycinylcarbamoyl adenosine), t 6 A(N 6 -threonylcarbamoyl adenosine), ms 2 t 6 A(2-methylthio-N 6 - and leonylcarbamoyl adenosine), m 6 t 6 A(N 6 -methyl-N 6 -threonylcarbamoyl adenosine), hn 6 A(N 6 hydroxynorvalylcarbamoyl adenosine), ms 2 hn 6 A(2-methylthio-N 6 -hydroxynorvalylcarbamoyl adenosine), Ar(p) (2'-O-ribosyladenosine (phosphate)), I (inosine), m 1 I (l-methylinosine), m l Im (l,2'-O-dimethylinosine), m 3 C(3-methylcytidine), Cm(2'-O-methylcytidine), S 2 C (2 thiocytidine), ac 4 C(N 4 -acetylcytidine), f 5 C(5-formylcytidine), m 5 Cm (5,2'-O-dimethylcytidine), ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine), k 2 C (lycidin), m 1 G (l-methylguanosine), m 2 G(N 2 -methylguanosine), m 7 G (7-methylguanosine), Gm (2'-O-methylguanosine), m 2 2G(N 2 ,N 2 -dimethylguanosine), m 2 Gm(N 2 ,2'-O-dimethylguanosine), m 2 2Gm(N 2 ,N 2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(wybutosine), o2yW(peroxywybutosine), OHyW(hydroxywybutosine), OHyW * (unmodified hydroxywybutosine), imG (wybutosine), mimG (methylwybutosine), Q (keuosine), oQ (epoxykeuosine), galQ (galactosyl-keuosine), manQ (mannosylkeuosine), preQ0 (7-cyano-7-deazaguanosine), preQ l (7-aminomethyl-7-deazaguanosine), G + (archaeosine), D (dihydrouridine), m 5 Um (5,2'-O-dimethyluridine), S 4 U(4-thiouridine), m 5 s 2 U(5-methyl-2-thiouridine), s 2 Um (2-thio-2'-O-methyluridine), acp 3 U(3-(3-amino-3-carboxypropyl)uridine), ho 5 U (5-hydroxyuridine), mo 5 U(5-methoxyuridine), cmo 5 U (uridine 5-oxyacetic acid), mcmo 5 U (uridine 5-hydroxyacetic acid methyl ester), chm 5 U(5-(carboxyhydroxymethyl)uridine)), mchm 5 U(5(carboxyhydroxymethyl)uridine methyl ester), mcm 5 U (5-methoxycarbonylmethyluridine), mcm 5 Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine), nm 5 s 2 U (5-aminomethyl-2-thiouridine), mnm 5 U (5-methylaminomethyluridine), mnm 5 s 2U (5-methylaminomethyl-2-thiouridine), mnmse 2 U(5-methylaminomethyl-2-selenouridine), ncm 5 U (5-carbamoylmethyluridine), ncm 5 Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm 5 U (5-carboxymethylaminomethyluridine), cmnm 5 Um (5-carboxymethylaminomethyl-2'-methyluridine), cmnm 5 s 2 U (5-carboxymethylaminomethyl-2-thiouridine), m 6 2A(N 6 ,N 6 -dimethyladenosine), Im (2'-O-methylinosine), m 4 C(N 4 -methylcytidine), m 4 Cm(N 4 ,2'-O-dimethylcytidine), hm 5 C (5-hydroxymethylcytidine), m 3 U (3-methyluridine), cm 5 U (5-carboxymethyluridine), m 6 Am(N 6 ,2'-Odimethyladenosine), m 6 2Am(N 6 ,N 6 ,2'-O-trimethyladenosine), m 2,7 G(N 2 ,7-dimethylguanosine), m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine), m 3 Um (3,2'-O-dimethyluridine), m 5 D (5-methyldihydrouridine), f 5 Cm (5-formyl-2'-O-methylcytidine), m l Gm (l,2'-O-dimethylguanosine), m 1 Am(1,2'-O-dimethyladenosine), τm 5U (5-taurinomethyluridine), τm5s2U (5-taurinomethyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac 6 A(N 6 -acetyladenosine). Each possibility represents a separate embodiment of the present invention.

[0121] In another embodiment, the RNA molecules, oligoribonucleotide molecules, or polyribonucleotide molecules of the methods and compositions of the present invention comprise a combination of two or more of the above modifications. In another embodiment, the RNA molecules or oligoribonucleotide molecules comprise a combination of three or more of the above modifications. In another embodiment, the RNA molecules or oligoribonucleotide molecules comprise a combination of four or more of the above modifications. Each possibility represents a separate embodiment of the present invention.

[0122] In another embodiment, between 0.1% and 100% of the residues in an RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule of methods and compositions of the invention are modified (e.g., by the presence of either pseudouridine or a modified nucleoside base). In another embodiment, 0.1% of the residues are modified. In another embodiment, 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0123] In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0124] In another embodiment, 0.1% of the residues of a given nucleotide (uridine, cytidine, guanosine, or adenine) are modified. In another embodiment, the fraction of nucleotides is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0125] In another embodiment, the fraction of a given nucleotide is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0126] In another embodiment, the terms "ribonucleotide," "oligoribonucleotide," and "polyribonucleotide" refer to a sequence of at least two base-sugar-phosphate combinations. In another embodiment, the term includes compounds containing nucleotides whose sugar moiety is ribose. In another embodiment, the term includes both RNA and RNA derivatives with modified backbones. In another embodiment, "nucleotide" refers to a monomeric unit of a nucleic acid polymer. In another embodiment, RNA can be in the form of tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, small interfering RNA (siRNA), microRNA (miRNA), and ribozyme. The use of siRNA and miRNA has been described (Caudy AA et al., Genes & Devel 16: 2491-96 and references cited therein). In addition, these forms of RNA can be single-stranded, double-stranded, triple-stranded, or quadruple-stranded. The term also includes artificial nucleic acids that may contain other types of backbones but the same bases in other embodiments. In another embodiment, the artificial nucleic acid is a PNA (peptide nucleic acid). PNA contains a peptide backbone and nucleotide bases, and in another embodiment, can bind to both DNA and RNA molecules. In another embodiment, the nucleotide is oxetane-modified. In another embodiment, the nucleotide is modified by replacing one or more phosphodiester bonds with phosphorothioate bonds. In another embodiment, the artificial nucleic acid contains any other variant of the phosphate backbone of natural nucleic acids known in the art. Phosphothiolate nucleic acids and PNA are known to those skilled in the art and are described, for example, in Neilsen PE, Curr Opin Struct Biol 9:353-57 and Raz NK et al. Biochem Biophys Res Commun. 297:1075-84.The production and use of nucleic acids is known to those of skill in the art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and GC Fareed. Each nucleic acid derivative represents a separate embodiment of the present invention.

[0127] In another embodiment, the term "oligoribonucleotide" refers to a stretch comprising fewer than 25 nucleotides (nt). In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 24 nucleotides. In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 23 nucleotides. In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 22 nucleotides. In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 21 nucleotides. In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 20 nucleotides. In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 19 nucleotides. In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 18 nucleotides. In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 17 nucleotides. In another embodiment, "oligoribonucleotide" refers to a stretch of fewer than 16 nucleotides. Each possibility represents a separate embodiment of the present invention.

[0128] In another embodiment, the term "polyribonucleotide" refers to a stretch comprising more than 25 nucleotides (nt). In another embodiment, "polyribonucleotide" refers to a stretch of more than 26 nucleotides. In another embodiment, "polyribonucleotide" refers to a stretch of more than 28 nucleotides. In another embodiment, "the term" refers to a stretch of more than 30 nucleotides. In another embodiment, "the term" refers to a stretch of more than 32 nucleotides. In another embodiment, "the term" refers to a stretch of more than 35 nucleotides. In another embodiment, "the term" refers to a stretch of more than 40 nucleotides. In another embodiment, "the term" refers to a stretch of more than 50 nucleotides. In another embodiment, "the term" refers to a stretch of more than 60 nucleotides. In another embodiment, "the term" refers to a stretch of more than 80 nucleotides. In another embodiment, "the term" refers to a stretch of more than 100 nucleotides. In another embodiment, "the term" refers to a stretch of more than 120 nucleotides. In another embodiment, "the term" refers to a stretch of more than 150 nucleotides. In another embodiment, "the term" refers to a stretch of more than 200 nucleotides. In another embodiment, "the term" refers to a stretch of more than 300 nucleotides. In another embodiment, "the term" refers to a stretch of more than 400 nucleotides. In another embodiment, "the term" refers to a stretch of more than 500 nucleotides. In another embodiment, "the term" refers to a stretch of more than 600 nucleotides. In another embodiment, "the term" refers to a stretch of more than 800 nucleotides. In another embodiment, "the term" refers to a stretch of more than 1000 nucleotides. In another embodiment, "the term" refers to a stretch of more than 1200 nucleotides. In another embodiment, "the term" refers to a stretch of more than 1400 nucleotides. In another embodiment, "the term" refers to a stretch of more than 1600 nucleotides. In another embodiment, "the term" refers to a stretch of more than 1800 nucleotides. In another embodiment, "the term" refers to a stretch of more than 2000 nucleotides. Each possibility represents a separate embodiment of the present invention.

[0129] In another embodiment, the present invention provides a method for inducing a mammalian cell to produce a protein of interest, comprising contacting the mammalian cell with an in vitro synthesized RNA molecule containing pseudouridine or a modified nucleoside that encodes the recombinant protein, thereby inducing the mammalian cell to produce the protein of interest. In another embodiment, the protein of interest is a recombinant protein. Each possibility represents a separate embodiment of the present invention.

[0130] In another embodiment, "encoding" refers to an RNA molecule that encodes a protein of interest. In another embodiment, the RNA molecule comprises an open reading frame that encodes the protein of interest. In another embodiment, one or more other proteins are also encoded. In another embodiment, the protein of interest is the only protein encoded. Each possibility represents a separate embodiment of the present invention.

[0131] In another embodiment, the invention provides a method for inducing a mammalian cell to produce a recombinant protein, comprising contacting the mammalian cell with an in vitro transcribed RNA molecule encoding the recombinant protein, wherein the RNA molecule further comprises a pseudouridine or modified nucleoside, thereby inducing the mammalian cell to produce the recombinant protein.

[0132] In another embodiment, the RNA molecules, oligonucleotide molecules, or polyribonucleotide molecules of the methods and compositions of the present invention are translated in cells more efficiently than unmodified RNA molecules having the same sequence. In another embodiment, the RNA molecules, oligoribonucleotide molecules, or polyribonucleotide molecules exhibit an increased ability to be translated by target cells. In another embodiment, translation is increased by about 2-fold relative to their unmodified counterparts. In another embodiment, translation is increased by about 3-fold. In another embodiment, translation is increased by about 5-fold. In another embodiment, translation is increased by about 7-fold. In another embodiment, translation is increased by about 10-fold. In another embodiment, translation is increased by about 15-fold. In another embodiment, translation is increased by about 20-fold. In another embodiment, translation is increased by about 50-fold. In another embodiment, translation is increased by about 100-fold. In another embodiment, translation is increased by about 200-fold. In another embodiment, translation is increased by about 500-fold. In another embodiment, translation is increased by about 1000-fold. In another embodiment, translation is increased by as much as 2000-fold. In another embodiment, the fold is 10-1000-fold. In another embodiment, the fold is 10-100-fold. In another embodiment, the fold is 10-200-fold. In another embodiment, the fold is 10-300-fold. In another embodiment, the fold is 10-500-fold. In another embodiment, the fold is 20-1000-fold. In another embodiment, the fold is 30-1000-fold. In another embodiment, the fold is 50-1000-fold. In another embodiment, the fold is 100-1000-fold. In another embodiment, the fold is 200-1000-fold. In another embodiment, translation is increased by any other significant amount or range of amounts. Each possibility represents a separate embodiment of the present invention.

[0133] Methods for determining translation efficiency are well known in the art and include, for example, measuring the activity of an encoded reporter protein (e.g., luciferase or Renilla [Examples herein] or green fluorescent protein [Wall AA, Phillips AM et al., Effective translation of the second cistron in two Drosophila dicistronic transcripts is determined by the absence of in-frame AUG codons in the first cistron. J Biol Chem 2005;280(30):27670-8]), or measuring the activity of radioactive material incorporated into the translated protein. and measuring the label (Ngosuwan J, Wang NM et al, Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum. J Biol Chem 2003;278(9):7034-42). Each method represents a separate embodiment of the present invention.

[0134] In some expression studies provided herein, translation was measured from RNA complexed with Lipofectin® (Gibco BRL, Gaithersburg, MD, USA) and injected into the tail vein of mice. In spleen lysates, pseudouridine-modified RNA was translated significantly more efficiently than unmodified RNA (Figure 17B). Under the conditions utilized herein, the efficiency of the transfection-based method of the present invention correlates with the ability of the transfection reagent to penetrate the tissue, providing an explanation for why the effect was most pronounced in splenocytes. The splenic blood flow is an open system, and the blood content comes into direct contact with the red and white pulp elements, including lymphoid cells.

[0135] In another experiment, an in vitro phosphorylation assay was performed using recombinant human PKR and its substrate, eIF2α, in the presence of mRNA encoding capped Renilla reniformis (0.5 and 0.05 ng / μL). Pseudouridine (Ψ)-containing mRNA did not activate PKR, as detected by the lack of both PKR autophosphorylation and eIF2α phosphorylation, whereas RNA without nucleoside modifications and mRNA with m5C modifications did activate PKR. Phosphorylated eIF2α is known to block the initiation of mRNA translation; therefore, the lack of phosphorylation, in another embodiment, allows for enhanced translation of pseudouridine (Ψ)-containing mRNA.

[0136] In another embodiment, the enhanced translation is in a cell (compared to translation in the same cell of an unmodified RNA having the same sequence, Examples 13-14). In another embodiment, the enhanced translation is in vitro (e.g., in an in vitro translation mixture or reticulocyte lysate, Examples 13-14). In another embodiment, the enhanced translation is in vivo (Example 13). In each case, the enhanced translation is relative to an unmodified RNA having the same sequence under the same conditions. Each possibility represents a separate embodiment of the present invention.

[0137] In another embodiment, the RNA molecules, oligoribonucleotide molecules, or polyribonucleotide molecules of the methods and compositions of the present invention are significantly less immunogenic than unmodified in vitro synthesized RNA molecules having the same sequence. In another embodiment, the modified RNA molecules are 2-fold less immunogenic than their unmodified counterparts. In another embodiment, the immunogenicity is 3-fold less. In another embodiment, the immunogenicity is 5-fold less. In another embodiment, the immunogenicity is 7-fold less. In another embodiment, the immunogenicity is 10-fold less. In another embodiment, the immunogenicity is 15-fold less. In another embodiment, the immunogenicity is 20-fold less. In another embodiment, the immunogenicity is 50-fold less. In another embodiment, the immunogenicity is 100-fold less. In another embodiment, the immunogenicity is 200-fold less. In another embodiment, the immunogenicity is 500-fold less. In another embodiment, the immunogenicity is 1000-fold less. In another embodiment, the immunogenicity is 2000-fold less. In another embodiment, the immunogenicity is another fold less.

[0138] In another embodiment, "significantly lower immunogenicity" refers to a detectable reduction in immunogenicity. In another embodiment, the term refers to a fold reduction in immunogenicity (e.g., one of the fold reductions listed above). In another embodiment, the term refers to a reduction such that an effective amount of an RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule can be administered without eliciting a detectable immune response. In another embodiment, the term refers to a reduction such that an RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule can be repeatedly administered without eliciting an immune response sufficient to detectably reduce the expression of the recombinant protein. In another embodiment, the reduction is such that an RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule can be repeatedly administered without eliciting an immune response sufficient to eliminate the detectable expression of the recombinant protein.

[0139] An "effective amount" of an RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule, in another embodiment, refers to an amount sufficient to exert a therapeutic effect. In another embodiment, the term refers to an amount sufficient to induce expression of a detectable amount of recombinant protein. Each possibility represents a separate embodiment of the present invention.

[0140] The reduced immunogenicity of the RNA molecules, oligoribonucleotide molecules, and polyribonucleotide molecules of the present invention is demonstrated herein (Examples 4-11).

[0141] Methods for determining immunogenicity are well known in the art and include, for example, measuring the secretion of cytokines (e.g., IL-12, IFN-α, TNF-α, RANTES, MIP-1 α or β, IL-6, IFN-β, or IL-8, see the Examples herein), measuring the expression of dendritic cell activation markers (e.g., CD83, HLA-DR, CD80, and CD86, see the Examples herein), or measuring the ability to act as an adjuvant for an adaptive immune response. Each possibility represents a separate embodiment of the present invention.

[0142] In another embodiment, the relative immunogenicity of modified nucleotide and its unmodified counterpart is determined by determining the amount of modified nucleotide required to induce one of the above responses to the same extent as a given amount of unmodified nucleotide.For example, if modified nucleotide is 2 times less immunogenic than unmodified nucleotide, then modified nucleotide is 2 times less immunogenic than unmodified nucleotide.

[0143] In another embodiment, the relative immunogenicity of a modified nucleotide and its unmodified counterpart is determined by determining the amount of cytokines (e.g., IL-12, IFN-α, TNF-α, RANTES, MIP-1α or β, IL-6, IFN-β, or IL-8) secreted in response to administration of the modified nucleotide relative to the same amount of unmodified nucleotide. For example, if half the cytokines are secreted compared to unmodified nucleotides, the modified nucleotide is two times less immunogenic than the unmodified nucleotide. In another embodiment, the background level of stimulation is subtracted before calculating immunogenicity in the above method. Each possibility represents a separate embodiment of the present invention.

[0144] In another embodiment, the method of the present invention further comprises mixing the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule with a transfection reagent prior to the contacting step. In another embodiment, the method of the present invention further comprises administering the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule together with a transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent (Example 6).

[0145] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine-based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin® or Lipofectamine®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0146] In another embodiment, the transfection reagent forms liposomes. Liposomes, in another embodiment, have increased intracellular stability, increased uptake efficiency, and improved biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a manner similar to that of the lipids forming the cell membrane. In another embodiment, liposomes have an internal aqueous space for entrapment of water-soluble compounds and range in size from 0.05 to several microns in diameter. In another embodiment, liposomes can deliver RNA to cells in a biologically active form.

[0147] Each type of transfection reagent represents a separate embodiment of the present invention.

[0148] In another embodiment, the target cell of the method of the present invention is an antigen-presenting cell. In another embodiment, the cell is an animal cell. In another embodiment, the cell is a dendritic cell (Example 14). In another embodiment, the cell is a neuronal cell. In another embodiment, the cell is a brain cell (Example 16). In another embodiment, the cell is a splenocyte. In another embodiment, the cell is a lymphoid cell. In another embodiment, the cell is a lung cell (Example 16). In another embodiment, the cell is a skin cell. In another embodiment, the cell is a keratinocyte. In another embodiment, the cell is an endothelial cell. In another embodiment, the cell is an astrocyte, microglia, or neuron (Example 16). In another embodiment, the cell is an alveolar cell (Example 16). In another embodiment, the cell is a surface alveolar cell (Example 16). In another embodiment, the cell is an alveolar macrophage. In another embodiment, the cell is an alveolar pneumocyte. In another embodiment, the cell is a vascular endothelial cell. In another embodiment, the cell is a mesenchymal cell. In another embodiment, the cell is an epithelial cell. In another embodiment, the cell is a hematopoietic cell. In another embodiment, the cell is a colony epithelial cell. In another embodiment, the cell is a lung epithelial cell. In another embodiment, the cell is a bone marrow cell.

[0149] In other embodiments, the target cell is a Claudius cell, a Hensen cell, a Merkel cell, a Muller cell, a Paneth cell, a Purkinje cell, a Schwann cell, a Sertoli cell, an eosinophil, an acinar cell, a lipoblast, an adipocyte, a brown or white alpha cell, an axonal cell, a beta cell, a theca cell, a cementocyte, a chief cell, a chondroblast, a chondrocyte, a chromaffin cell, a chromophobe cell, a corticotroph, a delta cell, a Langerhans cell, a follicular dendritic cell, an enterochromaffin cell, an ependymal cell, an epithelial cell, a basal cell, a squamous cell, Endothelial cell, transitional cell, erythroblast, red blood cell, fibroblast, fibrocyte, follicular cell, germ cell, gamete, egg, sperm, oocyte, primary oocyte, secondary oocyte, immotile sperm, spermatocyte, primary spermatocyte, secondary spermatocyte, germinal epithelium, giant cell, glial cell, astroblast, astrocyte, oligodendrocyte, oligodendrocyte, glioblast, goblet cell, gonadotropin-secreting cell, granulosa cell, hemoblast, hair cell, hepatoblast, hepatocyte, vitreous cell, stromal cell, juxtaglomerular cell, keratinocyte, keratocyte, lemma cell cell), white blood cell, granulocyte, basophil, eosinophil, neutrophil, lymphoblast, B lymphoblast, T lymphoblast, lymphocyte, B lymphocyte, T lymphocyte, helper induced T-lymphocyte, Th1 T lymphocyte, Th2 T lymphocyte, natural killer cell, thymocyte, macrophage, Kupffer cell, alveolar macrophage, foam cell, histocyte, lutein cell, lymphoid stem cell, lymphoid cell, immune stem cell, astroglia, lactotroph, mast cell, medulloblast, megakaryoblast, megakaryocyte, melanoblast, melanocyte, melanocyte, mesangial cell, mesothelial cell, metamyelocyte, monoblast, monocyte, gastric adenocarcinoma cell, muscle cell, cardiac muscle cell, skeletal muscle cell, smooth muscle cell, myelocyte, The cell is a myeloid cell, bone marrow stem cell, myoblast, myoepithelial cell, myofibroblast, neuroblast, neuroepithelial cell, neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell, parafollicular cell, paraluteal cell, digestive cell, pericyte, peripheral blood mononuclear cell, chromaffin cell, supporting cell, pineal cell, posterior glial cell, plasma cell, platelet, tectal cell, proerythroblast, promonocyte, promyeloblast, promyelocyte, pronormoblast, stem cell, Sertoli cell, terminal glial cell, or zymogen cell.Each possibility represents a separate embodiment of the present invention.

[0150] A variety of disorders, including, inter alia, single gene disorders, infectious diseases, acquired diseases, cancer, and the like, can be treated by employing the methods of the present invention. Exemplary single gene disorders include adenosine deaminase deficiency, cystic fibrosis, familial hypercholesterolemia, hemophilia, chronic granulomatous disease, Duchenne muscular dystrophy, Fanconi anemia, sickle cell anemia, Gaucher disease, Hunter syndrome, X-linked severe combined immunodeficiency, and the like. In another embodiment, the disorder to be treated involves one of the proteins listed below. Each possibility represents a separate embodiment of the present invention.

[0151] In another embodiment, a recombinant protein encoded by an RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule of methods and compositions of the present invention is an ecto-nucleoside triphosphate diphosphohydrolase.

[0152] In another embodiment, the recombinant protein is erythropoietin (EPO). In other embodiments, the encoded recombinant protein is ABCA4, ABCD3, ACADM, AGL, AGT, ALDH4AI, ALPL, AMPD1, APOA2, AVSD1, BRCD2, C1QA, C1QB, C1QG, C8A, C8B, CACNA1S, CCV, CD3Z, CDC2L1, CHML, CHS1, CIAS1, CLCNKB, CMD1A, CMH2, CMM, COL11A1, COL8A2, COL9A2, CPT2, CRB1, CSE, CSF3R, CTPA, CTSK, DBT, DIO1, DISC 1, DPYD, EKV, ENO1, ENO1P, EPB41, EPHX1, F13B, F5, FCGR2A, FCGR2B, FCGR3A, FCHL, FH, FMO3, FMO4, FUCA1, FY, GALE, GBA, GFND, GJA8, GJB3, GLC3B, HF1, HMGCL, HPC1, HRD, HRPT2, HSD3B2, HSPG2, KCNQ4, KCS, KIF1B, LAMB3, LAMC2, LGMD1B, LMNA, LOR, MCKD1, MCL1, MPZ, MTHFR, MTR, MUTYH, MYOC, NB, NCF2, NEM1, NPHS2, NPPA, NRAS, NTRK1, OPTA2, PBX1, PCHC, PGD, PHA2A, PHGDH, PKLR, PKP1, PLA2G2A, PLOD, PPOX, PPT1, PRCC, PRG4, PSEN2, PTOS1, R EN, RFX5, RHD, RMD1, RPE65, SCCD, SERPINC1, SJS1, SLC19A2, SLC2A1, SPG23, SPTA1, TAL1, TNFSF6, TNNT2, TPM3, TSHB, UMPK, UOX, UROD, USH2A, VMG LOM, VWS, WS2B, ABCB11, ABCG5, ABCG8, ACADL, ACP1, AGXT, AHHR, ALMS1, ALPP, ALS2, APOB, BDE, BDMR, BJS, BMPR2, CHRNA1, CMCWTD, CNGA3, COL3A1, COL4A3, COL4A4, COL6A3, CPSI, CRYGA, CRYGEP1, CYP1B1, CYP27A1, DBI, DES, DYSF, EDAR, EFEMPI, EIF2AK3, ERCC3, FSHR, GINGF, GLC1B, GPD2, GYPC,HADHA, HADHB, HOXD13, HPE2, IGKC, IHH, IRSI, ITGA6, KHK, KYNU, LCT, LHCGR, LSFC, MSH2, MSH6, NEB, NMTC, NPHP1, PAFAH1P1, PAX3, PAX8, PMS1, PNKD, PP H1、PROC、REG1A、SAG、SFTPB、SLC11A1、SLC3A1、SOS1、SPG4、SRD5A2、TCL4、T GFA、TMD、TPO、UGT1A@、UV24、WSS、XDH、ZAP70、ZFHX1B、ACAA1、AGS1、AGTR1、A HSG、AMT、ARMET、BBS3、BCHE、BCPM、BTD、CASR、CCR2、CCR5、CDL1、CMT2B、COL 7A1、CP、CPO、CRV、CTNNB1、DEM、ETM1、FANCD2、FIH、FOXL2、GBE1、GLB1、GLC1C 、GNAI2、GNATI、GP9、GPX1、HGD、HRG、ITIH1、KNG、LPP、LRS1、MCCCI、MDS1、MH S4、MITF、MLH1、MYL3、MYMY、OPA1、P2RY12、PBXP1、PCCB、POU1F1、PPARG、PROS 1, PTHR1, RCA1, RHO, SCA7, SCLC1, SCN5A, SI, SLC25A20, SLC2A2, TF, TGFBR2, THPO, THRB, TKT, TM4SF1, TRH, UMPS, UQCRC1, USH3A, VHL, WS2A, XPC, ZNF35 、ADH1B、ADH1C、AFP、AGA、AIH2、ALB、ASMD、BFHD、CNGA1、CRBM、DCK、DSPP、DT DP2、ELONG、ENAM、ETFDH、EVC、F11、FABP2、FGA、FGB、FGFR3、FGG、FSHMD1A、GC 、GNPTA、GNRHR、GYPA、HCA、HCL2、HD、HTN3、HVBS6、IDUA、IF、JPD、KIT、KLKB1 、LQT4、MANBA、MLLT2、MSX1、MTP、NR3C2、PBT、PDE6B、PEE1、PITX2、PKD2、QDPR 、SGCB、SLC25A4、SNCA、SOD3、STATH、TAPVR1、TYS、WBS2、WFS1、WHCR、ADAMTS2、ADRB2、AMCN、AP3BI、APC、ARSB、B4GALT7、BHR1、C6、C7、CCAL2、CKN1、CMDJ、CRHBP、CSF1R、DHFR、DIAPH1、DTR、EOS、EPD、ERVR、F12、FBN2、GDNF、GHR、GLR A1、GM2A、HEXB、HSD17B4、ITGA2、KFS、LGMD1A、LOX、LTC4S、MAN2A1、MCC、MCC C2, MSH3, MSX2, NR3C1, PCSK1, PDE6A, PFBI, RASAI, SCZDI, SDHA, SGCD, SLC22A5, SLC26A2, SLC6A3, SM1, SMA@, SMN1, SMN2, SPINK5, TCOF1, TELAB1, TGFB I、ALDH5A1、ARG1、AS、ASSP2、BCKDHB、BF、C2、C4A、CDKN1A、COL10A1、COL11A 2、CYP21A2、DYX2、EJM1、ELOVL4、EPM2A、ESR1、EYA4、F13A1、FANCE、GCLC、GJ A1、GLYS1、GMPR、GSE、HCR、HFE、HLA-A、HLA-DPBI、HLA-DRA、HPFH、ICS1、IDD M1、IFNGR1、IGAD1、IGF2R、ISCW、LAMA2、LAP、LCA5、LPA、MCDR1、MOCS1、MUT、M YB、NEU1、NKS1、NYS2、OA3、ODDD、OFC1、PARK2、PBCA、PBCRA1、PDB1、PEX3、PE X6, PEX7, PKHD1, PLA2G7, PLG, POLH, PPAC, PSORS1, PUJO, RCD1, RDS, RHAG, R P14、RUNX2、RWS、SCA1、SCZD3、SIASD、SOD2、ST8、TAPl、TAP2、TFAP2B、TNDM、 TNF、TPBG、TPMT、TULP1、WISP3、AASS、ABCB1、ABCB4、ACHE、AQP1、ASL、ASNS、A UTS1、BPGM、BRAF、C7orf2、CACNA2D1、CCM1、CD36、CFTR、CHORDOMA、CLCN1、C MH6、CMT2D、COL1A2、CRS、CYMD、DFNA5、DLD、DYT11、EEC1、ELN、ETV1、FKBP6、 GCK, GHRHR, GHS, GLI3, GPDS1, GUSB, HLXB9, HOXA13, HPFH2, HRX, IAB, IMMP2L, KCNH2, LAMB1, LEP, MET, NCF1, NM, OGDH, OPN1SW, PEX1, PGAM2, PMS2, PON1PPP1R3A, PRSS1, PTC, PTPN12, RP10, RP9, SERPINE1, SGCE, SHFM1, SHH, SLC26A3, SLC26A4, SLOS, SMAD1, TBXAS1, TWIST, ZWS1, ACHM3, ADRB3, ANK1, CA1 CA2、CCAL1、CLN8、CMT4A、CNGB3、COH1、CPP、CRH、CYP11B1、CYP11B2、DECR1、 DPYS、DURS1、EBS1、ECA1、EGI、EXT1、EYA1、FGFR1、GNRH1、GSR、GULOP、HR、KCN Q3、KFM、KWE、LGCR、LPL、MCPH1、MOS、MYC、NAT1、NAT2、NBS1、PLAT、PLEC1、PR KDC、PXMP3、RP1、SCZD6、SFIPC、SGM1、SPG5A、STAR、TG、TRPS1、TTPA、VMD1、W RN, ABCA1, ABL1, ABO, ADAMTS13, AK1, ALAD, ALDH1A1, ALDOB, AMBP, AMCD1, ASS, BDMF, BSCL, C5, CDKN2A, CHAC, CLA1, CMD1B, COL5A1, CRAT, DBH, DNAI1, DY S, DYT1, ENG, FANCC, FBP1, FCMD, FRDA, GALT, GLDC, GNE, GSM1, GSN, HSD17B3, HSN1, IBM2, INVS, JBTS1, LALL, LCCS1, LCCS, LGMD2H, LMX1B, MLLT3, MROS MSSE, NOTCH1, ORM1, PAPPA, PIP5K1B, PTCH, PTGS1, RLN1, RLN2, RMRP, ROR2, RPD1, SARDH, SPTLC1, STOM, TDFA, TEK, TMC1, TRIM32, TSC1, TYRP1, XPA, CACN B2, COL17Al, CUBN, CXCL12, CYP17, CYP2C19, CYP2C9, EGR2, EMX2, ERCC6, FGFR2, HK1, HPS1, IL2RA, LGI1, LIPA, MAT1A, MBL2, MKI67, MXI1, NODAL, OAT, OA TL3、PAX2、PCBD、PEO1、PHYH、PNLIP、PSAP、PTEN、RBP4、RDPA、RET、SFTPA1、S FTPD、SHFM3、SIAL、THC2、TLX1、TNFRSF6、UFS、UROS、AA、ABCC8、ACAT1、ALX4、AMPD3、ANC、APOA1、APOA4、APOC3、ATM、BSCL2、BWS、CALCA、CAT、CCND1、CD3E 、CD3G、CD59、CDKN1C、CLN2、CNTF、CPT1A、CTSC、DDB1、DDB2、DHCR7、DLAT、DR D4、ECB2、ED4、EVR1、EXT2、F2、FSHB、FTH1、G6PT1、G6PT2、GIF、HBB、HBBP1、H BD、HBE1、HBG1、HBG2、HMBS、HND、HOMG2、HRAS、HVBS1、IDDM2、IGER、INS、JBS、 KCNJ11、KCNJ1、KCNQ1、LDHA、LRP5、MEN1、MLL、MYBPC3、MYO7A、NNO1、OPPG、O PTB1、PAX6、PC、PDX1、PGL2、PGR、PORC、PTH、PTS、PVRL1、PYGM、RAG1、RAG2、RO M1, RRAS2, SAA1, SCA5, SCZD2, SDHD, SERPING1, SMPD1, TCIRG1, TCL2, TECTA, TH, TREH, TSG101, TYR, USH1C, VMD2, VRNI, WT1, WT2, ZNFl45, A2M, AAAS, ACA DS、ACLS、ACVRL1、ALDH2、AMHR2、AOM、AQP2、ATD、ATP2A2、BDC、C1R、CD4、CDK 4、CNA1、COL2A1、CYP27B1、DRPLA、ENUR2、FEOM1、FGF23、FPF、GNB3、GNS、HAL、 HBP1, HMGA2, HMN2, HPD, IGF1, KCNA1, KERA, KRAS2, KRT1, KRT2A, KRT3, KRT4, KRT5, KRT6A, KRT6B, KRTHB6, LDHB, LYZ, MGCT, MPE, MVK, MYL2, OAP, PAH, PPK B、PRB3、PTPN11、PXR1、RLS、RSN、SAS、SAX1、SCA2、SCNN1A、SMAL、SPPM、SPSM A、TBX3、TBX5、TCF1、TPI1、TSC3、ULR、VDR、VWF、ATP7B、BRCA2、BRCD1、CLN5、C PB2、ED2、EDNRB、ENUR1、ERCC5、F10、F7、GJB2、GJB6、IPF1、MBS1、MCOR、NYS4 、PCCA、RB1、RHOK、SCZD7、SGCG、SLC10A2、SLC25A15、STARP1、ZNF198、ACHM1、ARVD1, BCH, CTAA1, DAD1, DFNB5, EML1, GALC, GCH1, IBGC1, IGH@, IGCgroup, IGHG1, IGHM, IGHHR, IV, LTBP2, MCOP, MJD, MNG1, MPD1, MPS3C, MYH6, MYH7, NP NPC2、PABPN1、PSEN1、PYGL、RPGRIP1、SERPINA1、SERPINA3、SERPINA6、SLC7 A7、SPG3A、SPTB、TCL1A、TGMI、TITF1、TMIP、TRA@、TSHR、USH1A、VP、ACCPN、AH O2、ANCR、B2M、BBS4、BLM、CAPN3、CDAN1、CDAN3、CLN6、CMH3、CYP19、CYP1A1、 CYP1A2、DYX1、EPB42、ETFA、EYCL3、FAH、FBN1、FES、HCVS、HEXA、IVD、LCS1、LI PC、MY05A、OCA2、OTSC1、PWCR、RLBP1、SLC12A1、SPG6、TPM1、UBE3A、WMS、ABC 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LCFS2, MADH4, MAFD1, MC2R, MCL, MYP2 NPC1, SPPK, TGFBRE, TGIF, TTR, AD2, AMH, APOC2, APOE, ATHS, BAX, BCKDHA, BCL3, BFIC, C3, CACNA1A, CCO, CEACAM5, COMP, CRX, DBA, DDU, DFNA4, DLL3, DM1 DMWD, E11S, ELA2, EPOR, ERCC2, ETFB, EXT3, EYCLI, FTL, FUT1, FUT2, FUT6, GAMT, GCDH, GPI, GUSM, HB1, HCL1, HHC2, HHC3, ICAM3, INSR, JAK3, KLK3, LDLR 、LHB、LIG1、LOH19CR1、LYL1、MAN2B1、MCOLN1、MDRV、MLLT1、NOTCH3、NPHS1、 OFC3、OPA3、PEPD、PRPF31、PRTN3、PRX、PSG1、PVR、RYR1、SLC5A5、SLC7A9、STK 11, TBXA2R, TGFB1, TNNI3, TYROBP, ADA, AHCY, AVP, CDAN2, CDPD1, CHED1, CHED2, CHRNA4, CST3, EDN3, EEGV1, FTLL1, GDF5, GNAS, GSS, HNF4A, JAG1, KCNQ2 、MKKS、NBIA1、PCK1、PI3、PPCD、PPGB、PRNP、THBD、TOP1、AIRE、APP、CBS、COL 6Al、COL6A2、CSTB、DCR、DSCR1、FPDMM、HLCS、HPE1、ITGB2、KCNE1、KNO、PRSS7 RUNX1, SOD1, TAM, ADSL, ARSA, BCR, CECR, CHEK2, COMT, CRYBB2, CSF2RB, CTHM, CYP2D6, CYP2D7P1, DGCR, DIA1, EWSR1, GGT1, MGCR, MN1, NAGA, NE2, OGS2PDGFB, PPARA, PRODH, SCO2, SCZD4, SERPIND1, SLC5AI, SOXI0, TCN2, TIMP3. TST, VCF, ABCD1, ACTL1, ADFN, AGMX2, AHDS, AIC, AIED, AIH3, ALAS2, AMCD, AM ELX、ANOP1、AR、ARAF1、ARSC2、ARSE、ARTS、ARX、ASAT、ASSP5、ATP7A、ATRX、A VPR2, BFLS, BGN, BTK, BZX, C1HR, CACNA1F, CALB3, CBBM, CCT, CDR1, CFNS, CGF 1, CHM, CHR39C, CIDX, CLA2, CLCN5, CLS, CMTX2, CMTX3, CND, COD1, COD2, COL 4A5, COL4A6, CPX, CVD1, CYBB, DCX, DFN2, DFN4, DFN6, DHOF, DIAPH2, DKC1, DM D, DSS, DYT3, EBM, EBP, ED1, ELK1, EMD, EVR2, F8, F9, FCP1, FDPSL5, FGD1, FG S1, FMR1, FMR2, G6PD, GABRA3, GATA1, GDI1, GDXY, GJB1, GK, GLA, GPC3, GRPR. GTD, GUST, HMS1, HPRT1, HPT, HTC2, HTR2C, HYR, IDS, IHG1, IL2RG, INDX, IP1 IP2, JMS, KAL1, KFSD, LICAM, LAMP2, MAA, MAFD2, MAOA, MAOB, MCF2, MCS, MEA X, MECP2, MF4, MGCI, MIC5, MID1, MLLT7, MLS, MRSD, MRX14, MRX1, MRX20, MRX 2, MRX3, MRX40, MRXA, MSD, MTMI, MYCL2, MYPI, NDP, NHS, NPHLI, NROBI, NSX, N YSI、NYX、OAI、OASD、OCRL、ODTI、OFD1、OPA2、OPD1、OPEM、OPN1LW、OPN1MW、O TC, P3, PDHA1, PDR, PFC, PFKFB1, PGK1, PGK1P1, PGS, PHEX, PHKA1, PHKA2, PHP PIGA, PLP1, POF1, POLA, POU3F4, PPMX, PRD, PRPSI, PRPS2, PRS, RCCP2, REN BP, RENS1, RP2, RP6, RPGR, RPS4X, RPS6KA3, RS1, S11, SDYS, SEDL, SERPINA7.SH2D1A, SHFM2, SLC25A5, SMAX2, SRPX, SRS, STS, SYN1, SYP, TAF1, TAZ, TBX22, TDD, TFE3, THAS, THC, TIMM8A, TIMP1, TKCR, TNFSF5, UBE1, UBE2A, WAS, WSN, WTS, WWS, XIC, XIST, XK, XM, XS, ZFX, ZIC3, ZNF261, ZNF41, ZNF6, AMELY, ASSP6, AZFI, AZF2, DAZ, GCY, RPS4Y, SMCY, SRY, ZFY, ABAT, AEZ, AFA, AFD1, ASAH1, ASD1, ASMT, CCAT, CECR9, CEPA, CLA3, CLN4, CSF2RA, CTSI, DF, DIH1, DWS, DYT2, DYT4, EBR3, ECT, EEF1A1L14, EYCL2, FANCB, GCSH, GCSL, GIP, GTS, HHG, HMI, HOAC, HOKPP2, HRPT1, HSD3B3, HTC1, HV1S, ICHQ, ICR1, ICR5, IL3RA, KAL2, KMS, KRT18, KSS, LCAT, LHON, LIMM, MANBB, MCPH2, MEB, MELAS, MIC2, MPFD, MS, MSS, MTATP6, MTCOI, MTCO3, MTCYB, MTND1, MTND2, MTND4, MTND5, MTND6, MTRNR1, MTRNR2, MTTE, MTTG, MTTI, MTTK, MTTL1, MTTL2, MTTN, MTTP, MTTS1, NAMSD, OCD1, OPD2, PCK2, PCLD, PCOS1, PFKM, PKD3, PRCA1, PRO1, PROP1, RBS, RFXAP, RP, SHOX, SLC25A6, SPG5B, STO, SUOX, THM, or TTD. Each recombinant protein represents an individual embodiment of the present invention.,

[0153] In another embodiment, the present invention provides a method for treating anemia in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule encoding erythropoietin, thereby treating anemia in the subject. In another embodiment, the in vitro synthesized RNA molecule further comprises pseudouridine or a modified nucleoside. Each possibility represents a separate embodiment of the present invention. In another embodiment, the cell is a subcutaneous tissue cell. In another embodiment, the cell is a lung cell. In another embodiment, the cell is a fibroblast. In another embodiment, the cell is a lymphoid cell. In another embodiment, the cell is a smooth muscle cell. In another embodiment, the cell is any other type of cell known in the art. Each possibility represents a separate embodiment of the present invention.

[0154] In another embodiment, the present invention provides a method for treating vasospasm in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule encoding inducible nitric oxide synthase (iNOS), thereby treating vasospasm in the subject.

[0155] In another embodiment, the present invention provides a method for improving the viability of a cell in a subject, comprising contacting the cell with an in vitro synthesized RNA molecule encoding a heat shock protein, thereby improving the viability of the cell in the subject.

[0156] In another embodiment, the cells with improved viability are ischemic cells. In another embodiment, the cells are not ischemic. In another embodiment, the cells are exposed to an ischemic environment. In another embodiment, the cells are exposed to environmental stress. Each possibility represents a separate embodiment of the present invention.

[0157] In another embodiment, the present invention provides a method for reducing the incidence of restenosis in a blood vessel after a blood vessel dilation procedure, comprising contacting cells of the blood vessel with an in vitro synthesized RNA molecule encoding a heat shock protein, thereby reducing the incidence of restenosis in the subject.

[0158] In another embodiment, the procedure is angioplasty. In another embodiment, the procedure is any other procedure known in the art for enlarging blood vessels. Each possibility represents a separate embodiment of the present invention.

[0159] In another embodiment, the present invention provides a method for increasing hair growth from hair follicles in the scalp of a subject, comprising contacting cells of the scalp with an in vitro synthesized RNA molecule encoding telomerase or an immunosuppressive protein, thereby increasing hair growth from the hair follicles.

[0160] In another embodiment, the immunosuppressive protein is α-melanocyte stimulating hormone (α-MSH). In another embodiment, the immunosuppressive protein is transforming growth factor-β1 (TGF-β1). In another embodiment, the immunosuppressive protein is insulin-like growth factor-I (IGF-I). In another embodiment, the immunosuppressive protein is any other immunosuppressive protein known in the art. Each possibility represents a separate embodiment of the present invention.

[0161] In another embodiment, the present invention provides a method for inducing expression of an enzyme having antioxidant activity in a cell, comprising contacting the cell with an in vitro synthesized RNA molecule encoding the enzyme having antioxidant activity, thereby inducing expression of the enzyme having antioxidant activity in the cell.

[0162] In one embodiment, the enzyme is catalase. In another embodiment, the enzyme is glutathione peroxidase. In another embodiment, the enzyme is phospholipid hydroperoxide glutathione peroxidase. In another embodiment, the enzyme is superoxide dismutase-1. In another embodiment, the enzyme is superoxide dismutase-2. In another embodiment, the enzyme is any other enzyme known in the art that has antioxidant activity. Each possibility represents a separate embodiment of the present invention.

[0163] In another embodiment, the invention provides a method for treating cystic fibrosis in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule encoding the cystic fibrosis transmembrane conductance regulator (CFTR), thereby treating cystic fibrosis in the subject.

[0164] In another embodiment, the invention provides a method for treating X-linked agammaglobulinemia in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule encoding Bruton's tyrosine kinase, thereby treating the X-linked agammaglobulinemia.

[0165] In another embodiment, the present invention provides a method for treating adenosine deaminase severe combined immunodeficiency (ADA SCID) in a subject, comprising contacting cells of the subject with an in vitro synthesized RNA molecule encoding adenosine deaminase, thereby treating the ADA SCID.

[0166] In another embodiment, the present invention provides a method for reducing immune responsiveness of the skin and ameliorating a skin condition, comprising contacting cells of a subject with an in vitro synthesized RNA molecule encoding an ecto-nucleoside triphosphate diphosphohydrolase, thereby reducing immune responsiveness of the skin and ameliorating a skin condition.

[0167] In another embodiment, the RNA molecules or ribonucleotide molecules of the invention are encapsulated in nanoparticles. Methods for nanoparticle encapsulation are well known in the art, and are described, for example, in Bose S, et al. (Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells. J. Virol. 78:8146. 2004), Dong Y et al. Poly(d,l-lactide-co-glycolide) / montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 26:6068. 2005), Lobenberg R. et al. (Improved body distribution of 14C-labeled AZT bound to nanoparticles in rats determined by radioluminography. J. Drug Target 5:171. 1998), and Sakuma SR et al. (Mucoadhesion of polystyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract. Int. J. Pharm 177:161. 1999), Virovic L et al. Novel delivery methods for treatment of viral hepatitis: an update. Expert Opin Drug Deliv 2:707. 2005), and Zimmermann E et al., Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions in artificial gastrointestinal media. Eur J Pharm Biopharm 52:203. 2001).Each method represents a separate embodiment of the present invention.

[0168] Various embodiments of the dosage range of the compounds of the present invention can be used in the methods of the present invention. In one embodiment, the dosage is in the range of 1 to 10 μg / day. In another embodiment, the dosage is 2 to 10 μg / day. In another embodiment, the dosage is 3 to 10 μg / day. In another embodiment, the dosage is 5 to 10 μg / day. In another embodiment, the dosage is 2 to 20 μg / day. In another embodiment, the dosage is 3 to 20 μg / day. In another embodiment, the dosage is 5 to 20 μg / day. In another embodiment, the dosage is 10 to 20 μg / day. In another embodiment, the dosage is 3 to 40 μg / day. In another embodiment, the dosage is 5 to 40 μg / day. In another embodiment, the dosage is 10 to 40 μg / day. In another embodiment, the dosage is 20 to 40 μg / day. In another embodiment, the pharmaceutical dose is 5 to 50 μg / day. In another embodiment, the pharmaceutical dose is 10 to 50 μg / day. In another embodiment, the pharmaceutical dose is 20 to 50 μg / day. In one embodiment, the pharmaceutical dose is 1 to 100 μg / day. In another embodiment, the pharmaceutical dose is 2 to 100 μg / day. In another embodiment, the pharmaceutical dose is 3 to 100 μg / day. In another embodiment, the pharmaceutical dose is 5 to 100 μg / day. In another embodiment, the pharmaceutical dose is 10 to 100 μg / day. In another embodiment, the pharmaceutical dose is 20 to 100 μg / day. In another embodiment, the pharmaceutical dose is 40 to 100 μg / day. In another embodiment, the pharmaceutical dose is 60 to 100 μg / day.

[0169] In another embodiment, the dosage is 0.1 μg / day. In another embodiment, the dosage is 0.2 μg / day. In another embodiment, the dosage is 0.3 μg / day. In another embodiment, the dosage is 0.5 μg / day. In another embodiment, the dosage is 1 μg / day. In another embodiment, the dosage is 2 mg / day. In another embodiment, the dosage is 3 μg / day. In another embodiment, the dosage is 5 μg / day. In another embodiment, the dosage is 10 μg / day. In another embodiment, the dosage is 15 μg / day. In another embodiment, the dosage is 20 μg / day. In another embodiment, the dosage is 30 μg / day. In another embodiment, the dosage is 40 μg / day. In another embodiment, the dosage is 60 μg / day. In another embodiment, the dosage is 80 μg / day. In another embodiment, the dosage is 100 μg / day.

[0170] In another embodiment, the dosage is 10 μg / dose. In another embodiment, the dosage is 20 μg / dose. In another embodiment, the dosage is 30 μg / dose. In another embodiment, the dosage is 40 μg / dose. In another embodiment, the dosage is 60 μg / dose. In another embodiment, the dosage is 80 μg / dose. In another embodiment, the dosage is 100 μg / dose. In another embodiment, the dosage is 150 μg / dose. In another embodiment, the dosage is 200 μg / dose. In another embodiment, the dosage is 300 μg / dose. In another embodiment, the dosage is 400 μg / dose. In another embodiment, the dosage is 600 μg / dose. In another embodiment, the dosage is 800 μg / dose. In another embodiment, the dosage is 1000 μg / dose. In another embodiment, the dosage is 1.5 mg / dose. In another embodiment, the dosage is 2 mg / dose. In another embodiment, the dosage is 3 mg / dose. In another embodiment, the dosage is 5 mg / dose. In another embodiment, the dosage is 10 mg / dose. In another embodiment, the dosage is 15 mg / dose. In another embodiment, the dosage is 20 mg / dose. In another embodiment, the dosage is 30 mg / dose. In another embodiment, the dosage is 50 mg / dose. In another embodiment, the dosage is 80 mg / dose. In another embodiment, the dosage is 100 mg / dose.

[0171] In another embodiment, the pharmaceutical dose is 10 to 20 μg / dose. In another embodiment, the pharmaceutical dose is 20 to 30 μg / dose. In another embodiment, the pharmaceutical dose is 20 to 40 μg / dose. In another embodiment, the pharmaceutical dose is 30 to 60 μg / dose. In another embodiment, the pharmaceutical dose is 40 to 80 μg / dose. In another embodiment, the pharmaceutical dose is 50 to 100 μg / dose. In another embodiment, the pharmaceutical dose is 50 to 150 μg / dose. In another embodiment, the pharmaceutical dose is 100 to 200 μg / dose. In another embodiment, the pharmaceutical dose is 200 to 300 μg / dose. In another embodiment, the pharmaceutical dose is 300 to 400 μg / dose. In another embodiment, the pharmaceutical dose is 400 to 600 μg / dose. In another embodiment, the pharmaceutical dose is 500 to 800 μg / dose. In another embodiment, the pharmaceutical dose is 800 to 1000 μg / dose. In another embodiment, the pharmaceutical dose is 1000 to 1500 μg / dose. In another embodiment, the pharmaceutical dose is 1500 to 2000 μg / dose. In another embodiment, the pharmaceutical dose is 2 to 3 mg / dose. In another embodiment, the pharmaceutical dose is 2 to 5 mg / dose. In another embodiment, the pharmaceutical dose is 2 to 10 mg / dose. In another embodiment, the pharmaceutical dose is 2 to 20 mg / dose. In another embodiment, the pharmaceutical dose is 2 to 30 mg / dose. In another embodiment, the pharmaceutical dose is 2 to 50 mg / dose. In another embodiment, the pharmaceutical dose is 2 to 80 mg / dose. In another embodiment, the pharmaceutical dose is 2 to 100 mg / dose. In another embodiment, the pharmaceutical dose is 3 to 10 mg / dose. In another embodiment, the pharmaceutical dose is 3 to 20 mg / dose. In another embodiment, the dosage is 3 to 30 mg / dose. In another embodiment, the dosage is 3 to 50 mg / dose. In another embodiment, the dosage is 3 to 80 mg / dose. In another embodiment, the dosage is 3 to 100 mg / dose. In another embodiment, the dosage is 5 to 10 mg / dose. In another embodiment, the dosage is 5 to 20 mg / dose. In another embodiment, the dosage is 5 to 30 mg / dose.In another embodiment, the pharmaceutical dose is 5 to 50 mg / dose. In another embodiment, the pharmaceutical dose is 5 to 80 mg / dose. In another embodiment, the pharmaceutical dose is 5 to 100 mg / dose. In another embodiment, the pharmaceutical dose is 10 to 20 mg / dose. In another embodiment, the pharmaceutical dose is 10 to 30 mg / dose. In another embodiment, the pharmaceutical dose is 10 to 50 mg / dose. In another embodiment, the pharmaceutical dose is 10 to 80 mg / dose. In another embodiment, the pharmaceutical dose is 10 to 100 mg / dose.

[0172] In another embodiment, the dosage is a daily dosage.In another embodiment, the dosage is a weekly dosage.In another embodiment, the dosage is a monthly dosage.In another embodiment, the dosage is a yearly dosage.In another embodiment, the dosage is one in a series of a specified number of dosages.In another embodiment, the dosage is a single dosage.As will be explained below, in another embodiment, the advantage of the RNA molecule, oligoribonucleotide molecule or polyribonucleotide molecule of the present invention is the greater efficacy of the molecule, which allows the use of smaller dosages.

[0173] In another embodiment, the present invention provides a method for producing a recombinant protein, comprising contacting an in vitro translation tool with an in vitro synthesized RNA molecule that includes a pseudouridine or modified nucleoside, thereby producing the recombinant protein.

[0174] In another embodiment, the present invention provides a method for producing a recombinant protein, comprising contacting an in vitro translation tool with an in vitro transcribed RNA molecule that includes a pseudouridine or modified nucleoside, thereby producing the recombinant protein.

[0175] In another embodiment, the present invention provides an in vitro transcription tool comprising unmodified nucleotides, nucleotides containing pseudouridine or modified nucleosides, and a polymerase. In another embodiment, the present invention provides an in vitro transcription kit comprising unmodified nucleotides, nucleotides containing pseudouridine or modified nucleosides, and a polymerase. Each possibility represents a separate embodiment of the present invention.

[0176] In another embodiment, the in vitro translation tool comprises a reticulocyte lysate. In another embodiment, the reticulocyte lysate is a rabbit reticulocyte lysate.

[0177] In another embodiment, the present invention provides a method for reducing the immunogenicity of an oligoribonucleotide molecule or an RNA molecule, comprising replacing a nucleotide of the oligoribonucleotide molecule or an RNA molecule with a modified nucleoside or a modified nucleotide containing a pseudouridine, thereby reducing the immunogenicity of the oligoribonucleotide molecule or the RNA molecule.

[0178] In another embodiment, the invention provides a method for reducing the immunogenicity of a gene therapy vector comprising a polyribonucleotide molecule or an RNA molecule, comprising the step of replacing nucleotides of the polyribonucleotide molecule or RNA molecule with modified nucleosides or modified nucleotides containing pseudouridine, thereby reducing the immunogenicity of the gene therapy vector.

[0179] In another embodiment, the invention provides a method for enhancing in vivo translation from a gene therapy vector comprising a polyribonucleotide molecule or an RNA molecule, comprising the step of replacing nucleotides of the polyribonucleotide molecule or RNA molecule with modified nucleosides or modified nucleotides containing pseudouridine, thereby enhancing in vivo translation from the gene therapy vector.

[0180] In another embodiment, the present invention provides a method for increasing the efficiency of delivery of a recombinant protein by a gene therapy vector comprising a polyribonucleotide molecule or an RNA molecule, comprising the step of replacing nucleotides of the polyribonucleotide molecule or RNA molecule with modified nucleosides or modified nucleotides containing pseudouridine, thereby increasing the efficiency of delivery of the recombinant protein by the gene therapy vector.

[0181] In another embodiment, the invention provides a method for increasing the in vivo stability of a gene therapy vector, comprising replacing nucleotides of a polyribonucleotide molecule or an RNA molecule with modified nucleosides or modified nucleotides containing pseudouridine, thereby increasing the in vivo stability of the gene therapy vector.

[0182] In another embodiment, the invention provides a method for synthesizing in vitro transcribed RNA molecules containing pseudouridine nucleosides, comprising contacting an isolated polymerase with a mixture of unmodified and modified nucleotides (Examples 5 and 10).

[0183] In another embodiment, the in vitro transcription method of the present invention utilizes an extract from an animal cell. In another embodiment, the extract is derived from a reticulocyte or a cell with similar efficiency of in vitro transcription. In another embodiment, the extract is derived from any other type of cell known in the art. Each possibility represents a separate embodiment of the present invention.

[0184] Any of the RNA molecules or oligoribonucleotide molecules of the present invention may, in alternative embodiments, be used in any of the methods of the present invention.

[0185] In another embodiment, the present invention provides a method for enhancing an immune response to an antigen, comprising administering the antigen in combination with mitochondrial (mt)RNA (Examples 4 and 8).

[0186] In another embodiment, the present invention provides a method for reducing the ability of an RNA molecule to stimulate dendritic cells (DCs), comprising modifying the nucleosides of the RNA molecule by a method of the present invention (see, e.g., Examples).

[0187] In another embodiment, the DC is a DC1 cell. In another embodiment, the DC is a DC2 cell. In another embodiment, the DC is a subtype of a DC1 cell or a DC2 cell. Each possibility represents a separate embodiment of the present invention.

[0188] In another embodiment, the present invention provides a method for reducing the ability of an RNA molecule to stimulate signaling by TLR3, comprising modifying the nucleosides of the RNA molecule by a method of the present invention. In another embodiment, the present invention provides a method for reducing the ability of an RNA molecule to stimulate signaling by TLR7, comprising modifying the nucleosides of the RNA molecule by a method of the present invention. In another embodiment, the present invention provides a method for reducing the ability of an RNA molecule to stimulate signaling by TLR8, comprising modifying the nucleosides of the RNA molecule by a method of the present invention. Each possibility represents a separate embodiment of the present invention.

[0189] In another embodiment, all of the internucleoside or internucleotide linkages in the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule are phosphodiester. In another embodiment, the internucleotide linkages are predominantly phosphodiester. In another embodiment, most of the internucleotide linkages are phosphorothioate. In another embodiment, most of the internucleotide linkages are phosphodiester. Each possibility represents a separate embodiment of the present invention.

[0190] In another embodiment, the percentage of internucleotide linkages in the RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule that are phosphodiester is greater than 50%. In another embodiment, the percentage is greater than 10%. In another embodiment, the percentage is greater than 15%. In another embodiment, the percentage is greater than 20%. In another embodiment, the percentage is greater than 25%. In another embodiment, the percentage is greater than 30%. In another embodiment, the percentage is greater than 35%. In another embodiment, the percentage is greater than 40%. In another embodiment, the percentage is greater than 45%. In another embodiment, the percentage is greater than 55%. In another embodiment, the percentage is greater than 60%. In another embodiment, the percentage is greater than 65%. In another embodiment, the percentage is greater than 70%. In another embodiment, the percentage is greater than 75%. In another embodiment, the percentage is greater than 80%. In another embodiment, the percentage is greater than 85%. In another embodiment, the percentage is greater than 90%. In another embodiment, the percentage is greater than 95%.

[0191] In another embodiment, the methods of the invention involve increasing the number, proportion, or frequency of modified nucleosides in an RNA molecule to reduce immunogenicity or increase translation efficiency. As provided herein (see, e.g., the Examples), the number of modified residues in an RNA molecule, oligoribonucleotide molecule, or polyribonucleotide molecule, in another embodiment, determines the extent of the effect observed in the invention.

[0192] In another embodiment, the invention provides a method for introducing a recombinant protein into a cell of a subject, comprising contacting a subject with an in vitro transcribed RNA molecule encoding the recombinant protein, the RNA molecule further comprising pseudouridine or another modified nucleoside, thereby introducing the recombinant protein into the cell of the subject.

[0193] In another embodiment, the invention provides a method for reducing TNF-α production in a subject in response to a gene therapy vector, comprising the step of engineering the gene therapy vector to contain pseudouridine or a modified nucleoside base, thereby reducing TNF-α production in the subject in response to the gene therapy vector.

[0194] In another embodiment, the invention provides a method for reducing IL-12 production in a subject in response to a gene therapy vector, comprising the step of engineering the gene therapy vector to contain pseudouridine or a modified nucleoside base, thereby reducing IL-12 production in the subject in response to the gene therapy vector.

[0195] In another embodiment, the present invention provides a method for reducing the immunogenicity of a gene therapy vector comprising introducing modified nucleosides into the gene therapy vector, thereby reducing the immunogenicity of the gene therapy vector.

[0196] As provided herein, the findings of the present invention show that primary DCs have additional RNA signaling entities that recognize m5C- and m6A-modified RNAs, and that their signaling is inhibited by modification of U residues.

[0197] In another embodiment, the advantage of the RNA molecules, oligoribonucleotide molecules, and polyribonucleotide molecules of the present invention is that (contrary to DNA-based vectors), RNA does not integrate into the genome.In another embodiment, the advantage is that RNA translation, and therefore the appearance of the encoded product, is instantaneous.In another embodiment, the advantage is that the amount of protein produced from mRNA can be adjusted by delivering more or less RNA.In another embodiment, the advantage is that repeated delivery of purified pseudouridine or other modified RNA molecules, oligoribonucleotide molecules, or polyribonucleotide molecules does not induce immune responses, while repeated delivery of unmodified RNA can induce signaling pathways through RNA sensors.

[0198] In another embodiment, the advantage is the lack of immunogenicity, allowing repeated delivery without the generation of inflammatory cytokines. In another embodiment, RNA stability is increased by cyclization, which reduces degradation by exonucleases.

[0199] In another embodiment, the present invention provides a method for treating a subject having a disease involving an immune response to a self-RNA molecule, comprising administering to the subject an antagonist of a TLR-3 molecule, thereby treating the subject having a disease involving an immune response to a self-RNA molecule.

[0200] In another embodiment, the present invention provides a method of treating a subject having a disease involving an immune response to a self-RNA molecule, comprising administering to the subject an antagonist of a TLR-7 molecule, thereby treating the subject having a disease involving an immune response to a self-RNA molecule.

[0201] In another embodiment, the present invention provides a method of treating a subject having a disease involving an immune response to a self-RNA molecule, comprising administering to the subject an antagonist of a TLR-8 molecule, thereby treating the subject having a disease involving an immune response to a self-RNA molecule.

[0202] In another embodiment, the present invention relates to the disease that comprises immune response to self-RNA molecules is autoimmune disease.In another embodiment, the disease is systemic lupus erythematosus (SLE).In another embodiment, the disease is another disease known in the art that comprises immune response to self-RNA molecules.Each possibility represents a separate embodiment of the present invention.

[0203] In another embodiment, the invention provides kits containing reagents for use in practicing the methods of the invention. In another embodiment, the invention provides kits containing the compositions, tools, or instruments of the invention.

[0204] In another embodiment, the present invention provides kits for measuring or studying TLR-3, TLR-7, and signaling by the TLR-7 receptor, as exemplified in Example 7.

[0205] In another embodiment, the treatment protocol of the present invention is therapeutic. In another embodiment, the protocol is prophylactic. Each possibility represents a separate embodiment of the present invention.

[0206] In one embodiment, the phrase "contacting a cell" or "contacting a population" refers to a method of exposure that can be direct or indirect. In one method, such contacting includes direct injection of the cells through any means known in the art, such as microinjection. In another embodiment, the cells are supplied indirectly, such as through provision in the medium surrounding the cells, through administration to a subject, or through any route known in the art. In another embodiment, the term "contacting" means that a molecule of the present invention is introduced into a subject undergoing treatment, and the molecule is allowed to contact with cells in vivo. Each possibility represents a separate embodiment of the present invention.

[0207] Methods for quantifying reticulocyte frequency and for measuring EPO bioactivity are well known in the art and are described, for example, in Ramos, AS et al. (Biological evaluation of recombinant human erythropoietin in pharmaceutical products. Braz J Med Biol Res 36:1561). Each method represents a separate embodiment of the present invention.

[0208] In another embodiment, the compositions of the present invention can be administered to a subject by any method known to one of skill in the art, such as parenterally, intramuscularly, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intracerebroventricularly, intracranially, intravaginally, or intratumorally.

[0209] In another embodiment of the method and composition of the present invention, the composition is administered orally, and therefore is formulated in a form suitable for oral administration, i.e., as a solid or liquid preparation.Suitable solid oral preparations include tablets, capsules, pills, granules, pellets, and the like.Suitable liquid oral preparations include solutions, suspensions, dispersions, emulsions, oils, and the like.In another embodiment of the present invention, the active ingredient is formulated in a capsule.According to this embodiment, the composition of the present invention comprises a hard gelatinized capsule in addition to the active compound and an inert carrier or inert diluent.

[0210] In another embodiment, the pharmaceutical composition is administered by intravenous, intraarterial or intramuscular injection of a liquid preparation.Suitable liquid preparations include solutions, suspensions, dispersions, emulsions, oils, and the like.In another embodiment, the pharmaceutical composition is administered intravenously, and therefore is formulated in a form suitable for intravenous administration.In another embodiment, the pharmaceutical composition is administered intraarterially, and therefore is formulated in a form suitable for intraarterial administration.In another embodiment, the pharmaceutical composition is administered intramuscularly, and therefore is formulated in a form suitable for intramuscular administration.

[0211] In another embodiment, the pharmaceutical composition is administered topically to the body surface, and therefore is formulated in a form suitable for topical administration.Suitable topical formulations include gels, ointments, creams, lotions, drops, and the like.For topical administration, the composition or its physiologically tolerable derivatives are prepared and applied as a solution, suspension, or emulsion in a physiologically acceptable diluent, with or without a pharmaceutical carrier.

[0212] In another embodiment, the composition is administered as a suppository, such as a rectal suppository or a urethral suppository. In another embodiment, the pharmaceutical composition is administered by subcutaneous implantation of a pellet. In another embodiment, the pellet provides for sustained release of the drug over time.

[0213] In another embodiment, the active compound is delivered in a vesicle, such as a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0214] As used herein, "pharmaceutically acceptable carriers or diluents" are well known to those skilled in the art. In various embodiments, the carrier or diluent can be a solid carrier or diluent for a solid formulation, a liquid carrier or diluent for a liquid formulation, or a mixture thereof.

[0215] In another embodiment, solid carriers / diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0216] In other embodiments, the pharmaceutically acceptable carrier for liquid formulations can be aqueous or non-aqueous solution, suspension, emulsion, or oil.Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and ethyl oleate and other suitable organic esters.Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media.Examples of oils include oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish liver oil.

[0217] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of surfactants and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose, and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[0218] In another embodiment, the composition further comprises binders (e.g., acacia, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone), disintegrants (e.g., corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers of various pH and ionic strength (e.g., Tris-HCl, acetate, phosphate), additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), penetration enhancers, solubilizers (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose), thickeners (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow acids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film-forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates), and / or adjuvants. Each of the foregoing excipients represents a separate embodiment of the present invention.

[0219] In another embodiment, the pharmaceutical compositions provided herein are sustained-release compositions, i.e., compositions in which the compound is released over time after administration of 15. Slow-release or sustained-release compositions include formulations in lipophilic depots (e.g., fatty acids, waxes, oils). In another embodiment, the compositions are immediate-release compositions, i.e., compositions in which the entire compound is released immediately after administration.

[0220] In another embodiment, the molecules of the present invention are modified by the covalent attachment of a water-soluble polymer, such as polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline. Modified compounds are known to exhibit substantially longer serum half-lives after intravenous injection than the corresponding unmodified compounds (Abuchowski et al., 1981, Newmark et al., 1982, and Katre et al., 1987). In another embodiment, such modifications also increase the solubility of the compound in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and significantly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity can be achieved by administering such polymer-compound abducts less frequently or in smaller doses than the unmodified compound.

[0221] In another embodiment, the active ingredient is formulated into the composition as a neutralized pharmaceutically acceptable salt form.Pharmaceutical acceptable salts include, for example, acid addition salts (for example, formed with the free amino group of polypeptide or antibody molecule) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid and their analogues.In addition, the salt formed from free carboxyl group can be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

[0222] Each of the above additives, excipients, formulations, and methods of administration represents a separate embodiment of the present invention. [Example]

[0223] Unless otherwise stated, the following experimental protocols were employed in the examples provided below.

[0224] Materials and Methods for Examples 1-3 Cell culture.

[0225] Human neonatal foreskin fibroblast 1079 cells (Cat. No. CRL-2097, ATCC, Manassas, VA) and human IMR90 cells (Cat. No. CCL-186, ATCC) were cultured in Advanced MEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT), 2 mM Glutamax (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma, St. Louis, MO), and penicillin / streptomycin (Invitrogen). All cells were grown at 37°C and 5% CO. In some experiments, human iPS cells derived using the methods described herein were maintained in irradiated mouse embryonic fibroblasts (MEFs) (R&D Systems, Minneapolis, MN) in 10 cm plates pre-coated with 0.1% gelatin (Millipore, Phillipsburg, NJ) in DMEM / F12 medium supplemented with 20% knockout serum replacement, 0.1 mM L-glutamine (all from Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), and 100 ng / mL basic fibroblast growth factor (Invitrogen). In some experiments, human iPS cells derived using the methods described herein were maintained in MEF-conditioned medium collected as previously described (Xu et al. 2001).

[0226] Vector construction. cDNAs for the open reading frames (ORFs) of KLF4, LIN28, NANOG, and OCT4 were PCR-amplified from cDNA clones (Open Biosystems, Huntsville, AL) and cloned into plasmid vectors downstream of a T7 RNA polymerase promoter (Mackie 1988, Studier and Moffatt 1986) (e.g., various pBluescript™ vectors, Agilent, La Jolla, CA, or pGEM™ vectors, Promega, Madison, WI) and sequenced. The SOX2 ORF was PCR-amplified from a cDNA clone (Invitrogen), and the c-MYC ORF was isolated from HeLa cell total RNA by RT-PCR. Both the SOX2 and c-MYC ORFs were also cloned into plasmid vectors downstream of a T7 RNA polymerase promoter and sequenced.

[0227] Alternative plasmid vectors containing the human open reading frames (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) were cloned into pBluescriptII. These pBluescriptII vectors were constructed by ligating the preceding open reading frames into the EcoRV (cMyc) or EcoRV / SpeI (KLF4, LIN28, NANOG, OCT4, and SOX2) sites between the described 5' and 3' Xenopus β-globin untranslated regions (Krieg and Melton 1984).

[0228] mRNA production. Plasmid constructs containing the T7 RNA polymerase promoter (pT7-KLF4, pT7-LIN28, pT7-c-MYC, pT7-OCT4, pT7-SOX2, or pT7-XBg-KLF4, pT7-XBg-LIN28, pT7-XBg-c-MYC, pT7-XBg-OCT4, and pT7-XBg-SOX2) were linearized with BamHI, and pT7-NANOG and pT7-XBg-NANOG were linearized with XbaI. The mSCRIPT™ mRNA production system (EPICENTRE or CellScript, Madison, WI, USA) was used to produce mRNA with a 5′ Cap 1 structure and a 3′ poly(A) tail (e.g., with approximately 150 A residues), with the exception that pseudouridine-5′-triphosphate (TRILINK, San Diego, CA) was used instead of uridine-5′-triphosphate in the T7 RNA polymerase in vitro transcription reaction.

[0229] Purification and Analysis of mRNA. In some experimental embodiments, mRNA was purified by HPLC, column fractions were collected, and the mRNA fractions were analyzed for immunogenicity as described in and / or as described and shown in Figures 22-24. In some experimental embodiments, purified RNA preparations comprising or consisting of mRNA encoding one or more reprogramming factors that exhibit little or no immunogenicity were used in experiments to reprogram human somatic cells into iPS cells.

[0230] Reprogramming of human somatic cells in MEFs.

[0231] 1079 fibroblasts were cultured at 1 × 10 in 6-well dishes pre-coated with 0.1% gelatin (Millipore). 5Cells were seeded at 1079 cells / well and grown overnight. 1079 fibroblasts were transfected with equal amounts of each reprogramming factor mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) using TransIT mRNA transfection reagent (MirusBio, Madison, WI). A total of three transfections were performed, one transfection every other day, and medium was changed the day after the first and second transfections. The day after the third transfection, cells were trypsinized and 3.3 × 10 5 The day before, 7.5 x 10 5 MEFs were seeded in 1079 medium onto 10 cm plates pre-coated with 0.1% gelatin. The day after transfected 1079 fibroblasts were seeded onto MEFs, the medium was replaced with iPS cell medium. iPS cell medium was replaced daily. Eight days after transfected cells were seeded onto MEFs, MEF-conditioned medium was used. MEF-conditioned medium was collected as previously described (Xu et al. 2001). Plates were screened daily for the presence of colonies with iPS morphology using an inverted microscope.

[0232] An alternative protocol for reprogramming 1079 and IMR90 fibroblasts on MEFs was also used. MEFs were grown in 1.25 x 10 cells per well in 6-well dishes pre-coated with 0.1% gelatin. 5 1079 or IMR90 fibroblasts were seeded at 3 × 10 cells / well in the 6-well dishes seeded with MEFs the day before and incubated overnight in complete fibroblast medium. 4Cells were seeded at 100 cells / well and grown overnight at 37°C / 5% CO2. Next, capped / polyadenylated mRNAs were generated from the following vectors using the mScript Kit (pT7-Xβg-KLF4, pT7-Xβg-LIN28, pT7-Xβg-c-MYC, pT7-Xβg-NANOG, pT7-Xβg-OCT4, and pT7-Xβg-SOX2) and used in these daily transfections. All six reprogramming mRNAs were diluted to 100 ng / µL of each mRNA. Equimolar concentrations of each mRNA were added to each other using the following conversion factors (OCT4 was set to 1, and all other mRNAs were multiplied by the following conversion factors to obtain equimolar concentrations in each mRNA mixture): KLF = 1.32, LIN28 = 0.58, c-MYC = 1.26, NANOG = 0.85, OCT4 = 1, and SOX2 = 0.88. To obtain equimolar amounts of each factor, 132 μL of KLF4, 58 μL of LIN28, 126 μL of c-MYC, 85 μL of NANOG, 100 μL of OCT4, and 88 μL of SOX2 mRNA (100 ng / μL each) would be added to each other. A total dose of 600 μg for transfection would mean using 100 ng of each of the six reprogramming mRNAs (using the molar concentration conversion above). These mRNA doses were transfected using Trans-IT mRNA transfection reagent. For all transfections, the mRNA pool was added to 250 μL of either DMEM / F12 medium without supplements or Advanced MEM medium without supplements. Five microliters of mRNA stimulation reagent and five microliters of TransIT transfection reagent were added to each tube. After incubation at room temperature for 2 minutes, the transfection mixture was added to 2.5 mL of either Advanced MEM medium with 10% FBS and 100 ng / mL hFGFb or iPS medium containing 100 ng / mL hFGFb. Transfections were repeated daily for 10 to 16 days. Medium was changed 4 hours after each transfection. In some embodiments, 5 to 8 days after the initial transfection, cells were trypsinized and replated onto fresh MEF plates.1079 cells were split 1 / 6 or 1 / 12 onto fresh MEF plates, whereas IMR90 cells were split 1 / 3 or 1 / 6 onto fresh MEF plates.

[0233] Reprogramming of human somatic cells in MEF-conditioned medium. 1079 or IMR90 fibroblasts were cultured at 3 × 10 per 10 cm dish (Millipore) pre-coated with 0.1% gelatin. 5 Cells were seeded at 1000 x 1000 cells per well and grown overnight. 1079 or IMR90 fibroblasts were transfected with equal amounts of reprogramming factor mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) using TransIT mRNA transfection reagent (MirusBio, Madison, WI). For each transfection, 6 μg, 18 μg, or 36 μg of each reprogramming mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) was used per 10 cm dish. A total of three transfections were performed, one transfection per day, and medium was changed the day after the first and second transfections. All transfections were performed in MEF-conditioned medium. The day after the third transfection, cells were trypsinized and seeded onto fresh 10 cm dishes (Millipore) pre-coated with 0.1% gelatin. Cells were grown in MEF-conditioned medium for the duration of the experiment.

[0234] Similar daily mRNA transfections were also performed as described in the previous paper, with the only difference being that MEFs were not used as a feeder layer, but only MEF-conditioned medium was used.

[0235] Immunofluorescence. 1079 cells or 1079-derived iPS cells were washed with PBS and fixed in 4% paraformaldehyde in PBS for 30 minutes at room temperature. The iPS cells were then washed three times with PBS for 5 minutes each, followed by three washes with PBS + 0.1% Triton X-100. The iPS cells were then blocked in blocking buffer (PBS + 0.1% Triton, 2% FBS, and 1% BSA) for 1 hour at room temperature. Cells were then incubated with primary antibodies (mouse anti-human OCT4 Catalog No. sc-5279, Santa Cruz Biotechnology, Santa Cruz, CA; rabbit anti-human NANOG Catalog No. 3580; rabbit anti-human KLF4 Catalog No. 4038; mouse anti-human LIN28 Catalog No. 5930; rabbit anti-human c-MYC Catalog No. 5605; rabbit anti-human SOX2 Catalog No. 3579; and mouse anti-TRA-1-60, all from Cell Signaling Technology, Beverly, MA) at a 1:500 dilution in blocking buffer for 2 hours at room temperature. After washing five times with PBS + 0.1% Triton X-100, iPS cells were incubated for 2 hours with anti-rabbit Alexa Fluor 488 antibody (catalog no. 4412, Cell Signaling Technology), anti-mouse FITC secondary antibody (catalog no. F5262, Sigma), or anti-mouse Alexa Fluor 555 (catalog no. 4409, Cell Signaling Technology) at a 1:1000 dilution in blocking buffer. Images were taken on a Nikon TS100F inverted microscope (Nikon, Tokyo, Japan) equipped with a 2-megapixel monochrome digital camera (Nikon) using NIS-elements software (Nikon).

[0236] Example 1 This example describes studies to determine whether transfection with mRNAs encoding KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2 resulted in the expression and appropriate subcellular localization of each individual protein product in neonatal foreskin 1079 fibroblasts. The mRNA used in this experiment was prepared with pseudouridine-5'-triphosphate, replacing uridine-5'-triphosphate (Kariko et al. 2008). 1079 fibroblasts were transfected with 4 μg of each mRNA per well of a 6-well dish, and immunofluorescence analysis was performed 24 h posttransfection. Endogenous KLF4, LIN28, NANOG, OCT4, and SOX2 protein levels were undetectable by immunofluorescence in untransfected 1079 cells (Figure 1B, F, N, R, V). Endogenous levels of c-MYC were relatively high in untransfected 1079 cells (Fig. 1J). Transfection of mRNAs encoding the transcription factors KLF4, c-MYC, NANOG, OCT4, and SOX2 all resulted in predominantly nuclear localization of the respective proteins 24 hours after mRNA transfection (Fig. 1D, L, P, T, and X). The cytoplasmic mRNA-binding protein LIN28 was localized to the cytoplasm (Fig. 1H).

[0237] Example 2 Having demonstrated efficient mRNA transfection and appropriate subcellular localization of reprogramming proteins, this example describes the development of a protocol for generating iPS cells from somatic fibroblasts. Equal amounts (by weight) of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2 mRNA were transfected into 1079 fibroblasts three times (once every other day). The day after the third transfection, cells were plated onto irradiated MEF feeder cells and grown in iPS cell medium. Six days after plating 1079 fibroblasts onto irradiated MEFs, two putative iPS cell colonies were generated by transfecting 1079 fibroblasts with 3 μg of each reprogramming factor mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2). SOX2) were evident in 10 cm plates transfected with iPS cell colonies. Colonies were allowed to grow for 12 days after the final transfection, after which they were fixed for immunofluorescence analysis. The inner cell mass-specific marker NANOG is often used to assay whether iPS cell colonies are truly iPS cell colonies (Gonzalez et al. 2009, Huangfu et al. 2008). NANOG expression resulting from mRNA transfection 12 days earlier would be negligible based on previous reports regarding mRNA stability and duration of expression (Kariko et al. 2008). Staining for NANOG showed that both iPS cell colonies were NANOG-positive (Figure 2B, D, and not shown). Surrounding fibroblasts not part of the iPS cell colonies were NANOG-negative, suggesting that the fibroblasts were not reprogrammed into iPS cells.

[0238] In subsequent experiments using the same protocol, both 1079 and human IMR90 fibroblasts were transfected with the same reprogramming mRNAs. Multiple colonies were detected as early as 4 days after seeding the transfected cells onto irradiated MEFs. When 6 μg of each mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) was used in transfections in 6-well dishes, three putative iPS cell colonies were subsequently detected in both cell lines after seeding onto MEFs in 10 cm plates (Figure 3). In addition to analyzing these colonies for expression of NANOG, TRA-1-60 (Chan et al. 2009), a more stringent marker of fully reprogrammed iPS cells, was also used for immunofluorescence analysis. iPS colonies arising from 1079 fibroblasts (Figure 3A-F) and IMR90 fibroblasts (Figure 3G-I) were positive for both NANOG and TRA-1-60, indicating that these colonies were fully reprogrammed type III iPS cell colonies. Our protocol, involving transfection of mRNA encoding all six reprogramming factors and seeding onto MEF feeder cells, resulted in a similar reprogramming efficiency (1 × 10), as previously reported by a protocol involving delivery of the same reprogramming factors by transfection of expression plasmids (Aoi et al. 2008). 6 The resulting iPS cells were cultured in a 200-well plate (3-6 iPS colonies per 1000 cells).

[0239] Example 3 This example describes attempts to improve the efficiency of reprogramming differentiated cells using mRNA. In one approach, we used a protocol involving transfecting 1079 or IMR90 fibroblasts three times (once every other day) with mRNA encoding six reprogramming factors in MEF-conditioned medium rather than in fibroblast medium, and then growing the treated 1079 fibroblasts in MEF-conditioned medium after treatment rather than plating them on MEF feeder layers. At the highest transfection dose utilized (36 μg of each reprogramming factor per 10 cm dish), 208 iPS cell colonies were detected 3 days after the final transfection (Figures A–F). Interestingly, no iPS cell colonies were detected in dishes transfected with either 6 μg or 18 μg of each reprogramming factor at day 3, suggesting that doses above 18 μg are critical for iPS cell colony formation to occur within 3 days in MEF-conditioned medium under these conditions. IMR90 cells displayed even greater numbers of iPS cell colonies, with approximately 200 colonies 8 days after the final transfection in plates transfected with three 6 μg doses of each of the six reprogramming factor mRNAs and over 1,000 colonies in IMR90 cells transfected with three 18 μg or 36 μg doses of each of the six reprogramming factor mRNAs (Figure 4, G-I). Colonies were visible 3 days after the final transfection in 1079 cells, whereas colonies became visible 6 to 7 days after the final transfection in IMR90 cells. Thus, more mature colonies derived from 1079 cells were larger and denser, and were darker in bright-field images compared to IMR90 colonies (Figure 4). All colonies on 1079 plates transfected three times with 36 μg of each reprogramming mRNA were positive for both NANOG and TRA-1-60 8 days after the final mRNA transfection (Fig. 5A-I).All of the more immature IMR90 iPS colonies were also positive for both NANOG and TRA-1-60 (Fig. 5J-O), but showed less robust staining for both markers due to the less dense cellular nature of the more immature IMR90 iPS colonies compared to the more mature 1079 colonies (Fig. 5A-I). The present protocol, involving delivery of the mRNA to 1079 or IMR90 cells in MEF-conditioned medium, yielded 3 x 10 5 The present protocol for deriving iPS cells was faster and approximately 2-3 orders of magnitude more efficient than a published protocol involving transfecting fibroblasts with DNA plasmids encoding these same six reprogramming factors in fibroblast medium (Aoi et al. 2008). Furthermore, the present protocol produced 6 x 10 iPS cells. 5 This protocol is 7-40 times more efficient than a published protocol involving lentiviral delivery of reprogramming factors into 1079 neonatal fibroblasts (Aoi et al. 2008), based on published data on lentiviral delivery of reprogramming factors, resulting in approximately 57 iPS cell colonies per input cell. This protocol is also significantly faster than the published method.

[0240] Example 4: Natural RNA molecules exhibit differentiation potential to activate dendritic cells: Materials and Experimental Methods (Plasmids and Reagents) Plasmids pT7T3D-MART-1 and pUNO-hTLR3 were obtained from ATCC (Manassas, VA) and InvivoGen (San Diego, CA), respectively. pTEVluc was obtained from Dr. Daniel Gallie (UC Riverside) and contains pT7-TEV (tobacco etch virus genomic RNA leader sequence)-luciferase-A50, as described in Gallie, DR et al., 1995. The tobacco etch viral 5' leader and poly(A) tail are functionally synergistic regulators of translation. Gene 165:233. pSVren was derived from p2luc (Grentzmann G, Ingram JA, et al., A dual-luciferase reporter system for studying recoding signals. RNA 1998;4(4):479-86) by removal of the firefly luciferase coding sequence by BamHI and NotI digestion, end-filling, and religation.

[0241] A synthetic ODN encoding a 20-nt-long shRNA homologous to human TLR3 (nt 703-722, accession number: NM_003265) was first cloned into the plasmid pSilencer 4.1-CMV-neo (Ambion, Austin, TX), followed by NheI-HindIII digestion and subcloning into the corresponding sites of pcDNA3.1 (Invitrogen) to obtain the human TLR3-specific siRNA, pTLR3-sh. LPS (Escherichia coli 055:B5) was obtained from Sigma Chemical Co., St. Louis, MO. CpG ODN2006 and R-848 were obtained from InvivoGen.

[0242] (Cells and cell cultures) Human embryonic kidney 293 cells (ATCC) were grown in DMEM supplemented with glutamine (Invitrogen) and 10% FCS (Hyclone, Ogden, UT) (complete medium). In all cases herein, "293 cells" refers to human embryonic kidney (HEK) 293 cells. The 293-hTLR3 cell line was generated by transforming 293 cells with pUNO-hTLR3. Cell lines 293-hTLR7, 293-hTLR8, and 293-hTLR9 (InvivoGen) were grown in complete medium supplemented with blasticidin (10 μg / ml) (Invivogen). The cell lines 293-ELAM-Iuc and TLR7-293 (M. Lamphier, Eisai Research Institute, Andover MA), and TLR3-293 cells were cultured as described (Kariko et al., 2004, mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem 279: 12542-12550). Cell lines 293, 293-hTLR7, and 293-hTLR8 were stably transfected with pTLR3-sh and selected with G-418 (400 μg / ml) (Invitrogen). Neo-resistant colonies were screened, and only those that did not express TLR3, as determined by the lack of IL-8 secretion in response to poly(I):(C), were used in further studies. Leukopheresis samples were obtained from HIV-uninfected volunteers through an IRB-approved protocol.

[0243] (Mouse DC production) Murine DCs were generated by harvesting bone marrow cells from the tibia and femur of 6- to 8-week-old C57BL / 6 mice and lysing the red blood cells. The cells were cultured at 10x in 2 mL of DMEM + 10% FVS and 20 ng / mL mu GM-CSF (R&D Systems). 6muDCs were seeded at 1000 cells / well in a 6-well plate. After 3 days, 2 mL of fresh medium containing muGM-CSF was added. After 6 days, 2 mL of medium / well was removed, and the cells were pelleted and resuspended in fresh medium containing muGM-CSF. After 7 days of culture, muDCs were harvested and washed.

[0244] (natural RNA) Mitochondria were isolated from platelets obtained from the University of Pennsylvania Blood Bank using a fractional lysis procedure (Mitochondria Isolation Kit; Pierce, Rockford, IL). RNA was isolated from purified mitochondrial, cytoplasmic, and nuclear fractions of 293 cells, unfractionated 293 cells, rat liver, the mouse cell line TUBO, and the DH5α strain of E. coli using Master Blaster® (BioRad, Hercules, CA). Bovine tRNA, wheat tRNA, yeast tRNA, E. coli tRNA, poly(A)+ mRNA from mouse heart, and poly(I):(C) were purchased from Sigma, and total RNA from human spleen and E. coli RNA were purchased from Ambion. Oligoribonucleotide-5'-monophosphate was chemically synthesized (Dharmacon, Lafayette, CO).

[0245] Aliquots of RNA samples were incubated in the presence of Benzonase nuclease (1 microgram per microliter (μg / μL) at 1 U per 5 μL of RNA for 1 hour). Aliquots of RNA-730 were digested with alkaline phosphatase (New England Biolabs). For quality assurance, RNA samples were analyzed by denaturing agarose electrophoresis or polyacrylamide gel electrophoresis. Assays for LPS in RNA preparations using the Limulus Amebocyte Lysate gel mass assay were negative with a sensitivity of 3 picograms per milliliter (pg / mL) (University of Pennsylvania, Core Facility).

[0246] (HPLC analysis) Nucleoside monophosphates were separated and visualized via HPLC. To release free nucleoside 3'-monophosphates, a 5 μg aliquot of RNA was purified by HPLC with 0.1 U of RNase A in 10 μL of 50 mM NaOAc and 2 mM EDTA buffer (pH 4.5). After overnight digestion with T2 (Invitrogen), the sample was injected onto an Agilent 1100 HPLC using a Waters Symmetry C18 column (Waters, Milford, MA). A gradient from 100% Buffer A (30 mM KH2P04 and 10 mM tetraethylammonium phosphate [PicA Reagent, Waters], pH 6.0) to 30% Buffer B (acetonitrile) was run over 60 min at a flow rate of 1 mL / min. Nucleotides were detected at 254 nm using a photodiode array. Their identity was authenticated by retention time and spectrum.

[0247] (Dendritic cell assay) Dendritic cells (approximately 1.1 x 10) in a 96-well plate 5 Cells (cells / well) were treated with R-848, Lipofectin®, or Lipofectin®-RNA for 1 hour, followed by a medium change. At the end of 8 hours (unless otherwise noted), cells were harvested for either RNA isolation or flow cytometry, while collected medium was subjected to cytokine ELISA. IL-12(p70) (BD Biosciences Pharmingen, San Diego, CA), IFN-α, TNF-α, and IL-8 (Biosource International, Camarillo, CA) were measured in the supernatants by sandwich ELISA. Cultures were performed in triplicate or quadruplicate and measured in duplicate.

[0248] (Northern blot analysis) RNA was isolated from MDDCs after 8 hours of incubation following treatment, as previously described. Where applicable, cells were treated with 2.5 μg / mL cycloheximide (Sigma) 30 minutes prior to stimulation and throughout the entire length of incubation. RNA samples were processed and analyzed in Northern blots as described (Kariko et al. 2004, ibid.) using human TNF-α and GAPDH probes derived from plasmids obtained from ATCC (pE4 and pHcGAP, respectively).

[0249] (result) To determine the immunostimulatory potential of different cellular RNA subtypes, RNA was isolated from different subcellular compartments: cytoplasm, nucleus, and mitochondria. These RNA fractions, as well as total RNA, tRNA, and poly(A)-tail-selected mRNAs, all derived from mammalian sources, were complexed with Lipofectin® and added to MDDCs. Mammalian total RNA, nuclear RNA, and cytoplasmic RNA all stimulated MDDCs, as evidenced by detectable TNF-α secretion, although the TNF-α levels were significantly lower than those induced by in vitro-synthesized mRNA (Figure 6). Furthermore, mammalian tRNA did not induce any detectable levels of TNF-α, whereas mitochondrial (mt)RNA induced significantly more TNF-α than other mammalian RNA subtypes. Bacterial total RNA was also a potent activator of MDDCs; in contrast, bacterial tRNA induced only low levels of TNF-α. tRNA from other sources (yeast, wheat embryo, bovine) was nonstimulatory. Similar results were observed when RNA from other mammalian sources was tested. When RNA samples were digested with Benzonase, which cleaves single- and double-stranded RNA, RNA signaling was abolished in MDDCs, verifying that TNF-α secretion was due to RNA in the preparation. The activation potential of the tested RNA types correlated negatively with the degree of nucleoside modification. Similar results were obtained in the experiments described in this example for both types of cytokine-producing DCs.

[0250] These findings indicate that the immunogenicity of RNA is influenced by the degree of nucleoside modification, with greater degrees of modification tending to decrease immunogenicity.

[0251] Example 5 In Vitro Synthesis of RNA Molecules Using Modified Nucleosides: Materials and Experimental Methods (in vitro transcribed RNA) Using an in vitro transcription assay (MessageMachine kit and MegaScript kit; Ambion), the following long RNAs were generated by T7 RNA polymerase (RNAP) as described (Kariko et al., 1998, Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA. Biochim Biophys Acta 1369, 320-334). (Note: Template names are shown in parentheses, and numbers in the RNA names designate length.) RNA-1866 (pTEVluc linearized with NdeI) encodes firefly luciferase and is a 50-nt poly(A) tail. RNA-1571 (pSVren linearized with SspI) encodes Renilla luciferase. RNA-730 (pT7T3D-MART-l linearized with HindIII) encodes the human melanoma antigen MART-I. RNA-713 (pTIT3D-MART-1 linearized with EcoR I) corresponds to the antisense sequence of MART-1, and RNA497 (pCMV-hTLR3 linearized with Bgl II) encodes a partial 5' fragment of hTLR3. The sequences of the RNA molecules are as follows: RNA-I866:

[0252] RNA-1571:

[0253] RNA-730: Gggaauuuggcccucgaggccaagaauucggcacgaggcacgcggccagccagcagacagaggacucucauuaaggaagguguccugugcccugacccuacaagaugccaagagaagaugcucacuucaucuaugguuaccccaagaaggggcacggccacucuuacaccacggcugaagaggccgcugggaucggcauccugacagugauccugggagucuuacugcucaucggcuguugguauuguagaagacgaaauggauacagagccuugauggauaaaagucuucauguuggcacucaaugugccuuaacaagaagaugcccacaagaaggguuugaucaucgggacagcaaagugucucuucaagagaaaaacugugaaccugugguucccaaugcuccaccugcuuaugagaaacucucugcagaacagucaccaccaccuuauucaccuuaagagccagcgagacaccugagacaugcugaaauuauuucucucacacuuuugcuugaauuuaauacagacaucuaauguucuccuuuggaaugguguaggaaaaaugcaagccaucucuaauaauaagucaguguuaaaauuuuaguagguccgcuagcaguacuaaucaugugaggaaaugaugagaaauauuaaauugggaaaacuccaucaauaaauguugcaaugcaugauaaaaaaaaaaaaaaaaaaaacugcggccgca (SEQ ID NO: 3).

[0254] RNA-713 Gggaauaagcuugcggccgcaguuuuuuuuuuuuuuuuuuuuaucaugcauugcaacauuuauugauggaguuuucccaauuuaauauuucucaucauuuccucacaugauuaguacugcuagcggaccuacuaaaauuuuaacacugacuuauuauuagagauggcuugcauuuuuccuacaccauuccaaaggagaacauuagaugucuguauaaauucaagcaaaagugugagagaaauaauuucagcaugucucaggugucucgcuggcucuuaaggugaauaaggugguggugacuguucugcagagaguuucucauaagcagguggagcauugggaaccacagguucacaguuuuucucuugaagagacacuuugcugucccgaugaucaaacccuucuugugggcaucuucuuguuaaggcacauugagugccaacaugaagacuuuuauccaucaaggcucuguauccauuucgucuucuacaauaccaacagccgaugagcaguaagacucccaggaucacugucaggaugccgaucccagcggccucuucagccgugguguaagaguggccgugccccuucuugggguaaccauagaugaagugagcaucuucucuuggcaucuuguagggucagggcacaggacaccuuccuuaaugagaguccucugucugcuggcuggccgcgugccucgugccgaauu(SEQ ID NO: 4).

[0255] RNA-497: Gggagacccaagcuggcuagcagucauccaacagaaucaugagacagacuuugccuuguaucuacuuuugggggggccuuuugcccuuugggaugcugugugcauccuccaccaccaagugcacu guuagccaugaaguugcugacugcagccaccugaaguugacuccagguacccgaugaucuacccacaaacauaacaguguugaaccuuacccauaaucaacucagaagauuaccagccgccaacuuc acaagguauagccagcuaacuagcuuggauguaggauuuaacaccaucucaaaacuggagccagaauugugccagaaacuucccaucuuaaaaguuuugaaccuccagcacaaugagcuaucuca acuuucugauaaaaccuuugccuucugcacgaauuugacugaacuccaucucauguccaacucaauccagaaaauuaaaaauaaucccuuugucaagcagaagaauuuaaucacauua (SEQ ID NO: 5).

[0256] To obtain modified RNA, the transcription reaction is performed using one (or two) of the basic NTPs and the modified nucleotides 5-methylcytidine, 5-methyluridine, 2-thiouridine, N-methyluridine, and N-methyluridine. 6 ORNs were assembled by replacing α-methyladenosine or pseudouridine with the corresponding triphosphate derivative(s) (TriLink, San Diego, CA). In each transcription reaction, all four nucleotides or their derivatives were present at a concentration of 7.5 millimolar (mM). In selected experiments, 6 mM of the m7GpppG cap analog (New England BioLabs, Beverly, MA) was also included to obtain capped RNA, as indicated. ORN5 and ORN6 were generated using a DNA oligodeoxynucleotide template and T7 RNAP (Silencer® siRNA Construction Kit, Ambion).

[0257] (result) To further examine the effect of nucleoside modifications on immunogenicity, an in vitro system was developed to produce RNA molecules using pseudouridine or modified nucleosides. In vitro transcription reactions were performed in which one or two of the four nucleotide triphosphates (NTPs) were replaced with the corresponding nucleoside-modified NTPs. Several sets of RNAs with different primary sequences, ranging in length from 0.7 to 1.9 kb and containing either zero, one, or two of the modified nucleosides, were transcribed. The modified RNAs were indistinguishable from their unmodified counterparts in terms of their mobility in denaturing gel electrophoresis, indicating that the RNAs were intact and otherwise unmodified (Figure 7A). This procedure worked efficiently with either T7, SP6, or T3 phage polymerases and is therefore generalizable to a wide variety of RNA polymerases.

[0258] These discoveries provide a novel in vitro system for the production of RNA molecules using modified nucleosides.

[0259] Example 6 In vitro transcribed RNA stimulates human TLR3 and nucleoside modifications reduce the immunogenicity of the RNA: Materials and Experimental Methods Parental 293, 293-hTLR7, and 293-hTLR8 cells, all expressing TLR3-specific siRNA, were seeded in 96-well plates (5 × 10 4 Cells were cultured at 1000 x g (1000 x g) in the absence of antibiotics. The next day, cells were exposed to R-848 or RNA complexed with Lipofectin® (Invitrogen) as described (Kariko et al., 1998, ibid.). RNA was removed after 1 hour, and cells were further incubated for 7 hours in complete medium. Supernatants were collected for IL-8 measurement.

[0260] (result) To determine whether nucleoside modifications affect RNA-mediated activation of TLRs, human embryonic kidney 293 cells were stably transformed to express human TLR3. The cell line was treated with RNA complexed with Lipofectin®, and TLR activation was monitored by interleukin (IL)-8 release, as shown in Figure 10. Several different RNA molecules were tested. Unmodified in vitro transcribed RNA induced high levels of IL-8 secretion. In contrast, however, RNA containing m6A or s2U nucleoside modifications did not induce detectable IL-8 secretion (Figure 7B). Other nucleoside modifications tested (i.e., m5C, m5U, Ψ, and m5C / Ψ) had a smaller inhibitory effect on TLR3 stimulation (Figure 7B). "Ψ" refers to pseudouridine.

[0261] Thus, nucleoside modifications such as m5C, m5U, Ψ, and m5C / Ψ reduce the immunogenicity of RNA as mediated by TLR3 signaling.

[0262] Example 7: In vitro transcribed RNA stimulates human TLR7 and TLR8, and nucleoside modifications reduce the immunogenicity of the RNA. To test the possibility that 293 expresses endogenous TLR3, which could interfere with assessing the effects of RNA on specific TLR receptors, we eliminated endogenous TLR3 expression from the 293-TLR8 cell line by stably transfecting the cells with a plasmid expressing a TLR3-specific short hairpin (sh)RNA (also known as siRNA). This cell line did not respond to poly(I):(C), LPS, or CpG-containing oligodeoxynucleosides (ODNs), indicating the absence of TLR3, TLR4, and TLR9, but did respond to R-848, the cognate ligand of human TLR8 (Figure 7B), and was therefore used for further studies. When 293-hTLR8 cells expressing shRNA targeted to TLR3 (293-hTLR8 shRNA-TLR3 cells) were transfected with in vitro transcribed RNA, the cells secreted large amounts of IL-8. In contrast, nucleoside modifications (m5 Cm 5 U, Ψ, and m 5 C / Ψ, S 2 RNA containing most of the m6A (U) eliminated stimulation (IL-8 production below the negative control, i.e., empty vector). The m6A modification had mixed effects, eliminating IL-8 release in some cases and reducing it in others (Figure 7B).

[0263] The results of this and the preceding examples demonstrate that (a) RNA with natural phosphodiester internucleotide linkages (i.e., in vitro transcribed RNA) stimulates human TLR3, TLR7, and TLR8, and (b) nucleoside modifications such as m6A, m5C, m5U, s2U, and Ψ, alone and in combination, reduce the immunogenicity of RNA mediated by TLR3, TLR7, and TLR8 signaling. Additionally, these results provide a novel system for studying signaling by specific TLR receptors.

[0264] Example 8 Nucleoside Modifications Reduce the Immunogenicity of RNA Mediated by TLR7 and TLR8 Signaling. The next set of experiments tested the ability of RNA isolated from natural sources to stimulate TLR3, TLR7, and TLR8. RNA from different mammalian species was transfected into the 293 cell line expressing TLR3, TLR7, and TLR8 described in the previous examples. None of the mammalian RNA samples induced IL-8 secretion above negative control levels. In contrast, bacterial total RNA obtained from two different E. coli sources induced robust IL-8 secretion in cells transfected with TLR8, TLR7, and TLR8, but not TLR9 (Figure 7C). Neither LPS nor unmethylated DNA (CpG ODN), a potential contaminant in bacterial RNA isolates, activated the tested TLR3, TLR7, or TLR8. Mitochondrial RNA isolated from human platelets stimulated human TLR8, but not TLR3 or TLR7.

[0265] These results demonstrate that unmodified in vitro transcribed bacterial RNA is an activator of TLR3, TLR7, and TLR8, and that mitochondrial RNA stimulates TLR8. Additionally, these results confirm the finding that nucleoside modifications of RNA reduce its ability to stimulate TLR3, TLR7, and TLR8.

[0266] Example 9 Nucleoside modifications reduce the ability of RNA to induce cytokine secretion and activation marker expression by DCs. (Materials and Experimental Methods) DC stimulation assay After 20 hours of incubation with RNA, DCs were stained with CD83-phycoerythrin mAb (Research Diagnostics Inc, Flanders, NJ), HLA-DR-Cy5PE, and CD80 or CD86-fluorescein isothiocyanate mAb and analyzed on a FACScalibur® flow cytometer (BD Biosciences) using CellQuest® software. Cell culture supernatants were collected at the end of the 20-hour incubation and subjected to cytokine ELISA. IL-12(p70) (BD Biosciences Pharmingen, San Diego, CA), IFN-α, and TNF-α (Biosource International, Camarillo, CA) levels were measured in the supernatants by ELISA. Cultures were performed in triplicate or quadruplicate, and each sample was measured in duplicate.

[0267] (result) The next experiment tested the ability of RNAs containing modified or unmodified nucleosides to stimulate MDDCs to produce cytokines. Nucleoside modifications reproducibly reduced the ability of 5 RNAs to induce TNF-α and IL-12 secretion by both GM-CSF / IL-4-producing MDDCs and (GMCSF) / IFN-α-producing MDDCs, often to levels below those of the negative control (Figure 8A and B). Results were similar when other sets of RNAs with the same base modifications but different primary sequences and lengths were tested, or when the RNAs were further modified by adding a 5' cap structure and / or a 3' poly(A) tail or by removing the 5' triphosphate moiety. RNAs of different lengths and sequences induced varying amounts of TNF-α from DCs, typically by less than two-fold differences (Figure 8C).

[0268] The assay was then performed on primary DC1 and DC2. Primary monocyte-like (DC1, BDCA1 + ) and plasmacytoid (DC2, BDCA4 + DCs were purified from peripheral blood. Both cell types produced TNF-α upon exposure to R-848, but only DC1 responded to poly(I):(C) at very low levels, indicating the absence of TLR3 activity in DC2. In vitro transcript transfection induced TNF-α secretion in both DC1 and DC2, whereas transcripts modified with m5U, Ψ, or s2U were not stimulatory (Figure 8D). In contrast to cytokine-producing DCs, m5C and m6A modifications of RNA did not reduce its stimulatory capacity in primary DC1 and DC2. RNA molecules with a single type of modification (m6A + Ψ) were potent cytokine inducers. Thus, uridine modifications exerted a dominant-negative effect on RNA molecules in cis in primary DCs. These results were consistent across all donors tested.

[0269] These findings indicate that in vitro transcribed RNA stimulates cytokine production by DCs. In addition, because DC2s do not express TLR3 or TLR8, and the m5C and m6A modification of RNA reduced its stimulatory ability for TLR7, these findings indicate that primary DCs have an additional RNA signaling entity that recognizes m5C and m6A-modified RNA and whose signaling is inhibited by modification of U residues.

[0270] As further indicators of immunogenicity, cell surface expression of CD80, CD83, CD86, and MHC class II molecules, as well as secretion of TNF-α, were measured by FACS analysis of MDDCs treated with RNA-1571 and its modified versions. Pseudouridine and modified nucleosides (m5C, m6A, s2U, and m6A / Ψ) reduced these markers (Figure 9), confirming previous findings.

[0271] In summary, the ability of RNA to induce DCs to mature and secrete cytokines depends on the subtype of DC and on the characteristics of the nucleoside modifications present in the RNA. Increasing amounts of modifications decrease the immunogenicity of the RNA.

[0272] Example 10: Inhibition of RNA-mediated immune stimulation is proportional to the number of modified nucleosides present in the RNA. (Materials and Experimental Methods) (human DCs) For cytokine-producing DCs, monocytes were purified from peripheral blood mononuclear cells by discontinuous Percoll gradient centrifugation. The low-density fraction (enriched in monocytes) was depleted from B, T, and NK cells using magnetic beads specific for CD2, CD16, CD19, and CD56 (Dynal, Lake Success, NY), resulting in highly purified monocytes as determined by flow cytometry using anti-CD14 (>95%) or anti-CD11c (>98%) monoclonal antibodies.

[0273] To generate immature DCs, purified monocytes were cultured in AIM V medium (Invitrogen) supplemented with GM-CSF (50 ng / mL) and IL-4 (100 ng / mL) (R & D Systems, Minneapolis, Minnesota) as described for the generation of monocyte-derived DCs (MDDCs) (Weissman, D et al., 2000. J Immunol 165: 4710-4717). DCs were cultured in AIM V serum-free medium (Life Technologies) supplemented with GM-CSF (50 ng / mL) and IFN-α (1,000 ng / mL) (R & D Systems) to obtain IFN-αMDDCs (Santini et al., 2000). Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in vivo. -PBL-SCID mice. J Exp Med 191: 1777-178).

[0274] Primary myeloid DCs and plasmacytoid DCs (DC1 and DC2) were obtained from peripheral blood using BDCA-1 and BDCA-4 cell isolation kits (Miltenyi Biotec Auburn, Calif.), respectively.

[0275] (result) Up until now, most of the nucleoside-modified RNAs used contain one type of modification that accounts for approximately 25% of all nucleotides in RNA (for example, all uridine bases).In order to define the minimum frequency of specific modified nucleosides that is sufficient to reduce immunogenicity under the conditions used herein, RNA molecules with a limited number of modified nucleosides were generated.In the first set of experiments, RNA was transcribed in vitro in the presence of varying ratios of m6A, Ψ (pseudouridine), or m5C and their corresponding unmodified NTPs.The amount of modified nucleoside phosphate incorporated into RNA is expected to be proportional to the ratio contained in the transcription reaction, because the RNA yield obtained with T7 RNAP showed that the enzyme utilizes m6A, Ψ, or m5C NTPs almost as efficiently as basic NTPs. To confirm this expectation, we digested transcribed RNA in the presence of UTP:Ψ at a 50:50 ratio and found it to contain UTP and Ψ in an approximately 50:50 ratio (Figure 10A).

[0276] RNA molecules with increasing modified nucleoside content were transfected into MDDCs, and TNF-α secretion was assessed. Each modification (m6A, Ψ, and m5C) inhibited TNF-α secretion proportionally to the fraction of modified bases. Even the smallest amounts of modified bases tested (0.2–0.4%, corresponding to 3–6 modified nucleosides per 1571-nt molecule) were sufficient to measurably inhibit cytokine secretion (Figure 10B). RNA with 1.7–3.2% modified nucleoside levels (14–29 modifications per molecule) exhibited a 50% reduction in the induction of TNF-α expression. In TLR-expressing 293 cells, a higher percentage (2.5%) of modified nucleoside content was required to inhibit RNA-mediated signaling events.

[0277] Thus, pseudouridine and modified nucleosides, even when present as minor minor residues, reduce the immunogenicity of RNA molecules.

[0278] In additional experiments, 21-mer oligoribonucleotides (ORNs) with phosphodiester internucleotide linkages were synthesized in which modified nucleosides (mC, Ψ, or 2'-O-methyl-U [Um]) were substituted at specific positions (Figure 11A). Unmodified ORNs induced TNF-α secretion, whereas this effect was abolished by the presence of a single nucleoside modification (Figure 11B). Similar results were obtained using TLR-7 and TLR-8-transfected 293 cells expressing siRNA targeting TLR3.

[0279] The previous results were confirmed by measuring TNF-α mRNA levels in MDDCs by Northern blot assay using both the previous 21-mer ORN (ORN1) and the 31-mer in vitro synthesized transcripts (ORN5 and ORN6). To amplify the signal, cycloheximide, which blocks the degradation of selected mRNAs, was added to some samples as shown in the figure. Unmodified ODNs increased TNF-α mRNA levels, whereas ORNs containing a single modified nucleoside were significantly less stimulatory, with ORN2-Um exhibiting the greatest reduction in TNF-α production (Figure 11C). Similar results were observed in mouse macrophage-like RAW cells and in human DCs.

[0280] In summary, each of the modifications tested (m6A, m5C, m5U, s2U, Ψ, and 2'-O-methyl) suppressed RNA-mediated immune stimulation even when present as a small percentage of residues, with further suppression observed as the ratio of modified nucleosides increased.

[0281] Example 11 Pseudouridine modification of RNA reduces its immunogenicity in vivo. To determine the effect of pseudouridine modification on the in vivo immunogenicity of RNA, 0.25 μg of RNA was complexed with Lipofectin® and injected intratracheally into mice. Mice were bled 24 hours later, and circulating levels of TNF-α and IFN-α were assayed from serum samples. Capped pseudouridine-modified mRNA induced significantly less TNF-α and IFN-α mRNA than did unmodified mRNA (Figure 12A-B).

[0282] These results provide further evidence that pseudouridine-modified mRNA is significantly less immunogenic in vivo than unmodified RNA.

[0283] Example 12: Pseudouridine-containing RNA exhibits reduced ability to activate PRK. (Materials and Experimental Methods) (PKR phosphorylation assay) An aliquot of activated PKR agarose (Upstate) was diluted with magnesium / ATP cocktail (Upstate), kinase buffer, and [γ 32 The reaction was incubated for 30 minutes at 30°C in the presence of [P]ATP mixture and RNA molecules. Unmodified RNA and RNA with nucleoside modifications (m5C, pseudouridine, m6A, m5U) as well as double-stranded RNA were tested. Human recombinant eIF2a (BioSource) was added and the samples were further incubated for 5 minutes at 30°C. The reaction was then incubated for 30 minutes at 30°C in NuPage with reducing agent. The results were stopped by adding LDS sample buffer, denatured at 70°C for 10 min, and analyzed on a 10% PAGE. The gel was dried and exposed to film. Heparin (1 U / µL), a PKR activator, was used as a positive control.

[0284] (result) To determine whether pseudouridine-containing mRNA activates double-stranded RNA-dependent protein kinase (PKR), in vitro phosphorylation assays were performed using recombinant human PKR and its substrate, eIF2α (eukaryotic translation initiation factor 2α), in the presence of capped Renilla-encoding mRNA (0.5 and 0.05 ng / μL). Pseudouridine-containing mRNA did not activate PKR, as detected by the lack of both PKR autophosphorylation and eIF2α phosphorylation, whereas RNA without nucleoside modifications and mRNA without nucleoside modifications did. 5 mRNA with the C modification activated PKR (Figure 13). Thus, pseudouridine modifications reduce RNA immunogenicity.

[0285] Example 13 Pseudouridine and m 5 Enhanced in vitro translation of proteins from RNA containing C (Materials and Experimental Methods) In vitro translation of mRNA in rabbit reticulocyte lysates In vitro translation was performed in rabbit reticulocyte lysates (Promega, Madison, WI). A 9 μL aliquot of lysate was supplemented with 1 μL (1 μg) of mRNA and incubated at 30° C. for 60 minutes. A 1 μL aliquot was removed for analysis using firefly and Renilla assay systems (Promega, Madison, WI) and a LUMAT LB950 luminometer (Berthold / EG&G Wallac, Gaithersburg, MD) with a 10-second measurement time.

[0286] (result) To determine the in vitro effect of pseudouridine modification on RNA translation efficiency, pseudouridine-modified uncapped mRNA (0.1 μg / μL) encoding firefly luciferase was incubated in rabbit reticulocyte lysate at 30° C. for 1 hour, and luciferase activity was determined. Pseudouridine-containing mRNA was translated more than two-fold more efficiently than RNA without pseudouridine in rabbit reticulocyte lysate, but not in wheat extract or E. coli lysate (FIG. 14), indicating that pseudouridine modification enhances RNA translation efficiency. Similar results were obtained in m 5 C. When a polyA tail was added to the pseudouridine-containing mRNA, a further 10-fold increase in translation efficiency was observed (Example 10).

[0287] Therefore, pseudouridine and m 5 Modification with C increases the efficiency of RNA translation, and addition of a poly(A) tail to pseudouridine-containing mRNA further increases translation efficiency.

[0288] Example 14 Enhancement of protein translation from pseudouridine-containing RNA in cultured cells (Materials and Experimental Methods) (Translation assay in cells) 5 x 10 cells per well in a 96-well plate 4 Cells were seeded at 1000 x 1000 cells / well one day before transfection. Lipofectin®-mRNA complexes were assembled and added directly to the cell monolayer after removing the medium (0.2 μg mRNA-0.8 μg Lipofectin in 50 μL per well). After incubating the cells with the transfection mixture for 1 hour in a 37°C, 5% CO2 incubator, the mixture was replaced with fresh pre-warmed medium containing 10% FCS, and the cells were analyzed as described in the previous example.

[0289] (result) To determine the effect of pseudouridine modification on RNA translation in cultured cells, 293 cells were transfected with in vitro transcribed, nucleoside-modified, and capped mRNA encoding the reporter protein Renilla. Cells were lysed 3 hours after the start of transfection, and Renilla levels were measured by enzymatic assay. In 293 cells, pseudouridine- and m5C-modified DNA was translated approximately 10- and 4-fold more efficiently than unmodified mRNA, respectively (Figure 15A).

[0290] Next, this experiment was performed using primary bone marrow-derived mouse DCs, in which the cells were lysed 3 and 8 hours after transfection. RNA containing pseudouridine modifications was translated 15–30 times more efficiently than unmodified RNA (Figure 15B).

[0291] Similar expression results were obtained with human DCs and other primary cells, as well as established cell lines, including CHO cells and mouse macrophage-like RAW cells. In all cell types, pseudouridine modification produced the greatest enhancement of the modifications tested.

[0292] Thus, pseudouridine modification increased RNA translation efficiency in all cell types tested, including different types of professional and non-professional antigen-presenting cells, providing further evidence that pseudouridine modification enhances the efficiency of RNA translation.

[0293] Example 15. 5' and 3' elements enhance translation of Ψ mRNA in mammalian cells. To test the effect of additional RNA structural elements on translation enhancement by pseudouridine modifications, we synthesized a set of Ψ mRNAs encoding firefly luciferase containing a combination of the following modifications: 1) a unique 5' untranslated sequence (TEV, a capping-independent translation enhancer), 2) capping, and 3) a poly(A) tail. The ability of these modifications to enhance translation of Ψ mRNA or conventional mRNA was assessed (Figure 16A). These structural elements additionally enhanced the translation efficiency of both conventional and Ψ mRNAs, with Ψ mRNA exhibiting higher protein production from all constructs.

[0294] Next, the ability to express protein from a rapid firefly luciferase Ψ mRNA construct, capTEVlucA50 (containing TEV, capping, and an extended poly(A) tail), was examined in 293 cells over a 24-hour period (Figure 16B). The Ψ mRNA produced higher amounts of protein at each time point tested and conferred more sustained luciferase expression than the equivalent conventional mRNA construct, indicating that the Ψ modification stabilizes the mRNA.

[0295] To test whether the Ψ modification of mRNA improved translation efficiency in mammalian cells in situ, we added an extended poly(A) tail (A n We generated caplacZ-Ψ mRNA constructs encoding β-galactosidase (lacZ) with or without caplacZ-Ψ and used them to transfect 293 cells. 24 hours after mRNA delivery, a significant increase in β-galactosidase levels was detected by X-gal visualization of caplacZ and caplacZ-Ψ. n Both were detected compared to the corresponding control (conventional) transcripts (Figure 16C). This trend was observed when analyzing either the number of cells expressing detectable levels of β-galactosidase or the magnitude of the signal in individual cells.

[0296] Example 13 Enhancement of in vivo protein translation from pseudouridine-containing RNA: Materials and Experimental Methods (Intracerebral RNA injection) All animal procedures conformed to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Male Wistar rats (Charles River Laboratories, Wilmington, MA) were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg / kg body weight). The head was placed in a stereotaxic frame, and eight equally spaced 1.5 mm diameter burr holes were made bilaterally (coordinates relative to bregma: anterior / posterior +3, 0, -3, -6 mm, lateral ±2.5 mm), leaving the dura intact. Intracerebral injections were performed using a 25 μL syringe (Hamilton, Reno, NV) with a 30-gauge, 1-inch sterile needle (Beckton 25 Dickinson Labware, Franklin Lakes, NJ) secured to a large probe holder and stereotaxic arm. To avoid air spaces in the syringe, the needle was loaded with 55 μL of complex before the needle was attached, and the remainder of the sample was withdrawn through the needle. The injection depth (2 mm) was determined relative to the surface of the dura, and 4 μL of complex (32 ng of mRNA) was administered in a single bolus injection. After 3 hours, the rats were euthanized with halothane and the brains were removed into refrigerated phosphate buffered saline.

[0297] (RNA injection into mouse tail vein) Female BALB / c mice (Charles River Laboratories) were injected (bolus) with 60 μL of Lipofectin®-complexed RNA (0.26 μg) via the tail vein. Organs were removed and homogenized in luciferase or Renilla lysis buffer in microcentrifuge tubes using a mixing rod. The homogenates were centrifuged, and the supernatants were analyzed for activity.

[0298] (RNA delivery to the lungs) Female BALB / c mice were anesthetized with ketamine (100 mg / kg) and xylazine (20 mg / kg). A small incision was made in the skin near the trachea. Once the trachea was exposed, 501-11 RNA (0.2 μg) complexed with Lipofectin® was instilled into the trachea toward the lungs. The incision was closed, and the animals were allowed to recover. Three hours after RNA delivery, the mice were sacrificed by cervical dislocation, and the lungs were removed, homogenized in luciferase or Renilla lysis buffer (250 μL), and assayed for activity. In different sets of animals, blood samples (100 μL / animal) were collected from the tail vein, allowed to clot, and centrifuged. Serum fractions were used to determine TNF and IFNα levels by ELISA using mouse-specific antibodies, as described in previous studies.

[0299] (result) To determine the effect of pseudouridine modification on RNA translation in vivo, we injected either capped pseudouridine-modified or unmodified Renilla-encoding RNA into each hemisphere of rat brains and measured RNA translation. The pseudouridine-modified RNA was translated significantly more efficiently than the unmodified RNA (Figure 17A).

[0300] Expression studies were then carried out in mice. Firefly luciferase-encoding mRNA was used because endogenous mammalian enzymes do not interfere with its detection. The transcripts (unmodified mRNA and Ψ mRNA) were capped with TEV (capTEVA). 50) and an extended (-200 nt) poly(A) tail. 0.25 μg of RNA complexed with Lipofectin® was injected into mice (intravenously (iv) via the tail vein). A range of organs was investigated for luciferase activity to determine the optimal measurement site. Administration of 0.3 μg of capTEVlucAnΨ mRNA induced high luciferase expression in the spleen, moderate expression in the bone marrow, but little expression in the lung, liver, heart, kidney, or brain (Figure 17B). In subsequent studies, the spleen was examined.

[0301] The translation efficiencies of conventional and Ψ mRNA (0.015 mg / kg, 0.3 μg / animal given intravenously) were then compared in a time-course experiment. Luciferase activity was readily detectable 1 hour after administration of either conventional or Ψ mRNA, peaked at 4 hours, and declined by 24 hours, but was substantially greater in animals given Ψ mRNA at all times (Figure 17C, left panel). By 24 hours, only animals injected with Ψ mRNA showed detectable spleen luciferase activity (4-fold above background). Similar relative patterns of expression (between modified and unmodified mRNA) were obtained when animals were injected with mRNA encoding Renilla luciferase (capRen, with or without the Ψ modification) instead of firefly luciferase, or when isolated mouse splenocytes were exposed to the mRNA in culture.

[0302] In the next experiment, 0.25 μg of mRNA-Lipofectin® was delivered to mouse lungs by intratracheal injection. Capped RNA modified with pseudouridine was translated more efficiently than capped RNA without pseudouridine modification (Figure 17D).

[0303] Thus, pseudouridine modification enhances RNA translation efficiency in vitro in cultured cells and in vivo in multiple animal models and by multiple routes of administration, demonstrating its broad application as a means to increase the efficiency of RNA translation.

[0304] Example 17: Pseudouridine modifications enhance in vivo stability. Northern analysis of spleen RNA 1 and 4 hours after injection in animals from the previous example revealed that the administered mRNA was readily detectable in its intact and partially degraded form (Figure 17C, right panel). In contrast, at 24 hours, unmodified capTEVlucAn mRNA was below detection levels, whereas capTEVlucAnΨ mRNA was partially degraded but still clearly detectable. Thus, Ψ mRNA is more stably stored in vivo than control mRNA.

[0305] To test whether in vivo protein production quantitatively depends on the concentration of intravenously delivered mRNA, mRNA was administered to mice at 0.015–0.150 mg / kg (0.3–3.0 μg of capTEVlucAn per animal), and spleens were analyzed 6 h later as previously described. Expression correlated quantitatively with the amount of RNA injected (FIG. 18) at each concentration.

[0306] These findings confirm the results of Example 15 and indicate that Ψ-mRNA is more stable than unmodified RNA. The immunogenicity of Ψ-mRNA was also lower than unmodified RNA, as previously described herein (FIGS. 12 and 17C, right panels).

[0307] To summarize Examples 16-17, the three advantages of Ψ-mRNA compared to conventional mRNA observed in vitro (enhanced translation, increased stability, and reduced immunogenicity) are also observed in vivo.

[0308] Example 18: Ψ mRNA delivered via the airways behaves similarly to intravenously administered mRNA. To test the ability of Ψ mRNA to be delivered by inhalation, mRNA encoding firefly luciferase complexed with Lipofectin® or PEI was delivered to mice via the intratracheal route, in which a needle was placed in the trachea and the mRNA solution was sprayed into the lungs. Similar to intravenous delivery, significantly greater luciferase expression was observed with Ψ mRNA compared to unmodified mRNA (Figure 19), but significantly less protein was produced using the intratracheal route compared to the intravenous route. Unmodified mRNA administered via the intratracheal route was associated with significantly higher concentrations of inflammatory cytokines (IFN-α and TNF-α) compared to vehicle controls.

[0309] Thus, Ψ mRNA can be delivered by inhalation without activating the innate immune response.

[0310] Example 19 Delivery of EPO-Ψ mRNA to 293 cells Ψ mRNA was generated from a plasmid containing human EPO cDNA. 0.25 μg of EPO-Ψ mRNA was 6 The modified RNA molecules of the present invention were transfected into cultured 293 cells and produced more than 600 mU / mL of EPO protein. Thus, the modified RNA molecules of the present invention are effective in delivering recombinant proteins into cells.

[0311] Example 20 Preparation of an improved Ψ mRNA construct encoding EPO (Materials and Experimental Methods) The EPO coding sequence was cloned using restriction enzyme techniques to generate two new plasmids, pTEV-EPO and pT7TS-EPO, which were used as templates for EPO-Ψ mRNA production. EPO-Ψ mRNA was produced from these templates by in vitro transcription (MessageMachine® and MegaScript® kit; Ambion) using T7 RNA polymerase (RNAP) incorporating nucleosides at equimolar concentrations (7.5 mM). To incorporate the nucleoside modifications, Ψ triphosphate (TriLink, San Diego, CA) replaces UTP in the transcription reaction. To confirm capping of Ψ mRNA, 6 mM of the irreversible capping analog 3'-O-Mem7GpppG (New England BioLabs, Beverly, MA) was also included. Ψ mRNA is poly(A)-terminated in a reaction of ~1.5 μg / μL RNA, 5 mM ATP, and 60 U / μL yeast poly(A) polymerase (USB, Cleveland, OH) mixed for 3-24 h at 30°C. The quality of Ψ mRNA is assessed by denaturing agarose gel electrophoresis. The mRNA preparation is also assayed for LPS using a Limulus Amebocyte Lysate gel clot assay, which has a sensitivity of 3 pg / mL.

[0312] (result) The proximal 3' untranslated region (3'UTR) of the EPO-Ψ mRNA preserves a ~90-nt-long pyrimidine-rich stabilizing element from the nascent EPO mRNA, which stabilizes EPO mRNA through specific association with the ubiquitous protein erythropoietin mRNA-binding protein (ERBP). To maximize EPO-Ψ mRNA stability, two modifications were incorporated into the EPO plasmid to improve the stability and translation efficiency of the transcribed mRNA: 1) the 5'UTR sequence of the tobacco etch virus (TEV) was incorporated upstream of the EPO coding sequence, resulting in pTEV-EPO; and 2) the plasmid pT7TS-EPO, in which the EPO cDNA is flanked by sequences corresponding to the 5' and 3'UTRs of Xenopus β-globin mRNA.

[0313] Additionally, the length of the poly(A) tail during the production of Ψ mRNA from these plasmid templates can be extended by extending the incubation time of the poly(A) polymerase reaction. A longer poly(A) tail reduces the rate at which Ψ mRNA is degraded during translation.

[0314] These improvements result in increased translation efficiency in vivo and minimize the therapeutic dose of the final product.

[0315] Example 21 In Vitro Analysis of Protein Production from EPO mRNA Constructs (Materials and Experimental Methods) (Mammalian cell preparation) Human embryonic kidney 293 cells (ATCC) are grown in DMEM (complete medium) supplemented with glutamine (Invitrogen) and 10% FCS (Hyclone, Odgen, UT). Leukopheresis samples are obtained from HIV-uninfected volunteers through an IRB-approved protocol. DCs were prepared as previously described and cultured with GM-CSF (50 ng / mL) + IL-4 (100 ng / mL) (R & D Systems) in AIM V Medium® (Invitrogen).

[0316] Murine splenocytes and DCs were obtained according to published procedures. Briefly, spleens from BALB / c mice were aseptically removed and minced with tweezers in complete medium. Tissue fragments were allowed to settle by gravity, and single cell suspensions were washed and lysed in AKC lysis buffer (Sigma). Murine DCs were derived from bone marrow cells collected from the femurs and tibias of 6- to 9-week-old BALB / c mice. Cells were cultured in DMEM containing 10% FCS (Invitrogen) and 50 ng / mL mu GM-CSF (R&D) for 7 days before use.

[0317] (Cell Transfection and Detection of EPO and Proinflammatory Cytokines) Transfections were performed using Lipofectin in the presence of phosphate buffer, an effective delivery method for spleen and in vitro cell expression. EPO-Ψ mRNA (0.25 μg / well; 100,000 cells) was added to each cell type in triplicate for 1 hour, and the supernatant was replaced with fresh medium. After 24 hours, the supernatants were collected for ELISA measurements of EPO, IFN-α or β, and TNF-α.

[0318] (result) To evaluate the impact of the unique UTR on enhancing Ψ mRNA translation efficiency, EPO-Ψ mRNA containing or not containing each modification with a long poly(A) tail (5' TEV element, β-globin 5' and 3' UTR) is tested for in vitro protein production and in vitro immune activation using EPO mRNA containing conventional nucleosides as a control. The efficiency of protein production from each mRNA is evaluated for each mRNA in mammalian cell lines (HEK293, CHO), human and mouse primary DCs, and splenocytes. Total EPO produced in all cell types and immunogenicity (supernatant-associated proinflammatory cytokines) in primary cells are evaluated. The mRNA construct that exhibits the optimal combination of high EPO production and low cytokine induction (in one or more cell types) is used for subsequent studies. The modifications to the 5' and 3' UTRs and longer poly(A) tails of EPO-Ψ mRNA result in approximately a 2- to 10-fold enhancement in translation efficiency.

[0319] Example 22 Characterization of EPO Production and In Vivo Biological Response to EPO-Ψ mRNA (Materials and Experimental Methods) (Administration of EPO-Ψ mRNA to mice) All animal studies described herein were approved by the NIH Guide for the Administration and Use of Laboratory Animals and the University of Pennsylvania Institutional Animal Care and Use Committee. Female BALB / c mice (n = 5 per experimental condition, 6 weeks old, 18-23 g, Charles River Laboratories) were anesthetized with 3.5% halothane in a mixture of NO and O (70:30), then the halothane was reduced to 1% and anesthesia was maintained using a nasal mask. Animal body temperature was maintained throughout the procedure using a heating pad heated to 37°C. EPO-Ψ mRNA-lipofectin complexes (constructed by mixing varying amounts of nucleic acid with 1 μL of lipofectin in a final volume of 60 μL) were injected into the lateral tail vein. Blood samples are collected three times daily for three days after mRNA injection during the time course study, at one optimal time point in the dose-response study, and daily after days 2-6 in the study for reticulocytosis.

[0320] (Determination of reticulocytes by flow cytometry) Blood samples are stained with Retic-COUNT reagent (BD Diagnostics) and data events are acquired on a FACScan flow cytometer. Red blood cells (RBCs) are selected by forward and side scatter properties and analyzed for thiazole orange uptake. Retic-COUNT reagent-stained cells are detected by fluorescence, and reticulocytes are expressed as a percentage of total RBCs. At least 50,000 events are counted per sample.

[0321] (result) To optimize the production of biologically functional human EPO protein (hEPO) in response to EPO-encoding mRNA, the following studies are performed: (Time course of EPO production after a single injection of EPO-Ψ mRNA) After intravenous administration of 1 µg of EPO-Ψ mRNA, hEPO is serially measured 1 to 96 h after EPO-Ψ mRNA administration by ELISA to determine the half-life of EPO protein in serum. This half-life is the result of both the half-life of EPO protein and the functional half-life of EPO-Ψ mRNA. The resulting optimal time point for measuring EPO protein after EPO-Ψ mRNA administration is utilized for subsequent studies.

[0322] Dose-response of EPO production after a single injection of EPO-Ψ mRNA. To determine the correlation between the amount of EPO protein produced and the amount of EPO-Ψ mRNA administered, increasing concentrations of EPO-Ψ mRNA (0.01-1 μg / individual) are administered and EPO measured at optimal time points.

[0323] (Association between hEPO production and reticulocytosis) To measure the effect of EPO-Ψ mRNA on the biological correlates of EPO activity, flow cytometry is used to determine reticulocyte frequency in the blood. Flow cytometry has a coefficient of variation of less than 3%. Mice receive a single dose of EPO-Ψ mRNA, and blood is collected from the mice 2–6 days later. The association between EPO-Ψ mRNA dose and reticulocyte frequency is then assessed at the time of maximum reticulocytosis. The dose of EPO-Ψ mRNA that results in at least a 5% increase in reticulocyte count is used in subsequent studies. An estimated serum hEPO concentration of 50 mU / mL and / or an estimated 5% increase in reticulocyte frequency in mice is obtained.

[0324] Example 23 In vivo measurement of immune response to EPO-Ψ mRNA (Materials and Experimental Methods) (Detection of cytokines in plasma) Serum samples obtained from blood collected at different time points during and after seven daily administrations of mRNA complexed with Lipofectin are analyzed for murine IFN-α, TNF-α, and IL-12 using ELISA kits.

[0325] (Northern blot analysis) Aliquots (2.0 μg) of RNA samples isolated from spleens were separated by 1.4% agarose gel electrophoresis and transferred to charged membranes (Schleicher and Schuell) and hybridized in MiracleHyb® (Stratagene). Membranes are probed for TNF-α, downstream IFN signaling molecules (e.g., IRF7, IL-12 p35 and p40, and GAPDH), and other markers of immune activation. The specificity of all probes is confirmed by sequencing. To probe the membranes, Redivue [α- 32 50 ng of DNA is labeled with [P]dCTP® (Amersham). The hybridized membrane is exposed to Kodak BioMax MS film at -70°C using an MS intensifier screen.

[0326] (Histopathology) Spleens from mice treated with EPO-Ψ mRNA and mice treated with positive and negative controls were collected, fixed, sectioned, stained with hematoxylin and eosin, and examined by a veterinary pathologist for signs of immune activation.

[0327] (result) To confirm the reduced immunogenicity of the RNA molecules of the present invention, mice (n=5) receive a daily dose of EPO-Ψ mRNA for 7 days, and then are evaluated for immune-mediated adverse events as indicated by serum cytokine levels, splenic expression of mRNA encoding inflammatory proteins, and pathological examination. The maximum dose administered is 3 μg or 5 times the single effective dose, as previously determined. Unmodified mRNA and Lipofectin® alone are used as positive and negative controls, respectively.

[0328] These studies confirm the reduced immunogenicity of the RNA molecules of the invention.

[0329] Example 24 (Further Improvement of the EPO-Ψ mRNA Delivery Method) (Nanoparticle complex formation) The polymer and Ψ mRNA solutions are mixed to form a complex. Various formulation conditions are tested and optimized: (1) Polyethyleneimine (PEI) / mRNA complexes of less than 22 nm are made by adding 25 volumes of mRNA to 1 volume of PEI in water without mixing for 15 minutes. (2) Rod-shaped poly-L-lysine-polyethylene glycol (PLL-PEG) with an average size of 12 × 150 nm is added to 9 volumes of mRNA in 1 volume of CK in acetate counterion buffer. 30 -PEG 10k (3) For the synthesis of biodegradable gene carrier polymers, polyaspartic anhydride-co-ethylene glycol (PAE) is synthesized by ring-opening polycondensation of N-(benzyloxycarbonyl) L-aspartic anhydride and ethylene glycol. The branched amines of aspartic acid are then deprotected, protonated by acidification with hydrogen chloride, and condensed with mRNA. (4) For the final generation of nanoparticles, an aliquot of stock CK as ammonium acetate is added. 30 PEG 10k (1.25 mL; 6.4 mg / mL) was added to a siliconized Eppendorf tube. 30 PEG 10k (2.5 mg in 11.25 mL RNase-free H2O) slowly over 1-2 minutes. After 15 minutes, dilute 1:2 in RNase-free H2O.

[0330] (intratracheal delivery) Mice were anesthetized with 3% halothane (70% NO + 30% O) in an anesthesia chamber and maintained with 1% halothane (70% NO + 30% O) during the procedure using a nose cone. The trachea was exposed, and 50 μL of the mRNA complex was infused into the lungs with 150 μL of gas through the trachea using a 250 μL Hamilton syringe with a 27G 1 / 2" needle (Hamilton, Injection was performed using a syringe (Reno, NV).

[0331] (result) To improve the efficiency of delivery and expression of Ψ mRNA administered via the intratracheal (it) route, Ψ mRNA is encapsulated in nanoparticles. Nanoparticle packaging involves using chemicals including poly-L-lysine and polyethylene glycol to condense (for example) DNA and encapsulate it into particles smaller than the nuclear pore. RNA was encapsulated in four different nanoparticle formulations (PEI, PLL, PAE, and CK). 30 PEG 10k ) and the efficiency of Ψ mRNA delivery is compared to Ψ mRNA encoding luciferase (Luc-Ψ mRNA). Delivery kinetics and dose response are then characterized using EPO-Ψ mRNA.

[0332] Example 25 Prevention of restenosis by delivery of modified mRNA encoding recombinant heat shock proteins to the carotid artery (Materials and Experimental Methods) (Experimental Design) RNA is administered to the carotid artery of rats by intra-arterial injection at approximately the same time as balloon angioplasty, and then blood flow is restored. The rats are sacrificed 3 hours after injection, the carotid artery sections are removed, the vascular endothelial cells are collected and homogenized, and luciferase activity is determined as described in the previous example.

[0333] (result) Pseudouridine-modified RNA encoding luciferase is administered into the carotid artery of rats. Three hours later, the RNA is detectable at the delivery site but not at adjacent sites.

[0334] This protocol is then used to prevent vascular restenosis after balloon angioplasty in animal restenosis models by delivering modified RNA encoding heat shock proteins such as HSP70, growth factors (e.g., platelet-derived growth factor (PDGF)), vascular endothelial growth factor (V-EGF), or insulin-like growth factor (IGF), or proteins that downregulate or antagonize growth factor signaling. Administration of the modified RNA reduces the incidence of restenosis.

[0335] Example 26 Treatment of Cystic Fibrosis by Delivery of Modified mRNA Molecules Encoding CFTR to Respiratory Epithelia Pseudouridine- or nucleoside-modified RNA encoding CFTR was delivered to the lungs of animal models of cystic fibrosis as described in Example 16, and its effect on the disease was evaluated as described in Scholte BJ, et al. (Animal models of cystic fibrosis. J Cyst Fibros 2004; 3 Suppl 2: 183-90) or Copreni E, et al. (Lentivirus-mediated gene transfer to the respiratory epithelium: a promising approach to gene therapy of cystic fibrosis. Gene Ther 2004; 11 Suppl 1: S67-75). Administration of the RNA ameliorates cystic fibrosis.

[0336] In additional experiments, the modified mRNA molecules of the present invention are used to deliver other recombinant proteins of therapeutic value to the lung, for example, via an inhaler that delivers RNA.

[0337] Example 27 Treatment of XLA by delivery of modified mRNA molecules encoding ADA to hematopoietic cells Pseudouridine- or nucleoside-modified RNA encoding ADA is delivered to hematopoietic cells of X-linked agammaglobulinemia, and its effect on the disease is assessed as described in Tanaka M, Gunawan F, et al., Inhibition of heart transplant injury and graft coronaryartery disease after prolonged organ ischemia by selective protein kinase C regulators. J Thorac Cardiovasc Surg 2005;129(5):1160-7 or Zonta S, Lovisetto F, et al., Uretero-neocystostomy in a swine model of kidney transplantation: a new technique. J Surg Res. 2005 Apr;124(2):250-5. Administration of RNA has been found to ameliorate XLA.

[0338] Example 28 Prevention of organ rejection by delivery to the transplant site of modified mRNA molecules encoding immunomodulatory proteins Pseudouridine- or nucleoside-modified RNA encoding a cytokine, chemokine, or interferon IS (e.g., IL-4, IL-13, IL-10, or TGF-β) is delivered to the graft site in an animal model of organ transplant rejection, and its effect on the incidence of rejection is assessed as described in Yu PW, Tabuchi RS et al., Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer. Blood. 2004 104(5):1281-90 or Satoh M, Mizutani A et al., X-linked immunodeficient mice spontaneously produce lupus-related anti-20 RNA helicase A autoantibodies, but are resistant to pristane-induced lupus. Int Immunol 2003, 15(9):1117-24. Administration of RNA reduces the incidence of graft rejection.

[0339] Example 29: Treatment of Niemann-Pick disease, mucopolysaccharidoses, and other inborn errors of metabolism by delivery of modified mRNA to body tissues. Pseudouridine- or nucleoside-modified RNA encoding sphingomyelinase is delivered to the lung, brain, or other tissues of animal models of Niemann-Pick disease types A and B, and its effect on the disease is assessed as described in Passini MA, Macauley SL, et al., AAV vector-mediated correction of brain pathology in a mouse model of Niemann-Pick A disease. Mol Ther 2005;11(5):754-62 or Buccoliero R, Ginzburg L, et al., Elevation of lung surfactant phosphatidylcholine in mouse models of Sandhoff and of Niemann-Pick A disease. J Inherit Metab Dis 2004;27(5):641-8. Administration of RNA has been found to ameliorate the disease.

[0340] Pseudouridine- or nucleoside-modified RNA encoding α-L-iduronidase, iduronate-2-sulfatase, or related enzymes is delivered to the body tissues of an animal model of mucopolysaccharidosis, and its effect on the disease is evaluated as described in Simonaro CM, D'Angelo M, et al., Joint and bone disease in mucopolysaccharidoses VI and VII: identification of new therapeutic targets and biomarkers using animal models. Pediatr Res 2005;57(5 Pt 1): 701-7 or McGlynn R, Dobrenis K, et al., Differential subcellular localization of cholesterol, gangliosides, and glycosaminoglycans in murine models of mucopolysaccharide storage disorders. J Comp Neurol 2004 20;480(4): 415-26. Administration of RNA ameliorates the disease.

[0341] In additional experiments, the modified mRNA molecules of the present invention are used to provide clotting factors (e.g., for hemophiliacs). In additional experiments, the modified mRNA molecules of the present invention are used to provide acid-β-glucosidase for the treatment of Gaucher. In additional experiments, the modified mRNA molecules of the present invention are used to provide α-galactosidase for the treatment of Fabry's disease. In additional experiments, the modified mRNA molecules of the present invention are used to provide cytokines for the treatment of infectious diseases.

[0342] The results are indicated by the presence of mRNA scaffolds. ABCA4, ABCD3, ACADM, AGL, AGT, A LDH4Al, ALPL, AMPD1, APOA2, AVSD1, BRCD2, C1QA, C1QB, C1QG, C8A, C8B, CAC NA1S, CCV, CD3Z, CDC2L1, CHML, CHS1, CIAS1, CLCNKB, CMD1A, CMH2, CMM, COL 11AI, COL8A2, COL9A2, CPT2, CRB1, CSE, CSF3R, CTPA, CTSK, DBT, DIO1, DISC1 DPYD, EKV, ENO1, ENO1P, EPB41, EPHX1, F13B, F5, FCGR2A, FCGR2B, FCGR3A. FCHL, FH, FMO3, FMO4, FUCA1, FY, GALE, GBA, GFND, GJA8, GJB3, GLC3B, HF1, HM GCL, HPC1, HRD, HRPT2, HSD3B2, HSPG2, KCNQ4, KCS, KIF1B, LAMB3, LAMC2, LG MD1B, LMNA, LOR, MCKD1, MCL1, MPZ, MTHFR, MTR, MUTYH, MYOC, NB, NCF2, NEM1 NPHS2, NPPA, NRAS, NTRK1, OPTA2, PBX1, PCHC, PGD, PHA2A, PHGDH, PKLR, PKP 1, PLA2G2A, PLOD, PPOX, PPT1, PRCC, PRG4, PSEN2, PTOS1, REN, RFX5, RHD, RMD 1. RPE65, SCCD, SERPINC1, SJS1, SLC19A2, SLC2A1, SPG23, SPTA1, TAL1, TNF SF6, TNNT2, TPM3, TSHB, UMPK, UOX, UROD, USH2A, VMGLOM, VWS, WS2B, ABCB11. ABCG5, ABCG8, ACADL, ACP1, AGXT, AHHR, ALMS1, ALPP, ALS2, APOB, BDE, BDMR BJS, BMPR2, CHRNA1, CMCWTD, CNGA3, COL3A1, COL4A3, COL4A4, COL6A3, CPS1 CRYGA, CRYGEP1, CYP1B1, CYP27A1, DBI, DES, DYSF, EDAR, EFEMP1, EIF2AK3 ERCC3, FSHR, GINGF, GLC1B, GPD2, GYPC, HADHA, HADHB, HOXD13, HPE2, IGKC.IHH、IRS1、ITGA6、KHK、KYNU、LCT、LHCGR、LSFC、MSH2、MSH6、NEB、NMTC、NPHP 1、PAFAH1P1、PAX3、PAX8、PMS1、PNKD、PPH1、PROC、REGIA、SAG、SFTPB、SLC11A 1, SLC3Al, SOS1, SPG4, SRD5A2, TCL4, TGFA, TMD, TPO, UGT1A@, UV24, WSS, XDH, ZAP70, ZFHX1B, ACAA1, AGS1, AGTR1, AHSG, AMT, ARMET, BBS3, BCHE, BCPM, B TD、CASR、CCR2、CCR5、CDL1、CMT2B、COL7A1、CP、CPO、CRY、CTNNB1、DEM、ETM1 、FANCD2、F1H、FOXL2、GBE1、GLB1、GLC1C、GNAI2、GNAT1、GP9、GPX1、HGD、HRG、 ITIH1、KNG、LPP、LRS1、MCCC1、MDS1、MHS4、MITF、MLH1、MYL3、MYMY、OPA1、P2 RY12、PBXPI、PCCB、POU1FI、PPARG、PROS1、PTHR1、RCA1、RHO、SCA7、SCLC1、SC N5A、SI、SLC25A20、SLC2A2、TF、TGFBR2、THPO、THRB、TKT、TM4SF1、TRH、UMPS 、UQCRC1、USH3A、VHL、WS2A、XPC、ZNF35、ADH1B、ADH1C、AFP、AGA、AIH2、ALB、A 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SLC25A6, SPG5B, STO, SUOX, THM, or TTD.

[0343] Example 30 Treatment of Vasospasm by Delivery of Modified mRNA Molecules Encoding iNOS to Body Tissues The pseudouridine- or nucleoside-modified RNA encoding inducible nitric oxide synthase (iNOS) is delivered to the vascular endothelium of animal models of vasospasm (e.g., subarachnoid hemorrhage), and its effect on the disease is evaluated as described in Pradilla G, Wang PP, et al., Prevention of vasospasm by anti-CD11 / CD18 monoclonal antibody therapy following subarachnoid hemorrhage in rabbits. J Neurosurg 2004;101(1):88-92 or Park S, Yamaguchi M, et al., Neurovascular protection reduces early brain injury after subarachnoid hemorrhage. Stroke 2004;35(10):2412-7. The administration of RNA ameliorates the disease.

[0344] Example 31 Restoration of Hair Growth by Delivery of Modified mRNA Encoding an Immunosuppressive Protein Pseudouridine- or nucleoside-modified mRNA encoding telomerase or immunosuppressive proteins (e.g., α-MSH, TGF-β1, or IGF-I) is evaluated as described in Jiang J, Tsuboi R, et al., Topical application of ketoconazole stimulates hair growth in C3H / HeN mice. J Dermatol 2005;32(4):243-7 or McElwee KJ, Freyschmidt-Paul P, et al., Transfer of CD8(+) cells induces localized hair loss whereas CD4(+) / CD25(-) cells promote systemic alopecia areata and CD4(+) / CD25(+) cells blockade disease onset in the C3H / HeJ mouse model. J Invest Dermatol 2005;124(5):947-57. RNA administration restores hair growth.

[0345] Example 32 Synthesis of in vitro transcribed RNA molecules with altered nucleosides containing siRNA Double-stranded RNA (dsRNA) molecules containing pseudouridine or modified nucleosides and further containing small interfering RNA (siRNA) or short hairpin RNA (shRNA) are synthesized by the following procedure: An RNA strand complementary to the desired sequence containing uridine or one or more modified nucleosides is synthesized by in vitro transcription (e.g., using T7, SP6, or T3 phage RNA polymerase) as described in Example 5. Double-stranded RNA molecules exhibit reduced immunogenicity. In other experiments, double-stranded RNA molecules are designed to be processed by cellular enzymes to produce the desired siRNA or shRNA. Because double-stranded RNA molecules of several hundred nucleotides can be easily synthesized, each double-stranded RNA can also be designed to contain several siRNA or shRNA molecules, facilitating the delivery of multiple siRNAs or shRNAs to a single target cell.

[0346] Example 33 Use of in vitro transcribed RNA molecules with altered nucleosides to deliver siRNA The double-stranded RNA molecules of the preceding examples are complexed with a transfection reagent (e.g., a cationic transfection reagent, a lipid-based transfection reagent, a protein-based transfection reagent, a polyethyleneimine-based transfection reagent, or calcium phosphate) and delivered to a target cell of interest. Enzymes in or on the surface of the target cell degrade the double-stranded RNA into the desired siRNA or shRNA molecule(s). This method effectively silences the transcription of one or more cellular genes corresponding to the siRNA or shRNA sequence(s).

[0347] Example 34 Testing the effect of additional nucleoside modifications on RNA immunogenicity and translation efficiency Additional nucleoside modifications are introduced into in vitro transcribed RNA using the methods described above in Examples 5 and 10, and their effects on immunogenicity and translation efficiency are tested as described in Examples 4-11 and 12-18, respectively. Certain additional modifications have been found to reduce immunogenicity and enhance translation. These modifications are additional embodiments of the methods and compositions of the invention.

[0348] Modifications to be tested include, for example, m l A, m 2 A, Am, ms 2 m 6 A, i 6 A,ms 2 i6A, io 6 A,ms 2 i0 6 A, g 6 A, t 6 A,ms 2 t 6 A, m 6 t 6 A,hn 6 A,ms 2 hn 6 A, Ar(p), I, m 1 I, m l Im, m 3 C, Cm, S 2 C, ac 4 C, f 5 cm 5 Cm, ac 4 Cm, k 2 cm 1 G, m 2 G, m 7 G, Gm, m 2 2G, m 2 Gm, m 2 2Gm, Gr(p), yW, o2yW, OHyW, OHyW*, imG, mimG, Q, oQ, galQ, manQ, preQ0, preQ1, G + ,D,m 5 Um',m 1 Ψ, Ψm, S 4 U, m 5 s 2 U', S 2 Um, 'acp 3 U·, ho5 u., mo 5 U·, cmo 5 U·, mcmo 5 U, chm 5 U, mchm 5 U·, mcm 5 U, mcm 5 Um, mcm 5 s 2 U, nm 5 s 2 U, mnm 5 U, mnm 5 s 2 U, mnm 5 se 2 U, ncm 5 U, ncm 5 Um, cmnm 5 U, cmnm 5 Um, cmnm 5 s 2 U, m 6 2A, Im, m 4 Cm 4 Cm, hm 5 Cm 3 U, m 1 acp3Ψ, cm 5 U, m 6 Am, m 6 2Am,m 2,7 G, m 2,2,7 G, m 3 Um, m 5 D, m 3 Ψ, f 5 Cm, m l Gm, m l Am, τm 5 U, τm 5 s 2 U, imG-14, imG2, and ac 6 Contains A.

[0349] Materials and Methods for Examples 35-38 (HPLC purification of RNA): mRNA produced by T7 polymerase transcription was purified by HPLC using a column matrix of alkylated nonporous polystyrene-divinylbenzene (PS-DVB) copolymer microspheres (2.1 μm) (21 mm × 100 mm column) and a buffer system of triethylammonium acetate (TEAA) with an acetonitrile gradient. Buffer A contained 0.1 M TEAA, and buffer B contained 0.1 M TEAA and 25% acetonitrile. The column was equilibrated with 38% buffer B in buffer A, and the RNA was loaded. It was then run at 5 mL / min over 30 min using a linear gradient to 55% buffer B. Fractions corresponding to the desired peak were collected. RNA analysis was performed using the same column matrix and buffer system, but with a 7.8 mm × 50 mm column at 1.0 mL / min and a 25-minute gradient time.

[0350] (RNA isolation from column fractions): The collected fractions were combined, and their RNA content was first concentrated using an Amicon Ultra-15 centrifugal filter unit with a 30K membrane (Millipore). The filter device was loaded with a 15 mL sample and spun at 4,000 × g for 10 minutes (4 °C) in a Thermo Scientific Sorvall ST16R centrifuge using a swinging bucket rotor. Under these conditions, approximately 98% of the solvent volume could be removed. If the collected fraction had a volume greater than 15 mL, the filter unit was recycled by loading it with additional column fractions and centrifuging again until all the RNA fit into one tube. To remove salts and solvent from the concentrated RNA, nuclease-free water was added (up to 15 mL), and the filter device was spun again. The "washing" process was repeated until the acetonitrile concentration was less than 0.001%. The desalted, solvent-free sample was removed from the filter device, and the RNA was recovered by precipitation in NaOAc (0.3 M, pH 5.5), isopropanol (1 volume), and glycogen (3 μL) overnight at −20° C. The precipitated RNA was recovered, washed twice with ice-cold 75% ethanol, and reconstituted in water.

[0351] (Double-stranded RNA dot blot): RNA (25–100 ng) was blotted onto a nitrocellulose membrane, allowed to dry, blocked with 5% nonfat dry milk in TBS buffer supplemented with 0.05% Tween-20 (TBS-T), and incubated with double-stranded RNA-specific mAb J2 or K1 (English & Scientific Consulting) for 60 min. The membrane was washed six times with TBS-T and then reacted with HRP-conjugated donkey anti-mouse antibody (Jackson Immunology). After six washes, double-stranded RNA was stained with SuperSignal West Pico Detection was achieved by addition of a chemiluminescent substrate (Pierce), and images were captured for 30 seconds to 2 minutes on a Fujifilm LAS1000 digital imaging system.

[0352] (Dendritic cell generation): Monocytepheresis samples were obtained from normal volunteers through an IRB-approved protocol. Human DCs were produced by treating monocytes with GM-CSF (50 ng / mL) and IL-4 (100 ng / mL) in AIM V medium (Invitrogen) for 7 days. After 3 and 6 days, 50% volume of fresh medium with cytokines was added.

[0353] Murine DCs were generated by isolating bone marrow mononuclear cells from Balb / c mice and culturing them in RPMI+10% FBS medium supplemented with mouse GM-CSF (20 ng / mL, Peprotech). After 3 and 6 days, 50% volume of fresh medium containing GM-CSF was added. Non-adherent cells were used after 7 days of culture.

[0354] (Lipofectin complexation of RNA): Stock phosphate buffer was added to serum-free DMEM to give a final concentration of 20 mM potassium phosphate and 100 ng / mL BSA, pH 6.4. For three wells of a 96-well plate, lipofectin-complexed RNA was prepared in the following ratio: 2.4 μL of lipofectin was added to 21.3 μL of serum-free DMEM medium with phosphate buffer and incubated at room temperature for 10 minutes. Next, 0.75 μg of RNA in 9.9 μL of serum-free DMEM was added, and the mixture was incubated at room temperature for an additional 10 minutes. Finally, 116.4 mL of serum-free DMEM was added to bring the final volume to 150 mL. The mixture was vortexed.

[0355] (RNA TransIT complex formation): For each well of a 96-well plate, 0.25 μg of RNA was added to 17.3 μL of serum-free DMEM on ice. The mRNA reagent (0.3 μL) was added ...

Claims

1. 1. A method of inducing mammalian cells present in vitro to produce a recombinant protein, comprising: contacting the mammalian cells with a preparation of in vitro synthesized mRNA comprising an open reading frame encoding the recombinant protein; The in vitro synthesized mRNA is obtained by in vitro transcription (IVT) and contains Ψ (5-β-D-ribofuranosyluracil), with 0.1 to 100% of the uridine residues in the RNA being replaced with Ψ (5-β-D-ribofuranosyluracil), including cases where all uridine residues are replaced with Ψ (5-β-D-ribofuranosyluracil); and the in vitro synthesized mRNA preparation is purified using a purification process that removes RNA contaminant molecules that are immunogenic and toxic to the cells by inducing an innate immune response; the RNA contaminant molecules include double-stranded RNA contaminant molecules; The purification of the mRNA is determined by measuring reduced secretion of IFN-α or TNF-α cytokines by dendritic cells transfected with the purified in vitro synthesized mRNA preparation compared to secretion of the cytokines by dendritic cells transfected with an unpurified in vitro synthesized mRNA preparation.

2. The method described in claim 1, wherein all of the uridine residues are replaced with Ψ (5-β-D-ribofuranosyluracil).

3. 3. The method of claim 1 or 2, wherein less than 0.01% of the total RNA in said purified in vitro synthesized mRNA preparation consists of double-stranded RNA contaminant molecules.

4. 4. The method of any one of claims 1 to 3, wherein the purification step results in the in vitro synthesized mRNA preparation being capable of being administered without eliciting an immune response sufficient to eliminate detectable expression of the recombinant protein, or the in vitro synthesized mRNA preparation lacks immunogenicity, allowing for repeated delivery without the generation of proinflammatory cytokines.

5. The in vitro synthesized mRNA is A. Poly A tail; B. a 5' cap or cap-independent translation enhancer; or C. The modified nucleoside 5-methylcytidine (m-methylcytidine) instead of cytidine ５ C) 5. The method of claim 1, comprising:

6. The purification step comprises: A. Purification of said in vitro synthesized mRNA preparation using HPLC or gravity flow column purification; and / or B. Treating the in vitro synthesized mRNA preparation with ribonuclease III (RNase III) enzyme to produce short RNase III digestion products, and purifying the mRNA preparation to remove the short RNase III digestion products.

6. The method of claim 1, comprising:

7. The recombinant protein encoded by the in vitro synthesized mRNA may be selected from erythropoietin (EPO); a detection enzyme selected from firefly luciferase, Renilla luciferase, bacterial β-galactosidase (lacZ), and green fluorescent protein (GFP); a transcriptional regulator selected from MYC and SRY or MCOP; a transcription factor selected from platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), insulin-like growth factor (IGF), α-melanocyte-stimulating hormone (α-MSH), or a transcription factor (TRF). a growth factor or cytokine selected from the group consisting of insulin-like growth factor-I (IGF-I), IL-4, IL-13, and IL-10; inducible nitric oxide synthase (iNOS); cystic fibrosis transmembrane conductance regulator (CFTR); an enzyme having antioxidant activity selected from catalase, phospholipid hydroperoxide glutathione peroxidase, superoxide dismutase-1, and superoxide dismutase-2; Bruton's tyrosine kinase; adenosine deaminase; ecto-nucleoside triphosphate diphosphohydrolase; ABCA4; ABCD3; ACADM; AGL; AGT; ALDH4Al; ALPL; AMPD1; APOA2; AVSD1; BR CD2; C1QA; C1QB; C1QG; C8A; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CI AS1; CLCNKB; CMD1A; CMH2; CMM; COL11A1; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK; DBT; DIO1; DISC1; DPYD; EKV; ENO1; ENO1P; EPB41; EPHX1; F13B; F5; FCGR2A; FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALE; G BA; GFND; GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2; K CNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ; MTHF R; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS; NTRK1; OPTA2; PBX1;PCHC;PGD;PHA2A;PHGDH;PKLR;PKP1; PLA2G2A;PLOD;PPOX;PPT1;PRCC;PRG 4;PSEN2;PTOS1;REN;RFX5;RHD;RMD1 ;RPE65;SCCD;SERPINC1;SJS1;SLC19 A2;SLC2A1;SPG23;SPTA1;TAL1;TNFS F6;TNNT2;TPM3;TSHB;UMPK;UOX;URO D;USH2A;VMGLOM;VWS;WS2B;ABCB11; ABCG5;ABCG8;ACADL;ACP1;AGXT;AHHR ;ALMS1;ALPP;ALS2;APOB;BDE;BDMR; BJS;BMPR2;CHRNA1;CMCWTD;CNGA3;C OL3A1;COL4A3;COL4A4;COL6A3;CPS1 ;CRYGA;CRYGEP1;CYP1B1;CYP27A1;DB I;DES;DYSF;EDAR;EFEMP1;EIF2AK3;ERCC3;FSHR;GINGF;GLC1B;GPD2;GYP C;HADHA;HADHB;HOXD13;HPE2;IGKC; IHH;IRS1;ITGA6;KHK;KYNU;LCT;LHCG R;LSFC;MSH2;MSH6;NEB;NMTC;NPHP1 ;PAFAH1P1;PAX3;PAX8;PMS1;PNKD;P PH1;PROC;REG1A;SAG;SFTPB;SLC11A 1;SLC3Al;SOS1;SPG4;SRD5A2;TCL4;T GFA;TMD;TPO;UGT1A;UV24;WSS;XDH; AP70;FHX1B;ACAA1;AGS1;AGTR1;A HSG;AMT;ARMET;BBS3;BCHE;BCPM;BT D;CASR;CCR2;CCR5;CDL1;CMT2B;COL7 A1;CP;CPO;CRV;CTNNB1;DEM;ETM1;F ANCD2;FIH;FOXL2;GBE1;GLB1;GLC1C ;GNAI2;GNAT1;GP9;GPX1;HGD;HRG;I TIH1;KNG;LPP;LRS1;MCCC1;MDS1;MHS 4;MITF;MLH1;MYL3;MYMY;OPA1;P2RY 12;PBXP1;PCCB;POU1FI;PPARG;PROS 1;PTHR1;RCA1;RHO;SCA7;SCLC1;SCN 5A;SI;SLC25A20;SLC2A2;TF;TGFBR2;THPO;THRB;TKT;TM4SF1;TRH;UMPS;U QCRC1;USH3A;VHL;WS2A;XPC;ZNF35; ADH1B;ADH1C;AFP;AGA;AIH2;ALB;ASMD;BFHD;CNGA1;CRBM;DCK;DSPP;DTD P2;ELONG;ENAM;ETFDH;EVC;F11;FAB P2;FGA;FGB;FGFR3;FGG;FSHMD1A;GC ;GNPTA;GNRHR;GYPA;HCA;HCL2;HD;H TN3;HVBS6;IDUA;IF;JPD;KIT;KLKB1; LQT4;MANBA;MLLT2;MSX1;MTP;NR3C2;PBT;PDE6B;PEE1;PITX2;PKD2;QDPR ;SGCB;SLC25A4;SNCA;SOD3;STATH;TAPVR1;TYS;WBS2;WFS1;WHCR;ADAMTS 2;ADRB2;AMCN;AP3BI;APC;ARSB;B4G ALT7;BHR1;C6;C7;CCAL2;CKN1;CMDJ ;CRHBP;CSF1R;DHFR;DIAPH1;DTR;EO S;EPD;ERVR;F12;FBN2;GDNF;GHR;GLR A1;GM2A;HEXB;HSD17B4;ITGA2;KFS; LGMD1A;LOX;LTC4S;MAN2A1;MCC;MCC C2;MSH3;MSX2;NR3C1;PCSK1;PDE6A;PFBI;RASA1;SCZD1;SDHA;SGCD;SLC2 2A5;SLC26A2;SLC6A3;SM1;SMA;SMN1;SMN2;SPINK5;TCOF1;TELAB1;TGFBI ;ALDH5A1;ARG1;AS;ASSP2;BCKDHB;B F;C2;C4A;CDKN1A;COL10A1;COL11A2; CYP21A2;DYX2;EJM1;ELOVL4;EPM2A; ESR1;EYA4;F13A1;FANCE;GCLC;GJA1 ;GLYS1;GMPR;GSE;HCR;HFE;HLA-A;H LA-DPB1;HLA-DRA;HPFH;ICS1;IDDM1; IFNGR1;IGAD1;IGF2R;ISCW;LAMA2;L AP;LCA5;LPA;MCDR1;MOCS1;MUT;MYB ;NEU1;NKS1;NYS2;OA3;ODDD;OFC1;P ARK2;PBCA;PBCRA1;PDB1;PEX3;PEX6;PEX7;PKHD1;PLA2G7;PLG;POLH;PPAC ;PSORS1;PUJO;RCD1;RDS;RHAG;RP14 ;RUNX2;RWS;SCA1;SCD3;SIASD;SOD 2;ST8;TAP1;TAP2;TFAP2B;TNDM;TNF ;TPBG;TPMT;TULP1;WISP3;AASS;ABC B1;ABCB4;ACHE;AQP1;ASL;ASNS;AUT S1;BPGM;BRAF;C7orf2;CACNA2D1;CC M1;CD36;CFTR;CHORDOMA;CLCN1;CMH6 ;CMT2D;COL1A2;CRS;CYMD;DFNA5;DL D;DYT11;EEC1;ELN;ETV1;FKBP6;GCK ;GHRHR;GHS;GLI3;GPDS1;GUSB;HLXB 9;HOXA13;HPFH2;HRX;IAB;IMMP2L;KC NH2;LAMB1;LEP;MET;NCF1;NM;OGDH; OPN1SW;PEX1;PGAM2;PMS2;PON1;PPP 1R3A; PRSS1; PATC; PATN12; RAB10; RAB9; SERPAIN1; SGHEC; SHAFM1; SHAH; SLAC26A3; SLC26A4;SLOS;SMAD1;TBXAS1;TWIST ;, CCAL1;CLN8;CMT4A;CNGB3;COH1;CPP ;CRH;CYP11B1;CYP11B2;DECR1;DPYS; DURS1;EBS1;ECA1;EGI;EXT1;EYA1;F GFR1;GNRH1;GSR;GULO;HR;KCNQ3;K FM;KWE;LGCR;LPL;MCPH1;MOS;MYC;N AT1;NAT2;NBS1;PLAT;PLEC1;PRKDC;P XMP3;RP1;SCD6;SFTPC;SGM1;SPG5A ;STAR;TG;TRPS1;TTPA;VMD1;WRN;AB CA1;ABL1;ABO;ADAMTS13;AK1;ALAD; ALDH1A1;ALDOB;AMBP;AMCD1;ASS;BDM F;BSCL;C5;CDKN2A;CHAC;CLA1;CMD1 B;COL5A1;CRAT;DBH;DNAI1;DYS;DYT 1;ENG;FANCC;FBP1;FCMD;FRDA;GALT ;GLDC;GNE;GSM1;GSN;HSD17B3;HSN1;IBM2;INVS;JBTS1;LALL;LCCS1;LCCS;LGMD2H;LMX1B;MLLT3;MROS;MSSE;N OTCH1;ORM1;PAPPA;PIP5K1B;PTCH;P TGS1;RLN1;RLN2;RMRP;ROR2;RPD1;S ARDH;SPTLC1;STOM;TDFA;TEK;TMC1; TRIM32;TSC1;TYRP1;XPA;CACNB2;CO Ll7A1;CUBN;CXCL12;CYP17;CYP2C19 ;CYP2C9;EGR2;EMX2;ERCC6;FGFR2;HK 1;HPSI;IL2RA;LGI1;LIPA;MAT1A;MB L2;MKI67;MXI1;NODAL;OAT;OATL3;P AX2;PCBD;PEO1;PHYH;PNLIP;PSAP;P TEN;RBP4;RDPA;RET;SFTPA1;SFTPD;S HFM3;SIAL;THC2;TLX1;TNFRSF6;UFS;UROS;AA;ABCC8;ACAT1;ALX4;AMPD3 ;ANC;APOA1;APOA4;APOC3;ATM;BSCL 2;BWS;CALCA;CAT;CCND1;CD3E;CD3G; CD59;CDKN1C;CLN2;CNTF;CPT1A;CTS C;DDB1;DDB2;DHCR7;DLAT;DRD4;ECB 2;ED4;EVR1;EXT2;F2;FSHB;FTH1;G6 PT1;G6PT2;GIF;HBB;HBBP1;HBD;HBE1 ;HBG1;HBG2;HMBS;HND;HOMG2;HRAS; HVBS1;IDDM2;IGER;INS;JBS;KCNJ11 ;KCNJ1;KCNQ1;LDHA;LRP5;MEN1;MLL ;MYBPC3;MYO7A;NNO1;OPPG;OPTB1;PA X6;PC;PDX1;PGL2;PGR;PORC;PTH;PT S;PVRL1;PYGM;RAG1;RAG2;ROM1;RRA S2;SAA1;SCA5;SCZD2;SDHD;SERPING1;SMPD1;TCIRG1;TCL2;TECTA;TH;TRE H;TSG101;TYR;USH1C;VMD2;VRNI;WT 1;WT2;ZF145;A2M;AAS;ACADS;ACL S;ACVRL1;ALDH2;AMHR2;AOM;AQP2;A TD;ATP2A2;BDC;C1R;CD4;CDK4;CNA1;COL2A1;CYP27B1;DRPLA;ENUR2;FEOM 1;FGF23;FPF;GNB3;GNS;HAL;HBP1;HM GA2;HMN2;HPD;IGF1;KCNA1;KERA;KR AS2;KRT1;KRT2A;KRT3;KRT4;KRT5;K; RT6A;KRT6B;KRTHB6;LDHB;LYZ;MGCT ;MPE;MVK;MYL2;OAP;PAH;PPKB;PRB3 ;PTPN11;PXR1;RLS;RSN;SAS;SAX1;S CA2;SCNN1A;SMAL;SPPM;SPSMA;TBX3 ;TBX5;TCF1;TPI1;TSC3;ULR;VDR;VW F;ATP7B;BRCA2;BRCD1;CLN5;CPB2;E D2;EDNRB;ENUR1;ERCC5;F10;F7;GJB2;GJB6;IPF1;MBS1;MCOR;NYS4;PCCA; RB1;RHOK;SCD7;SGCG;SLC10A2;SLC 25A15;STARP1;ZFl98;ACHM1;ARVDI ;BCH;CTAA1;DAD1;DFNB5;EML1;GALC;GCH1;IBGC1;IGH;IGHCgroup;IGHG1; IGHM;IGHR;IV;LTBP2;MJD;MNG1;MPD1;MPS3C;MYH6;MYH7;NP;NPC2;PABN1 ;PSEN1;PYGL;RPGRIP1;SERPINA1;SE RPINA3;SERPINA6;SLC7A7;SPG3A;SPT B;TCL1A;TGMI;TITF1;TMIP;TRA;TSH R;USH1A;VP;ACCPN;AHO2;ANCR;B2M; BBS4;BLM;CAPN3;CDAN1;CDAN3;CLN6 ;CMH3;CYP19;CYP1A1;CYP1A2;DYX1;E PB42;ETFA;EYCL3;FAH;FBN1;FES;HC VS;HEXA;IVD;LCS1;LIPC;MYO5A;OCA 2;OTSC1;PWCR;RLBP1;SLC12A1;SPG6 ;TPM1;UBE3A;WMS;ABCC6;ALDOA;APRT ;ATP2A1;BBS2;CARD15;CATM;CDH1;C ETP;CHST6;CLN3;CREBBP;CTH;CTM;C YBA;CYLD;DHS;DNASE1;DPEP1;ERCC4 ;FANCA;GALNS;GAN;HAGH;HBA1;HBA2; HBHR;HBQ1;HB, L4R;LIPB、MC1R;MEFV;MHC2TA;MLYCD ;MMVP1;PHKB;PHKG2;PKD1;PKDTS;PM M2;PXE;SALL1;SCA4;SCNN1B;SCNN1G;SLC12A3;TAT;TSC2;VDI;WT3;ABR;ACACA;ACADVL;ACE;ALDH3A2;APOH;ASP A;AXIN2;BCL5;BHD;BLMH;BRCA1;CAC D;CCA1;CCS;CHRNB1;CHRNE;CMT1A;C OL1A1;CORD5;CTNS;EPX;ERBB2;G6PC ;GAA;GALK1;GCGR;GFAP;GH1;GH2;GP 1BA;GPSC;GUCY2D;ITGA2B;ITGB3;IT GB4;KRT10;KRT12;KRT13;KRT14;KRT1 4L1;KRT14L2;KRT14L3;KRT16;KRT16 L1;KRT16L2;KRT17;KRT9;MAPT;MDB; MDCR;MGI;MHS2;MKS1;MPO;MYO15A;N AGLU;NAPB;NF1;NME1;P4HB;PAFAH1B1 ;PECAM1;PEX12;PHB;PMP22;PRKAR1A ;PRKCA;PRKWNK4;PRP8;PRPF8;PTLAH ;RARA;RCV1;RMSA1;RP17;RSS;SCN4A;SERPINF2;SGCA;SGSH;SHBG;SLC2A4; SLC4A1;SLC6A4;SMCR;SOST;SOX9;SS TR2;SYM1;SYNS1;TCF2;THRA;TIMP2; TOC;TOP2A;TP53;TRIM37;VBCH;ATP8 B1;BCL2;CNSN;CORD1;CYB5;DCC;F5F8 D;FECH;FEO;LAMA3;LCFS2;MADH4;MA FD1;MC2R;MCL;MYP2;NPC1;SPPK;TGF BRE;TGIF;TTR;AD2;AMH;APOC2;APOE ;ATHS;BAX;BCKDHA;BCL3;BFIC;C3;CA CNA1A;CCO;CEACAM5;COMP;CRX;DBA; DDU;DFNA4;DLL3;DM1;DMWD;E11S;EL A2;EPOR;ERCC2;ETFB;EXT3;EYCL1;FTL;FUT1;FUT22;FUT6;GAMT;GCDH;GPI; GUSM;HB1;HCL1;HHC2;HHC3;ICAM3;I NSR;JAK3;KLK3;LDLR;LHB;LIG1;LOH1 9CR1;LYL1;MAN2B1;MCOLN1;MDRV;ML LT1;NOTCH3;NPHS1;OFC3;OPA3;PEPD;PRPF31;PRTN3;PRX;PSG1;PVR;RYR1; SLC5A5;SLC7A9;STK11;TBXA2R;TGFB 1;TNNI3;TYROBP;ADA;AHCY;AVP;CDA N2;CDPD1;CHED1;CHED2;CHRNA4;CST 3;EDN3;EEGV1;FTLL1;GDF5;GNAS;GS S;HNF4A;JAG1;KCNQ2;MKKS;NBIA1;P CK1;PI3;PPCD;PPGB;PRNP;THBD;TOP 1;AIRE;APP;CBS;COL6A1;COL6A2;CST B;DCR;DSCR1;FPDMM;HLCS;HPE1;ITG B2;KCNE1;KNO;PRSS7;RUNX1;SOD1;T AM;ADSL;ARSA;BCR;CECR;CHEK2;COMT;CRYBB2;CSF2RB;CTHM;CYP2D6;CYP2 D7P1;DGCR;DIA1;EWSR1;GGT1;MGCR; MN1;NAGA;NF2;OGS2;PDGFB;PPARA;P RODH;SCO2;SCZD4;SERPIND1;SLC5A1;SOX10;TCN2;TIMP3;TST;VCF;ABCD1; ACTL1;ADFN;AGMX2;AHDS;AIC;AIED; AIH3;ALAS2;AMCD;AMELX;ANOP1;AR; ARAF1;ARSC2;ARSE;ARTS;ARX;ASAT; ASSP5;ATP7A;ATRX;AVPR2;BFLS;BGN; BTK;BZX;C1HR;CACNA1F;CALB3;CBBM ;CCT;CDR1;CFNS;CGF1;CHM;CHR39C; CIDX;CLA2;CLCN5;CLS;CMTX2;CMTX3 ;CND;COD1;COD2;COL4A5;COL4A6;CPX ;CVD1;CYBB;DCX;DFN2;DFN4;DFN6;D HOF;DIAPH2;DKC1;DMD;DSS;DYT3;EB M;EBP;ED1;ELK1;EMD;EVR2;F8;F9;F CP1;FDPSL5;FGD1;FGS1;FMR1;FMR2;G 6PD;GABRA3;GATA1;GDI1;GDXY;GJB1 ;GK;GLA;GPC3;GRPR;GTD;GUST;HMS1 ;HPRT1;HPT;HTC2;HTR2C;HYR;IDS;I HG1;IL2RG;INDX;IP1;IP2;JMS;KAL1;KFSD;L1CAM;LAMP2;MAA;MAFD2;MAOA ;MAOB;MCF2;MCS;MEAX;MECP2;MF4;M GC1;MIC5;MID1;MLLT7;MLS;MRSD;MR X14;MRX1;MRX20;MRX2;MRX3;MRX40; MRXA;MSD;MTM1;MYCL2;MYP1;NDP;NH S;NPHL1;NR0B1;NSX;NYS1;NYX;OA1; OASD;OCRL;ODT1;OFD1;OPA2;OPD1;O PEM;OPN1LW;OPN1MW;OTC;P3;PDHA1;P DR;PFC;PFKFB1;PGK1;PGK1P1;PGS;P HEX;PHKA1;PHKA2;PHP;PIGA;PLP1;P OF1;POLA;POU3F4;PPMX;PRD;PRPS1; PRPS2;PRS;RCCP2;RENBP;RENS1;RP2 ;RP6;RPGR;RPS4X;RPS6KA3;RS1;S11 ;SDYS;SEDL;SERPINA7;SH2D1A;SHFM 2;SLC25A5;SMAX2;SRPX;SRS;STS;SY N1;SYP;TAF1;TA;TBX22;TDD;TFE3;T HAS;THC;TIMM8A;TIMP1;TKCR;TNFSF 5;UBE1;UBE2A;WAS;WSN;WTS;WWS;XI C;XIST;XK;XM;XS;ZFX;ZIC3;ZNF261 ;: F2;DAZ;GCY;RPS4Y;SMCY;ZFY;ABAT;AEZ;AFA;AFD1;ASAH1;ASD1;ASMT;CC AT;CECR9;CEPA;CLA3;CLN4;CSF2RA; CTS1;DF;DIH1;DWS;DYT2;DYT4;EBR3; ECT;EEF1A1L14;EYCL2;FANCB;GCSH; GCSL;GIP;GTS;HHG;HMI;HOAC;HOKPP 2;HRPT1;HSD3B3;HTC1;HV1S;ICHQ;I CR1;ICR5;IL3RA;KAL2;KMS;KRT18;KS S;LCAT;LHON;LIMM;MANBB;MCPH2;ME B;MELAS;MIC2;MPFD;MS;MSS;MTATP6 ;MTCO1;MTCO3;MTCYB;MTND1;MTND2; MTND4;MTND5;MTND6;MTRNR1;MTRNR2;7. The method of any one of claims 1 to 6, wherein the target gene is selected from the group consisting of MTTE; MTTG; MTTI; MTTK; MTTL1; MTTL2; MTTN; MTTP; MTTS1; NAMSD; OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1; RBS; RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; and TTD.

8. The mammalian cells may be antigen presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglial cells, neurons, spleen cells, lymphoid cells, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, superficial alveolar cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colony epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Muller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, axonal cells, beta cells, thecal cells, cement cells, chief cells, chondroblasts, chondrocytes, chromaffin cells , chromophobe cells, corticotropes, delta cells, Langerhans cells, follicular dendritic cells, enterochromaffin cells, ependymal cells, basal cells, squamous cells, transitional cells, erythroblasts, red blood cells, fibroblasts, fibrocytes, follicular cells, germ cells, gametes, eggs, sperm, oocytes, primary oocytes, secondary oocytes, immotile sperm, spermatocytes, primary spermatocytes, secondary spermatocytes, germ epithelium, giant cells, glial cells, astroblasts, oligodendrocytes, oligodendrocytes, glioblasts, goblet cells, gonadotropins, granulosa cells, hemoblasts, hair cells, hepatoblasts, hepatocytes, vitreous cells, stromal cells, juxtaglomerular cells, keratinocytes, keratocytes, lemma cells cell), leukocytes, granulocytes, basophils, eosinophils, neutrophils, lymphoblasts, B lymphoblasts, T lymphoblasts, lymphocytes, B lymphocytes, T lymphocytes, helper induced T-lymphocytes, Th1 T lymphocytes, Th2T lymphocyte, natural killer cell, thymocyte, Kupffer cell, alveolar macrophage, foam cell, histiocyte, lutein cell, lymphoid stem cell, lymphoid cell, immune stem cell, astroglia, lactotroph, mast cell, medulloblast, megakaryoblast, megakaryocyte, melanoblast, melanocyte, mesangial cell, mesothelial cell, metamyelocyte, monoblast, monocyte, gastric mucous cell, myoblast, myocyte, cardiac muscle cell, skeletal muscle cell, smooth muscle cell, myelocyte, myeloid cell, bone marrow stem cell, 8. The method of any one of claims 1 to 7, wherein the cell is a myoblast, myoepithelial cell, myofibroblast, neuroblast, neuroepithelial cell, neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell, parafollicular cell, paraluteal cell, digestive cell, pericyte, peripheral blood mononuclear cell, pheochromocytocyte, supporting cell, pineal cell, pituitary cell, plasma cell, platelet, tectal cell, proerythroblast, promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte, stem cell, Sertoli cell, terminal glial cell, or zymogen cell.

9. 1. Use of a preparation of in vitro synthesized mRNA in the preparation of a medicament for the production of a recombinant protein in mammalian cells present in vitro in gene therapy, comprising: The in vitro synthesized mRNA comprises an open reading frame encoding the recombinant protein, is obtained by in vitro transcription (IVT), and contains Ψ (5-β-D-ribofuranosyluracil), with 0.1 to 100% of the uridine residues in the RNA being replaced with Ψ (5-β-D-ribofuranosyluracil), including cases where all uridine residues are replaced with Ψ (5-β-D-ribofuranosyluracil); and the in vitro synthesized mRNA preparation is purified using a purification process that removes RNA contaminant molecules that are immunogenic and toxic to the cells by inducing an innate immune response; the RNA contaminant molecules include double-stranded RNA contaminant molecules; The mRNA is determined to be purified by measuring reduced secretion of IFN-α or TNF-α cytokines by dendritic cells transfected with the purified in vitro synthesized mRNA preparation compared to secretion of the cytokines by dendritic cells transfected with an unpurified in vitro synthesized mRNA preparation.

10. The use according to claim 9, wherein all of the uridine residues are replaced with Ψ (5-β-D-ribofuranosyluracil).

11. The recombinant protein may be selected from erythropoietin (EPO); a detection enzyme selected from firefly luciferase, Renilla luciferase, bacterial β-galactosidase (lacZ), and green fluorescent protein (GFP); a transcriptional regulator selected from MYC and SRY or MCOP; platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), insulin-like growth factor (IGF), α-melanocyte-stimulating hormone (α-MSH), insulin-like growth factor-I (IGF-I), IL-4 a growth factor or cytokine selected from the group consisting of IL-13, IL-10, and IL-13; inducible nitric oxide synthase (iNOS); cystic fibrosis transmembrane conductance regulator (CFTR); an enzyme having antioxidant activity selected from catalase, phospholipid hydroperoxide glutathione peroxidase, superoxide dismutase-1, and superoxide dismutase-2; Bruton's tyrosine kinase; adenosine deaminase; ecto-nucleoside triphosphate diphosphohydrolase; ABCA4; ABCD3; ACADM; AGL; A GT; ALDH4Al; 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CNA1;COL2A1;CYP27B1;DRPLA;ENUR2;FEOM1;FGF23;FPF;GNB3;GNS;HAL;HB P1;HMGA2;HMN2;HPD;IGF1;KCNA1;KE RA;KRAS2;KRT1;KRT2A;KRT3;KRT4;K RT5;KRT6A;KRT6B;KRTHB6;LDHB;LYZ ;MGCT;MPE;MVK;MYL2;OAP;PAH;PPKB ;PRB3;PTPN11;PXR1;RLS;RSN;SAS;S AX1;SCA2;SCNN1A;SMAL;SPPM;SPSMA;TBX3;TBX5;TCF1;TPI1;TSC3;ULR;V DR;VWF;ATP7B;BRCA2;BRCD1;CLN5;C PB2;ED2;EDNRB;ENUR1;ERCC5;F10;F 7;GJB2;GJB6;IPF1;MBS1;MCOR;NYS4 ;PCCA;RB1;RHOK;SCD7;SGCG;SLC10A 2;SLC25A15;STARP1;NFl98;ACHM1; ARVDI;BCH;CTAA1;DAD1;DFNB5;EML1 ;GALC;GCH1;IBGC1;IGH;IGHCgroup; IGHG1;IGHM;IGHR;IV;LTBP2;MJD;MNG 1;MPD1;MPS3C;MYH6;MYH7;NP;NPC2; PABN1;PSEN1;PYGL;RPGRIP1;SERPIN A1;SERPINA3;SERPINA6;SLC7A7;SPG3A;SPTB;TCL1A;TGMI;TITF1;TMIP;TR A;TSHR;USH1A;VP;ACCPN;AHO2;ANCR ;B2M;BBS4;BLM;CAPN3;CDAN1;CDAN3 ;CLN6;CMH3;CYP19;CYP1A1;CYP1A2;DYX1;EPB42;ETFA;EYCL3;FAH;FBN1; FES;HCVS;HEXA;IVD;LCS1;LIPC;MYO 5A;OCA2;OTSC1;PWCR;RLBP1;SLC12A 1;SPG6;TPM1;UBE3A;WMS;ABCC6;ALD OA;APRT;ATP2A1;BBS2;CARD15;CATM; CDH1;CETP;CHST6;CLN3;CREBBP;CTH ;CTM;CYBA;CYLD;DHS;DNASE1;DPEP1 ;ERCC4;FANCA;GALNS;GAN;HAGH;HBA 1;HBA2;HBHR;HBQ1;HBZ;HBZP;HP;HSD 11B2;IL4R;LIPB、MC1R;MEFV;MHC2TA ;MLYCD;MMVP1;PHKB;PHKG2;PKD1;PK DTS;PMM2;PXE;SALL1;SCA4;SCNN1B; 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The use according to claim 9 or 10, wherein the target gene is selected from the group consisting of MTTL1; MTTL2; MTTN; MTTP; MTTS1; NAMSD; OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1; RBS; RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; and TTD.

12. 1. A purified preparation of in vitro synthesized mRNA for use in gene therapy, comprising: The in vitro synthesized mRNA is obtained by in vitro transcription (IVT) and contains Ψ (5-β-D-ribofuranosyluracil), with 0.1 to 100% of the uridine residues in the RNA being replaced with Ψ (5-β-D-ribofuranosyluracil), including cases where all uridine residues are replaced with Ψ (5-β-D-ribofuranosyluracil); and the in vitro synthesized mRNA preparation is purified using a purification process that removes RNA contaminant molecules that are immunogenic and toxic to the cells by inducing an innate immune response; the RNA contaminant molecules include double-stranded RNA contaminant molecules; the mRNA is determined to be purified by measuring reduced secretion of IFN-α or TNF-α cytokines by dendritic cells transfected with the purified in vitro synthesized mRNA preparation compared to secretion of the cytokines by dendritic cells transfected with an unpurified in vitro synthesized mRNA preparation; A purified preparation of in vitro synthesized mRNA for use in gene therapy, wherein the gene therapy comprises administering the purified preparation of in vitro synthesized mRNA to a subject.

13. A purified preparation of in vitro synthesized mRNA for use in gene therapy as described in claim 12, wherein all of the uridine residues have been replaced with Ψ (5-β-D-ribofuranosyluracil).

14. 12. The use of claim 9, 10 or 11, wherein less than 0.01% of the total RNA in said preparation of purified in vitro synthesized mRNA consists of double-stranded RNA contaminant molecules.

15. 15. The use of claim 9, 10, 11 or 14, wherein as a result of the purification process, the in vitro synthesized mRNA preparation can be administered repeatedly without eliciting an immune response sufficient to eliminate detectable expression of the recombinant protein, or the in vitro synthesized mRNA preparation lacks immunogenicity such that repeated delivery is possible without the generation of proinflammatory cytokines.

16. The purification step comprises: A. Purification of said in vitro synthesized mRNA preparation using HPLC or gravity flow column purification; and / or B. Treating the in vitro synthesized mRNA preparation with ribonuclease III (RNase III) enzyme to produce short RNase III digestion products, and purifying the mRNA preparation to remove the short RNase III digestion products.

16. The use according to claim 9, 10, 11, 14 or 15, comprising:

17. The in vitro synthesized mRNA contains 5-methylcytidine (m ５ 17. The use of claim 9, 10, 11, 14, 15 or 16, further comprising C).

18. 11. The use of claim 9 or 10, wherein the immunogenicity is further determined by measuring the secretion of IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1β, IL-6, IFN-β, or IL-8.

19. 11. The use according to claim 9 or 10, wherein the immunogenicity is determined by measuring the secretion of a cytokine, preferably IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1β, IL-6, IFN-β or IL-8.

20. 1. A method for producing a pharmaceutical composition comprising a purified preparation of in vitro synthesized mRNA molecules that are immunogenic in mammalian cells, comprising: The in vitro synthesized mRNA is obtained by in vitro transcription (IVT) and contains Ψ (5-β-D-ribofuranosyluracil), with 0.1 to 100% of the uridine residues in the RNA being replaced with Ψ (5-β-D-ribofuranosyluracil), including cases where all uridine residues are replaced with Ψ (5-β-D-ribofuranosyluracil); and purifying said in vitro synthesized mRNA preparation with a step of removing RNA contaminant molecules that are immunogenic and toxic to said cells by inducing an innate immune response; the RNA contaminant molecules include double-stranded RNA contaminant molecules; The purification of the mRNA is determined by measuring reduced secretion of IFN-α or TNF-α cytokines by dendritic cells transfected with the purified in vitro synthesized mRNA preparation compared to secretion of the cytokines by dendritic cells transfected with an unpurified in vitro synthesized mRNA preparation.

21. The method of claim 20, wherein all of the uridine residues are replaced with Ψ (5-β-D-ribofuranosyluracil).

22. 14. A purified preparation of in vitro synthesized mRNA for use in gene therapy according to claim 12 or 13, wherein less than 0.01% of the total RNA in said purified in vitro synthesized mRNA preparation consists of double-stranded RNA contaminant molecules.

23. 23. A purified preparation of in vitro synthesized mRNA for use in gene therapy according to claim 12, 13 or 22, wherein as a result of the purification process, the purified preparation of in vitro synthesized mRNA can be administered without eliciting an immune response sufficient to eliminate detectable expression of the recombinant protein, or the in vitro synthesized mRNA lacks immunogenicity such that delivery is possible without the generation of pro-inflammatory cytokines.

24. The purification step comprises: A. Purification of said in vitro synthesized mRNA preparation using HPLC or gravity flow column purification; and / or B. Treating the in vitro synthesized mRNA preparation with ribonuclease III (RNase III) enzyme to produce short RNase III digestion products, and purifying the mRNA preparation to remove the short RNase III digestion products.

24. A purified preparation of in vitro synthesized mRNA for use in gene therapy according to claim 12, 13, 22 or 23, comprising:

25. The in vitro synthesized mRNA contains 5-methylcytidine (m ５ 25. A purified preparation of in vitro synthesized mRNA for use in gene therapy according to claim 12, 13, 22, 23 or 24, further comprising: C).

26. 14. The purified preparation of in vitro synthesized mRNA for use in gene therapy of claim 12 or 13, wherein the immunogenicity is further determined by measuring the secretion of IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1β, IL-6, IFN-β, or IL-8.

27. 14. A purified preparation of in vitro synthesized mRNA for use in gene therapy according to claim 12 or 13, wherein the immunogenicity is determined by measuring the secretion of a cytokine, preferably IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1β, IL-6, IFN-β or IL-8.

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