WO2013071047A1

WO2013071047A1 - Compositions and methods for in vitro transcription of rna

Info

Publication number: WO2013071047A1
Application number: PCT/US2012/064359
Authority: WO
Inventors: Derrick Rossi; Wataru EBINA; Pankaj MANDAL
Original assignee: Children's Medical Center Corporation
Priority date: 2011-11-11
Filing date: 2012-11-09
Publication date: 2013-05-16

Abstract

Provided herein are IVT templates and constructs for rapidly and efficiently generating synthesized RNAs.

Description

COMPOSITIONS AND METHODS FOR IN VITRO TRANSCRIPTION OF RNA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional

Application Serial No.: 61/558, 563 filed November 11, 2011, the contents of which are herein incorporated by reference in their entireties.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in

ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 6, 2012, is named 03339307.txt and is 128,526 bytes in size.

FIELD OF THE INVENTION

[0003] The field of the invention relates to compositions, kits, and methods for synthesis and in vitro transcription of RNAs, such as modified RNAs.

BACKGROUND

[0004] In vitro transcription (IVT) is a biochemical process of mRNA synthesis in a cell-free transcription system in which an RNA polymerase assembles ribonucleotides using DNA as template. The template typically contains an open reading frame (ORF) that is often, but not exclusively, comprised of the complete coding sequence of a gene, along with 5' and 3' regulatory elements and a polyA tail. Therefore, a quality template DNA is a prerequisite for efficient IVT of standard mRNAs or those mRNAs synthesized with modified nucleosides. However, template DNA construction is a serious bottleneck in the process of mRNA synthesis.

SUMMARY

[0005] The present invention is based on the novel construction of a vector system or a nucleic acid construct which allows making nucleic acid constructs comprising open reading frames (ORF) for in vitro transcription simple, efficient and scalable. The vector system of the invention allows the use of essentially identical conditions for cloning any nucleic acid into the newly constructed vector thereby significantly increasing efficiency of the cloning process.

[0006] Accordingly, provided herein are compositions, methods, and kits for in vitro transcription (IVT) template construction that are simple, efficient, and readily scalable. These compositions, methods, and kits can be used to generate RNAs, such as modified RNAs, that can be used, for example, to express a desired protein in a cell or tissue, or to change the differentiated phenotype of a cell to that of another, desired cell type. [0007] In some aspects, provided herein are isolated nucleic acid sequence comprising, in the

5' to 3' direction: a first nucleic acid sequence comprising a forward universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence, a second blunt-ended restriction enzyme digestion site, a 3' UTR sequence, and a second nucleic acid sequence comprising a sequence complementary to a reverse universal primer sequence.

[0008] In some embodiments of these aspects and all such aspects described herein, the 5'

UTR sequence comprises the sequence of SEQ ID NO: 20.

[0009] In some embodiments of these aspects and all such aspects described herein, the first blunt-ended restriction enzyme digestion site comprises the sequence of SEQ ID NO: 60.

[0010] In some embodiments of these aspects and all such aspects described herein, the second blunt-ended restriction enzyme digestion site comprises the sequence of SEQ ID NO: 61.

[0011] In some embodiments of these aspects and all such aspects described herein, the 3'

UTR sequence comprises the sequence of SEQ ID NO: 21.

[0012] In some embodiments of these aspects and all such aspects described herein, the nucleic acid sequence further comprises a vector backbone sequence.

[0013] In some embodiments of these aspects and all such aspects described herein, the 3'

UTR followed by a second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence is not operably linked to a poly-A tail sequence.

[0014] In one aspect of all the embodiments and aspects of the invention, the isolated nucleic acid sequence further comprises a nucleic acid encoding an open reading frame (ORF) sequence between the first and the second blunt-ended restriction enzyme digestion sites.

[0015] In some embodiments of these aspects and all such aspects described herein, the ORF sequence excludes the 5' adenine nucleotide prior to inserting it between the first and the second blunt-ended restriction sites.

[0016] Also provided herein, in some aspects, are libraries of nucleic acids. Accordingly, in some aspects, provided herein are a plurality of isolated nucleic acids encoding a plurality of open reading frames comprised within a vector, each said isolated nucleic acid comprising: in the 5' to 3' direction: a first nucleic acid sequence comprising a forward universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence, a second blunt-ended restriction enzyme digestion site, a 3' UTR sequence, a second nucleic acid sequence comprising a sequence complementary to a reverse universal primer sequence, and a vector backbone sequence.

[0017] In some embodiments of these aspects and all such aspects described herein, the 5'

UTR sequence of at least one of said plurality of isolated nucleic acids comprises the sequence of SEQ ID NO: 20. [0018] In some embodiments of these aspects and all such aspects described herein, the first blunt-ended restriction enzyme digestion site of at least one of said plurality of isolated nucleic acids comprises the sequence of SEQ ID NO: 60.

[0019] In some embodiments of these aspects and all such aspects described herein, the second blunt-ended restriction enzyme digestion site of at least one of said plurality of isolated nucleic acids comprises the sequence of SEQ ID NO: 61.

[0020] In some embodiments of these aspects and all such aspects described herein, the 3'

UTR sequence of at least one of said plurality of isolated nucleic acids comprises the sequence of SEQ ID NO: 21.

[0021] In some aspects, the invention provides kits comprising the isolated nucleic acid of any one of any of the aspects or embodiments described herein in a suitable container. The kit can optionally, in some embodiments, comprise instructions for cloning nucleic acid fragments into the vector part, namely the nucleic acid sequence which does not comprise an ORF, and/or instructions for using the constructs for in vitro transcription.

[0022] In some embodiments of these aspects and all such aspects described herein, the kit further comprises a first and a second blunt-ended restriction enzyme specific for the first and second blunt-ended restriction enzyme digestion sites respectively.

[0023] In some embodiments of these aspects and all such aspects described herein, the kit further comprises a first universal primer comprising the forward universal primer sequence and a second universal primer comprising a poly-T sequence.

[0024] In some aspects, the invention provides a kit comprising a plurality of nucleic acids in a suitable container.

[0025] In some embodiments of these kits and all such kits described herein, the 5' UTR sequence of at least one of said plurality of isolated nucleic acids comprises the sequence of SEQ ID NO: 20.

[0026] In some embodiments of these kits and all such kits described herein, the first blunt- ended restriction enzyme digestion site of at least one of said plurality of isolated nucleic acids comprises SEQ ID NO: 60.

[0027] In some embodiments of these kits and all such kits described herein, the second blunt-ended restriction enzyme digestion site of at least one of said plurality of isolated nucleic acids comprises SEQ ID NO: 61.

[0028] In some embodiments of these kits and all such kits described herein, the 3' UTR sequence of at least one of said plurality of isolated nucleic acids comprises the sequence of SEQ ID NO: 21.

[0029] In some embodiments of these kits and all such kits described herein, at least one isolated nucleic acid further comprises a vector backbone sequence. [0030] In some embodiments of these kits and all such kits described herein, the 3' UTR followed by the second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence of at least one of said plurality of isolated nucleic acids is not operably linked to a poly-A tail sequence.

[0031] In other aspects, the invention also provides methods of synthesizing a nucleic acid construct for transcribing a gene of interest in vitro. Such methods comprise the steps of:

a. amplifying an open reading frame (ORF) sequence of a gene of interest to form an ORF amplification product using a phosphorylated forward primer and a

phosphorylated reverse primer, and omitting the 5 'adenosine of the ORF from the ORF amplification product;

b. digesting a vector comprising, in the 5' to 3' direction: a first nucleic acid sequence comprising a first universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence, a second blunt-ended restriction enzyme digestion site operably linked to a 3' UTR sequence, a second nucleic acid sequence comprising a sequence

complementary to a second universal primer sequence, and a vector backbone sequence; with a first and a second blunt-ended restriction enzyme specific for the first and second blunt-ended restriction enzyme digestion sites respectively;

c. dephosphorylating the digested vector with a phosphatase enzyme; and

d. contacting the phosphorylated ORF amplification product with the digested and

dephosphorylated vector in the presence of a DNA ligase, thereby incorporating the ORF amplification product into the digested vector and generating the nucleic acid construct.

[0032] In some embodiments of these methods and all such methods described herein, the method further comprises screening for proper orientation of the ORF sequence comprising the steps of:

e. amplifying the ORF sequence with a forward primer comprising the first universal primer sequence and a reverse primer comprising a sequence specific for the 3' end of the ORF sequence; and

f. selecting the nucleic acid constructs wherein the ORF sequence is in the proper 5' to 3' orientation.

[0033] In some embodiments of these methods and all such methods described herein, the method further comprises a step of adding a poly-A tail to the ORF sequence by amplifying the nucleic acid construct with a forward primer comprising the first universal primer sequence and a reverse primer sequence comprising a poly-T sequence.

Definitions [0034] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" generally refer to any polyribonucleotide or poly-deoxyribonucleotide, and includes unmodified RNA, unmodified DNA, modified RNA, and modified DNA. Polynucleotides include, without limitation, single- and double-stranded DNA and RNA polynucleotides. The term polynucleotide, as it is used herein, embraces chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the naturally occurring chemical forms of DNA and RNA found in or characteristic of viruses and cells, including for example, simple (prokaryotic) and complex (eukaryotic) cells. A nucleic acid polynucleotide or oligonucleotide as described herein retains the ability to hybridize to its cognate complimentary strand.

[0036] Accordingly, as used herein, the terms "nucleic acid," "polynucleotide," and

"oligonucleotide" also encompass primers and probes, as well as oligonucleotide fragments, and is generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides

(containing D-ribose), and to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including, but not limited to, abasic sites). There is no intended distinction in length between the term "nucleic acid," "polynucleotide," and "oligonucleotide," and these terms are used interchangeably. These terms refer only to the primary structure of the molecule. An oligonucleotide is not necessarily physically derived from any existing or natural sequence, but can be generated in any manner, including chemical synthesis, DNA replication, DNA amplification, n vitro transcription, reverse transcription or any combination thereof

[0037] As used herein, the terms "synthetic, modified RNA" or "modified RNA" refer to an

RNA molecule produced in vitro, using any of the IVT templates and methods of use thereof described herein, which comprises at least one modified nucleoside as that term is defined herein below. The synthetic, modified RNAs do not encompass mRNAs that are isolated from natural sources such as cells, tissue, organs etc., having those modifications, but rather only synthetic, modified RNAs that are synthesized using in vitro techniques, as described herein.

[0038] The terms "nucleotide" or "mononucleotide," as used herein, refer to a phosphate ester of a nucleoside, e.g., mono-, di-, tri-, and tetraphosphate esters, wherein the most common site of esterification is the hydroxyl group attached to the C-5 position of the pentose (or equivalent position of a non-pentose "sugar moiety"). The term "nucleotide" includes both a conventional nucleotide and a non-conventional nucleotide which includes, but is not limited to, phosphorothioate, phosphite, ring atom modified derivatives, and the like.

[0039] As used herein, the term "conventional nucleotide" refers to one of the "naturally occurring" deoxynucleotides (dNTPs), including dATP, dTTP (or TTP), dCTP, dGTP, dUTP, and dITP.

[0040] As used herein, the term "non-conventional nucleotide" refers to a nucleotide that is not a naturally occurring nucleotide The term "naturally occurring" refers to a nucleotide that exists in nature without human intervention. In contradistinction, the term "non-conventional nucleotide" refers to a nucleotide that exists only with human intervention, i.e., an "artificial nucleotide." A "non- conventional nucleotide" can include a nucleotide in which the pentose sugar and/or one or more of the phosphate esters is replaced with a respective analog. Exemplary phosphate ester analogs include, but are not limited to, alkylphosphonates, methylphosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates,

phosphoroanilothioates, phosphoroanilidates, phosphoroamidates, boronophosphates, etc., including any associated counterions, if present. A non-conventional nucleotide can show a preference of base pairing with another non-conventional or "artificial" nucleotide over a conventional nucleotide (e.g., as described in Ohtsuki et al. 2001, Proc. Natl. Acad. Sci., 98: 4922-4925, hereby incorporated by reference). The base pairing ability may be measured by the T7 transcription assay as described in Ohtsuki et al. (supra). Other non-limiting examples of "non-conventional" or "artificial" nucleotides can be found in Lutz et al. (1998) Bioorg. Med. Chem. Lett., 8: 1149-1152); Voegel and Benner (1996) Helv. Chim. Acta 76, 1863-1880; Horlacher et al. (1995) Proc. Natl. Acad. Sci., 92: 6329- 6333; Switzer et al. (1993), Biochemistry 32: 10489-10496; Tor and Dervan (1993) J. Am. Chem. Soc. 115: 4461-4467; Piccirilli et al. (1991) Biochemistry 30: 10350-10356; Switzer et al. (1989) J. Am. Chem. Soc. I l l : 8322-8323, all of which are hereby incorporated by reference. A "non-conventional nucleotide" can also be a degenerate nucleotide or an intrinsically fluorescent nucleotide.

[0041] As used herein the term "modified ribonucleoside" refers to a ribonucleoside that encompasses modification(s) relative to the standard guanine (G), adenine (A), cytidine (C), and uridine (U) nucleosides. Such modifications can include, for example, modifications normally introduced post-transcriptionally to mammalian cell mRNA, and artificial chemical modifications, as known to one of skill in the art.

[0042] As used herein, the term "nonextendable nucleotide" refers to nucleotides that prevent extention of a polynucleotide chain by a polymerase. Examples of such nucleotides include dideoxy nucleotides (ddA, ddT, ddG, ddC) that lack a 3'-hydroxyl on the ribose ring, thereby preventing 3' extension by DNA polymerases. Other examples of such nucleotides include, but are not limited to, inverted bases, which can be incorporated at the 3'-end of an oligo, leading to a 3'-3' linkage, which inhibits extension by DNA polymerases. [0043] Because mononucleotides are reacted to make poly- and oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring, and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also can be said to have 5' and 3' ends.

[0044] As used herein, "complementary" refers to the ability of a single strand of a nucleic acid (or portion thereof) to hybridize to an anti-parallel nucleic acid strand (or portion thereof) by contiguous base-pairing, i.e., hydrogen bonding, between the nucleotides of the anti-parallel nucleic acid single strands, thereby forming a double-stranded polynucleotide comprising the complementary strands. A first nucleic acid is said to be "completely complementary" to a second nucleic acid strand if each and every nucleotide of the first nucleic acid forms a hydrogen-bonded base-pair with nucleotides within the complementary region of the second nucleic acid. A first nucleic acid sequence is not completely complementary (i.e., "partially complementary") to a second nucleic acid sequence if at least one nucleotide in the first nucleic acid sequence does not base pair with the corresponding nucleotide in the second nucleic acid sequence.

[0045] The degree of complementarity between different nucleic acid strands has significant effects on the efficiency and strength of annealing or hybridization between polynucleotide strands. This is of particular importance in transcription and amplification reactions, such as those described herein, which depend upon binding and annealing between polynucleotide strands. Generally, the 3' terminal nucleotide of a primer must base pair with a corresponding nucleotide on the nucleic acid for which it is designed to be specific for a template-dependent polymerase enzyme to extend the primer. Accordingly, it is understood that a primer that is said to be "specific for" a nucleic acid sequence comprises at least a portion of sequence at its 3' end that is completely complementary to or has a high degree of complementarity to a portion of the sequence of the nucleic acid.

[0046] As used herein, "in vitro transcription" or "IVT" refer to the process whereby transcription occurs in vitro in a non-cellular system to produce "synthetic RNA molecules" for use in various applications, including the production of protein or polypeptides. The synthetic RNA molecules or "transcription products" generated can be translated in vitro or introduced directly into cells, where they can be translated. Such synthetic transcription products include mRNAs, antisense RNA molecules, shRNA molecules, ribozymes, and the like. An IVT reaction typically requires a purified linear DNA template comprising a promoter sequence and the sequence of the open reading frame of interest, ribonucleotide triphosphates or modified ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase. [0047] An "IVT template" or "IVT template sequence" refers to an isolated nucleic acid sequence that comprises the minimal component sequences required for in vitro transcription of an inserted open reading frame of interest.

[0048] The terms "promoter" or "promoter sequence," as used herein, refer to a nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving RNA polymerase- mediated transcription of the nucleic acid sequence, which can be a heterologous target gene, such as one encoding a protein or an RNA. A promoter is a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled.

Promoters can be constitutive, inducible, activateable, repressible, tissue-specific, or any combination thereof. A promoter can be said to drive expression or drive transcription of the nucleic acid sequence that it regulates.

[0049] The phrases "operably linked," "operatively positioned," "operatively linked," "under control," and "under transcriptional control" indicate that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence it regulates to control transcriptional initiation and/or expression of that sequence.

[0050] As used herein, the terms "five prime untranslated region" or "5' UTR" refer to the sequence of an mRNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region of an RNA.

[0051] As used herein, the terms "three prime untranslated region" or "3' UTR" refer to the sequence of an mRNA molecule that begins following the stop codon of the coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after the stop codon of the coding region of an open reading frame sequence. In other embodiments, the the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence

[0052] As used herein, "blunt-ended restriction sequences" or "blunt-ended restriction site sequences" refer to a specific sequence of nucleotides that can be recognized by a specific restriction enzyme or restruction endonuclease, and that upon cutting by the restriction enzyme generates a "blunt" or "non-sticky" end.

[0053] The term "blunt-end" in connection with restruction enzymes is a well-recognized term in the art. It refers to the end of a double-stranded nucleic acid which terminates in a complementary base pair. In contrast, a "sticky end" refers to overhangs or a stretch of non-base- paired nucleotides at the end of a double-stranded nucleic acid molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs.

[0054] As used herein, an "oligonucleotide primer" or "primer" refer to a polynucleotide molecule (i.e., DNA, RNA, artificial nucleotides or a combination thereof) capable of annealing to a portion of a sequence of a nucleic acid, such as a strand of an IVT template as described herein, and providing a 3' end substrate for a polymerase enzyme to produce an enzymatic extension product that is complementary to the nucleic acid to which the primer is annealed. An oligonucleotide primer can refer to more than one primer and can be naturally occurring, as in, for example, a purified restriction digest, or can refer to a molecule produced synthetically. The conditions for initiation and extension usually include the presence of four different deoxyribonucleoside triphosphates (dNTPs) or analogs thereof and a polymerization-inducing agent, such as a DNA polymerase, in a suitable buffer ("buffer" includes constituents that are cofactors for the enzymatic reactions, and/or which affect pH, ionic strength, etc.) and at a suitable temperature. "Primers" useful in the methods described herein are generally less than or equal to 200 nucleotides in length, e.g., less than or equal to 175 nucleotides in length, less than or equal to 150 nucleotides in length, less than or equal to 140 nucleotides in length, less than or equal to 130 nucleotides in length, less than or equal to 120 nucleotides in length, less than or equal to 110 nucleotides in length, less than or equal to 100 nucleotides in length, less than or equal to 90 nucleotides in length, less than or equal to 80 nucleotides in length, less than or equal to 70 nucleotides in length, less than or equal to 60 nucleotides in length, less than or equal to 50 nucleotides in length, less than or equal to 40 nucleotides in length, less than or equal to 30 nucleotides in length, less than or equal to 20 nucleotides in length, or less than or equal to 15 nucleotides in length, but preferably at least 10 nucleotides in length.

[0055] The terms "primer binding sequence," "primer site" or "primer binding site" refer to the segment of the sequence of a nucleic acid sequence to which a primer hybridizes, i.e., the primer is specific for or complementary to the primer binding site.

[0056] A "polymerase," as used herein, refers to an enzyme that catalyzes polynucleotide synthesis by addition of nucleotide units to a nucleotide chain using DNA or RNA as a template. The term refers to either a complete enzyme as it occurs in nature, or an isolated, active catalytic domain, or fragment. Generally, the polymerase enzyme initiates synthesis at the 3'-end of a primer or nucleic acid strand, or at a promoter sequence, and proceeds in the 5'-direction along the target nucleic acid to synthesize a strand complementary to the target nucleic acid until synthesis terminates.

[0057] When two different, non-overlapping oligonucleotides anneal or hybridize to different regions of the same linear complementary nucleic acid sequence, and the 3' end of the first oligonucleotide points toward the 5' end of the other, second oligonucleotide, the former can be called the "upstream" oligonucleotide and is considered "5"' of the second oligonucleotide, and the latter the "downstream" oligonucleotide and is "3"' of the first oligonucleotide.

[0058] As used herein, the terms a "spacer" or a "spacer sequence" refer to a heterologous or random nucleotide sequence containing a known number of nucleotides. The number of nucleotides, or analogues thereof, in the spacer can range from at least 2 nucleotides, or analogues thereof up to and including at least 175 nucleotides or analogues thereof.

[0059] As used herein, an "open reading frame" or "ORF" or "open reading frame sequence" or "open reading frame DNA" refers to a series of nucleotides that comprises a sequence of bases that can encode a polypeptide or protein or an non-translated RNA product. An open reading frame is flanked by and includes the start-code sequence (initiation codon or start codon) and the stop-codon sequence (termination codon).

[0060] As used herein, the term " polypeptide " refers to a polymer of amino acids comprising at least 2 amino acids (e.g. , at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 amino acids or more). The terms "protein" and "polypeptide" are used interchangeably herein. As used herein, the term "peptide" refers to a relatively short polypeptide, typically between about 2 and 60 amino acids in length.

[0061] As used herein, the term "added co-transcriptionally" refers to the addition of a feature, e.g., a 5' diguanosine cap or other modified nucleoside, to a synthetic, modified RNA during transcription of the RNA molecule (i.e. , the RNA is not fully transcribed prior to the addition of the 5' cap).

[0062] As used herein, "isolated" or "purified" when used in reference to a polynucleotide or nucleic acid means that a naturally occurring sequence has been removed from its normal cellular environment or is in a non-natural environment. Thus, an "isolated" or "purified" sequence can be in a cell-free solution or placed in a different cellular environment. The terms isolated or partially purified can refer, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g. , nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and/or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation is considered "isolated."

[0063] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, translation, folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. In some embodiments, an expression product is transcribed from a sequence that does not encode a polypeptide, such as a microRNA.

[0064] As used herein, a "vector" refers to a nucleic acid molecule, such as a dsDNA molecule, that provides a useful biological or biochemical property to an inserted nucleotide sequence, such as the IVT templates described herein. Examples include plasmids, bacteriophage nucleic acid molecules, viral or modified viral nucleic acid molecules, autonomously replicating sequences (ARS), centromeres, and other sequences which are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell. [0065] The term "transfection" as used herein refers to the use of methods, such as chemical methods, to introduce exogenous nucleic acids, such as, for example, synthetic, modified RNAs generated using the constructs and methods described herein, into a cell, preferably a eukaryotic cell. As used herein, the term transfection does not encompass viral-based methods of introducing exogenous nucleic acids into a cell. Methods of transfection include physical treatments

(electroporation, nanoparticles, magnetofection), and chemical-based transfection methods. Chemical- based transfection methods include, but are not limited to, cyclodextrin, polymers, liposomes, and nanoparticles. In some embodiments, cationic lipids or mixtures thereof can be used to transfect the synthetic RNAs generated using the constructs and methods described herein, into a cell, such as DOPA, Lipofectamine and UptiFectin. In some embodiments, cationic polymers such as DEAE- dextran or polyethylenimine, can be used to transfect a synthetic RNA.

[0066] The term "transduction" as used herein refers to the use of viral particles or viruses to introduce exogenous nucleic acids into a cell.

[0067] As used herein the term "comprising " or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0068] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0069] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

BRIEF DESCRIPTION OF THE FIGURES

[0070] FIGS. 1A-1C demonstrate exemplary template construction for in vitro transcription

(IVT). FIG. 1A is a Schematic representation of pORFin comprising a 5' (SEQ ID NO: 202) and a 3' (SEQ ID NO: 203) UTR. The open reading frame (lacking a starting A nucleotide) was cloned between Alel and Afel restriction sites by blunt cloning. The recognition sites for both the enzymes are depicted. Unique restriction sites flanking UTRs are shown (Drawn not to scale). FIG. IB shows that screening of clones in correct orientation was done by colony PCR using primer pairs XU-Fl and gene specific reverse primers. If a right size product was identified, the clone was in the correct orientation. Lower panel shows results of colony PCR. Clones in correct orientation are marked by (*). Unspecific or nor amplification will result if the clone is in the wrong orientation. FIG. 1C depicts tail PCR carried out to add poly-adenylation signal using primer pairs XU-Fl and XU-(T)₁₂o ("(T)₁₂₀" disclosed as SEQ ID NO: 189). Bottom panel shows amplicons from various templates after tail PCR.

[0071] FIGS. 2A-2C show quality control parameters for in vitro transcribed mRNA. FIG.

2A shows a representative nano-drop reading of a typical IVT preparation showing a high yield sample. A poor yield reaction is included for comparison purpose. In FIG. 2B, IVT was performed using tailed template and the in vitro transcribed mRNA quality was confirmed by agarose gel electrophoresis. Bottom panel shows a graphical representation of 81 independent IVT preparations. Typical yield of in vitro transcribed mRNA was between 400-500 ng/ μΐ in total volume of 100 μΐ. mRNA yield is not affected by the size of ORF (in Kb) (n= number of independent preparation). FIG. 2C shows data from in vitro transcribed mRNA that were functionally verified for expression of respective proteins by fluorescence microscopy (left panel), FACS analysis (middle panel) or Western blot (right panel).

[0072] FIGS. 3A-3F demonstrate important factors that determine mRNA transfection efficiency. As shown in FIG. 3A, the most important determinant for mRNA transfection efficiency is target cells. lxlO⁵ indicated cell types of human origin were transfected with 250 ng of GFP mRNA and as shown fibroblasts were readily transfected while blood cells were refractory to transfection (right panel represents quantified data of histogram done in triplicate). In FIG. 3B, indicated transfection reagents were tested on human dermal fibroblasts. Transfection was done as per manufacturer's instruction. Among the tested reagents, Lipofectamine RNAiMax showed outstanding transfection efficiency (mean+SD). However, this may not be true for other cell types. FIG. 3C demonstrates how cell culture medium and its serum content influence the transfection efficiency. With increase in FCS content there was a notable reduction in % transfectactibility (right panel represents mean+SD of triplicate samples for each conditions). However, this could be cell type dependent phenomenon. FIG. 3D shows a typical representative matrix for optimization of RNA to transfection reagent ratio. At indicated RNA/Lipofectamine ratio, transfection efficiency is shown as % eGFP positive cells (in green). FIG. 3E demonstrates that transfection efficiency improves with increasing amount of RNA transfected unless the saturation point is reached. FIG. 3F shows that irrespective of transfection reagent, longer incubation time increases the transfection efficiency but could lead to increase in cell death. For FIGS. 3B, 3C, and 3F, lxlO⁵ human dermal fibroblasts were tranfected with 250 ng of GFP mRNA while for FIG. 3E indicated amount of mRNA was used for transfection. Experiments were carried out in triplicate and data was analyzed by FACS 14 hours post-transfection (data represents mean±SD).

[0073] FIG. 4 depicts an exemplary embodiment of an IVT template vector sequence, generated using the methods described herein, into which a desired open reading frame sequence of interest can be inserted and subsequently transcribed. The IVT template vector sequence of FIG. 4 comprises M13 forward and reverse universal primer sequences, a 5' UTR sequence from TEV, a T7 promoter sequence, Alel and Afel blunt-ended restriction enzyme recognition sites separated by a spacer sequence, a 3' UTR sequence from human oc-globin, and a vector backbone sequence from pZero. FIG. 4 discloses the top nucleotide sequence as SEQ ID NO: 204, the first coded protein as SEQ ID NO: 205, the bottom nucleotide sequence as SEQ ID NO: 206 and the second coded protein as SEQ ID NO: 207

[0074] FIG. 5 depicts an open-reading frame sequence of B 19R (SEQ ID NO: 208) inserted into an embodiment of the IVT templates described herein, as shown in FIG. 4, for in vitro transcription of a B 19R RNA.

[0075] FIG. 6 depicts an open-reading frame sequence of CD4 (SEQ ID NO: 209) inserted into an embodiment of the IVT templates described herein, as shown in FIG. 4, for in vitro transcription of a CD4 RNA.

[0076] FIG. 7 depicts an open-reading frame sequence of LNGFR (SEQ ID NO: 210) inserted into an embodiment of the IVT templates described herein, as shown in FIG. 4, for in vitro transcription of a LNGFR RNA.

[0077] FIG. 8 depicts an open-reading frame sequence of GFP-Lamin A (SEQ ID NO: 211) inserted into an embodiment of the IVT templates described herein, as shown in FIG. 4, for in vitro transcription of a GFP-Lamin A RNA.

[0078] FIG. 9 depicts an open-reading frame sequence of GFP-Progerin (SEQ ID NO: 212) inserted into an embodiment of the IVT templates described herein, as shown in FIG. 4, for in vitro transcription of a GFP-Progerin RNA.

[0079] FIG. 10 depicts an open-reading frame sequence of human Pax5 (SEQ ID NO: 213) inserted into an embodiment of the IVT templates described herein, as shown in FIG. 4, for in vitro transcription of a human Pax5 RNA.

[0080] FIG. 11 depicts an open-reading frame sequence of Stat3(c) (SEQ ID NO: 214) inserted into an embodiment of the IVT templates described herein, as shown in FIG. 4, for in vitro transcription of a Stat3(c) RNA.

[0081] FIG. 12 depicts an open-reading frame sequence of Leptin (SEQ ID NO: 215) inserted into an embodiment of the IVT templates described herein, as shown in FIG. 4, for in vitro transcription of a Leptin RNA.

[0082] FIG. 13 depicts an open-reading frame sequence of G-Luciferase (SEQ ID NO: 216) inserted into an embodiment of the IVT templates described herein for in vitro transcription of a G- Luciferase RNA.

[0083] FIG. 14 demonstrates successful in vivo delivery and expression of a Firefly lucif erase reporter polypeptide generated from intra-muscular (left panel) or intra-tracheal (right panel) delivery of a modified RNA synthesized using an embodiment of the IVT templates and methods thereof described herein.

DETAILED DESCRIPTION

[0084] Described herein are novel compositions, methods, and kits for the rapid and efficient synthesis of RNA molecules, such as modified RNA molecules comprising one or more modified nucleosides. By using the IVT templates and constructs described herein, RNA molecules encoding for an expression product of interest, including RNA molecules comprising one or more modified nucleosides, can be synthesized in an efficient and high-throughput manner for use in a variety of applications, including cellular reprogramming and modifying cellular phenotypes. The specific benefits of the methods and constructs described herein include, for example, that one can use a single vector system to clone in any nucleic acid sequence, such as an open reading frame, for IVT without separately optimizing cloning or PCR conditions for each template. This allows a significant increase in output, as well as standardization of conditions, for making template nucleic acids for IVT. Thus, the novel constructs and methods described herein provide a solution to the bottleneck in IVT template production.

[0085] The IVT templates or constructs thereof comprise the following parts: a) primer sequences, b) a promoter sequence, c) a 5' UTR sequence, d) restriction enzyme site sequences, e) an open reading frame sequence, and f) a 3' UTR sequence (see, for example, FIG. 1A). The IVT template can further comprise a vector backbone. The poly-A tail is not included in the IVT template sequence and is introduced in a separate PCR reaction (see, for example, FIG. 1C). In some embodiments, the IVT templates or constructs thereof consist essentially of the following parts: a) primer sequences, b) a promoter sequence, c) a 5' UTR sequence, d) restriction enzyme site sequences, e) an open reading frame sequence, and f) a 3' UTR sequence (see, for example, FIG. 1A). The IVT template can further comprise a vector backbone, in some embodiments. In some such embodiments, the poly-A tail is not included in the IVT template sequence and is introduced in a separate PCR reaction (see, for example, FIG. 1C). In yet some embodiments, the IVT templates or constructs thereof consist of the following parts: a) primer sequences, b) a promoter sequence, c) a 5' UTR sequence, d) restriction enzyme site sequences, e) an open reading frame sequence, and f) a 3' UTR sequence (see, for example, FIG. 1 A). The IVT template can further comprise a vector backbone, in some embodiments. In some such embodiments, the poly-A tail is not included in the IVT template sequence and is introduced in a separate PCR reaction (see, for example, FIG. 1C).

[0086] The IVT templates or constructs thereof provided herein permit easy cloning of any desired open reading frame (ORF) of interest, without the additional steps of, for example, adding flanking restriction enzyme recognition sequences to the ORF, which can then subsequently be transcribed. The IVT templates described herein comprise various specific component sequences or elements including primer recognition sequences, promoter sequences, 5' UTR sequences, restriction enzyme site sequences, 3' UTR sequences, vector backbone sequences, and optionally open reading frame sequences, as described in more detail herein. These novel IVT template nucleic acids and constructs thereof allow rapid and high-throughput insertion of practically any open reading frame of interest for use in a subsequent in vitro transcription reaction, thereby easily generating synthetic RNA molecules for use in a variety of applications, such as, for example, cellular reprogramming. [0087] Accordingly, provided herein are methods of designing and making the IVT templates and constructs described throughout the specification, and for generating synthetic RNAs using these IVT templates. These methods can comprise PCR steps, cloning steps, digestion steps, and in vitro transcription steps, as known to one of skill in the art. By using the IVT template design described herein, any open reading frame of interest can be inserted, via blunt-end ligation, into the IVT template, to generate a synthetic RNA upon in vitro transcription, as demonstrated herein.

[0088] Some exemplary embodiments of the steps comprising these methods are briefly described below. Because an IVT template is necessarily a double-stranded DNA sequence, all the templates and methods of generating synthetic RNA sequences provided herein are described in reference to the coding strand of the IVT template DNA, such that the complementary RNA sequence generated from the complementary template strand of the IVT template DNA, reads the same in the 5' to 3' direction but includes uracil (U) or an modified nucleotide equivalent thereof in all instances where thymine (T) occurs in the coding DNA strand.

[0089] To construct an IVT DNA template, selected 5' and 3' UTR coding sequences can be de novo synthesized using synthetic oligonucleotides, for example, and annealed together and amplified using forward and reverse primers comprising restriction site sequences, preferably sticky- end sequences, to correspond to the cloning site restriction site sequences of a vector backbone of interest, for example, pZErO-2. Blunt-end restriction site sequences, such as, for example, Alel and Afel, can be introduced between the selected 5' and 3' UTR sequences to provide the entry sites for the given open reading frame sequence of interest. In some embodiments, the 5' UTR sequence can further comprise the sequence of the promoter sequence. In other embodiments, the promoter sequence is, for example, de novo synthesized along with the 5' and 3' UTR sequences, or can be PCR amplified and ligated to the 5' UTR coding sequence. A spacer sequence can also be included between the blunt-end restriction site sequences to allow for optimal digestion prior to insertion of the ORF sequence.

[0090] In further embodiments, a forward universal primer sequence is included 5' of the sequence coding for the 5' UTR sequence, and a sequence complementary to a reverse universal primer is included 3' of the sequence coding for the 3' UTR sequence, thus allowing for easy and rapid amplification of the IVT template sequence for subsequent cloning. For example, the primer sequences can be deisgned to anneal to an Ml 3 forward primer sequence and an Ml 3 reverse primer sequence, as described herein the Examples. Accordingly, an IVT DNA template is generated comprising, in the 5' to 3' direction of its coding strand, (a) a sequence comprising a forward universal primer sequence, (b) a promoter sequence operably linked to a sequence encoding a 5' UTR sequence, (c) a 5' UTR sequence, (d) a first blunt-ended restriction enzyme sequence, (e) a spacer sequence, (f) a second blunt-ended restriction enzyme sequence, (g) a sequence encoding a 3 'UTR sequence, and (h) a sequence comprising a sequence complementary to a reverse universal primer sequence. In some embodiments, the IVT template can be constructed to include at its 5' and 3' sticky-end restriction site sequences for cloning into a vector backbone of interest having the same sticky-end restriction site sequences.

[0091] The vector backbone of interest can then be digested with restriction enzymes specific for the sticky-end restriction site sequences of interest, as described herein in the Examples for the pZErO-2 vector using Hindlll and Notl restriction enzymes. The amplified IVT template sequence is then ligated into the vector backbone of interest, thereby generating the "IVT template vector" for insertion of a desired open reading frame sequence of interest. FIG. 1A describes an exemplary embodiment of an IVT template vector termed "pORFin." The cloned or inserted IVT template can be verified, for example, by sequencing.

[0092] To generate and insert an open reading frame sequence of a gene of interest, the ORF sequence can be amplified using a forward and reverse primer pair. In some embodiments, one or more nucleotides of the 5' end of the open reading frame sequence can be omitted from the forward primer sequence as it can be provided by the first blunt-ended restriction site sequence. For example, as described herein in the Examples section, the adenine nucleotide (A) of the 1^st codon (ATG) of the open reading frame sequence was provided by the Alel site of the IVT template. Therefore, the ORFs cloned into the vector used in the Examples, pORFin, are provided without the first "A" nucleotide. In some embodiments, the forward and reverse primers are phosphorylated prior to the PCR reaction, as described herein in the Examples.

[0093] The IVT template vector can be digested using enzymes specific for the blunt-ended restriction site sequences of the IVT template to linearize the vector. The IVT template vector can also be dephosphorylated. The amount of IVT template vector can also be quantified, using any technique known to one of ordinary skill in the art. The amplified ORF fragment can be cloned or inserted into the linearized and dephosphorylated IVT template vector by blunt-end ligation. Screening for clones in which amplified ORF fragment is in the right-orientation can be performed, for example, using colony PCR (see, for example, FIG. IB).

[0094] In some embodiments, the polyadenylation template sequence or "tail" is added after verifying that the ORF is in a correct orientation and using a poly-T- template construct or primer. This will allow addition of a specified length of the poly-A sequence. If a sequence is added into the vector by PCR, the length of the tail can be harder to control.

[0095] In some embodiments, to add a polyadenylation tail to the ORF sequence, the sequence -verified clone can be used as a DNA template for a "Tail PCR," which appends a polyadenylation template to the IVT template construct, for example, as depicted in FIG. 1C.

Accordingly, the IVT template vector comprising the ligated ORF sequence is amplified via PCR using: a forward universal primer comprising at its 3' end a sequence of the forward universal primer sequence of the IVT template, and a reverse universal primer sequence comprising a sequence complementary to the reverse primer sequence of the IVT template at its 3' end and a poly-T sequence at the primer's 5' end, generating an "amplified IVT template vector fragment." Therefore, the amplified IVT template vector fragment comprises the necessary template for generating a polyadenylation tail upon in vitro transcription. The conditions for this reaction can be optimized by one of skill in the art according to the DNA polymerase used for the PCR and the length of the ORF sequence. The methylated vector DNA can then be digested. In other embodiments, the IVT template vector can be linearized prior to the Tail-PCR reaction to eliminate any circular templates that would generate run-on transcripts.

[0096] Following the Tail PCR, the amplified IVT template vector fragment comprising the

ORF sequence of interest, see, for example, FIG. ID, can undergo in vitro transcription using any method known to one of ordinary skill in the art. An in vitro transcription reaction typically comprises ribonucleotide triphosphates or NTPS (e.g., ATP, GTP, CTP, and UTP), an RNA polymerase specific for the promoter sequence of the amplified IVT template vector fragment, and an appropriate buffer mixture for the RNA polymerase being used. In some embodiments, the ribonucleotide triphosphates used in the in vitro transcription reaction comprises one or more modified nucleotides, such as pseudo-UTP or methyl-CTP. Any residual DNA in the reaction mixture following the IVT reaction can be removed by addition of a DNase enzyme, and the synthesized RNA can be eluted using any method known to one of ordinary skill in the art.

[0097] The purified synthesized RNA can be treated with a phosphatase, which prevents recognition of the uncapped synthesized RNA by the RIG-I complex. The RNA can then be utilized to transfect any cell type of interest. Optimal transfection conditions can be determined by a skilled artisan. In addition, highlighted in the Examples section are some important issues related with transfection to be considered, and related guidelines, including target cell type, transfection reagents, cell culture media and their FCS or protein content, optimal ratio of RNA to transfection reagent, and dosing and incubation time.

[0098] Having outlined the various elements of an IVT template and methods thereof required for generating a synthetic RNA, such as a synthetic, modified RNA, these elements and methods are described in more detail herein below:

Transcription and Synthesis of RNA Molecules

[0099] In vivo production of an RNA molecule, such as an mRNA molecule, begins with transcription and ultimately ends in degradation. An RNA molecule can further be processed, edited, and transported prior to translation. As used herein, "transcription" refers to the process of creating a complementary RNA copy of a sequence of DNA. During transcription, RNA polymerase reads a DNA sequence and produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in a complementary RNA sequence that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. Eukaryotic RNA polymerase associates with mRNA processing enzymes during transcription so that processing can proceed quickly after the start of transcription. If the gene transcribed encodes a protein, the result of transcription is a messenger RNA (mRNA), which is then used to create that protein via the process of translation. Alternatively, a transcribed gene sequence can encode for ribosomal RNA (rRNA) or transfer RNA (tRNA), other components of the protein-assembly process, antisense RNA molecules, or ribozymes.

[00100] As described in more detail herein, a DNA transcription unit encoding for a protein comprises not only the sequence that is eventually directly translated into the protein (the coding sequence) but also regulatory sequences that direct and regulate the synthesis of that protein. The regulatory sequence before (or upstream from) the coding sequence is termed the "five prime untranslated region" or "5' UTR," and the sequence following (or downstream of) the coding sequence is termed the "three prime untranslated region" or "3' UTR." During transcription, DNA is read from the 3'→ 5' direction, and the complementary RNA is created from the 5'→ 3' direction. Although DNA is arranged as two antiparallel strands in a double helix, only one of the two DNA strands, termed the "template strand," is used for transcription. The other DNA strand is termed the "coding strand," because its sequence is the same as the newly created RNA transcript (except for the substitution of uracil for thymine).

[00101] Accordingly, the sequences of the various components of the IVT templates and constructs provided herein reflect the sequence of the coding strand, such that the complementary RNA sequence reads the same in the 5' to 3' direction but includes uracil (U) or an modified nucleotide equivalent thereof in all instances where thymine (T) occurrs in the coding strand.

[00102] Briefly, the five stages of cellular transcription in vivo are as follows:

[00103] Pre -initiation. In eukaryotes, RNA polymerase and the initiation of transcription, requires the presence of sequence termed the "core promoter sequence" in the DNA. Core promoter sequences or promoter consensus sequences refer to sequences within the promoter that are essential for transcription initiation. RNA polymerase is able to bind to core promoters in the presence of various specific transcription factors. The most common type of core promoter in eukaryotes is a short DNA sequence known as a TATA box, found 25-30 base pairs upstream from the transcription start site or TSS.

[00104] Initiation. In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. In eukaryotes, eukaryotic RNA polymerases do not directly recognize the core promoter sequences. Instead, transcription factors mediate the binding of RNA polymerases and the initiation of transcription. Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it. The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex.

[00105] Promoter clearance. After the first bond is synthesized, the RNA polymerase must clear the promoter. During this time there is a tendency to release the RNA transcript and produce truncated transcripts, which is called abortive initiation. Once the transcript reaches approximately 23 nucleotides, it no longer slips and elongation can occur. This, like most of the remainder of transcription, is an energy-dependent process, consuming adenosine triphosphate (ATP). [00106] Elongation. One strand of the DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3'→ 5', the coding (non-template) strand and newly-formed RNA can also be used as reference points, so transcription can be described as occurring 5'→ 3'. This produces an RNA molecule from 5'→ 3', which is an exact copy of the coding strand, except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone.

[00107] mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gene.

[00108] Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this can correspond with short pauses during transcription that allow appropriate RNA editing factors to bind.

[00109] Termination. Bacteria use two different strategies for transcription termination. In

Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C-rich hairpin loop followed by a run of uracils. In the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.

[00110] Transcription termination in eukaryotes involves cleavage of the new transcript followed by template-independent addition of A nucleotides at its new 3' end, in a process termed "polyadenylation."

[00111] While cellular transcription processes, as described above, involve a variety of factors, such as RNA polymerases, transcription factors, etc., and many steps, transcription can occur in a non-cellular system by utilizing bacteriophage or viral RNA polymerases in a process termed "in vitro transcription." Accordingly, as used herein, "in vitro transcription" or "IVT" refer to the process whereby transcription as described above occurs in vitro in a non-cellular system to produce

"synthetic RNA molecules" for use in various applications, including the production of protein or polypeptides. The synthetic RNA molecules or "transcription products" generated can be translated in vitro or introduced directly into cells, where they can be translated. Such synthetic transcription products include mRNAs, antisense RNA molecules, shRNA molecules, ribozymes, and the like. Further, the synthetic RNA molecules generated by IVT can comprise natural or unmodified nucleosides or one or more modified nucleosides, such as, for example, 5-methylcytidine or pseudouridine. An IVT reaction typically requires a purified linear DNA template comprising a promoter sequence and the sequence of the open reading frame of interest, ribonucleotide triphosphates or modified ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase.

In vitro Transcription Templates

[00112] Despite the great advances and progress in recombinant nucleic acid methodologies, including the ability to transcribe DNA into RNA molecules in cell-free systems, a number of time- consuming steps are typically required to generate a DNA template having the desired properties for use in an in vitro transcription reaction. Generation of such templates or constructs typically require multiple restriction enzyme digestion and ligation steps, specific for each open reading frame of interest, in addition to numerous purification and characterization steps. All these steps require ORF- specific optimization.

[00113] We provide herein novel IVT templates or constructs comprising such templates for in vitro transcription of an open reading frame sequence of interest. By using these templates or constructs, any desired open reading frame (ORF) of interest can be easily, and without additional optimization, cloned and subsequently transcribed without the additional steps of, for example, adding flanking restriction enzyme recognition sequences to the ORF. Such IVT templates comprise various component sequences including primer sequences, promoter sequences, 5' UTR sequences, restriction enzyme site sequences, open reading frame sequences, 3' UTR sequences, and vector backbone sequences, as described in more detail herein, and analyzed and selected in a novel way to provide a simple, scaleable system for IVT template production for different ORFs using the same vector and essentially the same conditions for cloning and amplification. These novel IVT template nucleic acids and constructs thereof allow rapid and high-throughput insertion of an open reading frame of interest for use in a subsequent in vitro transcription reaction, thereby easily generating synthetic RNA molecules for use in a variety of applications, such as, for example, cellular reprogramming.

[00114] As used herein, an "IVT template" or "IVT template sequence" refers to an isolated nucleic acid sequence that comprises the minimal component sequences required for in vitro transcriprion of an open reading frame of interest. At a minimum, an IVT template for use in the compositions, kits, and methods described herein, comprises, in the 5' to 3' direction of its coding strand, (a) a nucleic acid sequence comprising a first universal primer sequence, (b) a promoter sequence, (c) a 5' UTR sequence, (d) a first blunt-ended restriction site, (e) a spacer sequence, (f) a second blunt-ended restriction site, (g) a 3' UTR sequence, and (h) a nucleic acid sequence comprising a sequence complementary to a second universal primer sequence.

[00115] In some aspects and embodiments described herein, the first blunt-ended restriction site sequence overlaps with or comprises one or more 3' terminal nucleotides of the 5' UTR sequence.

[00116] In some aspects and embodiments described herein, the second blunt-ended restriction site sequence overlaps with or comprises one or more of the 5' terminal nucleotides of the 3' UTR sequence. [00117] In some aspects and embodiments, the IVT template further comprises one or more open reading frame sequences between the first blunt-ended restriction site and the second blunt- ended restriction site.

[00118] It is preferred that an IVT template as described herein does not comprise a sequence encoding a poly-adenylation template, as such a sequence can be added to the IVT template in a subsequent step prior to in vitro transcription in a PCR reaction termed herein as "Tail PCR." Tail PCR provides the advantages described herein of, for example, allowing the length of the poly-A tail to be controlled. Accordingly, in some embodiments of the aspects described herein, the IVT template does not comprise a sequence encoding a polyadenylation sequence.

[00119] The various components or elements comprising an IVT template for use in the compositions, methods, and kits described herein are described in more detail below:

Promoters

[00120] Promoter sequences are required in the IVT templates described herein to allow in vitro transcription of an open reading frame of interest. By engineering the IVT template sequence to include a promoter sequence, any open reading frame sequence inserted into the IVT template can be transcribed under the appropriate conditions and in the presence of the required reagents, without depending or relying on a promoter sequence found in the sequence of the vector backbone.

[00121] Accordingly, provided herein are promoter sequences for use in in vitro transcription templates or constructs comprising such templates. The terms "promoter" or "promoter sequence," as used herein, refer to a nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving RNA polymerase-mediated transcription of the nucleic acid sequence, which can be a heterologous target gene, such as one encoding a protein or an RNA. A promoter is a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. Promoters can be constitutive, inducible, activateable, repressible, tissue-specific, or any combination thereof. Both weak and strong promoters can be used. Typically, strong promoters are preffered. Typically, a promoter for use in the in vitro transcription templates or constructs described here are constitutive. A promoter can also contain one or mroe genetic elements at which regulatory proteins and molecules can bind. Such regulatory proteins include RNA polymerase and other transcription factors.

[00122] A promoter can be said to "drive expression" or "drive transcription" of the nucleic acid sequence that it regulates. The phrases "operably linked," "operatively positioned," "operatively linked," "under control," and "under transcriptional control" indicate that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence it regulates to control transcriptional initiation and/or expression of that sequence. In addition, in some embodiments described herein, a promoter can be used in conjunction with an "enhancer," which refers to a cis- acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence downstream of the promoter. The enhancer can be located at any functional location before or after the promoter, and/or the encoded nucleic acid.

[00123] A promoter for use in the templates and methods described herein can be one naturally associated with a gene or sequence, and can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon of a given natural genomic gene or sequence. Such a promoter can be referred to as "endogenous." Similarly, an enhancer can be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.

[00124] Alternatively, and preferably, as described herein, certain advantages can be gained by positioning a coding nucleic acid segment or an "open reading frame" under the control of or operably linked to a "recombinant promoter" or "heterologous promoter," which refers to a promoter that is not normally associated with the encoded nucleic acid sequence in its natural environment. Similarly, the terms a "recombinant enhancer" or "heterologous enhancer" refer to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes; promoters or enhancers isolated from any other viral, prokaryotic, or eukaryotic genes; and synthetic promoters or enhancers that are not "naturally occurring", i.e., comprise different elements of different transcriptional regulatory regions, and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences can be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference).

[00125] Accordingly, provided herein are promoter sequences for use in the IVT templates and constructs thereof described herein. In preferred embodiments of the aspects described herein, a promoter sequence used in the IVT templates described herein comprises a bacteriophage RNA polymerase promoter sequence, such as an SP6 promoter sequence, T7 promoter sequence, or a T3 promoter sequence that can be specifically recognized by SP6, T7, or T3 RNA polymerases, respectively, in order to initiate in vitro transcription.

[00126] In some embodiments of the aspects described herein, a promter sequence comprises a T7 promoter sequence. As used herein, a "T7 promoter sequence" refers to any promoter sequence that comprises a sequence to which the T7 RNA polymerase can specifically bind and initiate transcription from, i.e. , the T7 consensus promoter sequence. A list of exemplary, but not limiting, T7 promoter sequences for use in the IVT templates described herein is provided in Table 1.

Table 1: Exemplary T7 Promoter Sequences

SEQ ID NO: 2 T7 Consensus Promoter Sequence TAATACGACTCACTATAGGGAGA

SEQ ID NO: 3 T7 Promoter GAATTTAATACGACTCACTATAGGGAGA

SEQ ID NO: 4 T7 consensus -10 and rest TAATACGACTCACTATAGG

SEQ ID NO: 5 overlapping T7 promoter GAGTCGTATTAATACGACTCACTATAGGGG

SEQ ID NO: 6 more overlapping T7 promoter AGTGAGTCGTACTACGACTCACTATAGGGG

SEQ ID NO: 7 weaken overlapping T7 promoter GAGTCGTATTAATACGACTCTCTATAGGGG

SEQ ID NO: 8 T7 RNAP promoter TTATACGACTCACTATAGGGAGA

SEQ ID NO: 9 T7 RNAP promoter GAATACGACTCACTATAGGGAGA

SEQ ID NO: 10 T7 RNAP promoter TAATACGTCTCACTATAGGGAGA

SEQ ID NO: 11 T7 RNAP promoter TCATACGACTCACTATAGGGAGA

SEQ ID NO: 12 T7 strong promoter TAATACGACTCACTATAGGGAGACCACAAC

SEQ ID NO: 13 T7 weak binding and processivity TAATTGAACTCACTAAAGGGAGACCACAGC

SEQ ID NO: 14 T7 weak binding promoter CGAAGTAATACGACTCACTATTAGGGAAGA

SEQ ID NO: 15 T7 14.3 m ATTAACCCTCACTAAAGGGAGA

[00127] In other embodiments of the aspects described herein, an SP6 promoter sequence can be used as the promoter sequence in the IVT templates or constructs. As used herein, an "SP6 promoter sequence" refers to any promoter sequence that comprises a sequence to which the SP6 RNA polymerase can specifically bind and initiate transcription from. The consensus SP6 promoter sequence is ATTTAGGTGACACTATAGA (SEQ ID NO: 16), and represents the minimal sequence required for efficient binding and transcription by the SP6 RNA polymerase.

[00128] In other embodiments of the aspects described herein, a T3 promoter sequence can be used as the promoter sequence in the IVT templates or constructs. As used herein, a "T3 promoter sequence" refers to any promoter sequence that comprises a sequence to which the T3 RNA polymerase can specifically bind and initiate transcription from. The consensus T3 promoter sequence is AATTAACCCTCACTAAAGG (SEQ ID NO: 17), and represents the minimal sequence required for efficient binding and transcription by the T7 RNA polymerase.

' and 3 ' UTR Sequences

[00129] Untranslated regions or UTRs refer to sections of an mRNA sequence prior to the start codon and after the stop codon, termed the five prime untranslated region (5' UTR) and three prime untranslated region (3' UTR), respectively, that are not translated, but that are transcribed as part of an mRNA sequence. Several roles in gene expression have been attributed to the untranslated regions, including mRNA stability, mRNA localization, and translational efficiency. The ability of a UTR to perform these functions depends on the sequence of the UTR and can differ between mRNAs. These UTR regions are transcribed with the coding region and thus are exonic as they are present in the mature mRNA. The stability of mRNAs can be controlled by the 5' UTR and/or 3' UTR due to varying affinity for RNA degrading enzymes or "ribonucleases" and for ancillary proteins that can promote or inhibit RNA degradation. Translational efficiency, including sometimes the complete inhibition of translation, can be controlled by UTRs. Proteins that bind to either the 3' or 5' UTR can affect translation by influencing the ribosome's ability to bind to the mRNA. MicroRNAs bound to the 3' UTR also can also affect translational efficiency or mRNA stability. Some of the elements contained in untranslated regions form a characteristic secondary structure when transcribed into RNA. These structural mRNA elements are involved in regulating the mRNA. Some, such as the SECIS element, are targets for proteins to bind. One class of mRNA element, the riboswitches, directly binds small molecules, changing their fold to modify levels of transcription or translation.

[00130] The IVT templates described herein comprise both a 5 'UTR sequence operably linked to a promoter sequence and a 3' UTR sequence that occurs 3' of the second blunt-ended restriction enzyme site, thereby providing an open reading frame transcribed from the IVT templates additional stability and translational efficiency. By including standard and characterized 5' and 3' UTR sequences in the IVT templates, as described herein, any open reading frame of interest inserted into the template, upon transcription, comprises 5' and 3' UTR sequences that are known to provide optimal stability and allow for optimal translational efficiency.

[00131] As used herein, the terms "five prime untranslated region" or "5' UTR" refer to the sequence of an mRNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region of an RNA. A 5' UTR can comprise genetic elements or sequence for controlling gene expression by way of regulatory elements. In prokaryotes, the 5' UTR usually contains a ribosome binding site (RBS), also known as the Shine Dalgarno sequence (AGGAGGU, SEQ ID NO: 18). The 5' UTR in eukaryotes typically has a median length of -150 nt, but can be as long as several thousand bases. Some viruses and cellular genes have unusually long and structured 5' UTRs which can impact gene expression. On average, 3' UTR sequences tend to be twice as long as the 5' UTR. In prokaryotic mRNAs, the 5' UTR is normally shorter.

[00132] Regulatory sequences that can be found in a 5' UTR include, for example, binding sites for proteins, which can affect the mRNA's stability or translation; riboswitches; and sequences that promote or inhibit translation initiation.

[00133] As used herein, the terms "three prime untranslated region" or "3' UTR" refer to the sequence of an mRNA molecule that begins following, but not necessarily immediately after, the stop codon of the coding region of an open reading frame sequence. Cytoplasmic localization of mRNA is believed to be a function of the 3' UTR. In the case of proteins that undergo translation at a particular location in a cell where they are needed, the 3' UTR can contain sequences that allow the transcript to be localized to this region for translation. [00134] Regulatory sequences typically found in the 3' UTR include, for example: (i) a polyadenylation signal, usually AAUAAA (SEQ ID NO: 19), or a slight variant thereof. This marks the site of cleavage of the transcript approximately 30 base pairs past the signal, followed by the several hundred adenine residue (poly-A tail); (ii) binding sites for proteins, that can affect the mRNA's stability or location in the cell, like SECIS elements (which direct the ribosome to translate the codon UGA as selenocysteines rather than as a stop codon), or AU-rich elements (AREs), stretches consisting of mainly adenine and uracil nucleotides (which can either stabilize or destabilize the mRNA depending on the protein bound to it), (iii) binding sites for miRNAs, and/or (iv) microRNA seed sequences.

[00135] Accordingly, in some embodiments, a 3' UTR sequence for use in the IVT templates and constructs described herein does not comprise a Group I ARE having the sequence

AUUUAUUUAUUUAUUUAUUUA (SEQ ID NO: 163). In some embodiments, the 3' UTR sequence for use in the IVT templates and constructs described herein does not comprise a Group II ARE having the sequence AUUUAUUUAUUUAUUUA (SEQ ID NO: 164). In some embodiments, the 3' UTR sequence for use in the IVT templates and constructs described herein does not comprise a Group III ARE having the sequence WAUUUAUUUAUUUAW(SEQ ID NO: 165). In some embodiments, the 3' UTR sequence for use in the IVT templates and constructs described herein does not comprise a Group IV ARE having the sequence WWAUUUAUUUAWW (SEQ ID NO: 166). In some embodiments, the 3' UTR sequence for use in the IVT templates and constructs described herein does not comprise a Group V ARE having the sequence WWWWAUUUAWWWW (SEQ ID NO: 167).

[00136] Provided herein are sequences encoding 5' and 3' UTR sequences for use in IVT templates and constructs. In some embodiments of the aspects described herein, the 5' and 3' UTR sequences used in the IVT templates can be the naturally occurring or endogenous 5' and 3' UTR sequences for the gene encoding the open reading frame of interest. In general, the length of the 3' UTR exceeds 100 nucleotides, and therefore, in some embodiments, a 3' UTR sequence longer then 100 nucleotides is preferred. The length of the 5' UTR is typically not as important as the length of the 3' UTR and can be shorter. For example, the 5' UTR is between 1 and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to an open reading frame sequence can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[00137] The 5' and 3' UTRs can, in some embodiments, comprise the naturally occurring, endogenous 5' and 3' UTRs sequences for the gene encoding the open reading frame of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA sequence transcribed. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[00138] In some embodiments, the 5' UTR sequence can comprise the Kozak sequence of the endogenous gene encoding the open reading frame sequence. Alternatively, when a 5' UTR that is not endogenous to the gene encoding the open reading frame sequence of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences increase the efficiency of translation of some RNA transcripts, but are not required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the transcribed RNA sequence, as described elsewhere herein.

[00139] A large number of 5' and 3' UTR sequences have been identified, sequenced, and curated, such as, for example, those found on the worldwide web at utrdb.ba.itb.cnr.it. Such 5' and 3' UTR sequences can be selected for use in the IVT templates and contrsucts described herein to have constitutive and/or tissue specific properties, lack inhibitory elements, etc. In some aspects and embodiments, the 5' and 3' UTR sequences used in the IVT templates are those described herein as SEQ ID NO: 20 and SEQ ID NO: 21, respectively. As demonstrated herein, these particular UTR sequences are particularly useful in designing and making IVT contacts and templates as they permit enhanced stability and efficient translation efficiency of any open reading frame sequence of interest, and work well with blunt-end restriction sites used in the IVT templates, such as, for example, those recognized by the Alel and Afel restriction enzymes.

[00140] SEQ ID NO: 20 (TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGG

GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC) encodes for a sequence comprising the 5' UTR sequence of the Tobacco Etch Virus and further comprises the T7 consensus promoter sequence. The 5' UTR sequence of the Tobacco Etch Virus (TEV) has a long history of use in recombinant engineering methods as an exogenous 5' UTR element to increase translation efficiency of mRNA sequences. The 5' UTR sequence of TEV is a 144 nucleotide sequence that has been shown to be largely responsible for directing efficient translation of an operably linked mRNA sequence. The 5' UTR sequence of TEV has also been demonstrated to act optimally and function synergistically as a regulator of translation when the RNA sequence comprises a poly-adenylation tail.

[00141] Accordingly, in some embodiments of the aspects described herein, a sequence encoding a 5' UTR sequence for use in the IVT templates comprises a sequence encoding the 5' UTR sequence of TEV. In some such embodiments, the 5' UTR sequence comprises SEQ ID NO: 20. In some such embodiments, the 5' UTR sequence consists essentially of SEQ ID NO: 20. In some such embodiments, the 5' UTR sequence consists of SEQ ID NO: 20.

[00142] Other exemplary 5' UTR sequences for use in the IVT templates described herein include, but are not limited to, Xenopus or human 5' UTR sequences of a-globin, or Xenopus or human 5' UTR sequences of β-globin.

[00143] SEQ ID NO: 21

(GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCT

CTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG) encodes for a sequence comprising the 3' UTR sequence of a-globin. The 3' UTR sequence of a-globin is known as a stability determinant for a-globin messenger RNAs, and mutations in the 3' UTR sequence have been shown be a causal factor for a-thalassemia by causing a global loss in a-globin production. The 3' UTR sequence of a-globin is a 109 nucleotide sequence that has been shown to comprise three cytosine-rich (C-rich) segments that contribute to α-globin mRNA stability. In addition, in vitro and in vivo analyses have indicated that the a-complex stabilizes α-globin mRNA by two mechanisms: control of 3'-terminal deadenylation and steric protection of an endoribonuclease-sensitive site.

[00144] Accordingly, in some embodiments of the aspects described herein, a sequence encoding a 3' UTR sequence for use in the IVT templates comprises a sequence encoding the 3' UTR sequence of a-globin. In some such embodiments, the 3' UTR sequence comprises SEQ ID NO: 21. In some such embodiments, the 3' UTR sequence consists essentially of SEQ ID NO: 21. In some such embodiments, the 3' UTR sequence consists of SEQ ID NO: 21. Other exemplary 3' UTR sequences for use in the IVT templates described herein include, but are not limited to, Xenopus or human 3' UTR sequences of β-globin.

[00145] Other exemplary 3' UTR sequences for use in the IVT templates described herein include 3' UTR sequences found in housekeeping genes, such as GAPDH and UBB. For example, in some embodiments, a 3' UTR sequence for use in the IVT templates comprises a sequence encoding the 3' UTR sequence of GAPDH or SEQ ID NO: 168

(GACCCCTGGACCACCAGCCCCAGCAAGAGCACAAGAGGAAGAGAGAGACCCTCACTGC TGGGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGC CATGTAGACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATC AATAAAGTACCCTGTGCTCAACC). In some embodiments, a 3' UTR sequence for use in the IVT templates comprises a sequence encoding the 3' UTR sequence of ACTB or SEQ ID NO: 169

(GCGGACTATGACTTAGTTGCGTTACACCCTTTCTTGACAAAACCTAACTTGCGCAGAAA

ATAGTCATTCCAAATATGAGATGCGTTGTTACAGGAAGTCCCTTGCCATCCTAAAAGCCA CCCCACTTCTCTCTAAGGAGAATGGCCCAGTCCTCTCCCAAGTCCACACAGGGGAGGTGA TAGCATTGCTTTCGTGTAAATTATGTAATGCAAAATTTTTTTAATCTTCGCCTTAATACTT TTTTATTTTGTTTTATTTTGAATGATGAGCCTTCGTGCCCCCCCTTCCCCCTTTTTTGTCCC CCAACTTGAGATGTATGAAGGCTTTTGGTCTCCCTGGGAGTGGGTGGAGGCAGCCAGGG CTTACCTGTACACTGACTTGAGACCAGTTGAATAAAAGTGCACACCTT). In some embodiments, a 3' UTR sequence for use in the IVT templates comprises a sequence encoding the 3' UTR sequence of UBB or SEQ ID NO: 170

(TTCTTCAGTCATGGCATTCGCAGTGCCCAGTGATGGCATTACTCTGCACTATAGCCATTT

GCCCCAACTTAAGTTTAGAAATTACAAGTTTCAGTAATAGCTGAACCTGTTCAAAATGTT

AATAAAGGTTTCGTTGCATGGTA). In some embodiments, a 3' UTR sequence for use in the IVT templates comprises a sequence encoding the 3' UTR sequence of UBB or SEQ ID NO: 171

(TTCTTCAGTCATGGCATTCGCAGTGCCCAGTGATGGCATTACTCTGCACTATAGCCATTT

GCCCCAACTTAAGTTTAGAAATTACAAGTTTCAGTAATAGCTGAACCTGTTCAAAATGTT

AATAAAGGTTTCGTTGCATGGTA).

[00146] Other exemplary 3' UTR sequences for use in the IVT templates described herein include 3' UTR sequences found in tissue-specific genes, such as albumin and myosin. For example, in some embodiments, a 3' UTR sequence for use in the IVT templates comprises a sequence encoding the 3' UTR sequence of Albumin or SEQ ID NO: 172

(CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATC AAAAGCTTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAA CATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAA GAATCTAATAGAGTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGCTATCCTGAAAAT TCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGGTAGAGGATTTCTA GTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCT). In some embodiments, a 3' UTR sequence for use in the IVT templates comprises a sequence encoding the 3' UTR sequence of MYL2 (Cardiac myosin) or SEQ ID NO: 173

(GAGGGGGCTCGCTGCTGCGCCCTGGGCTCGTCTTTGCAGAGTGGTCCCTGCCCTCATCTC

TCTCCCCCGAGTACCGCCTCTGTCCCTACCTTGTCTGTTAGCCATGTGGCTGCCCCATTTA

TCCACCTCCATCTTCTTTGCAGCCTGGGTGGCTATGGGTACTTCGTGGCCGCACATCCTAC

AGTTGGAAATCCATCCAGAGGCCATGTTCCAATAAACAGGAGGTCGTGTATTTGGTCACG

ACATTTCTCTGAC). In some embodiments, a 3' UTR sequence for use in the IVT templates comprises a sequence encoding the 3' UTR sequence of MYH2 (Skeletal myosin) or SEQ ID NO:

174

(TCATGTCCTGATGCCATGGAATGACTGAAGACAGGCACAAAATGTGACATCTTTGGTCA TTTCCCTCTGTAATTATTGTGTATTCTACCCTGTTGCAAAGGAAATAAAGCATAGGGTAG TTTGC A A AC A A) . Blunt-ended Restriction Sites and Enzymes

[00147] A key feature of the IVT templates described herein is the use of blunt-ended restriction sites that are designed to flank the sequence of the open-reading frame sequence of interest to be transcribed in the IVT reactions. By adding blunt-ended restriction site sequences into the IVT template itself, the need to incorporate restriction site sequences into the open reading frame sequence(s) is eliminated, thereby removing additional steps, such as PCR-mediated incorporation of restriction site sequences into each open reading frame of interest to be expressed, and additional digestion steps. Further, by selecting combinations of blunt-ended restriction sites that comprise part of the 5' or 3' UTR sequence being used in the IVT templates, as demonstrated herein, simple PCR based cloning methods can be used to generate and amplify a desired sequence comprising both the 5' and the first blunt-ended restriction site and/or both the 3' UTR sequence and the second blunt-ended restriction site, respectively. For example, in those embodiments where the sequence encoding the 5' UTR sequence is SEQ ID NO: 20, the four 3' terminal nucleotides of SEQ ID NO: 20 are the same as the first four nucleotides of the Ale I blunt-ended restriction sequence. Similarly, in those embodiments where the sequence encoding the 3' UTR sequence is SEQ ID NO: 21, the first or 5' three nucleotides of SEQ ID NO: 21 are the same as the three terminal nucleotides of the Afel blunt-ended restriction sequence.

[00148] Accordingly, provided herein are blunt-ended restriction sequences for use in the IVT templates described herein. As used herein, "blunt-ended restriction sequences" or "blunt-ended restriction site sequences" refer to a specific sequence of nucleotides that can be recognized by a specific restriction enzyme or restruction endonuclease, and that upon cutting by the restriction enzyme generates a "blunt" or "non-sticky" end. As used herein, a "blunt-end" refers to the end of a double-stranded nucleic acid which terminates in a complementary base pair. In contrast, a "sticky end" refers to overhangs or a stretch of non-base-paired nucleotides at the end of a double-stranded nucleic acid molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs. When generated using a restriction enezyme, these overhangs are in most cases palindromic.The simplest case of an overhang is a single nucleotide. Longer overhangs (i.e. , greater than one nucleotide) are called cohesive ends or sticky ends, and are most often created by restriction endonucleases when they cut DNA. Such ends are called cohesive since they can be easily joined back together by a ligase. Accordingly, by identifying restriction enzymes that produce blunt-ends upon digestion of a double-stranded nucleic acid sequence, blunt-ended restriction sequences can be selected for use in the IVT templates described herein.

[00149] "Restriction enzymes," as used herein, refer to those enzymes that recognize a specific sequence of nucleotides and produce a double-stranded cut in a DNA molecule. Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. [00150] Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and are typically the only class used in the laboratory for DNA analysis and gene cloning. Rather than forming a single family of related proteins, Type II enzymes are a collection of unrelated proteins of many different sorts. Type II enzymes frequently differ so utterly in amino acid sequence from one another, and indeed from every other known protein, that they exemplify the class of rapidly evolving proteins that are often indicative of involvement in host-parasite interactions.

[00151] The most common Type II enzymes are those like Hhal, Hindlll and Notl that cleave

DNA within their recognition sequences. Enzymes of this kind are the principle ones available commercially. Most recognize DNA sequences that are symmetric because they bind to DNA as homodimers, but a few, (e.g., BbvCI) recognize asymmetric DNA sequences because they bind as heterodimers. Some enzymes recognize continuous sequences (e.g., EcoRI) in which the two half- sites of the recognition sequence are adjacent, while others recognize discontinuous sequences (e.g., Bgll) in which the half-sites are separated. Cleavage leaves a 3'-hydroxyl on one side of each cut and a 5 '-phosphate on the other. They require only magnesium for activity and the corresponding modification enzymes require only S-adenosylmethionine. They tend to be small, with subunits in the 200-350 amino acid range.

[00152] The next most common Type II enzymes, usually referred to as 'Type IIS", are those like Fokl and Alwl that cleave outside of their recognition sequence to one side. These enzymes are intermediate in size, 400-650 amino acids in length, and they recognize sequences that are continuous and asymmetric. They comprise two distinct domains, one for DNA binding the other for DNA cleavage.

[00153] Type IIG restriction enzymes, the third major kind of Type II enzyme, are large, combination restriction-and-modification enzymes, 850-1250 amino acids in length, in which the two enzymatic activities reside in the same protein chain. These enzymes cleave outside of their recognition sequences; those that recognize continuous sequences (e.g., Acul: CTGAAG) cleave on just one side; those that recognize discontinuous sequences (e.g., Bcgl: CGANNNNNNTGC) (SEQ ID NO: 190) cleave on both sides releasing a small fragment containing the recognition sequence. The amino acid sequences of these enzymes are varied but their organization are consistent. They comprise an N-terminal DNA -cleavage domain joined to a DNA-modification domain and one or two DNA sequence-specificity domains forming the C-terminus, or present as a separate subunit. When these enzymes bind to their substrates, they switch into either restriction mode to cleave the DNA, or modification mode to methylate it.

[00154] Type III enzymes are also large combination restriction-and-modification enzymes.

They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests. [00155] Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of E. coli.

[00156] Restriction enzymes that generate blunt-ends for use with the IVT templates and constructs described herein are Type II restriction enzymes. Typical type II restriction enzymes comprise a dimer of only one type of subunit; typically have undivided and palindromic recognition site sequences of 4-8 nucleotides in length; they recognize and cleave DNA at the same site; and they do not use ATP or AdoMet for their activity and usually only require Mg2+ as a cofactor. These are the most commonly available and used restriction enzymes. Sub-groups of Type II restriction enzymes have also been categorized, as described herein.

[00157] When selecting the first and second blunt-ended restriction site sequences used in the

IVT templates described herein, it is important to ensure that the first and second blunt-ended restriction site sequences are not found at any other location in the IVT template, open reading frame sequence, or vector backbone sequence, such that contacting with the restriction enzymes specific for the first and second blunt-ended restriction site sequences does not generate additional cleavages of the double-stranded nucleic acid sequence. Identifying such potential cleavage sequences in the IVT templates or open reading frame sequences is known to those of ordinary skill in the art and can be done, for example, using any of a number of tools on the world wide web. For example, New England Biolabs provides a web-based tool termed NEBcutter (V2.0) to identify sites for all Type II and commercially available Type III restriction enzymes that cut only once a sequence submitted to their website. Therefore, it is preferred that rare or very rare cutters are used as the first and second blunt- ended restriction site sequences used in the IVT templates described herein. Alel and Afel are examples of such rare cutter restriction enzyme sites that are ideal for a vector that is designed to be used for cloning hundreds of different ORFs.

[00158] Additional considerations to factor when selecting the first and second blunt-ended restriction sites to use when designing the IVT templates described herein include the choice of which 5' and 3' UTR sequences are being used. As described herein, for example, if the sequence encoding the 5' UTR sequence is selected to be SEQ ID NO: 20, the 4 3' terminal nucleotides of SEQ ID NO: 20 are the same as the first or 5' 4 nucleotides of the Alel blunt-ended restriction sequence (SEQ ID NO: 60). Similarly, if the sequence encoding the 3' UTR sequence is designed to be SEQ ID NO: 21, the first or 5' 3 nucleotides of SEQ ID NO: 21 are the same as the 3 terminal (3') nucleotides of the Afel blunt-ended restriction sequence (SEQ ID NO: 30).

[00159] A list of exemplary, but non-limiting, restriction site sequences (the corresponding complementary sequence is easily identifiable and determined by one of skill in the art) that generate blunt-ends upon digestion, and their corresponding restriction enzymes are provided herein as Table 2. As described, one preferably uses an enzyme that is a rare cutting enzyme to allow cloning of as many different ORFs into the construct as possible. Naturally enzymes or cutters that can cut the construct nucleic acid sequence cannot be used. Accordngly, enzymes like Afel and Alel were selected the exemplary nucleic acid construct shown in the examples.

Table 2: Examples of Blunt-end Generating Restriction Enzymes and Restriction Sequences

Restriction Enzyme Restriction SEQ ID NO Sequence

BspANI GG/CC SEQ ID NO 73

BspFNI CG/CG SEQ ID NO 74

BstFNI CG/CG SEQ ID NO 75

BsuRI GG/CC SEQ ID NO 76

CviJI RG/CY SEQ ID NO 77

CviRI TG/CA SEQ ID NO 78

CviTI RG/CY SEQ ID NO 79

FnuDII CG/CG SEQ ID NO 80

Glal GC/GC SEQ ID NO 81

Mall GA/TC SEQ ID NO 82

Mvnl CG/CG SEQ ID NO 83

Pall GG/CC SEQ ID NO 84

Thai CG/CG SEQ ID NO 85

Cdil CATC/G SEQ ID NO 86

Aatl AGG/CCT SEQ ID NO 87

Accini AGT/ACT SEQ ID NO 88

Accl6I TGC/GCA SEQ ID NO 89

AccBSI CCG/CTC SEQ ID NO 90

Acvl CAC/GTG SEQ ID NO 91

Ajil CACGTC SEQ ID NO 92

Aor51HI AGCGCT SEQ ID NO 93

Assl AGTACT SEQ ID NO 94

Avtll TGCGCA SEQ ID NO 95

Ball TGGCCA SEQ ID NO 96

BprPI CACGTG SEQ ID NO 97

BmcAI AGTACT SEQ ID NO 98

Bmil GGNNCC SEQ ID NO 99

BscBI GGNNCC SEQ ID NO 100

Bsp68I TCGCGA SEQ ID NO 101

BspLI GGNNCC SEQ ID NO 102

BssNAI GTATAC SEQ ID NO 103

Bstll07I GTATAC SEQ ID NO 104

BstBAI YACGTR SEQ ID NO 105

BstC8I GCNNGC SEQ ID NO 106

BstHPI GTTAAC SEQ ID NO 107

BstSNI TACGTA SEQ ID NO 108

Btrl CACGTC SEQ ID NO 109

BtuMI TCGCGA SEQ ID NO 110

Dinl GGCGCC SEQ ID NO 111

Ecll36II GCGCTC SEQ ID NO 112

Ecol05I TACGTA SEQ ID NO 113

Ecol47I AGGCCT SEQ ID NO 114

Eco32I GACTATC SEQ ID NO 115

Eco47lll AGCGCT SEQ ID NO 116

Eco72I CACGTG SEQ ID NO 117

EcilCRI GAGCTC SEQ ID NO 118

Egel GGCGCC SEQ ID NO 119

Ehel GGCGCC SEQ ID NO 120

Funl AGCGCT SEQ ID NO 121

Hael WGGCCW SEQ ID NO 122

Hindll GTYRAC SEQ ID NO 123

Hpy8I GTNNAC SEQ ID NO 124

KspAI GTTACC SEQ ID NO 125

Mbil CCGCTC SEQ ID NO 126

MM TGGCCA SEQ ID NO 127

MluNI TGGCCA SEQ ID NO 128 Restriction Enzyme Restriction SEQ ID NO

Sequence

Msp20I TGGCCA SEQ ID NO 129

Mstl TGCGCA SEQ ID NO 130

Nsbl TGCGCA SEQ ID NO 131

NspBII CMGCKG SEQ ID NO 132

Peel AGGCTT SEQ ID NO 133

Pdil GCCGGC SEQ ID NO 134

PmaCI CACGTG SEQ ID NO 135

Ppu21I YACGTR SEQ ID NO 136

Psil TTATAA SEQ ID NO 137

PspCI CACGTG SEQ ID NO 138

PspN4I GGNNCC SEQ ID NO 139

SseBI AGGCCT SEQ ID NO 140

Zral GACGTC SEQ ID NO 141

Zrml AGTACT SEQ ID NO 142

FspAI RTGCGCAY SEQ ID NO 143

Mssl GTTTAAAC SEQ ID NO 144

Smil ATTTAAAT SEQ ID NO 145

Srfl GCCCGGGC SEQ ID NO 146

Asp700I GAANNNNTTC SEQ ID NO 147

Boxl GACNNNNGTC SEQ ID NO 148

Bse8I GATNNNNATC SEQ ID NO 149

BoxJI GATNNNNATC SEQ ID NO 150

BsiBI GATNNNNATC SEQ ID NO 151

BsrBRI GATNNNNATC SEQ ID NO 152

BstPAI GACNNNNGTC SEQ ID NO 153

Maml GATNNNNNATC SEQ ID NO 154

MroXI GAANNNNTTC SEQ ID NO 155

Olil CACNNNNGTG SEQ ID NO 156

Pdml GAANNNNTTC SEQ ID NO 157

Rsel CAYNNNNRTG SEQ ID NO 158

SmiMI CAYNNNNRTG SEQ ID NO 159

Schl GAGTCNNNN/ SEQ ID NO 160

Spacer Sequences

[00160] The IVT templates described herein are further designed to include a spacer sequence between the first and second blunt-ended restriction site sequences in order to allow efficient utilization of and access to the restriction site sequences by their corresponding restriction enzymes during a digestion reaction.

[00161] Accordingly, a "spacer" or a "spacer sequence" refer to a heterologous or random nucleotide sequence containing a known number of nucleotides that can be used to separate or 'space' two sequences that are not desired to be adjacent or consecutive. The number of nucleotides, or analogues thereof, in the spacer can range from at least 2 nucleotides or analogues thereof, up to and including at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, or at least 175 nucleotides or analogues thereof.

[00162] In some embodiments, the number of nucleotides, or analogues thereof, in the spacer sequence can be in a range from about 2-5, from about 2-10, from about 2-15, from about 2-20, from about 2-165, from about 5-25, from about 5-50, from about 5-100, from about 10-50, from about 10- 100, from about 10-150, from about 10-175 nucleotides or analogues thereof.

Primers and Primer Sequences

[00163] Primer sequences and primer binding sequences are used in the IVT constructs described herein and in the methods of synthesizing RNAs, such as modified RNAs, using these constructs. More specifically, in some embodiments, primer pair sequences are designed to flank the promoter sequence and 3' UTR sequence of an IVT template and are thus useful for amplifying a desired DNA sequence comprising, for example, the promoter sequence, the 5' UTR sequence, the ORF sequence of interest, and the 3' UTR sequence prior to addition of the poly-A tail sequence in a tail-PCR reaction, as described herein (see, for example, FIG. 1C). In addition, primers and primer pairs can be used in the amplification of a given ORF sequence of interest, in screening experiments to identify clones in the correct orientation, in constructing UTR sequences for use in an IVT construct, and various combinations thereof.

[00164] As used herein, a "primer" refers to any polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as a substrate or point of initiation of nucleic acid synthesis. Oligonucleotide primers for use with the compositions and in the kits and methods described herein can be prepared using any suitable method known to those skilled in the art, such as, for example, methods using phosphotriesters and phosphodiesters. In some embodiments, one or more phosphorothioate linkages can be included in the primers. An oligonucleotide primer can also be modified at the base moiety, sugar moiety, or phosphate backbone with minor groove binders, intercalating agents and the like, so long as its ability to specifically bind a template and serve as a substrate for polymerase extension are maintained.

[00165] Primers can be designed according to known algorithms. Where amplification is desired, the primers are designed to hybridize to sequences that flank the target nucleic acid sequence being amplified, i.e. , a "primer pair." Typically, commercially available or custom software use algorithms to design primers such that the annealing temperatures of the primers are close to melting temperature. Primers can be of a variety of lengths and are preferably less than 50 nucleotides in length and greater than 6 nucleotides in length, preferably 6-35 nucleotides, more preferably 12-30 nucleotides, and most preferably 15-25 nucleotides in length. Oligonucleotide primers are usually at least 6 bases, at least 8 bases, at least 10 bases, at least 12 bases, more often about 15 bases, about 16 bases, about 17 bases, about 18 bases, about 19 bases, about 20 bases, about 21 bases, about 22 bases, about 23 bases, about 24 bases, or about 25 bases in length. Primers are typically designed so that all primers participating in a particular reaction have melting temperatures that are within 10 °C, preferably within 5°C, and most preferably within 2°C of each other. Primers are further designed to avoid priming on themselves or another primer as templates in a reaction, and to avoid intra- and intermolecular complementarity. In some embodiments, the oligonucleotide primers for use in the methods described herein have a GC content similar to that of the template nucleic acid. It is preferred that oligonucleotide primers do not comprise unusual sequence runs, such as stretches of polypurines or polypyrimidines, as such stretches can result in secondary structures that inhibit amplification steps, such as PCR. It is also preferred a given set of oligonucleotide primers do not have complementarity to each other in their 3' ends.

[00166] Primers must be sufficiently complementary to their respective target nucleic acid strands to anneal or hybridize selectively and form stable duplexes. In some embodiments, oligonucleotide primers are designed to be exactly complementary to a target nucleic acid sequence. In other embodiments, base-pair mismatches or sites of non-complementarity can be included. In those embodiments where one or more mismatches are to be included in an oligonucleotide primer or primer set, it is preferred that the mismatches or non-complementary sites occur at the 5' end of the primer, as the closer a mismatch is to the 3' end of a primer, the more likely it is to prevent extension of the annealed primer.

[00167] As understood by one of skill in the art, when a first DNA molecule is said to be

"complementary" to a second DNA sequence, any C, G, A, or T nucleotides on the first DNA molecule is base-paired with the complementary G, C, T, or A, respectively, on the second DNA molecule, and vice versa. Similarly, when a DNA molecule is said to be "complementary" to an RNA sequence, any C, G, or A nucleotides on the RNA molecule is base -paired with the complementary G, C, and T, respectively, on the DNA molecule, while any U nucleotides on the RNA molecule are base-paired with A nucleotides on the DNA molecule.

[00168] In some embodiments of the methods described herein, a primer can comprise a 5' end sequence of "n" nucleotides that is not complementary to a target sequence and a 3' end that is highly complementary to or exactly complementary to a target nucleic acid sequence, such that extension of the primer hybridized to a target RNA or DNA sequence generates a product comprising an extra "n" complmentary nucleotides. In some such embodiments, the primer comprises an extra "n" T nucleotides, thereby adding on a poly-adenylation tail of desired length.

[00169] In the case of an amplification reaction, primer concentrations should be sufficient to bind to the amount of target sequences that are amplified. Those of skill in the art will recognize that the amount or concentration of primer should vary according to the binding affinity of the primers as well as the quantity of sequence to be bound. Typical primer concentrations range from, for example, Ο.ΟΙμΜ to Ι .ΟμΜ in a reaction.

[00170] The PCR amplification reactions described herein are performed under conditions in which the primers hybridize to the target sequence template and are extended by a polymerase. As appreciated by those of skill in the art, such reaction conditions can vary, depending on the target nucleic acid of interest and the composition of the primer. Amplification reaction cycle conditions are selected so that the primers hybridize specifically to the desired target sequence and are extended, if the appropriate polymerase is present. Primers that hybridize specifically to a target sequence enable amplification of the target sequence preferentially in comparison to other nucleic acids that can be present in the sample that is analyzed.

[00171] Exemplary primers used in the pORFin system described herein provide a specific and robust PCR. Similar primers can be used in alternative embodiments or sequences can be designed to result in a specific product with other sequences. Agains, it is preferable that the primer sequences are long enough, typically about 12-25 nucleotides, to provide a rare or very rare sequence that can be used for a specific amplification of any number of ORFs without the concern of recognizing an identical sequence within the ORF sequence. This is easy to also verify prior to cloning any given ORF to the template, using software programs and the like that are well-known to those of ordinary skill in the art.

Tail PCR and Tail PCR Primers

[00172] On a linear DNA template, such as a digested IVT template as described herein, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003). This can lead to runoff transcript bending followed by template exchange with the second DNA strand or transcription of RNA itself (Triana-Alonso et al., J. Biol. Chem., 270:6298-307 (1995); Dunn and Studier, J. Mol. Biol., 166:477-535 (1983); Arnaud-Barbe et al., Nuc. Acids Res., 26:3550-54 (1998); Macdonald et al., 1993), and then to aberrant transcription in a reverse direction and accumulation of double stranded RNA, which can inhibit gene expression. Thus, as known by those of skill in the art, DNA linearization itself is not sufficient for correct transcription (Triana-Alonso et al., J. Biol. Chem., 270:6298-307 (1995); Dunn and Studier, J. Mol. Biol., 166:477-535 (1983); Arnaud-Barbe et al, 1998 Nuc. Acids Res., 26:3550-54 (1998);

Macdonald et al., J. Mol. Biol., 232:1030-47 (1993); Nakano et al., Biotechnol. Bioeng., 64: 194-99 (1999).

[00173] However, a DNA sequence, such as an IVT template sequence, linearized

downstream of a poly(A/T) stretch of 64-100 nucleotides results in good templates for in vitro transcription (Saeboe-Larssen et al., J. Immunol. Meth., 259: 191-203 (2002); Boczkowski et al., Cancer Res., 60: 1028-34 (2000); Elango et al., Biochem Biophys Res Commun., 330:958-966 2005). Thus, in order to terminate transcription and prevent aberrant transcription, it is preferred that the IVT template used in an IVT reaction comprises a "poly-A" tail that, upon transcription, becomes an extended poly-U tail that acts to terminate transcription.

[00174] Accordingly, as used herein, a "poly(A)-tail" refers to the series of adenosines attached by polyadenylation to generate a 3' homopolymeric tail of adenine nucleotides, which can vary in length {e.g., at least 5 adenine nucleotides) and can be up to several hundred adenine nucleotides. The inclusion of a 3' poly(A) tail can protect RNA from degradation in the cell, and also facilitates extra-nuclear localization to enhance translation efficiency, and can thus be added to an RNA generated using the IVT templates and methods thereof as described herein to promote effective translation and stability of an RNA sequence of interest.

[00175] In some embodiments, the poly(A) tail comprises between 1 and 500 adenine nucleotides (SEQ ID NO: 191); in other embodiments the poly(A) tail comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 adenine nucleotides or more. In some embodiments, the poly(A) tail comprises between 1 and 150 adenine nucleotides (SEQ ID NO: 192). In other embodiments, the poly (A) tail comprises between 90 and 120 adenine nucleotides (SEQ ID NO: 193). In some such embodiments, the poly(A) tail comprises one or more modified nucleosides.

[00176] One conventional method of integration of polyA/T stretches into a DNA template is molecular cloning by adding the poly-A/T stretch to the end of the IVT template sequence to be transcribed, for example. However, integration of a polyA/T sequence into a plasmid DNA sequence can cause plasmid instability, often resulting in deletions and other aberrations. Further, such cloning procedures add additional laborious and time consuming steps.

[00177] Accordingly, as described herein, in preferred embodiments, a poly-adenylation template is added by PCR, in a process referred to herein as a "Tail PCR," to an open reading frame sequence inserted into an IVT template, as described herein. In such embodiments, following the dephosphorylation and linearization of an IVT template comprising an inserted ORF sequence, and prior to the in vitro transcription reaction, the IVT template sequence comprising the inersted ORF sequence is amplified using forward and reverse universal primer sequences. The forward universal primer sequence is identical to, or comprises at its 3' end a sequence identical to, the sequence termed the "first universal primer sequence" of the IVT template coding strand. In some such embodiments, the forward universal primer sequence further comprises, at its 5' end, an additional sequence, such as, for example, a tag sequence.

[00178] The reverse universal primer sequence is complementary to, or comprises a sequence at its 3' end complementary to, the sequence termed the "sequence complementary to the second universal primer sequence" of the IVT template coding strand. Importantly, the reverse universal primer sequence further comprises at its 5' end a poly-T sequence of "n" T nucleotides, such that upon amplification of the IVT template sequence during the Tail PCR, a poly-adenylation sequence or poly- A tail is added to the 3' end of the IVT template coding strand sequence, thereby generating the IVT template to be used in the subsequent in vitro transcription reaction. The universal reverse primer can comprise a poly-T sequence of, typically 50-5000 T nucleotides (SEQ ID NO: 194), for example, about 50-1000 (SEQ ID NO: 195), about 50-500 T nucleotides (SEQ ID NO: 196), about 50-250 T nucleotides (SEQ ID NO: 197), about 75-150 T nucleotides (SEQ ID NO: 198), or at least 50 T nucleotides, at least 60 T nucleotides, at least 70 T nucleotides, at least 80 T nucleotides, at least 90 T nucleotides, at least 100 T nucleotides, at least 110 T nucleotides, at least 120 T nucleotides, at least 130 T nucleotides, at least 140 T nucleotides, at least 150 T nucleotides, at least 160 T nucleotides, at least 170 T nucleotides, at least 180 T nucleotides, at least 190 T nucleotides, at least 200 T nucleotides, at least 225 T nucleotides, at least 250 T nucleotides, at least 275 T nucleotides, at least 300 T nucleotides, at least 325 T nucleotides, at least 350 T nucleotides, at least 375 T nucleotides, at least 400 T nucleotides, at least 425 T nucleotides, at least 450 T nucleotides, at least 475 T nucleotides, at least 500 T nucleotides, or more. The examples provided herein demonstrate that, for example, a 120 base pair stretch of poly(A) (SEQ ID NO: 199) in a transcribed RNA molecule generated using the IVT templates and methods described herein is sufficient to enable efficient translation of a synthetic RNA transcript.

[00179] In other embodiments, if desired, poly(A) tails of synthesized RNAs can be further extended following in vitro transcription with the use of a poly (A) polymerase, such as E. coli poly A polymerase (E-PAP) or yeast polymerase.

[00180] In addition, the attachment of different chemical groups to the 3' end can increase

RNA stability. Such an attachment can comprise modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA. Suitable ATP analogs include, but are not limited to, cordiocipin and 8-azaadenosine.

Open Reading Frame Sequences

[00181] The IVT templates described herein are specifically designed and constructed to permit the easy and rapid introduction or insertion of an open reading frame sequence of interest into the template. By including known and characterized sequences encoding for 5' and 3' UTR sequences in the templates that permit the synthesis of stable RNA molecules in vitro, the sequence of interest to be transcribed does not necessarily need to include any endogenous sequences encoding 5' and 3' UTR sequences, or any enhancer elements. By designing the templates to use only pairs of blunt- ended restriction site sequences at the site in the IVT template where a desired open reading frame sequence is to be inserted, any suitable open reading frame can be ligated into the IVT template and subsequently transcribed.

[00182] Accordingly, an open reading frame sequence can be from any DNA source, including, but not limited to, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other appropriate source of DNA. A open reading frame DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that comprises portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be, for example, from a single organism or from more than one organism. Genes that can be used as sources of DNA for PCR include genes that encode peptides that are important for regulating cellular differentiation, trans-differentiation and reprogramming. Preferred genes include those encoding for transcription factors and mRNA-binding proteins, for example, transcription factors that regulate, for example, the self-renewal and/or proliferation of stem cells. Exemplary open reading frame sequences that have been ligated into an embodiment of the IVT templates described herein in FIG. 4 are provided in FIGS. 5-13, and are further described herein.

[00183] In some embodiments, the open reading frame DNA encodes an mRNA that undergoes translation into a peptide or polypeptide. In some embodiments, the open reading frame DNA encodes inhibitory RNAs, such as small interfering RNAs (siRNA) or micro RNAs (miRNA). For example, the open reading frame DNA can encode an interfering RNA that prevents expression of an mRNA. The open reading frame DNA can encode an RNA that is a pre -RNA, for example pre- miRNA, or a mature RNA, for example mature miRNA. The open reading frame DNA can encode an RNA that is a fragment or variant of an RNA that retains the biological activity of the RNA.

[00184] Accordingly, as used herein, an "open reading frame" or "ORF" or "open reading frame sequence" or "open reading frame DNA" refers to a series of nucleotides that comprises a sequence of bases that can encode a RNA sequence that is translated into a polypeptide or protein or an non-translated RNA product, such as an miRNA. An open reading frame is flanked by and includes the start-code sequence (initiation codon or start codon) and the stop-codon sequence (termination codon). It is preferred that an open reading frame sequence of interest does not comprise a sequence encoding a poly-adenylation tail upon insertion into an IVT template described herein.

[00185] As described herein, PCR can be used to generate or amplify a desired open reading frame sequence to be inserted between the blunt-ended restriction site sequences of a given IVT template. Methods for performing PCR and designing PCR primers are well known in the art and are described elsewhere herein (see, for example, the sub-section entitled Primers and Primer Sequences). Upon selection and/or generation of an open reading frame sequence of interest, using, for example, phosphorylated ORF-specific PCR primers to amplify the desired open reading frame sequence of interest, the amplified open reading frame fragment can be inserted into a digested IVT template by blunt-end ligation, as described herein. PCR screening can then be performed to identify those clones in which the open reading frame sequence has been inserted into the digested IVT template in the correct orientation.

[00186] In those embodiments where the open reading frame sequence encodes for an mRNA, the mRNA can encode or be translated into essentially any polypeptide or peptide that is desired to be expressed. Such polypeptides include, but are not limited to, transcription factors, targeting moieties and other cell-surface polypeptides, cell-type specific polypeptides, differentiation factors, death receptors, death receptor ligands, structural proteins, enzymes, hormones, reprogramming factors, de- differentiation factors, cytokines, and any combination thereof. Further, such polypeptides or peptides to be expressed can include fusion proteins, truncated variants, protein domains, allelic variants and the like of any polypeptide. Additionally, the open reading frame sequences can encode essentially any non-translated RNA molecule that it is desired to synthesize, including, for example, shRNA molecules, siRNA molecules, dsRNA molecules, ribozymes, and any combinations thereof.

[00187] In some embodiments, an open reading frame sequence encodes for a transcription factor. As used herein the term "transcription factor" refers to a protein that binds to specific DNA sequences and thereby controls the transfer (or transcription) of genetic information from DNA to mRNA. In one embodiment, the transcription factor encoded by the synthetic, modified RNA is a human transcription factor, such as those described in e.g., Messina DM, et al. (2004) Genome Res. 14(10B):2041-2047, which is herein incorporated by reference in its entirety. Some non-limiting examples of human transcription factors (and their mRNA IDs and sequence identifiers) for use in the aspects and embodiments described herein include those listed in Table 3 of International Patent Publication PCT/US2011/032679, the contents of which are herein incorporated by reference in their entireties.

[00188] An exemplary ORF encoding a transcription factor molecule is provided herein as

SEQ ID NO: 175. SEQ ID NO: 175 is an ORF encoding human PAX5 that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGGATTTAGAGAAAAATTATCCGACTCCTCGGACCAGCAGGACAGGACATGGAGGAGT

GAATCAGCTTGGGGGGGTTTTTGTGAATGGACGGCCACTCCCGGATGTAGTCCGCCAGA

GGATAGTGGAACTTGCTCATCAAGGTGTCAGGCCCTGCGACATCTCCAGGCAGCTTCGGG

TCAGCCATGGTTGTGTCAGCAAAATTCTTGGCAGGTATTATGAGACAGGAAGCATCAAG

CCTGGGGTAATTGGAGGATCCAAACCAAAGGTCGCCACACCCAAAGTGGTGGAAAAAAT

CGCTGAATATAAACGCCAAAATCCCACCATGTTTGCCTGGGAGATCAGGGACCGGCTGC

TGGCAGAGCGGGTGTGTGACAATGACACCGTGCCTAGCGTCAGTTCCATCAACAGGATC

ATCCGGACAAAAGTACAGCAGCCACCCAACCAACCAGTCCCAGCTTCCAGTCACAGCAT

AGTGTCCACTGGCTCCGTGACGCAGGTGTCCTCGGTGAGCACGGATTCGGCCGGCTCGTC

GTACTCCATCAGCGGCATCCTGGGCATCACGTCCCCCAGCGCCGACACCAACAAGCGCA

AGAGAGACGAAGGTATTCAGGAGTCTCCGGTGCCGAACGGCCACTCGCTTCCGGGCAGA

GACTTCCTCCGGAAGCAGATGCGGGGAGACTTGTTCACACAGCAGCAGCTGGAGGTGCT

GGACCGCGTGTTTGAGAGGCAGCACTACTCAGACATCTTCACCACCACAGAGCCCATCA

AGCCCGAGCAGACCACAGAGTATTCAGCCATGGCCTCGCTGGCTGGTGGGCTGGACGAC

ATGAAGGCCAATCTGGCCAGCCCCACCCCTGCTGACATCGGGAGCAGTGTGCCAGGCCC

GCAGTCCTACCCCATTGTGACAGGCCGTGACTTGGCGAGCACGACCCTCCCCGGGTACCC

TCCACACGTCCCCCCCGCTGGACAGGGCAGCTACTCAGCACCGACGCTGACAGGGATGG

TGCCTGGGAGTGAGTTTTCCGGGAGTCCCTACAGCCACCCTCAGTATTCCTCGTACAACG

ACTCCTGGAGGTTCCCCAACCCGGGGCTGCTTGGCTCCCCCTATTATTATAGCGCTGCCG CCCGAGGAGCCGCCCCACCTGCAGCCGCCACTGCCTATGACCGTCACTGA (SEQ ID NO: 175)

[00189] Another exemplary ORF encoding a transcription factor molecule is provided herein as SEQ ID NO: 176. SEQ ID NO: 176 is an ORF encoding mouse STAT3 that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGGCTCAGTGGAACCAGCTGCAGCAGCTGGACACACGCTACCTGAAGCAGCTGCACCA

GCTGTACAGCGACAGCTTCCCCATGGAGCTGCGGCAGTTCCTGGCACCTTGGATTGAGAG

TCAAGACTGGGCATATGCAGCCAGCAAAGAGTCACATGCCACGTTGGTGTTTCATAATCT

CTTGGGTGAAATTGACCAGCAATATAGCCGATTCCTGCAAGAGTCCAATGTCCTCTATCA

GCACAACCTTCGAAGAATCAAGCAGTTTCTGCAGAGCAGGTATCTTGAGAAGCCAATGG

AAATTGCCCGGATCGTGGCCCGATGCCTGTGGGAAGAGTCTCGCCTCCTCCAGACGGCA

GCCACGGCAGCCCAGCAAGGGGGCCAGGCCAACCACCCAACAGCCGCCGTAGTGACAG

AGAAGCAGCAGATGTTGGAGCAGCATCTTCAGGATGTCCGGAAGCGAGTGCAGGATCTA

GAACAGAAAATGAAGGTGGTGGAGAACCTCCAGGACGACTTTGATTTCAACTACAAAAC

CCTCAAGAGCCAAGGAGACATGCAGGATCTGAATGGAAACAACCAGTCTGTGACCAGAC

AGAAGATGCAGCAGCTGGAACAGATGCTCACAGCCCTGGACCAGATGCGGAGAAGCATT

GTGAGTGAGCTGGCGGGGCTCTTGTCAGCAATGGAGTACGTGCAGAAGACACTGACTGA

TGAAGAGCTGGCTGACTGGAAGAGGCGGCAGCAGATCGCGTGCATCGGAGGCCCTCCCA

ACATCTGCCTGGACCGTCTGGAAAACTGGATAACTTCATTAGCAGAATCTCAACTTCAGA

CCCGCCAACAAATTAAGAAACTGGAGGAGCTGCAGCAGAAAGTGTCCTACAAGGGCGAC

CCTATCGTGCAGCACCGGCCCATGCTGGAGGAGAGGATCGTGGAGCTGTTCAGAAACTT

AATGAAGAGTGCCTTCGTGGTGGAGCGGCAGCCCTGCATGCCCATGCACCCGGACCGGC

CCTTAGTCATCAAGACTGGTGTCCAGTTTACCACGAAAGTCAGGTTGCTGGTCAAATTTC

CTGAGTTGAATTATCAGCTTAAAATTAAAGTGTGCATTGATAAAGACTCTGGGGATGTTG

CTGCCCTCAGAGGGTCTCGGAAATTTAACATTCTGGGCACGAACACAAAAGTGATGAAC

ATGGAGGAGTCTAACAACGGCAGCCTGTCTGCAGAGTTCAAGCACCTGACCCTTAGGGA

GCAGAGATGTGGGAATGGAGGCCGTGCCAATTGTGATGCCTCCTTGATCGTGACTGAGG

AGCTGCACCTGATCACCTTCGAGACTGAGGTGTACCACCAAGGCCTCAAGATTGACCTAG

AGACCCACTCCTTGCCAGTTGTGGTGATCTCCAACATCTGTCAGATGCCAAATGCTTGGG

CATCAATCCTGTGGTATAACATGCTGACCAATAACCCCAAGAACGTGAACTTCTTCACTA

AGCCGCCAATTGGAACCTGGGACCAAGTGGCCGAGGTGCTCAGCTGGCAGTTCTCGTCC

ACCACCAAGCGAGGGCTGAGCATCGAGCAGCTGACAACGCTGGCTGAGAAGCTCCTAGG

GCCTGGTGTGAACTACTCAGGGTGTCAGATCACATGGGCTAAATTCTGCAAAGAAAACA

TGGCTGGCAAGGGCTTCTCCTTCTGGGTCTGGCTAGACAATATCATCGACCTTGTGAAAA

AGTATATCTTGGCCCTTTGGAATGAAGGGTACATCATGGGTTTCATCAGCAAGGAGCGGG

AGCGGGCCATCCTAAGCACAAAGCCCCCGGGCACCTTCCTACTGCGCTTCAGCGAGAGC

AGCAAAGAAGGAGGGGTCACTTTCACTTGGGTGGAAAAGGACATCAGTGGCAAGACCCA GATCCAGTCTGTAGAGCCATACACCAAGCAGCAGCTGAACAACATGTCATTTGCTGAAA

TCATCATGGGCTATAAGATCATGGATTGTACCTGCATCCTGGTGTCTCCACTTGTCTACCT

CTACCCCGACATTCCCAAGGAGGAGGCATTTGGAAAGTACTGTAGGCCCGAGAGCCAGG

AGCACCCCGAAGCCGACCCAGGTAGTGCTGCCCCGTACCTGAAGACCAAGTTCATCTGT

GTGACACCAACGACCTGCAGCAATACCATTGACCTGCCGATGTCCCCCCGCACTTTAGAT

TCATTGATGCAGTTTGGAAATAACGGTGAAGGTGCTGAGCCCTCAGCAGGAGGGCAGTT

TGAGTCGCTCACGTTTGACATGGATCTGACCTCGGAGTGTGCTACCTCCCCCATGTGA

(SEQ ID NO: 176)

[00190] In some embodiments, an open reading frame sequence encodes for a reprogramming factor. The term a "reprogramming factor," as used herein, refers to a developmental potential altering factor, such as a protein, RNA, or small molecule, the expression of which contributes to the reprogramming of a cell, e.g. a somatic cell, to a less differentiated or undifferentiated state, e.g. to a cell of a pluripotent state or partially pluripotent state. A reprogramming factor can be, for example, transcription factors that can reprogram cells to a pluripotent state, such as SOX2, OCT3/4, KLF4, NANOG, LIN-28, c-MYC, and the like, including as any gene, protein, RNA or small molecule, that can substitute for one or more of these in a method of reprogramming cells in vitro. A reprogramming factor can also be termed a "de -differentiation factor," which refers to a developmental potential altering factor, such as a protein or RNA, that induces a cell to de-differentiate to a less differentiated phenotype, or, in other words, increases the developmental potential of a cell.

[00191] In some embodiments, an open reading frame sequence encodes for a differentiation factor. As used herein, the term "differentiation factor" refers to a developmental potential altering factor, such as a protein, RNA, or small molecule, that induces a cell to differentiate to a desired cell- type, i.e. , a differentiation factor reduces the developmental potential of a cell. In some embodiments, a differentiation factor can be a cell-type specific polypeptide, however this is not required.

[00192] In some embodiments, an open reading frame sequence encodes for a CD ("cluster of differentiation") molecules and/or other cell-surface/membrane bound molecule or receptor, such as transmembrane tyrosine kinase receptors, ABC transporters, and integrins, for example.

[00193] An exemplary ORF encoding a CD molecule is provided herein as SEQ ID NO: 177.

SEQ ID NO: 177 is an ORF encoding a truncated form of human CD4 lacking the intracellular domains that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGA

CCTGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATA

AAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGC

GCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTT

AAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGC AATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCC

TGACCCTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAA

GGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGAT

AGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGA

CATCGTGGTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAAC

AGGTGGAGTTCTCCTTCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGC

TGTGGTGGCAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGA

ACAAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAG

CTCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTC

ACCCTGGCCCTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGAT

GAGAGCCACTCAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTA

AGCTGATGCTGAGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAA

GGCGGTGTGGGTGCTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGG

GACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCCACATGGTCGACCCCGGTGCAGC

CAATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCA

TCTTCTTCTGTGTCAGGTGCCGGCACTGA (SEQ ID NO: 177)

[00194] Another exemplary ORF encoding a CD molecule is provided herein as SEQ ID NO:

178. SEQ ID NO: 178 is an ORF encoding a truncated form of human CD271 or LNGFR (low- affinity nerve growth factor receptor) lacking the intracellular domains, that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples. ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTT CTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAG CGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCA ACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCG ACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTG CGTGGAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGA CTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCC AGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCC AACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCG CGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACAC GGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCA CCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGG CAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTC CATCCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGA (SEQ ID NO: 178)

[00195] Another exemplary ORF encoding a CD molecule is provided herein as SEQ ID NO:

179. SEQ ID NO: 179 is an ORF encoding a truncated form of mouse CD118 or leukemia inhibitory factor receptor (LIFR) that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGGCAGCTTACTCATGGTGGAGACAGCCATCGTGGATGGTAGACAATAAAAGATCGAG

GATGACTCCAAACCTGCCATGGCTCCTGTCAGCTCTGACCCTCCTGCATCTGACGATGCA

TGCAAACGGTCTGAAGAGAGGGGTACAAGACTTGAAATGCACAACCAACAACATGCGA

GTGTGGGACTGCACGTGGCCAGCTCCCCTCGGGGTCAGCCCTGGAACTGTTAAAGATATT

TGCATTAAAGACAGGTTCCATTCTTGTCACCCATTAGAGACAACAAACGTTAAAATTCCA

GCTCTTTCACCTGGTGATCACGAAGTCACAATAAATTATCTAAATGGCTTTCAGAGTAAA

TTCACGTTGAATGAAAAAGATGTCTCTTTAATTCCAGAGACTCCCGAGATCCTGGATTTG

TCTGCTGACTTCTTCACCTCCTCCTTACTACTGAAGTGGAACGACAGAGGGTCTGCTCTGC

CTCACCCCTCCAATGCCACCTGGGAGATTAAGGTTCTACAGAATCCAAGGACGGAACCA

GTAGCACTCGTGTTACTCAACACAATGCTGAGTGGTAAAGATACCGTTCAGCACTGGAAC

TGGACCTCAGACCTGCCCTTGCAATGTGCCACTCACTCGGTGAGCATTCGATGGCACATT

GACTCGCCTCATTTCTCCGGTTACAAAGAGTGGAGTGACTGGAGCCCGCTGAAGAACATC

TCAAACATGACAATTTGTTGTATGAGTCCAACGAAAGTGCTTTCAGGACAGATCGGCAAT ACCCTTCGTCCTCTCATCCATCTGTACGGGCAAACCGTTGCGATCCATATCCTGAACATCC

CGGTGGTCTTTGCAGGCTATCCTCCCGATGTTCCTCAGAAGCTGAGCTGTGAGACACATG

ACTTAAAAGAGATTATATGTAGCTGGAATCCAGGAAGGATAACTGGACTGGTGGGCCCA

CGAAATACAGAATACACCCTGTTTGAAAGCATTTCAGGAAAATCGGCAGTATTTCACAG

GATTGAAGGACTTACAAACGAGACCTACCGGTTAGGCGTGCAAATGCATCCCGGCCAAG

AAATCCATAACTTCACCCTGACTGGTCGCAATCCACTGGGGCAGGCACAGTCAGCAGTG

GTCATCAATGTGACTGAGAGAGTTGCTCCTCATGATCCGACTTCGTTGAAAGTGAAGGAC

ATCAATTCAACAGTTGTTACATTTTCTTGGTATTTACCAGGAAATTTTACAAAGATTAATC

ATCAGAGGAGCCGAGGATTCAACTTACCATGTTGCTGTAGACAAATTAAATCCATACACT

GCATACACTTTCCGGGTTCGTTGTTCTTCCAAGACTTTCTGGAAGTGGAGCAGGTGGAGT

GATGAGAAGCGACATCTAACCACAGAAGCCACTCCTTCAAAGGGACCAGACACTTGGAG

AGAGTGGAGTTCTGATGGAAAAAATCTAATCGTCTACTGGAAGCCTTTACCTATTAATGA

AGCTAATGGAAAAATACTTTCCTACAATGTTTCGTGTTCATTGAACGAGGAGACACAGTC

ACATCATCAGTGTGGTGGCAAGAAATTCTGCTGGCTCATCACCACCTTCGAAAATAGCTA GTATGGAAATCCCAAATGACGACATCACAGTAGAGCAAGCGGTGGGGCTAGGAAACAG GATCTTCCTCACCTGGCGTCACGACCCCAACATGACTTGTGACTACGTAATTAAATGGTG CAACTCATCTCGGTCTGAGCCCTGCCTCCTGGACTGGAGAAAGGTTCCTTCAAACAGCAC TGGGTGCACTAACCAGGGATACCAACTGTTACGTTCCATAATTGGATACGTAGAAGAACT

GGCTCCCATTGTCGCGCCAAATTTCACCGTGGAAGATACGTCGGCAGACTCGATATTAGT

GAAATGGGACGACATCCCCGTGGAAGAGCTCCGAGGCTTCCTAAGAGGGTATTTATTTTA

CTTTCAGAAAGGAGAGAGAGATACGCCCAAGACGAGGAGCTTGGAGCCACATCATTCTG

ACATCAAGCTAAAGAACATCACTGACATATCCCAGAAGACACTGAGGATTGCTGACCTT

CAGGGTAAAACCAGTTACCATCTGGTCCTGCGAGCCTATACACATGGTGGTCTGGGCCCA

GAGAAGAGCATGTTTGTGGTGACCAAGGAAAACTCTGTGGGATTGATTATTGCCATCCTC

ATCCCAGTGACTGTGGCTGTCATTGTTGGCGTGGTAACGAGCATCCTTTGCTATCGGAAG

CGAGAATGGATTAAGGAAACATTCTACCCAGATATTCCCAATCCAGAAAACTGTAAGGC

GCTACAGTTTCAGAAGAGCGTCTGCGAGGGAAGCAATGCTCTTAAGACATTGGAAATGA

ACCCCTGTACCCCGAACAACGTTGAAGTCCTGGAATCTCGGTCGATAGTCCCTAAAATAG

AAGATACAGAAATAATTTCCCCAGTTGCTGAGCGTCCTGGGGAAAGATCTGAGGTGGAC

CCTGAGAACCATGTGGTCGTGTCTTACTGCCCACCCATCATCGAGGAGGAAATAACGAA

CCCCGCGGCCGATGAAGTGGGAGGGGCTTCCCAGGTCGTGTACATCGATGTGCAGTCCA

TGTATCAGCCGCAAGCCAAAGCAGAGGAAGAGCAGGACGTGGATCCTGTGGTGGTGGCA

GGCTATAAGCCACAGATGCGCCTTCCCATCAGCCCCGCTGTGGAAGACACAGCAGCAGA

AGATGAAGAGGGTAAGACCGCCGGTTACAGACCTCAGGCCAATGTAAACACTTGGAATT

GTCCGTGCTCCATCAATTCCAGGCAATTCTTGATTCCTCCTAAAGACGAAGACTCTCCTA AATCTAATGGAGGAGGGTGGTCCTTTACAAACTTCTTCCAGAACAAACCAAATGACTAA

(SEQ ID NO: 179)

[00196] In some embodiments, an open reading frame sequence encodes for a cell-type specific polypeptide. As used herein, the term "cell-type specific polypeptide" refers to a polypeptide that is expressed in a cell having a particular phenotype (e.g. , a muscle cell) but is not generally expressed in other cell types with different phenotypes. For example, MyoD is expressed specifically in muscle cells but not in non-muscle cells, thus MyoD is a cell-type specific polypeptide. As another example, albumin is expressed in hepatocytes and is thus a hepatocyte-specific polypeptide. Such cell- specific polypeptides are well known in the art or can be identified, for example, using a gene array analysis and comparison of at least two different cell types. Methods for gene expressional array analysis is well known in the art.

[00197] In some embodiments, an open reading frame sequence encodes for a death receptor or death receptor ligand. By "death receptor" is meant a receptor that induces cellular apoptosis once bound by a ligand. Death receptors include, for example, tumor necrosis factor (TNF) receptor superfamily members having death domains (e.g. , TNFRI, Fas, DR3, 4, 5, 6) and TNF receptor superfamily members without death domains LTbetaR, CD40, CD27, HVEM. Death receptors and death receptor ligands are well known in the art. Some non-limiting examples of death receptors include FAS (CD95, Apol), TNFRI (p55, CD120a), DR3 (Apo3, WSL-1 , TRAMP, LARD), DR4, DR5 (Apo2, TRAIL-R2, TRICK2, KILLER), CARl , and the adaptor molecules FADD, TRADD, and DAXX. Some non-limiting examples of death receptor ligands include FASL (CD95L), TNF, lymphotoxin alpha, Apo3L (TWEAK), and TRAIL (Apo2L).

[00198] In some embodiments, an open reading frame sequence encodes for a mitogen receptor. Mitogen receptors include those that bind ligands including, but not limited to: insulin, insulin-like growth factor (e.g. , IGF1 , IGF2), platelet derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), fibroblast growth factor (FGF), bone morphogenic proteins (BMPs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-alpha and -beta, among others.

[00199] In addition, cytokines that promote cell growth can also be encoded by an open reading frame sequence to be transcribed by the IVT templates and methods described herein. For example, cytokines such as erythropoietin, thrombopoietin and other cytokines from the IL-2 subfamily tend to induce cell proliferation and growth.

[00200] An exemplary ORF encoding a polypeptide having structural activity is provided herein as SEQ ID NO: 180. SEQ ID NO: 180 is an ORF encoding human Lamin A that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATCGTGCCCATCCTGATCGAGCTGAA

TGGCGATGTGAATGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGATGCCACCT

ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCTGTGCCCTGGCCCA

CCCTGGTGACCACCCTGAGCTACGGCGTGCAGTGCTTCTCACGCTACCCCGATCACATGA

AGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACATCCAGGAGCGCACCATC

TTCTTCGAGGATGACGGCAACTACAAGTCGCGCGCCGAGGTGAAGTTCGAGGGCGATAC

CCTGGTGAATCGCATCGAGCTGACCGGCACCGATTTCAAGGAGGATGGCAACATCCTGG

GCAATAAGATGGAGTACAACTACAACGCCCACAATGTGTACATCATGACCGACAAGGCC

AAGAATGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGATGGCAGCGTGCA

GCTGGCCGACCACTACCAGCAGAATACCCCCATCGGCGATGGCCCTGTGCTGCTGCCCGA

TAACCACTACCTGTCCACCCAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGCGATC

ACATGATCTACTTCGGCTTCGTGACCGCCGCCGCCATCACCCACGGCATGGATGAGCTGT

ACAAGTCCGGACTTAAGGCCTCTGTCGACAGCAGTCTCTGTCCTTCGACCCGAGCCCCGC

GCCCTTTCCGGGACCCCTGCCCCGCGGGCAGCGCTGCCAACCTGCCGGCCATGGAGACCC

CGTCCCAGCGGCGCGCCACCCGCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCC

ACCCGCATCACCCGGCTGCAGGAGAAGGAGGACCTGCAGGAGCTCAATGATCGCTTGGC

GGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCAGGGCTGCGCCTTCGCA

TCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCTACGAG

GCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCT GCAGCTGGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCA

AGAAGGAGGGTGACCTGATAGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTG

AACTCCAAGGAGGCCGCACTGAGCACTGCTCTCAGTGAGAAGCGCACGCTGGAGGGCGA

GCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGCAGCCCTAGGTGAGGCCAAGA

AGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTGCAGACCATG

AAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGC

GCCGTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGC

CGGCTGGCGGATGCGCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTA

TAAGAAGGAGCTGGAGAAGACTTATTCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTG

AGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGGAGCTGCAGCAGTCGCGCATCCGC

ATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGCAGCCAAGGAGGC

GAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTGC

TGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGA

CGAGTACCAGGAGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACC

GCAAGCTCTTGGAGGGCGAGGAGGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAG

CGCAGCCGTGGCCGTGCTTCCTCTCACTCATCCCAGACACAGGGTGGGGGCAGCGTCACC

AAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCTCACAGCACGCACGCAC

TAGCGGGCGCGTGGCCGTGGAGGAGGTGGACGAGGAGGGCAAGTTTGTCCGGCTGCGCA

ACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGAT

GATCCCTTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTG

ACGATCTGGGCTGCAGGAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAA

GGCACAGAACACCTGGGGCTGCGGGAACAGCCTGCGTACGGCTCTCATCAACTCCACTG

GGGAAGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGACGACGAG

GATGAGGATGGAGATGACCTGCTCCATCACCACCACGGCTCCCACTGCAGCAGCTCGGG

GGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGC

CTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGCGGACCCATCTCCTCT

GGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGG

GGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCC

CGAACCCAGAGCCCCCAGAACTGCAGCATCATGTAA (SEQ ID NO: 180)

[00201] Another exemplary ORF encoding a polypeptide having structural activity is provided herein as SEQ ID NO: 181 SEQ ID NO: 181 is an ORF encoding human progerin, which is a truncated mutant form of Lamin A involved in Hutchinson-Gilford progeria syndrome that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATCGTGCCCATCCTGATCGAGCTGAA

TGGCGATGTGAATGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGATGCCACCT

ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCTGTGCCCTGGCCCA CCCTGGTGACCACCCTGAGCTACGGCGTGCAGTGCTTCTCACGCTACCCCGATCACATGA

AGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACATCCAGGAGCGCACCATC

TTCTTCGAGGATGACGGCAACTACAAGTCGCGCGCCGAGGTGAAGTTCGAGGGCGATAC

CCTGGTGAATCGCATCGAGCTGACCGGCACCGATTTCAAGGAGGATGGCAACATCCTGG

GCAATAAGATGGAGTACAACTACAACGCCCACAATGTGTACATCATGACCGACAAGGCC

AAGAATGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGATGGCAGCGTGCA

GCTGGCCGACCACTACCAGCAGAATACCCCCATCGGCGATGGCCCTGTGCTGCTGCCCGA

TAACCACTACCTGTCCACCCAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGCGATC

ACATGATCTACTTCGGCTTCGTGACCGCCGCCGCCATCACCCACGGCATGGATGAGCTGT

ACAAGTCCGGACTTAAGGCCTCTGTCGACAGCAGTCTCTGTCCTTCGACCCGAGCCCCGC

GCCCTTTCCGGGACCCCTGCCCCGCGGGCAGCGCTGCCAACCTGCCGGCCATGGAGACCC

CGTCCCAGCGGCGCGCCACCCGCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCC

ACCCGCATCACCCGGCTGCAGGAGAAGGAGGACCTGCAGGAGCTCAATGATCGCTTGGC

GGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCAGGGCTGCGCCTTCGCA

TCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCTACGAG

GCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCT

GCAGCTGGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCA

AGAAGGAGGGTGACCTGATAGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTG

AACTCCAAGGAGGCCGCACTGAGCACTGCTCTCAGTGAGAAGCGCACGCTGGAGGGCGA

GCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGCAGCCCTAGGTGAGGCCAAGA

AGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTGCAGACCATG

AAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGC

GCCGTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGC

CGGCTGGCGGATGCGCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTA

TAAGAAGGAGCTGGAGAAGACTTATTCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTG

AGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGGAGCTGCAGCAGTCGCGCATCCGC

ATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGCAGCCAAGGAGGC

GAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTGC

TGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGA

CGAGTACCAGGAGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACC

GCAAGCTCTTGGAGGGCGAGGAGGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAG

CGCAGCCGTGGCCGTGCTTCCTCTCACTCATCCCAGACACAGGGTGGGGGCAGCGTCACC

AAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCTCACAGCACGCACGCAC

TAGCGGGCGCGTGGCCGTGGAGGAGGTGGATGAGGAGGGCAAGTTTGTCCGGCTGCGCA

ACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGAT

GATCCCTTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTG

ACGATCTGGGCTGCAGGAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAA GGCACAGAACACCTGGGGCTGCGGGAACAGCCTGCGTACGGCTCTCATCAACTCCACTG

GGGAAGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGACGACGAG

GATGAGGATGGAGATGACCTGCTCCATCACCACCATGGCTCCCACTGCAGCAGCTCGGG

GGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGC

CTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGAGCCCCCAGAACTGCAGCATC

ATGTAA (SEQ ID NO: 181)

[00202] An exemplary ORF encoding a polypeptide having enzymatic activity is provided herein as SEQ ID NO: 182. SEQ ID NO: 182 is an ORF encoding human DNA ligase 4 or LIG4 that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGGCTGCCTCACAAACTTCACAAATTGTTGCATCTCACGTTCCTTTTGCAGATTTGTGTT

CAACTTTAGAACGAATACAGAAAAGTAAAGGACGTGCAGAAAAAATCAGACACTTCAG

GGAATTTTTAGATTCTTGGAGAAAATTTCATGATGCTCTTCATAAGAACCACAAAGATGT

CACAGACTCTTTTTATCCAGCAATGAGACTAATTCTTCCTCAGCTAGAAAGAGAGAGAAT

GGCCTATGGAATTAAAGAAACTATGCTTGCTAAGCTTTATATTGAGTTGCTTAATTTACCT

AGATGCTGGAGACTTTGCAATGATTGCATATTTTGTGTTGAAGCCAAGATGTTTACAGAA

TGCTAAAAGAAAAGACCTAATAAAAAAGAGCCTTCTTCAACTTATAACTCAGAGTTCAG CACTTGAGCAAAAGTGGCTTATACGGATGATCATAAAGGATTTAAAGCTTGGTGTTAGTC

TCTGGAAAAAGTCTGTAGGCAACTGCATGATCCTTCTGTAGGACTCAGTGATATTTCTAT

CACTTTATTTTCTGCATTTAAACCAATGCTAGCTGCTATTGCAGATATTGAGCACATTGAG

AAGGATATGAAACATCAGAGTTTCTACATAGAAACCAAGCTAGATGGTGAACGTATGCA

AATGCACAAAGATGGAGATGTATATAAATACTTCTCTCGAAATGGATATAACTACACTG

ATCAGTTTGGTGCTTCTCCTACTGAAGGTTCTCTTACCCCATTCATTCATAATGCATTCAA

AGCAGATATACAAATCTGTATTCTTGATGGTGAGATGATGGCCTATAATCCTAATACACA

AACTTTCATGCAAAAGGGAACTAAGTTTGATATTAAAAGAATGGTAGAGGATTCTGATCT

GACTCTGAGAAAGAGGTATGAGATTCTTAGTAGTATTTTTACACCAATTCCAGGTAGAAT AGAAATAGTGCAGAAAACACAAGCTCATACTAAGAATGAAGTAATTGATGCATTGAATG AAGCAATAGATAAAAGAGAAGAGGGAATTATGGTAAAACAACCTCTATCCATCTACAAG CCAGACAAAAGAGGTGAAGGGTGGTTAAAAATTAAACCAGAGTATGTCAGTGGACTAAT GGATGAATTGGACATTTTAATTGTTGGAGGATATTGGGGTAAAGGATCACGGGGTGGAA

TGTTTCATACTCTCTCTCGTGTTGGGTCTGGCTGCACCATGAAAGAACTGTATGATCTGGG TTTGAAATTGGCCAAGTATTGGAAGCCTTTTCATAGAAAAGCTCCACCAAGCAGCATTTT ATGTGGAACAGAGAAGCCAGAAGTATACATTGAACCTTGTAATTCTGTCATTGTTCAGAT

ACGAATTGAAAAGATAAGAGATGACAAGGAGTGGCATGAGTGCATGACCCTGGACGAC CTAGAACAACTTAGGGGGAAGGCATCTGGTAAGCTCGCATCTAAACACCTTTATATAGGT GGTGATGATGAACCACAAGAAAAAAAGCGGAAAGCTGCCCCAAAGATGAAGAAAGTTA TTGGAATTATTGAGCACTTAAAAGCACCTAACCTTACTAACGTTAACAAAATTTCTAATA

TGGAGAACAGAATTGCAGAATTTGGTGGTTATATAGTACAAAATCCAGGCCCAGACACG TACTGTGTAATTGCAGGGTCTGAGAACATCAGAGTGAAAAACATAATTTTGTCAAATAA

ATGGCAGCCTCGCTTTATGATTCATATGTGCCCATCAACCAAAGAACATTTTGCCCGTGA

ATATGATTGCTATGGTGATAGTTATTTCATTGATACAGACTTGAACCAACTGAAGGAAGT

ATTCTCAGGAATTAAAAATTCTAACGAGCAGACTCCTGAAGAAATGGCTTCTCTGATTGC

TGATTTAGAATATCGGTATTCCTGGGATTGCTCTCCTCTCAGTATGTTTCGACGCCACACC

GTTTATTTGGACTCGTATGCTGTTATTAATGACCTGAGTACCAAAAATGAGGGGACAAGG

TTAGCTATTAAAGCCTTGGAGCTTCGGTTTCATGGAGCAAAAGTAGTTTCTTGTTTAGCTG

TTAGAAGAACTTTTAAGAGAAAGTTTAAAATCCTAAAAGAAAGTTGGGTAACTGATTCA ATAGACAAGTGTGAATTACAAGAAGAAAACCAGTATTTGATTTAA (SEQ ID NO: 182)

[00203] Another exemplary ORF encoding a polypeptide having enzymatic activity is provided herein as SEQ ID NO: 183. SEQ ID NO: 183 is an ORF encoding human DNA cross-link repair 1C or DCLRE1C that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGAGTTCTTTCGAGGGGCAGATGGCCGAGTATCCAACTATCTCCATAGACCGCTTCGAT AGGGAGAACCTGAGGGCCCGCGCCTACTTCCTGTCCCACTGCCACAAAGATCACATGAA AGGATTAAGAGCCCCTACCTTGAAAAGAAGGTTGGAGTGCAGCTTGAAGGTTTATCTAT

AACGAATTATATCTATTGAAATCGAGACTCCTACCCAGATATCTTTAGTGGATGAAGCAT CAGGAGAGAAGGAAGAGATTGTTGTGACTCTCTTACCAGCTGGTCACTGTCCGGGATCA

GCGCAAGGAGAAGCTGCTAGAATGGAGCTTCTGCACTCCGGGGGCAGAGTCAAAGACAT

CCAAAGTGTATATTTGGATACTACGTTCTGTGATCCAAGATTTTACCAAATTCCAAGTCG

GGAGGAGTGTTTAAGTGGAGTCTTAGAGCTGGTCCGAAGCTGGATCACTCGGAGCCCGT

ACCATGTTGTGTGGCTGAACTGCAAAGCGGCTTATGGCTATGAATATCTGTTCACCAACC

TTAGTGAAGAATTAGGAGTCCAGGTTCATGTGAATAAGCTAGACATGTTTAGGAACATG

CCTGAGATCCTTCATCATCTCACAACAGACCGCAACACTCAGATCCATGCATGCCGGCAT

CCCAAGGCAGAGGAATATTTTCAGTGGAGCAAATTACCCTGTGGAATTACTTCCAGAAAT AGAATTCCACTCCACATAATCAGCATTAAGCCATCCACCATGTGGTTTGGAGAAAGGAG

CAGAAAAACAAATGTAATTGTGAGGACTGGAGAGAGTTCATACAGAGCTTGTTTTTCTTT

TCACTCCTCCTACAGTGAGATTAAAGATTTCTTGAGCTACCTCTGTCCTGTGAACGCATAT

CCAAATGTCATTCCAGTTGGCACAACTATGGATAAAGTTGTCGAAATCTTAAAGCCTTTA

TGCCGGTCTTCCCAAAGTACGGAGCCAAAGTATAAACCACTGGGAAAACTGAAGAGAGC

TAGAACAGTTCACCGAGACTCAGAGGAGGAAGATGACTATCTCTTTGATGATCCTCTGCC

GACTGCAGTATCAGAAAAGCAGCCTGAAAAACTGAGACAAACCCCAGGATGCTGCAGA

GCAGAGTGTATGCAGAGCTCTCGTTTCACAAACTTTGTAGATTGTGAAGAATCCAACAGT

GAAAGTGAAGAAGAAGTAGGAATCCCAGCTTCACTGCAAGGAGATCTGGGCTCTGTACT

TCACCTGCAAAAGGCTGATGGGGATGTACCCCAGTGGGAAGTATTCTTTAAAAGAAATG

ATGAAATCACAGATGAGAGTTTGGAAAACTTCCCTTCCTCCACAGTGGCAGGGGGATCTC

CTTCCCAGTCAACACACATAACAGAACAAGGAAGTCAAGGCTGGGACAGCCAATCTGAT

ACTGTTTTGTTATCTTCCCAAGAGAGAAACAGTGGGGATATTACTTCCTTGGACAAAGCT

GACTACAGACCAACAATCAAAGAGAATATTCCTGCCTCTCTCATGGAACAAAATGTAATT

TGCCCAAAGGATACTTACTCTGATTTGAAAAGCAGAGATAAAGATGTGACAATAGTTCCT

AGTACTGGAGAACCAACTACTCTAAGCAGTGAGACACATATACCCGAGGAAAAAAGTTT

TCCAGAAGCTGAGTTACCTAAACGAGAGCATTTACAATATTTATATGAGAAGCTGGCAA CTGGTGAGAGTATAGCAGTCAAAAAAAGAAAATGCTCACTCTTAGATACCTAA (SEQ ID NO: 183)

[00204] In some embodiments, an open reading frame sequence encodes for a protein therapeutic. Some exemplary protein therapeutics include, but are not limited to: hormones, such as insulin, growth hormone, leptin, erythropoietin, granulocyte colony-stimulating factor (G-CSF), thrombopoietin, clotting factor VII, Factor IX, interferon, glucocerebrosidase, anti-HER2 monoclonal antibody, and Etanercept, among others.

[00205] An exemplary ORF encoding a hormone is provided herein as SEQ ID NO: 184.

SEQ ID NO: 184 is an ORF encoding mouse leptin that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGTGCTGGAGACCCCTGTGTCGGTTCCTGTGGCTTTGGTCCTATCTGTCTTATGTTCAAG

CAGTGCCTATCCAGAAAGTCCAGGATGACACCAAAACCCTCATCAAGACCATTGTCACC

AGGATCAATGACATTTCACACACGCAGTCGGTATCCGCCAAGCAGAGGGTCACTGGCTT

GGACTTCATTCCTGGGCTTCACCCCATTCTGAGTTTGTCCAAGATGGACCAGACTCTGGC

AGTCTATCAACAGGTCCTCACCAGCCTGCCTTCCCAAAATGTGCTGCAGATAGCCAATGA

CCTGGAGAATCTCCGAGACCTCCTCCATCTGCTGGCCTTCTCCAAGAGCTGCTCCCTGCCT

CAGACCAGTGGCCTGCAGAAGCCAGAGAGCCTGGATGGCGTCCTGGAAGCCTCACTCTA CTCCACAGAGGTGGTGGCTTTGAGCAGGCTGCAGGGCTCTCTGCAGGACATTCTTCAACA GTTGGATGTTAGCCCTGAATGCTGA (SEQ ID NO: 184)

[00206] In some embodiments, an open reading frame sequence encodes for an RNA molecule found in a non-human species, including other mammalian RNAs, avian RNA, reptilian RNAs, bacterial RNA, and viral RNAs. Such open reading frame sequences can encode for protein or peptides that have a desirable function, such as a reporter molecule, a secreted anti-microbial peptide, and the like.

[00207] An exemplary ORF encoding a viral molecule is provided herein as SEQ ID NO:

185. SEQ ID NO: 185 is an ORF encoding surface antigen S precursor or B 19R of the vaccinia virus that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGACGATGAAAATGATGGTACATATATATTTCGTATCATTATTGTTATTGCTATTCCACA

GTTACGCCATAGACATCGAAAATGAAATCACAGAATTCTTCAATAAAATGAGAGATACT

CTACCAGCTAAAGACTCTAAATGGTTGAATCCAGCATGTATGTTCGGAGGCACAATGAAT

GATATAGCCGCTCTAGGAGAGCCATTCAGCGCAAAGTGTCCTCCTATTGAAGACAGTCTT

TTATCGCACAGATATAAAGACTATGTGGTTAAATGGGAAAGGCTAGAAAAAAATAGACG

GCGACAGGTTTCTAATAAACGTGTTAAACATGGTGATTTATGGATAGCCAACTATACATC

TAAATTCAGTAACCGTAGGTATTTGTGCACCGTAACTACAAAGAATGGTGACTGTGTTCA

GGGTATAGTTAGATCTCATATTAGAAAACCTCCTTCATGCATTCCAAAAACATATGAACT

AGGTACTCATGATAAGTATGGCATAGACTTATACTGTGGAATTCTTTACGCAAAACATTA

TAATAATATAACTTGGTATAAAGATAATAAGGAAATTAATATCGACGACATTAAGTATTC

ACAAACGGGAAAGGAATTAATTATTCATAATCCAGAGTTAGAAGATAGCGGAAGATACG

ACTGTTACGTTCATTACGACGACGTTAGAATCAAGAATGATATCGTAGTATCAAGATGTA

AAATACTTACGGTTATACCGTCACAAGACCACAGGTTTAAACTAATACTAGATCCAAAA

ATCAACGTAACGATAGGAGAACCTGCCAATATAACATGCACTGCTGTGTCAACGTCATTA

TTGATTGACGATGTACTGATTGAATGGGAAAATCCATCCGGATGGCTTATAGGATTCGAT

TTTGATGTATACTCTGTTTTAACTAGTAGAGGCGGTATTACCGAGGCGACCTTGTACTTTG

AAAATGTTACTGAAGAATATATAGGTAATACATATAAATGTCGTGGACACAACTATTATT

TTGAAAAAACCCTTACAACTACAGTAGTATTGGAGTAA (SEQ ID NO: 185)

[00208] An exemplary ORF encoding a reporter molecule is provided herein as SEQ ID NO:

186. SEQ ID NO: 186 is an ORF encoding Gaussia lucif erase reporter molecule derived from the marine copepod Gaussia princeps that was successfully transcribed in an IVT reaction using the exemplary pORFin vector described herein in the Examples.

ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACC GAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCT CGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGA TGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCAC ATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGG

CGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTC

CTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGG

ACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGA

AGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAG

ATCAAGGGGGCCGGTGGTGACTAA (SEQ ID NO: 186)

[00209] In some embodiments, an open reading frame sequence encodes for a tRNA (transfer

RNA), an snRNA (small nuclear RNA), an rRNA (ribosomal RNA), an anti-sense RNA, a small interfering RNA (siRNA), a micro RNA (miRNA), or any other RNA sequence that does not encode a functional protein or peptide.

[00210] As used herein, an "antisense RNA" comprises one or more nucleotide sequences sufficient in identity, number and size to effect specific hybridization with a preselected nucleic acid sequence.

[00211] As used herein, "ribozymes" refer to RNA molecules having enzymatic activities usually associated with cleavage, splicing or ligation of nucleic acid sequences to which the ribozyme binds. Typical substrates for ribozymes include RNA molecules, although ribozymes can also catalyze reactions in which DNA molecules serve as substrates. Two distinct regions can be identified in a ribozyme: the binding region which gives the ribozyme its specificity through hybridization to a specific nucleic acid sequence, and a catalytic region which gives the ribozyme the activity of cleavage, ligation or splicing.

[00212] As used herein, "siRNA" refers to a 1-50 nucleotide double stranded RNA (dsRNA) molecule that has sequence-specific homology to its "target" nucleic acid sequences (Caplen, N. J., et al., Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001)) and is derived from the processing of a larger dsRNA by an RNase known as Dicer (Bernstein, E., et al., Nature 409:363-366 (2001)).

[00213] As used herein, "shRNA" molecules are single stranded nucleic acid molecules that comprise two sequences complementary to each other, oriented such that one of the sequences is inverted relative to the other, which allows the two complementary sequences to base pair with each other, thereby forming a hairpin structure. The two copies of the inverted repeat need not be contiguous. There may be "n" additional nucleotides between the hairpin forming sequences, wherein "n" is any number of nucleotides.

[00214] As used herein, "microRNA" refers to molecules which are structurally similar to shRNA molecules, as described herein, but, typically, contain one or more mismatches or insertion/deletions in their regions of sequence complementary. The binding of miRNA of perfect complementarity to a target sequence results in mRNA degradation; single base mismatches can block translation.

Libraries of Open Reading Frame Sequences [00215] Also provided herein, are combinatorial libraries comprising a plurality of different open reading frame sequences inserted into the IVT templates described herein that can be used to produce a plurality of different synthetic RNAs. Accordingly, in some aspects, open reading frame libraries can be generated by ligating each of a plurality of open reading frame sequences into an IVT template vector generated using the compositions and methods described herein. These IVT template vectors each comprising a unique open reading frame sequence can be maintained as libraries of isolated IVT template vector nucleic acids, or can be transfected into cells, such as bacterial cells that the vector can replicate in, to be maintained as individual clones each comprising a unique open reading frame sequence inserted or ligated into an IVT template vector.

[00216] In some embodiments of these aspects, each of the open reading frame sequences in a library is from the same species or subspecies of organism. Exemplary species of libaries include, but are not limited to, a library of human open reading frame sequences, a library of chimpanzee open reading frame sequences, a library of murine open reading frame sequences, a library of rat open reading frame sequences, a library of zebrafish open reading frame sequences, a library of

Caenorhabditis elegans open reading frame sequences, a library of Saccharomyces cerevisiae (yeast) open reading frame sequences, etc.

[00217] In other embodiments of these apects, each of the open reading frame sequences in a library encodes the same "type" of polypeptide or non-translated RNA. Exemplary libaries of this nature include, but are not limited to, libraries of open reading frame sequences encoding transcription factors, libraries of open reading frame sequences encoding reprogramming factors, libraries of open reading frame sequences encoding differentiation factors, libraries of open reading frame sequences encoding CD molecules, libraries of open reading frame sequences encoding cell-type specific polypeptides, libraries of open reading frame sequences encoding death receptors, libraries of open reading frame sequences encoding mitogen receptors, libraries of open reading frame sequences encoding cytokines, libraries of open reading frame sequences encoding protein therapeutics, libraries of open reading frame sequences encoding tRNAs, libraries of open reading frame sequences encoding snRNAs, libraries of open reading frame sequences encoding rRNAs, libraries of open reading frame sequences encoding anti-sense RNAs, libraries of open reading frame sequences encoding siRNAs, libraries of open reading frame sequences encoding miRNAs, or any combination thereof.

Additional Elements of IVT Templates

[00218] IRES sequences. The IVT templates described herein can further comprise an sequence encoding an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation.

[00219] In some embodiments of the aspects described herein, a sequence encoding an IRES sequence can be inserted, for example, between two different open reading frame sequences, thereby permitting the in vitro transcription of two or more open reading frame sequences from the same IVT template.

[00220] Self-cleaving Peptides. The IVT templates described herein can further comprise an sequence encoding an autonomous "self-cleaving peptides." Self-cleaving peptides were originally identified and characterized in apthovirus foot-and-mouth disease virus (FMDV), which were termed "2A peptides." Self-cleaving peptides are generally 18-22 amino acids, and 2A peptides contain a highly conserved c-terminal D(V/I)EXNPGP (SEQ ID NO: 200) motif that mediates "ribosomal skipping" at the terminal 2A proline and subsequent amino acid "2B" glycine. The most well- characterized 2A peptides are derived from FMDV, equine rhinitis A virus, porcine teschovirus-1, and insect Thosea asigna virus.

[00221] Accordingly, in some embodiments of the aspects described herein, a sequence encoding a self-cleaving peptide sequence can be inserted, for example, between two different open reading frame sequences, thereby permitting the in vitro transcription of two or more open reading frame sequences from the same IVT template.

[00222] Cap Nucleotides. To increase efficiency of expression in cells, a synthesized RNA can comprise a cap. A "cap" or a "cap nucleotide" refers to a nucleoside-5 '-triphosphate that, under suitable reaction conditions, is used as a substrate by a capping enzyme system and that is thereby joined to the 5'-end of an uncapped RNA comprising primary RNA transcripts or RNA having a 5'- diphosphate. The nucleotide that is so joined to the RNA is also referred to as a "cap nucleotide" herein. A "cap nucleotide" is a guanine nucleotide that is joined through its 5' end to the 5' end of a primary RNA transcript. The RNA that has the cap nucleotide joined to its 5' end is referred to as "capped RNA" or "capped RNA transcript" or "capped transcript." A common cap nucleoside is 7- methylguanosine or N⁷-methylguanosine (sometimes referred to as "standard cap"), which has a structure designated as "m⁷G," in which case the capped RNA or "m⁷G-capped RNA" has a structure designated as m⁷G(5')ppp(5')Ni(pN)_x— OH(3'), or more simply, as m. ⁷GpppNi(pN) _x or

m⁷G[5']ppp[5']N, wherein m⁷G represents the 7-methylguanosine cap nucleoside, ppp represents the triphosphate bridge between the 5' carbons of the cap nucleoside and the first nucleotide of the primary RNA transcript, Ni (pN) _x— OH(3') represents the primary RNA transcript, of which Ni is the most 5'-nucleotide, "p" represents a phosphate group, "G" represents a guanosine nucleoside, "m⁷" represents the methyl group on the 7-position of guanine, and "[5']" indicates the position at which the "p" is joined to the ribose of the cap nucleotide and the first nucleoside of the mRNA transcript ("N").

[00223] In addition to this "standard cap," a variety of other naturally-occurring and synthetic cap analogs are known in the art. RNA that has any cap nucleotide is referred to as "capped RNA." The capped RNA can be naturally occurring from a biological sample or it can be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (e.g., vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, the capped RNA can be obtained by in vitro transcription (IVT) of a DNA template that contains an RNA polymerase promoter, wherein, in addition to the GTP, the IVT reaction also contains a dinucleotide cap analog (e.g., a m GpppG cap analog or an N⁷-methyl, -O- methyl -GpppG ARC A cap analog or an N⁷-methyl, 3'-0-methyl-GpppG ARC A cap analog) using methods known in the art (e.g., using an e.g., using an AMPLICAP™ T7 Kit or a MESSAGEMAX™ T7 ARCA-CAPPED MESSAGE Transcription Kit; EPICENTRE or CellScript, Madison, Wis., USA).

[00224] RNA that results from the action of the RNA triphosphatase and the RNA

guanyltransierase enzymatic activities, as well as RNA that is additionally methylated by the guanine - 7-methyltransferase enzymatic activity, is referred to herein as "5' capped RNA" or "capped RNA", and a "capping enzyme system" or, more simply, a "capping enzyme" herein means any combination of one or more polypeptides having the enzymatic activities that result 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 that can convert uncapped RNA that has a 5' polyphosphate to capped RNA can be used to provide a 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

embodiments, the capping enzyme system is vaccinia virus capping enzyme. In some embodiments, the capping enzyme system is Saccharomyces cerevisiae capping enzyme. Also, in view of the fact that genes encoding RNA triphosphatase, RNA guanyltransierase and guanine -7-methyltransf erase from one source can complement deletions in one or all of these genes from another source, the capping enzyme system can originate from one source, or one or more of the RNA triphosphatase, RNA guanyltransierase, and/or guanine -7-methyltransferase activities can comprise a polypeptide from a different source.

[00225] Accordingly, in some embodiments, the synthesized RNA molecules provided herein are synthesized in vitro by incubating uncapped primary RNA in the presence a capping enzyme system. In other embodiments, capped RNA can be synthesized co-transcriptionally by using a dinucleotide cap analog in the IVT reaction (e.g., using an AMPLICAP™ T7 Kit or a

MESSAGEMAX™ T7 ARCA-CAPPED MESSAGE Transcription Kit; EPICENTRE or CellScript, Madison, Wis., USA). If capping is performed co-transcriptionally, in som embodiments, the dinucleotide cap analog is an anti-reverse cap analog (ARC A).

[00226] In some embodiments, a synthesized RNA generated using an IVT template as described herein has a cap with a capl structure, meaning that the 2' hydroxyl of the ribose in the penultimate nucleotide with respect to the cap nucleotide is methylated. In other embodiments, a synthesized RNA generated using an IVT template as described herein has a cap with a capO structure, meaning that the 2' hydroxyl of the ribose in the penultimate nucleotide with respect to the cap nucleotide is not methylated. With some, but not all transcripts, transfection of eukaryotic cells with synthesized RNA generated using an IVT template as described herein having a cap with a capl structure results in a higher level or longer duration of protein expression in the transfected cells compared to transfection of the same cells with the same mRNA but with a cap having a capO structure.

[00227] Tags. In some embodiments, an open reading frame sequence as described supra can further comprise a sequence encoding a tag, such that the protein or peptide encoded by the open reading frame sequence can be produced with an amino terminal and/or carboxy terminal tag. These tags can be used for any number of purposes, including (1) to increase the stability of the protein or peptide or (2) to allow for purification.

[00228] Thus, in some embodiments, proteins or peptides produced from the synthetic RNAs generated using the IVT templates and methods described herein, can comprise affinity purification tags (e.g., epitope tags such as the V5 epitope). As used herein, "affinity purification tags" refer to amino acid sequences that can interact with a binding partner immobilized on a solid support. Nucleic acids encoding multiple consecutive single amino acids, such as histidine, can be used for one-step purification of the recombinant protein by affinity binding to a resin column, such as nickel sepharose. A protease cleavage site can be engineered between the affinity tag and the desired protein to allow for removal of the tag, for example, after the purification process is complete or to induce release of the desired protein or peptide from the solid support. Affinity tags which can be used as described herein include tags such as the chitin binding domain (which binds to chitin), polyarginine, glutathione-S-transferase (which binds to glutathione), maltose binding protein (which binds maltose), FlAsH, biotin (which binds to avidin and strepavidin), and the like.

[00229] In other embodiments, proteins or peptides produced from the synthetic RNAs generated using the IVT templates and methods described herein, can comprise "epitope tags." Epitope tags refer to short amino acid sequences that are recognized by epitope specific antibodies. Proteins or peptides which comprise one or more epitope tags may be purified, for example, using a cognate antibody bound to a chromatography resin. The presence of the epitope tag furthermore allows the recombinant protein to be detected in subsequent assays, such as Western blots, without having to produce an antibody specific for the recombinant protein itself. Examples of commonly used epitope tags include V5, glutathione-S-transferase (GST), hemaglutinin (HA), the peptide Phe- His-His-Thr-Thr (SEQ ID NO: 161), chitin binding domain, and the like. As discussed above, these affinity tags can be removed from the desired protein or peptide by proteolytic cleavage.

[00230] In other embodiments, FlAsH tags can be used. "FlAsH tags" comprise the sequence a cys-cys-Xaa-Xaa-cys-cys (SEQ ID NO: 162), where Xaa and Xaa are amino acids. In many instances, Xaa and Xaa, which can be the same or different amino acids, are amino acids with high a- helical propensity. In some embodiments, X and Y are the same amino acid. These peptides have been shown to bind to biarsenical compounds. The FlAsH systems are described in U.S. Pat. No. 6,054,271, the entire disclosure of which is incorporated herein by reference. Vectors for the IVT Templates

[00231] Also provided herein are vectors or vector backbone sequences into which an IVT template, such as an IVT template comprising an open reading frame sequences or amplified fragment thereof, can be inserted or incorporated using standard molecular biology techniques.

[00232] As used herein, a "vector" refers to a nucleic acid molecule, such as a dsDNA molecule that provides a useful biological or biochemical property to an inserted nucleotide sequence, such as the IVT templates described herein. Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences which are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell. A vector can have one or more restriction endonuclease recognition sites (whether type I, II or lis) at which the sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a nucleic acid fragment can be spliced or inserted in order to bring about its replication and cloning. Vectors can also comprise one or more recombination sites that permit exchange of nucleic acid sequences between two nucleic acid molecules. Vectors can further provide primer sites, e.g., for PCR, transcriptional and/or translational initiation and/or regulation sites, recombinational signals, replicons, selectable markers, etc. A vector can further contain one or more selectable markers suitable for use in the identification of cells transformed with the vector.

[00233] Vectors known in the art and those commercially available (and variants or derivatives thereof) can be used with the IVT templates and methods of generating synthetic RNAs described herein. Such vectors can be obtained from, for example, Vector Laboratories Inc.,

Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen, and Research Genetics. General classes of vectors include prokaryotic and/or eukaryotic cloning vectors, expression vectors, fusion vectors, two-hybrid or reverse two-hybrid vectors, shuttle vectors for use in different hosts, mutagenesis vectors, transcription vectors, vectors for receiving large inserts and the like.

[00234] Exemplary prokaryotic vectors include, but are not limited to, pZErOl .1 , pZErO-2.1 , pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Corp.), pGEMEX-1, and pGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A, pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc.), pKK233-2 and pKK388-l (Clontech, Inc.), and pProEx-HT (Invitrogen, Corp.) and variants and derivatives thereof. Other vectors of interest include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-Cl, pPUR, pMAM, pMAMneo, pBIlOl, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCHl lO, and pKK232-8 (Pharmacia, Inc.), p3'SS, pXTl, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBacIII, pCDM8, pcDNAl, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.), pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(-)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pA0815, pPICZ, pPICZ.alpha., pGAPZ, pGAPZ.alpha., pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SPl), pVgRXR, pcDNA2.1, pYES2, , pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNAl.l, pcDNAl. l/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen; .lamda. ExCell, .lamda. gtl l, pTrc99A, pKK223-3, pGEX-l.lamda.T, pGEX-2T, pGEX-2TK, pGEX-4T-l, pGEX- 4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-l, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCHl lO, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia;

pSCREEN-lb(+), pT7Blue(R), pT7Blue-2, pCITE-4-abc(+), pOCUS-2, pTAg, pET-32LIC, pET- 30LIC, pBAC-2 cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC, pT7Blue-2, .lamda.SCREEN-1, .lamda.BlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pETl labcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-l, pBACgus4x-l, pBAC- 3cp, pBACgus-2cp, pBACsurf-1, pig, Signal pig, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg, and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-l, pGAD424, pACT2, pGAD GL, pGAD GH, pGADIO, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP ("6xHis" disclosed as SEQ ID NO: 201), pSEAP2-Basic, pSEAP2- Contral, pSEAP2-Promoter, pSEAP2-Enhancer, p gal-Basic, p gal-Control, pPgal-Promoter, p gal- Enhancer, pCMV , pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRESlneo, pIRESlhyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-l/2/3, pYEX-Sl, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTriplEx, λgtl0, XgtU, pWE15, and λΤήρΙΕχ from Clontech; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS +/-, pBluescript II SK +/-, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR- Script Direct, pBS +/-, pBC KS +/-, pBC SK +/-, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL- kc, pET-3abcd, pET-l labcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS, pOPI3 CAT, pXTl, pSG5, pPbac, pMbac, pMClneo, pMClneo Poly A, pOG44, pOG45, pFRT GAL, pNEO GAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 from Stratagene, and variants or derivatives thereof.

Methods of Synthesizing IVT Template Constructs

[00235] Also provided herein, in some aspects, are methods for generating IVT templates and

IVT template constructs comprising, in some embodiments, an open reading frame sequence.

Accordingly, provided herein are methods of designing and making the IVT templates and constructs described herein, and for generating synthetic RNAs using these IVT templates. These methods comprise PCR steps, cloning steps, digestion steps, and in vitro transcription steps, as known to one of skill in the art. By using the IVT template designs described herein, any open reading frame of interest can be inserted, via blunt-end ligation, into the IVT template, to generate a synthetic RNA upon in vitro transcription.

[00236] In some aspects, provided herein are methods of synthesizing a nucleic acid construct for transcribing a gene of interest in vitro. These methods comprises the steps of:

a. amplifying an open reading frame (ORF) sequence of a gene of interest to generate an ORF amplification product using a phosphorylated forward primer and a phosphorylated reverse primer;

b. digesting an IVT template vector comprising a vector backbone sequence and an IVT template sequence, wherein the IVT template sequence comprises, in the 5' to 3' direction of the coding strand: a first nucleic acid sequence comprising a forward universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence a second blunt-ended restriction enzyme digestion site, a 3' UTR sequence, second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence, with a first and a second blunt-ended restriction enzyme specific for the first and second blunt-ended restriction enzyme digestion sites respectively; and c. contacting the dephosphorylated ORF amplification product with the digested IVT template vector in the presence of a ligase, thereby incorporating the ORF into the vector and generating the nucleic acid construct.

[00237] To construct an IVT DNA template sequence for use in these methods, selected 5' and 3' UTR coding sequences can be de novo synthesized using synthetic oligonucleotides, for example, and ligated and annealed together and amplified using forward and reverse primers. These forward and reverse primers can further comprise restriction site sequences, preferably sticky-end sequences, to correspond to the cloning site restriction site sequences of a vector backbone of interest. For example, in some of the embodiments described herein, the vector backbone selected was pZErO- 2.

[00238] Blunt-end restriction site sequences can be introduced between the selected 5' and 3'

UTR sequences to provide the entry sites for the given open reading frame sequence of interest. In some embodiments, the 5' UTR sequence can further comprise the sequence of the promoter sequence. In other embodiments, the promoter sequence is, for example, de novo synthesized along with the 5' and 3' UTR sequences, or can be PCR amplified and ligated to the 5' UTR coding sequence. In some embodiments, the 5' UTR sequence comprises SEQ ID NO: 20. In some embodiments, the 3' UTR sequence comprises SEQ ID NO: 21. [00239] In some embodiments, a spacer sequence can also be included between the blunt-end restriction site sequences of the IVT template sequence to allow for optimal digestion prior to insertion of the ORF sequence.

[00240] In some mbodiments, a forward universal primer sequence is included 5' of the sequence coding for the 5' UTR sequence, and a sequence comprising a sequence complementary to a reverse universal primer is included 3' of the sequence coding for the 3' UTR sequence, thus allowing for the easy and rapid amplification of the IVT template sequence for subsequent cloning. For example, an M13 forward primer sequence and an M13 reverse primer sequence, as described herein the Examples.

[00241] Accordingly, an IVT DNA template is generated comprising, in the 5' to 3' direction of its coding strand, a sequence comprising a forward universal primer sequence, a promoter sequence operably linked to a sequence encoding a 5' UTR sequence, a first blunt-ended restriction enzyme sequence, a spacer sequence, a second blunt-ended restriction enzyme sequence, a sequence encoding a 3 'UTR sequence, and a sequence comprising a sequence complementary to a reverse universal primer sequence. In some embodiments, the IVT template can be constructed to include at its 5' and 3' sticky-end restriction site sequences for cloning into the vector backbone of interest having the same sticky-end restriction site sequences.

[00242] To insert the IVT template into a vector backbone, the vector backbone of interest is digested with restriction enzymes specific for the sticky-end restriction site sequences of interes. The IVT template sequence is amplified, and the amplified IVT template sequence is then ligated into the vector backbone of interest, thereby generating the "IVT template vector" for insertion of the desired open reading frame sequence of interest. The cloned or inserted IVT template can be verified by sequencing.

[00243] To generate the ORF amplification product, an ORF sequence is amplified using a forward and reverse primers specific for the ORF sequence. In some embodiments, the forward and reverse primers specific for the ORF sequence are phosphorylated.

[00244] In some embodiments, one or more nucleotides of the 5' end of the open reading frame sequence can be omitted from the forward primer sequence as it can be provided by the first blunt-ended restriction site sequence. For example, as described herein in the Examples section, the adenine nucleotide (A) of the 1^st codon (ATG) of the open reading frame sequence was provided by the Alel site of the IVT template.

[00245] The IVT template vector is digested using enzymes specific for the blunt-ended restriction site sequences of the IVT template to linearize the vector. The IVT template vector can also be dephosphorylated, in some embodiments. The amount of an IVT template vector can be quantified, using any technique known to one of ordinary skill in the art.

[00246] The ORF amplification product is cloned or inserted into the linearized IVT template vector by blunt-end ligation in the presence of a ligase. [00247] In some embodiments of the aspects described herein, the methods further comprise a screening for proper orientation of the ORF sequence comprising the steps of:

d. amplifying the ORF sequence with a forward primer comprising the first universal primer sequence and a reverse primer comprising a sequence specific for the 3' end of the ORF sequence; and

e. selecting the clones comprising the IVT template vectors where the ORF sequence is in the proper 5' to 3' orientation.

In some such embodiments, screening for clones in which the ORF amplification product fragment is in the right-orientation is performed using colony PCR (see, for example, FIG. IB).

[00248] In some embodiments of the aspects described herein, the methods further comprise a step of adding a poly-adenylation tail to the ORF sequence inserted into the IVT template vector. In order to add a polyadenylation tail to the ORF sequence, the sequence-verified clone can be used as a

DNA template for a "Tail PCR," which appends a polyadenylation template to the IVT template construct.

[00249] Accordingly, the IVT template vector comprising the ligated ORF sequence is amplified via PCR using: a forward universal primer comprising at its 3' end a sequence of the forward universal primer sequence of the IVT template, and a reverse universal primer sequence comprising a sequence complementary to the reverse primer sequence of the IVT template at its 3' end and a poly-T sequence at its 5' end, generating an "amplified IVT template vector fragment." Therefore, the amplified IVT template vector fragment comprises the necessary template for generating a polyadenylation tail upon in vitro transcription. The conditions for this reaction can be optimized by one of skill in the art according to the DNA polymerase used for the PCR and the length of the ORF sequence. The methylated vector DNA can then be digested. In other embodiments, the IVT template vector can be linearized prior to the Tail-PCR reaction to eliminate any circular templates that would generate run-on transcripts.

[00250] Following the Tail PCR, the amplified IVT template vector fragment comprising the

ORF sequence of interest can undergo in vitro transcription using any method known to one of ordinary skill in the art. An in vitro transcription reaction typically comprises ribonucleotide triphosphates or NTPS (e.g., ATP, GTP, CTP, and UTP), an RNA polymerase specific for the promoter sequence of the amplified IVT template vector fragment, and an appropriate buffer mixture for the RNA polymerase being used. In some embodiments, the ribonucleotide triphosphates used in the in vitro transcription reaction comprises one or more modified nucleotides, such as pseudo-UTP or methyl-CTP. Any residual DNA in the reaction mixture following the IVT reaction can then be removed by addition of a DNase enzyme, and the synthesized RNA can be eluted using any method known to one of ordinary skill in the art.

[00251] The purified synthesized RNA can then be treated with a phosphatase, which prevents recognition of the uncapped synthesized RNA by the RIG-I complex. The RNA can then be utilized to transfect any cell type of interest. Optimal transfection conditions can be determined by a skilled artisan. In addition, highlighted in the Examples section are some important issues related with transfection to be considered, and related guidelines, including target cell type, transfection reagents, cell culture media and their FCS or protein content, optimal ratio of RNA to transfection reagent, and dosing and incubation time.

Kits for Synthesizing IVT Template Constructs

[00252] Provided herein, in some aspects, are kits comprising IVT templates for preparing synthetic RNAs as described herein. These kits can be used in combination with currently commercially available reagents and kits to perform aspects and embodiments of the methods described herein. These kits can be used, for example, to generate modified, synthetic RNAs for altering the phenotype or the developmental potential of a cell. In addition, these kits can be used, for example, to generate libraries of open reading frame sequences that can be used to generate a plurality of synthetic RNAs.

[00253] Accordingly, in some aspects, provided herein are kits comprising an isolated nucleic acid encoding an IVT template described herein, in a suitable container. Such IVT template sequences comprise, in the 5' to 3' direction, a nucleic acid sequence comprising a forward universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence, a second blunt-ended restriction enzyme digestion site, a 3' UTR sequence, and a nucleic acid sequence comprising a sequence complementary to a reverse universal primer sequence.

[00254] In some embodiments of these aspects, the kit can further comprise open reading frame sequences to be inserted between the first blunt-ended restriction enzyme digestion site and the second blunt-ended restriction enzyme digestion site. Such open reading frame sequences can encode for any desired polypeptide or non-coding RNA, such as, for example, a transcription factor, a targeting moiety, a cell type-specific polypeptide, a cell-surface polypeptide, a differentiation factor, a reprogramming factor, a de-differentiation factor, an anti-sense RNA, an shRNA, a microRNA, etc. Optionally, the kit can further comprise one or more control open reading frame sequences, such as one encoding green fluorescent protein (GFP) or other marker molecule.

[00255] In some embodiments of these aspects, the kit further comprises a vector backbone sequence that the IVT template can be inserted into.

[00256] In some embodiments of these aspects, the kit further comprises a first and a second blunt-ended restriction enzyme specific for the first and second blunt-ended restriction enzyme digestion sites respectively.

[00257] In some embodiments of these aspects, the kits further comprise a forward universal primer comprising the forward universal primer sequence of the IVT template and a reverse universal primer comprising a poly-T sequence. [00258] In some embodiments of these aspects, the kits comprise one or more reagents required for performing a PCR reaction, such as buffers, MgCl₂, DNA polymerases,

deoxyribonucleotides or nucleotide analogs thereof, etc.

[00259] In some embodiments of these aspects, the kits comprise one or more reagents required for performing an in vitro transcription reaction, such as buffers, RNA polymerases, ribonucleotides or modified ribonucleotides, etc. For example, the kits can comprise at least one modified nucleoside, such as 5'-methylcytidine or pseudouridine, and an RNA polymerase. Other modified nucleosides that can be provided with kits described herein include, but are not limited to, 5- methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-0-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0-methyluridine (Um), 2'deoxy uridine (2' dU), 4- thiouridine (s4U), 5-methyluridine (m5U), 2'-0-methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'-0-trimethyladenosine (m62Am), 2'-0-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7- trimethylguanosine (m2,2,7G), and inosine (I). In some embodiments of this aspect, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

[00260] The kits can also comprise, for example, a 5' cap analog. The kits can also comprise a phosphatase enzyme (e.g., Calf intestinal phosphatase) to remove the 5' triphosphate.

[00261] In other aspects, provided herein are kits comprising a library of open reading frame sequences inserted into a plurality of any of the IVT template or IVT template constructs described herein, in a suitable container(s).

[00262] All kits described herein can further comprise a buffer, a cell culture medium, a transfection medium and/or a media supplement. In preferred embodiments, the buffers, cell culture mediums, transfection mediums, and/or media supplements are DNase- and/or RNase-free.

Synthetic RNAs and Synthetic, modified RNAs

[00263] The IVT templates, IVT template vectors, and methods of use thereof described herein are useful for the rapid and efficient synthesis of RNAs encoding a polypeptide or protein or non-translated RNA of interest for use in a variety of applications, including, but not limited to, changing the phenotype of a cell or altering the developmental potential of a cell. Such RNAs generated using the IVT constructs and templates and methods thereof described herein are termed "synthetic RNAs." When the RNAs generated include one or more modified nucleodides, such an RNA is termed a "synthetic, modified RNA."

[00264] As used herein, the term "synthetic, modified RNA" refers to a ribonucleicacid molecule which comprises at least one modified ribonucleoside and has at least the following characteristics as the term is used herein: (i) it can be generated by in vitro transcription and is not isolated from a cell; and (ii) it is translatable in a mammalian (and preferably human) cell. Ideally, a synthetic, modified RNA generated using the templates and methods described herein further does not provoke or provokes a significantly reduced innate immune response or interferon response in a cell to which it is introduced or contacted.

[00265] A synthetic, modified RNA can be generated by in vitro transcription of the IVT templates described herein. The transcribed, synthetic, modified RNA polymer can be modified further post-transcriptionally, e.g. , by adding a cap or other functional group, as described elsewhere herein.

[00266] To be suitable for in vitro transcription, the modified nucleoside(s) must be recognized as substrates by at least one RNA polymerase enzyme. Generally, RNA polymerase enzymes can tolerate a range of nucleoside base modifications, at least in part because the naturally occurring G, A, U, and C nucleoside bases differ from each other quite significantly. Thus, the structure of a modified nucleoside base for use in generating the synthetic, modified RNAs described herein can generally vary more than the sugar-phosphate moieties of the modified nucleoside. That said, ribose and phosphate-modified nucleosides or nucleoside analogs are known in the art that permit transcription by RNA polymerases. In some embodiments of the aspects described herein, the RNA polymerase is a phage RNA polymerase. The modified nucleotides pseudouridine, m5U, s2U, m6A, and m5C are known to be compatible with transcription using phage RNA polymerases, while Nl-methylguanosine, Nl-methyladenosine, N7-methylguanosine, 2'-)-methyluridine, and 2'-0- methylcytidine are not. Polymerases that accept modified nucleosides are known to those of skill in the art.

[00267] It is also contemplated that modified polymerases can be used to generate synthetic, modified RNAs, as described herein. Thus, for example, a polymerase that tolerates or accepts a particular modified nucleoside as a substrate can be used to generate a synthetic, modified RNA including that modified nucleoside.

[00268] Second, the synthetic, modified RNA must be translatable by the translation machinery of a eukaryotic, preferably mammalian, and more preferably, human cell. Translation generally requires at least a ribosome binding site, a methionine start codon, and an open reading frame encoding a polypeptide. Accordingly, as described herein, the synthetic, modified RNA can further comprise a 5' cap, a stop codon, a Kozak sequence, and/or a poly-A tail. In addition, because RNAs in a eukaryotic cell are regulated by degradation, a synthetic RNA generated using the IVT templates and methods as described herein can be further modified to extend its half -life in the cell by incorporating modifications to reduce the rate of RNA degradation {e.g., by increasing serum stability of a synthetic, modified RNA).

[00269] Nucleoside modifications can interfere with translation. To the extent that a given modification interferes with translation, those modifications are not encompassed by the synthetic, modified RNA as described herein. One can test a synthetic, modified RNA for its ability to undergo translation and translation efficiency using an in vitro translation assay {e.g. , a rabbit reticulocyte lysate assay, a reporter activity assay, or measurement of a radioactive label in the translated protein) and detecting the amount of the polypeptide produced using SDS-PAGE, Western blot, or immunochemistry assays etc. The translation of a synthetic, modified RNA comprising a candidate modification is compared to the translation of an RNA lacking the candidate modification, such that if the translation of the synthetic, modified RNA having the candidate modification remains the same or is increased then the candidate modification is contemplated for use with the compositions and methods described herein. It is noted that fluoro-modified nucleosides are generally not translatable and can be used herein as a negative control for an in vitro translation assay.

[00270] It is also preffered that the synthetic, modified RNAs generated using the IVT templates and methods described herein provokes a reduced (or absent) innate immune response or interferon response by the transfected cell or population of cells thereof. mRNA produced in eukaryotic cells, e.g. , mammalian or human cells, is heavily modified, the modifications permitting the cell to detect RNA not produced by that cell. The cell responds by shutting down translation or otherwise initiating an innate immune or interferon response. Thus, to the extent that an exogenously added RNA can be modified to mimic the modifications occurring in the endogenous RNAs produced by a target cell, the exogenous RNA can avoid at least part of the target cell's defense against foreign nucleic acids. Thus, in some embodiments, synthetic, modified RNAs as described herein include in vitro transcribed RNAs including modifications as found in eukaryotic/mammalian/human RNA in vivo. Other modifications that mimic such naturally occurring modifications can also be helpful in producing a synthetic, modified RNA molecule that will be tolerated by a cell. The various modifications contemplated or useful in the synthetic, modified RNAs described herein are discussed further herein below.

RNA Modifications

[00271] In some aspects, the IVT templates and methods of use thereof described herein generate synthetic, modified RNA molecules encoding polypeptides or non-translated RNAs, where the synthetic, modified RNA molecules comprise one or more modifications, such that introducing the synthetic, modified RNA molecules to a cell or organism results in a reduced innate immune response relative to a cell or organism contacted with or administered synthetic RNA molecules encoding the polypeptides or non-translated RNA not comprising the one or more modifications.

[00272] The synthetic, modified RNAs generated using the IVT templates and methods of use thereof described herein include modifications to prevent rapid degradation by endo- and exo- nucleases and to avoid or reduce the cell's innate immune or interferon response to the RNA.

Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications {e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. To the extent that such modifications interfere with translation (i.e. , results in a reduction of 50% or more in translation relative to the lack of the modification - e.g., in a rabbit reticulocyte in vitro translation assay), the modification is not contemplated to be generated using the IVT templates and methods of use thereof described herein. Specific examples of synthetic, modified RNA generated using the IVT templates and methods of use thereof described herein include, but are not limited to, RNA molecules containing modified or non-natural internucleoside linkages. Synthetic, modified RNAs having modified internucleoside linkages include, among others, those that do not have a phosphorus atom in the internucleoside linkage. In other embodiments, the synthetic, modified RNA has a phosphorus atom in its internucleoside linkage(s).

[00273] Non-limiting examples of modified internucleoside linkages include

phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters,

aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates , thionophosphoramidates , thionoalkylphosphonates ,

thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[00274] Representative U.S. patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821 ; 5,541,316; 5,550,111 5,563,253; 5,571,799; 5,587,361 ; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and US Pat RE39464, each of which is herein incorporated by reference in its entirety.

[00275] Modified internucleoside linkages that do not include a phosphorus atom therein have internucleoside linkages that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[00276] Representative U.S. patents that teach the preparation of modified oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference in its entirety.

[00277] Some embodiments of the synthetic, modified RNAs generated using the IVT templates and methods of use thereof described herein include nucleic acids with phosphorothioate internucleoside linkages and oligonucleosides with heteroatom internucleoside linkage, and in particular -CH2-NH-CH2-, -CH2-N(CH3)-0-CH2- [known as a methylene (methylimino) or MMI ], -CH2-0-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- [wherein the native phosphodiester internucleoside linkage is represented as -0-P-0-CH2-] of the above -referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above -referenced U.S. Pat. No. 5,602,240, both of which are herein incorporated by reference in their entirety. In some embodiments, the nucleic acid sequences featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506, herein incorporated by reference in its entirety.

[00278] Synthetic, modified RNAs generated using the IVT templates and methods of use thereof described herein can also contain one or more substituted sugar moieties. The nucleic acids featured herein can include one of the following at the 2' position: H (deoxyribose); OH (ribose); F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted CI to CIO alkyl or C2 to CIO alkenyl and alkynyl. Exemplary modifications include 0[(CH2)nO] mCH3, 0(CH2).nOCH3, 0(CH2)nNH2, 0(CH2) nCH3, 0(CH2)nONH2, and 0(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments, synthetic, modified RNAs include one of the following at the 2' position: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNA, or a group for improving the pharmacodynamic properties of a synthetic, modified RNA, and other substituents having similar properties. In some embodiments, the modification includes a 2' methoxyethoxy (2'-0- CH2CH20CH3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'- dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'- DMAEOE), i.e. , 2'-0-CH2-0-CH2-N(CH2)2.

[00279] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-

OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the nucleic acid sequence, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked nucleotides and the 5' position of 5' terminal nucleotide. A synthetic, modified RNA can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981 ,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811 ; 5,576,427; 5,591 ,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[00280] As non-limiting examples, synthetic, modified RNAs generated using the IVT templates and methods of use thereof described herein can include at least one modified nucleoside including a 2'-0-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a 2'-amino-modified nucleoside, 2'-alkyl-modified nucleoside, morpholino nucleoside, a

phosphor amidate or a non-natural base comprising nucleoside, or any combination thereof.

[00281] In some embodiments of this aspect and all other such aspects described herein, the at least one modified nucleoside is selected from the group consisting of 5-methylcytidine (5mC), N6- methyladenosine (m6A), 3,2'-0-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0-methyluridine (Um), 2' deoxyuridine (2' dU), 4-thiouridine (s4U), 5- methyluridine (m5U), 2'-0-methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'- O-trimethyladenosine (m6₂Am), 2'-0-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0- methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G), and inosine (I).

[00282] Alternatively, a synthetic, modified RNA generated using the IVT templates and methods of use thereof described herein can comprise at least two modified nucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the oligonucleotide. At a minimum, a synthetic, modified RNA molecule comprising at least one modified nucleoside comprises a single nucleoside with a modification as described herein. It is not necessary for all positions in a given synthetic, modified RNA to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single synthetic, modified RNA or even at a single nucleoside within a synthetic, modified RNA. However, it is preferred, but not absolutely necessary, that each occurrence of a given nucleoside in a molecule is modified (e.g. , each cytosine is a modified cytosine e.g. , 5mC). However, it is also contemplated that different occurrences of the same nucleoside can be modified in a different way in a given synthetic, modified RNA molecule generated using the IVT templates and methods of use thereof described herein (e.g. , some cytosines modified as 5mC, others modified as 2'-0- methylcytidine or other cytosine analog). The modifications need not be the same for each of a plurality of modified nucleosides in a synthetic, modified RNA. Furthermore, in some embodiments, a synthetic, modified RNA generated using the IVT templates and methods of use thereof described herein comprises at least two different modified nucleosides. In some such preferred embodiments of the aspects described herein, the at least two different modified nucleosides are 5-methylcytidine and pseudouridine. A synthetic, modified RNA generated using the IVT templates and methods of use thereof described herein can also contain a mixture of both modified and unmodified nucleosides.

[00283] As used herein, "unmodified" or "natural" nucleosides or nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). In some embodiments, a synthetic, modified RNA comprises at least one nucleoside ("base") modification or substitution. Modified nucleosides include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2 (amino)adenine, 2-(aminoalkyll)adenine, 2 (aminopropyl)adenine, 2 (methylthio) N6 (isopentenyl)adenine, 6 (alkyl)adenine, 6 (methyl)adenine, 7 (deaza)adenine, 8 (alkenyl)adenine, 8-(alkyl)adenine, 8 (alkynyl)adenine, 8 (amino)adenine, 8- (halo)adenine, 8-(hydroxyl)adenine, 8 (thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6 (methyl) adenine, N6, N6 (dimethyl)adenine, 2-(alkyl)guanine,2 (propyl)guanine, 6-(alkyl)guanine, 6 (methyl)guanine, 7 (alkyl)guanine, 7 (methyl)guanine, 7 (deaza)guanine, 8 (alkyl)guanine, 8- (alkenyl)guanine, 8 (alkynyl)guanine, 8-(amino)guanine, 8 (halo)guanine, 8-(hydroxyl)guanine, 8 (thioalkyl)guanine, 8-(thiol)guanine, N (methyl)guanine, 2-(thio)cytosine, 3 (deaza) 5 (aza)cytosine, 3-(alkyl)cytosine, 3 (methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5 (halo)cytosine, 5 (methyl)cytosine, 5 (propynyl)cytosine, 5 (propynyl)cytosine, 5 (trifluoromethyl)cytosine, 6- (azo)cytosine, N4 (acetyl)cytosine, 3 (3 amino-3 carboxypropyl)uracil, 2-(thio)uracil, 5 (methyl) 2 (thio)uracil, 5 (methylaminomethyl)-2 (thio)uracil, 4-(thio)uracil, 5 (methyl) 4 (thio)uracil, 5 (methylaminomethyl)-4 (thio)uracil, 5 (methyl) 2,4 (dithio)uracil, 5 (methylaminomethyl)-2,4 (dithio)uracil, 5 (2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5 (aminoallyl)uracil, 5 (aminoalkyl)uracil, 5 (guanidiniumalkyl)uracil, 5 (l,3-diazole-l-alkyl)uracil, 5- (cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5 (dimethylaminoalkyl)uracil, 5-(halo)uracil, 5- (methoxy)uracil, uracil-5 oxyacetic acid, 5 (methoxycarbonylmethyl)-2-(thio)uracil, 5

(methoxycarbonyl-methyl)uracil, 5 (propynyl)uracil, 5 (propynyl)uracil, 5 (trifluoromethyl)uracil, 6 (azo)uracil, dihydrouracil, N3 (methyl)uracil, 5-uracil (i.e., pseudouracil), 2 (thio)pseudouracil,4 (thio)pseudouracil,2,4-(dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)- 2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4 (thio)pseudouracil, 5-(methyl)-4 (thio)pseudouracil, 5-(alkyl)-2,4 (dithio)pseudouracil, 5-(methyl)-2,4 (dithio)pseudouracil, 1 substituted pseudouracil, 1 substituted 2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, 1 substituted 2,4-(dithio)pseudouracil, 1 (aminocarbonylethylenyl)-pseudouracil, 1

(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil, 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1 (arninoalkylaminocarbonylethylenyl)- pseudouracil, 1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1

(arninoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil, 1 (arninoalkylaminocarbonylethylenyl)- 2,4-(dithio)pseudouracil, l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, l-(aza)-2-(thio)-3-(aza)-phenoxazin-l- yl, l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, 7-substituted 1,3- (diaza)-2-(oxo)-phenoxazin-l-yl, 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7-substituted l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, 7- (aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3- (aza)-phenoxazin-l-yl, 7-(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7- (aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, 7-(guanidiniumalkylhydroxy)-l,3- (diaza)-2-(oxo)-phenoxazin-l-yl, 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin- 1 -yl, 7-(guanidiniumalkyl-hydroxy)- 1 ,3-(diaza)-2-(oxo)-phenthiazin- 1 -yl, 7- (guanidiniumalkylhydroxy)-l -(aza)-2-(thio)-3-(aza)-phenthiazin-l -yl, 1 ,3,5-(triaza)-2,6-(dioxa)- naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza- inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)- 7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)- imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7- (aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6- (methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5 nitroindole, 3 nitropyrrole, 6-(aza)pyrimidine, 2 (amino)purine, 2,6-(diamino)purine, 5 substituted pyrimidines, N2- substituted purines, N6-substituted purines, 06-substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-substituted-6-phenyl- pyrrolo-pyrimidin-2-on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho- substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-(aminoalkylhydroxy)- 6-phenyl-pyrrolo- pyrimidin-2-on-3-yl, ortho-(aminoalkylhydroxy)- 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho— (aminoalkylhydroxy)- 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino- pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivatives thereof. Modified nucleosides also include natural bases that comprise conjugated moieties, e.g. a ligand. As discussed herein above, the RNA containing the modified nucleosides must be translatable in a host cell (i.e. , does not prevent translation of the polypeptide encoded by the modified RNA). For example, transcripts containing s2U and m6A are translated poorly in rabbit reticulocyte lysates, while pseudouridine, m5U, and m5C are compatible with efficient translation. In addition, it is known in the art that 2'-fluoro-modified bases useful for increasing nuclease resistance of a transcript, leads to very inefficient translation. Translation can be assayed by one of ordinary skill in the art using e.g., a rabbit reticulocyte lysate translation assay.

[00284] Further modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley- VCH, 2008; those disclosed in Int. Appl. No. PCT/US09/038425, filed March 26, 2009; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[00285] Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191 ; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091 ; 5,614,617; 5,681,941 ; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference in its entirety, and U.S. Pat. No. 5,750,692, also herein incorporated by reference in its entirety.

[00286] Another modification for use with the synthetic, modified RNAs generated using the

IVT templates and methods of use thereof described herein involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the RNA. Ligands can be particularly useful where, for example, a synthetic, modified RNA is subsequently administered in vivo. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556, herein incorporated by reference in its entirety), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060, herein incorporated by reference in its entirety), a thioether, e.g. , beryl-S- tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al, Biorg. Med. Chem. Let., 1993, 3:2765-2770, each of which is herein incorporated by reference in its entirety), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538, herein incorporated by reference in its entirety), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10: 1111-1118; Kabanov et al, FEBS Lett, 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54, each of which is herein incorporated by reference in its entirety), a phospholipid, e.g. , di-hexadecyl-rac-glycerol or triethyl-ammonium 1 ,2-di-O-hexadecyl-rac-glycero- 3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783, each of which is herein incorporated by reference in its entirety), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973, herein incorporated by reference in its entirety), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654, herein incorporated by reference in its entirety), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237, herein incorporated by reference in its entirety), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937, herein incorporated by reference in its entirety).

[00287] The synthetic, modified RNAs generated using the IVT templates and methods of use thereof described herein can further comprise a 5' cap. In some embodiments of the aspects described herein, the synthetic, modified RNAs comprise a 5' cap comprising a modified guanine nucleotide that is linked to the 5' end of an RNA molecule using a 5'-5'triphosphate linkage. As used herein, the term "5' cap" is also intended to encompass other 5' cap analogs including, e.g., 5' diguanosine cap, tetraphosphate cap analogs having a methylene -bis(phosphonate) moiety (see e.g. , Rydzik, AM et al., (2009) Org Biomol Chem 7(22):4763-76), dinucleotide cap analogs having a phosphorothioate modification (see e.g. , Kowalska, J. et al., (2008) RNA 14(6): 1119-1131), cap analogs having a sulfur substitution for a non-bridging oxygen (see e.g. , Grudzien-Nogalska, E. et al., (2007) RNA 13(10): 1745-1755), N7-benzylated dinucleoside tetraphosphate analogs (see e.g., Grudzien, E. et al., (2004) RNA 10(9): 1479-1487), or anti-reverse cap analogs (see e.g. , Jemielity, J. et al., (2003) RNA 9(9): 1108-1122 and Stepinski, J. et al., (2001) RNA 7(10): 1486-1495). In one such embodiment, the 5' cap analog is a 5' diguanosine cap. In some embodiments, the synthetic, modified RNA does not comprise a 5' triphosphate.

[00288] The 5' cap is important for recognition and attachment of an mRNA to a ribosome to initiate translation and enhances translation efficiency. The 5' cap also protects the synthetic, modified RNA from 5' exonuclease mediated degradation and thus increases half-life. It is not an absolute requirement that a synthetic, modified RNA comprise a 5' cap, and thus in other embodiments the synthetic, modified RNAs lack a 5' cap.

[00289] It is contemplated that one or more modifications to the synthetic, modified RNAs generated using the IVT templates and methods of use thereof described herein permit greater stability of the synthetic, modified RNA in a cell. To the extent that such modifications permit translation and either reduce or do not exacerbate a cell's innate immune or interferon response to the synthetic, modified RNA with the modification, such modifications are specifically contemplated for use herein. Generally, the greater the stability of a synthetic, modified RNA, the more protein can be produced from that synthetic, modified RNA. Typically, the presence of AU-rich regions in mammalian mRNAs tend to destabilize transcripts, as cellular proteins are recruited to AU-rich regions to stimulate removal of the poly(A) tail of the transcript. Loss of a poly(A) tail of a synthetic, modified RNA can result in increased RNA degradation. Thus, in some embodiments, a synthetic, modified RNA as described herein does not comprise an AU-rich region. In particular, it is preferred that the 3' UTR substantially lacks AUUUA sequence elements.

[00290] In some embodiments, a ligand alters the cellular uptake, intracellular targeting or half-life of a synthetic, modified RNA generated using the IVT templates and methods of use thereof described herein into which it is incorporated. In some embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, intracellular compartment, e.g. , mitochondria, cytoplasm, peroxisome, lysosome, as, e.g., compared to a composition absent such a ligand. Preferred ligands do not interfere with expression of a polypeptide from the synthetic, modified RNA.

[00291] Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g. , a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether- maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N- isopropylacrylamide polymers, or polyphosphazine. Example of poly amines include:

polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[00292] Ligands can also include targeting groups, e.g. , a cell targeting agent, (e.g. , a lectin, glycoprotein, lipid or protein), or an antibody, that binds to a specified cell type such as a fibroblast cell. A targeting group can be, for example, a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic, among others.

[00293] Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross- linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g. , phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g. , cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03- (oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g. , antennapedia peptide, Tat peptide), alkylating agents, amino, mercapto, PEG (e.g. , PEG-40K), MPEG, [MPEGJ2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), and transport/absorption facilitators (e.g. , aspirin, vitamin E, folic acid).

[00294] Ligands can be proteins, e.g. , glycoproteins, or peptides, e.g. , molecules having a specific affinity for a co-ligand, or antibodies e.g. , an antibody, that binds to a specified cell type such as a fibroblast cell, or other cell useful in the production of polypeptides. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose.

[00295] The ligand can be a substance, e.g. , a drug, which can increase the uptake of the synthetic, modified RNA or a composition thereof into the cell, for example, by disrupting the cell's cytoskeleton, e.g. , by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[00296] One exemplary ligand is a lipid or lipid-based molecule. A lipid or lipid-based ligand can (a) increase resistance to degradation, and/or (b) increase targeting or transport into a target cell or cell membrane. A lipid based ligand can be used to modulate, e.g. , binding, of the modified RNA composition to a target cell.

[00297] In another aspect, the ligand is a moiety, e.g. , a vitamin, which is taken up by a host cell. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include B vitamin, e.g. , folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up, for example, by cancer cells. Also included are HSA and low density lipoprotein (LDL).

[00298] In another aspect, the ligand is a cell-permeation agent, preferably a helical cell- permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

[00299] A "cell permeation peptide" is capable of permeating a cell, e.g. , a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell- permeating peptide can be, for example, an a-helical linear peptide (e.g. , LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g. , a -defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g. , PR-39 or indolicidin). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.

31 :2717-2724, 2003).

Introduction of Synthetic RNAs into Target Cells

[00300] A synthetic RNA generated using the IVT templates and methods of use thereof described herein can be introduced into a cell in any manner that achieves intracellular delivery of the synthetic RNA, such that, for example, expression of the polypeptide encoded by the synthetic RNA can occur. As used herein, the term "transfecting a cell" refers to the process of introducing nucleic acids into cells using means for facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. As the term is used herein, "transfection" does not encompass viral- or viral particle based delivery methods. Absorption or uptake of a synthetic, modified RNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Further approaches are described herein below or known in the art.

[00301] A synthetic RNA generated using the IVT templates and methods of use thereof described herein can be introduced into a target cell, for example, by transfection, nucleofection, lipofection, electroporation (see, e.g. , Wong and Neumann, Biochem. Biophys. Res. Commun.

107:584-87 (1982)), microinjection (e.g. , by direct injection of a synthetic, modified RNA), biolistics, cell fusion, and the like. In an alternative embodiment, a synthetic RNA can be delivered using a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of a synthetic, modified RNA (negatively charged polynucleotides) and also enhances interactions at the negatively charged cell membrane to permit efficient cellular uptake. Cationic lipids, dendrimer s, or polymers can either be bound to synthetic RNAs, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2): 107-116) that encases the synthetic RNA. Methods for making and using cationic-modified RNA complexes are well within the abilities of those skilled in the art (see e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9: 1291-1300; Arnold, AS et al (2007) J. Hypertens. 25: 197-205, which are incorporated herein by reference in their entirety).

[00302] In some embodiments of the aspects described herein, the composition further comprises a reagent that facilitates uptake of a synthetic RNA into a cell (transfection reagent), such as an emulsion, a liposome, a cationic lipid, a non-cationic lipid, an anionic lipid, a charged lipid, a penetration enhancer or alternatively, a modification to the synthetic RNA to attach e.g., a ligand, peptide, lipophillic group, or targeting moiety.

[00303] The process for delivery of a synthetic RNA to a cell will necessarily depend upon the specific approach for transfection chosen. One preferred approach is to add the RNA, complexed with a cationic transfection reagent directly to the cell culture media for the cells.

[00304] It is further possible to transfect cells with one or more distinct RNAs. For example, the population of cells can be transfected with one or more distinct mRNAs, one or more distinct siRNAs, one or more distinct miRNAs, or combinations thereof. The population of cells can be transfected with multiple RNAs simultaneously in a single administration, or multiple administrations can be staggered minutes, hours, days, or weeks apart. Transfection of multiple distinct RNAs can be staggered. For example, if it is desirable for a first RNA to be expressed prior to expression of one or more additional RNAs.

[00305] The level of expression of the transfected synthetic RNA can be manipulated over a wide range by changing the amount of input RNA, making it possible to individually regulate the expression level of each transfected RNA. The effective amount of input RNA is determined based on the desired result. Thus, each of a plurality of synthetic RNAs generated using the IVT templates and methods of use thereof described herein can be administered at a separate time or at a different frequency interval to achieve the desired expression of a polypeptide. Typically, 100 fg to 100 pg of a synthetic RNA is administered per cell using cationic lipid-mediated transfection. Since cationic lipid-mediated transfection is highly inefficient at delivering synthetic, modified RNAs to the cytosol, other techniques can require less RNA. The entire transcriptome of a mammalian cell constitutes about 1 pg of mRNA, and a polypeptide (e.g., a transcription factor) can have a physiological effect at an abundance of less than 1 fg per cell. Transfection Reagents

[00306] In certain embodiments of the aspects described herein, a synthetic RNA generated using the IVT templates and methods of use thereof described herein, such as a synthetic, modified RNA can be introduced into target cells by transfection or lipofection. Suitable agents for transfection or lipofection include, for example, calcium phosphate, DEAE dextran, lipofectin, lipofectamine, DIMRIE C™, SUPERFECT™, and EFFECTIN™ (QIAGEN™), UNIFECTIN™, MAXIFECTIN™, DOTMA, DOGS™ (Transfectam; dioctadecylamidoglycylsperrnine), DOPE (1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine), DOTAP (l,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecylammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB (N-n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene,

poly(ethylenimine) (PEI), and the like. (See, e.g., Banerjee et al., Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6: 1380-88 (1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J. Pharm. 183: 195-207 (1999)).

[00307] A synthetic RNA generated using the IVT templates and methods of use thereof described herein can be transfected into target cells as a complex with cationic lipid carriers {e.g. , Oligofectamine™) or non-cationic lipid-based carriers {e.g., TRANSIT-TKOTM™, Minis Bio LLC, Madison, WI). Successful introduction of a modified RNA into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Successful transfection of a synthetic RNA can also be determined by measuring the protein expression level of the target polypeptide by e.g. , Western Blotting or immunocytochemistry.

[00308] In some embodiments of the aspects described herein, the synthetic RNA generated using the IVT templates and methods of use thereof described herein is introduced into a cell using a transfection reagent. Some exemplary transfection reagents include, for example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731). Examples of

commercially available transfection reagents include, for example LIPOFECTAMINE™ (Invitrogen; Carlsbad, CA), LIPOFECTAMINE 2000™ (Invitrogen; Carlsbad, CA), 293FECTIN™ (Invitrogen; Carlsbad, CA), CELLFECTIN™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FREESTYLE™ MAX (Invitrogen; Carlsbad, CA), RNAIMAX (Invitrogen; Carlsbad, CA), OLIGOFECTAMINE™ (Invitrogen; Carlsbad, CA), OPTIFECT™ (Invitrogen; Carlsbad, CA), X- TREMEGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP

Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or FUGENE (Grenzacherstrasse, Switzerland),

TRANSFECTAM® Reagent (Promega; Madison, WI), TRANSFAST™ Transfection Reagent (Promega; Madison, WI), TFX™-20 Reagent (Promega; Madison, WI), TFX™-50 Reagent (Promega; Madison, WI), DREAMFECT™ (OZ Biosciences; Marseille, France), ECOTRANSFECT (OZ Biosciences; Marseille, France), TRANSPASS³ Dl Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LYOVEC™/LIPOGEN™ (Invitrogen; San Diego, CA, USA), PERFECTIN Transfection Reagent (Genlantis; San Diego, CA, USA), NEUROPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GENEPORTER Transfection reagent (Genlantis; San Diego, CA, USA), GENEPORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), CYTOFECTIN Transfection Reagent (Genlantis; San Diego, CA, USA), BACULOPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TROGANPORTER™ transfection Reagent (Genlantis; San Diego, CA, USA ), RIBOFECT (Bioline; Taunton, MA, USA), PLASFECT (Bioline; Taunton, MA, USA), UNIFECTOR (B -Bridge International; Mountain View, CA, USA), SUREFECTOR (B -Bridge International; Mountain View, CA, USA), or HIFECT™ (B-Bridge International, Mountain View, CA, USA), among others.

[00309] In other embodiments, highly branched organic compounds, termed "dendrimers," can be used to bind the exogenous nucleic acid, such as the synthetic, modified RNAs described herein, and introduce it into the cell.

[00310] In other embodiments of the aspects described herein,, non-chemical methods of transfection are contemplated. Such methods include, but are not limited to, electroporation (methods whereby an instrument is used to create micro-sized holes transiently in the plasma membrane of cells under an electric discharge), sono-poration (transfection via the application of sonic forces to cells), and optical transfection (methods whereby a tiny (~1 μπι diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser). In other embodiments, particle-based methods of transfections are contemplated, such as the use of a gene gun, whereby the nucleic acid is coupled to a nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus; "magnetofection," which refers to a transfection method, that uses magnetic force to deliver exogenous nucleic acids coupled to magnetic nanoparticles into target cells;

"impalefection," which is carried out by impaling cells by elongated nanostructures, such as carbon nanofibers or silicon nano wires which have been coupled to exogenous nucleic acids.

[00311] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols, such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes, such as limonene and menthone.

[00312] Following transfection with one or more synthetic RNAs generated using the IVT templates and methods of use thereof described herein, the cells can be maintained or expanded in culture. Methods for culturing both transfected and non-transfected cells are known in the art, and may include providing additional reagents or supplements to enhance viability and/or growth, for example, growth factors or a feeder layer of cells.

Target Cells [00313] Cells suitable for transfections with the synthetic RNAs generated using the IVT templates and methods of use thereof described herein include, but are not limited to, primary cells and established cell lines, embryonic cells, immune cells, stem cells, and differentiated cells including, but not limited to, cells derived from ectoderm, endoderm, and mesoderm, including fibroblasts, parenchymal cells, hematopoietic cells, and epithelial cells. As used herein, stem cells include unipotent cells, multipotent cells, and pluripotent cells; embryonic stem cells, and adult stem cells such as hematopoietic stem cells, mesenchymal stem cells, epithelial stem cells, and muscle satellite cells. In one embodiment, somatic cells are de-differentiated or reprogrammed. Any suitable somatic cell can be used. Representative somatic cells include fibroblasts, keratinocytes, adipocytes, muscle cells, organ and tissue cells, and various blood cells including, but not limited to, hematopoietic cells including hematopoietic stem cells, and cells that provide short- or long-term hematopoietic engraftment. Cell types of particular utility with the IVT templates described herein include, but are not limited to, human fibroblasts, keratinocytes and hematopoietic stem cells. The synthetic RNAs generated using the IVT templates and methods of use thereof described herein are particularly useful for de -differentiating and optionally re-differentiating cells, without permanent alteration of cell genomes.

[00314] Other cell types suitable for transfection with the synthetic RNAs generated using the

IVT templates and methods of use thereof described herein include a: Claudius' cell, Hensen cell, Merkel cell, Muller cell, Paneth cell, Purkinje cell, Schwann cell, Sertoli cell, acidophil cell, acinar cell, adipoblast, adipocyte, brown or white alpha cell, amacrine cell, beta cell, capsular cell, cementocyte, chief cell, chondroblast, chondrocyte, chromaffin cell, chromophobic cell, corticotroph, delta cell, Langerhans cell, follicular dendritic cell, enterochromaffin cell, ependymocyte, epithelial cell, basal cell, squamous cell, endothelial cell, transitional cell, erythroblast, erythrocyte, fibroblast, fibrocyte, follicular cell, germ cell, gamete, ovum, spermatozoon, oocyte, primary oocyte, secondary oocyte, spermatid, spermatocyte, primary spermatocyte, secondary spermatocyte, germinal epithelium, giant cell, glial cell, astroblast, astrocyte, oligodendroblast, oligodendrocyte, glioblast, goblet cell, gonadotroph, granulosa cell, haemocytoblast, hair cell, hepatoblast, hepatocyte, hyalocyte, interstitial cell,' juxtaglomerular cell, keratinocyte, keratocyte, lemmal cell, leukocyte, granulocyte, basophil, eosinophil, neutrophil, lymphoblast, B-lymphoblast, T-lymphoblast, lymphocyte, B -lymphocyte, T- lymphocyte, helper induced T-lymphocyte, Thl T-lymphocyte, Th2 T-lymphocyte, natural killer cell, thymocyte, macrophage, Kupffer cell, alveolarmacrophage, foam cell, histiocyte, luteal cell, lymphocytic stem cell, lymphoid cell, lymphoid stem cell, macroglial cell, mammotroph, mast cell, medulloblast, megakaryoblast, megakaryocyte, melanoblast, melanocyte, mesangial cell, mesothelial cell, metamyelocyte, monoblast, monocyte, mucous neck cell, muscle cell, cardiac muscle cell, skeletal muscle cell, smooth muscle cell, myelocyte, myeloid cell, myeloid stem cell, myoblast, myoepithelial cell, myofibrobast, neuroblast, neuroepithelial cell, neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell, parafollicular cell, paraluteal cell, peptic cell, pericyte, peripheral blood mononuclear cell, phaeochromocyte, phalangeal cell, pinealocyte, pituicyte, plasma cell, platelet, podocyte, proerythroblast, promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte, retinal pigment epithelial cell, retinoblast, small cell, somatotroph, stem cell, sustentacular cell, teloglial cell, or zymogenic cell.

Applications Using Synthetic RNAs

[00315] The synthetic RNAs generated using the IVT templates and methods of use thereof described herein have a wide range of applications in therapy and research. The synthetic RNAs are useful for expressing one or multiple synthetic RNAs in different cell populations such as fully differentiated cells, partially differentiated cells, such as multipotent cells and non-differentiated cells, such as pluripotent cells, for modulating cellular phenotypes and developmental potential.

[00316] The synthetic RNAs generated using the IVT templates and methods of use thereof described herein, such as synthetic, modified RNAs encoding transcription factors, are particularly useful in the field of stem cell therapies and personalized medicine. In some embodiments, the methods are applied in the context of personalized therapy, for example, to generate iPS cells for introduction into a subject in need thereof. In vitro de-differentiation, re -differentiation, and/or reprogramming can be applied to a variety of different starting cell types and allows fast and safe generation of cells over a diverse range of de -differentiated or re-differentiated states. For example, target cells are first isolated from a donor using methods known in the art, contacted with one or more synthetic RNAs generated using the IVT templates and methods of use thereof described herein causing the cells to be de-differentiated, re -differentiated, or reprogrammed in vitro (ex vivo), and administered to a patient in need thereof. Sources or cells include, but are not limited to peripheral lymphocytes, fibroblasts, keratinocytes primary cell lines, or cells harvested directly from the patient or an allographic donor. In preferred embodiments, the target cells to be administered to a subject will be autologous, e.g. derived from the subject, or syngenic. Allogeneic cells can also be isolated from antigenically matched, genetically unrelated donors (identified through a national registry), or by using target cells obtained or derived from a genetically related sibling or parent.

[00317] In some embodiments, cells can be contacted with one or more synthetic RNAs generated using the IVT templates and methods of use thereof described herein that reprogram the cells to prevent expression of one or more antigens. For example, the RNA can be an interfering RNA that prevents expression of an mRNA encoding antigens as CTLA-4 or PD-1. These methods can be used to prepare universal donor cells. RNAs used to alter the expression of allogenic antigens can be used alone or in combination with RNAs that result in de -differentiation of the target cell.

[00318] Cells can be selected by positive and/or negative selection techniques. For example, antibodies binding a particular cell surface protein may be conjugated to magnetic beads and immunogenic procedures utilized to recover the desired cell type. It can be desirable to enrich the target cells prior to transient transfection. As used herein in the context of compositions enriched for a particular target cell, "enriched" indicates a proportion of a desirable element (e.g. the target cell) which is higher than that found in the natural source of the cells. A composition of cells can be enriched over a natural source of the cells by at least one order of magnitude, preferably two or three orders, and more preferably 10, 100, 200 or 1000 orders of magnitude. Once target cells have been isolated, they may be propagated by growing in suitable medium according to established methods known in the art. Established cell lines can also be useful for use with the synthetic RNAs generated using the IVT templates and methods of use thereof described herein. The cells can be stored frozen before transfection, if necessary.

[00319] Next, cells are contacted with one or more synthetic RNAs generated using the IVT templates and methods of use thereof described herein in vitro, for example using a transfection technique known in the art. De-differentiation, re -differentiation, and/or re-programming can be monitored, and the desired cell type, for example iPS cells, can be selected for therapeutic administration.

[00320] Following de -differentiation, and/or re-differentiation and/or reprogramming, the cells are administered to a patient in need thereof. In preferred embodiments, the cells are isolated from and administered back to the same patient. In alternative embodiments, the cells are isolated from one patient, and administered to a second patient. Scuh methods can also be used to produce frozen stocks of RNA-reprogrammed or dedifferentiated cells stored long-term, for later use. In some embodiments, fibroblasts, keratinocytes or hematopoietic stem cells are isolated from a patient and de -differentiation, and/or re-differentiated and/or reprogrammed in vitro to provide iPS cells for the patient.

[00321] The synthetic RNAs generated using the IVT templates and methods of use thereof described herein can also be used to reprogram somatic cells wherein synthetic, preferably modified RNAs are introduced into cells in order to modulate their viability. For example, mRNA coding dominant-negative mutant p53 protein can temporarily block p53 function. This mRNA can be introduced into cells to protect them from p53 -mediated apoptosis caused by metabolic disturbances during de-differentiation.

[00322] In some embodiments, cells are reprogrammed with the synthetic RNAs generated using the IVT templates and methods of use thereof described herein to modulate the immune response. For example, lymphocytes can be reprogrammed into regulatory T cells which can be administered to a patient in need thereof to increase or transfer immune tolerance, especially self- tolerance. The induction or administration of Foxp3 positive T cells may be useful in reducing autoimmune responses such graft rejection, and/or reducing, inhibiting or mitigating one or more symptoms of an autoimmune diseases or disorder such as diabetes, multiple sclerosis, asthma, inflammatory bowel disease, thyroiditis, renal disease, rheumatoid arthritis, systemic lupus erythematosus, alopecia greata, anklosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliierative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease, Dego's disease, dermatomyositis, dermatomyositis— juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia— fibromyositis, Grave's disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin dependent diabetes (Type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[00323] The synthetic RNAs generated using the IVT templates and methods of use thereof described herein can also be used to generate cells that can be useful in the treatment of a variety of diseases and disorders, including, but not limited to, neurodegenerative diseases such as Parkinson's, Alzheimer disease, and multiple sclerosis. The synthetic RNAs are also useful for organ regeneration, and for restoration or supplementation of the immune system. For example, cells at different stages of differentiation such as iPS cells, hematopoietic stem cells, multipotent cells or unipotent cells such as precursor cells, for example, epithelial precursor cells, and others can be administered intravenously or by local surgery. Such methods can be used in combination with other conventional methods, such as a prescription medication regime, surgery, hormone therapy, chemotherapy and/or radiotherapy.

[00324] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. In addition, the term 'cell' can be construed as a cell population, which can be either heterogeneous or homogeneous in nature, and can also refer to an aggregate of cells.

[00325] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[00326] All references cited herein in the specification are incorporated by reference in their entirety.

[00327] Embodiments of the various aspects described herein can be illustrated by the following numbered paragraphs.

1. An isolated nucleic acid sequence comprising, in the 5' to 3' direction: a first nucleic acid sequence comprising a forward universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence, a second blunt-ended restriction enzyme digestion site, a 3' UTR sequence, and a second nucleic acid sequence comprising a sequence complementary to a reverse universal primer sequence.

2. The isolated nucleic acid sequence of paragraph 1, wherein the 5' UTR sequence comprises the sequence of SEQ ID NO: 20.

3. The isolated nucleic acid sequence of any one of the preceding paragraphs, wherein the first blunt-ended restriction enzyme digestion site comprises the sequence of SEQ ID NO: 60.

4. The isolated nucleic acid sequence of any one of the preceding paragraphs, wherein the

second blunt-ended restriction enzyme digestion site comprises the sequence of SEQ ID NO: 61.

5. The isolated nucleic acid sequence of any one of the preceding paragraphs, wherein the 3' UTR sequence comprises the sequence of SEQ ID NO: 21.

6. The isolated nucleic acid sequence of any one of the preceding paragraphs, further comprising a vector backbone sequence.

7. The isolated nucleic acid sequence of any one of the preceding paragraphs, wherein the 3' UTR followed by a second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence is not operably linked to a poly-A tail sequence.

8. The isolated nucleic acid sequence of any one of the preceding paragraphs, further comprising a nucleic acid encoding an open reading frame (ORF) sequence between the first and the second blunt-ended restriction enzyme digestion sites.

9. The isolated nucleic acid sequence of paragraph 8, wherein the ORF sequence excludes the 5' adenine nucleotide prior to inserting it between the first and the second blunt-ended restriction sites.

10. A plurality of isolated nucleic acids encoding a plurality of open reading frames comprised within a vector, each said isolated nucleic acid comprising: in the 5' to 3' direction: a first nucleic acid sequence comprising a forward universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence, a second blunt-ended restriction enzyme digestion site, a 3' UTR sequence, a second nucleic acid sequence comprising a sequence complementary to a reverse universal primer sequence, and a vector backbone sequence.

A kit comprising the isolated nucleic acid of any one of paragraphs 1-9, in a suitable container. The kit of paragraph 11 , further comprising a first and a second blunt-ended restriction enzyme specific for the first and second blunt-ended restriction enzyme digestion sites respectively.

The kit of paragraphs 11 or 12, further comprising a first universal primer comprising the forward universal primer sequence and a second universal primer comprising a poly-T sequence.

A kit comprising the plurality of nucleic acids of paragraph 10, in a suitable container.

The kit of any of paragraphs 11-14, wherein the 5' UTR sequence comprises the sequence of SEQ ID NO: 20.

The kit of any of paragraphs 11-15, wherein the first blunt-ended restriction enzyme digestion site comprises SEQ ID NO: 60.

The kit of any of paragraphs 11-16, wherein the second blunt-ended restriction enzyme digestion site comprises SEQ ID NO: 61.

The kit of any of paragraphs 11-17, wherein the 3' UTR sequence comprises the sequence of SEQ ID NO: 21.

The kit of any of paragraphs 11-18, wherein the isolated nucleic acid further comprises a vector backbone sequence.

The kit of any of paragraphs 11-19, wherein the 3' UTR followed by the second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence is not operably linked to a poly- A tail sequence.

A method of synthesizing a nucleic acid construct for transcribing a gene of interest in vitro, the method comprising the steps of:

a. amplifying an open reading frame (ORF) sequence of the gene of interest to form an ORF amplification product using a phosphorylated forward primer and a

b. digesting a vector comprising, in the 5' to 3' direction: a first nucleic acid sequence comprising a first universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence, a second blunt-ended restriction enzyme digestion site operably linked to a 3' UTR sequence, a second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence, and a vector backbone sequence; with a first and a second blunt-ended restriction enzyme specific for the first and second blunt-ended restriction enzyme digestion sites respectively;

c. dephosphorylating the digested vector with a phosphatase enzyme; and

22. The method of paragraph 21, further comprising one or more steps of screening for proper orientation of the ORF sequence comprising the steps of:

a. amplifying the ORF sequence with a forward primer comprising the first universal primer sequence and a reverse primer comprising a sequence specific for the 3' end of the ORF sequence; and

b. selecting the nucleic acid constructs wherein the ORF sequence is in the proper 5' to 3' orientation.

23. The method of any one of paragraphs 21 or 22, further comprising a step of adding a poly-A tail to the ORF sequence by amplifying the nucleic acid construct with a forward primer comprising the first universal primer sequence and a reverse primer sequence comprising a poly-T sequence.

EXAMPLES

[00328] The use of in vitro transcribed mRNAs for ectopic expression of proteins in mammalian cells has largely been hampered by the induction of interferon-mediated innate immune responses that are associated with high cellular toxicity. However, transcripts synthesized with incorporation of certain non-canonical nucleosides, largely evade eliciting such immune responses. Transfection of such modified mRNAs into cells can be used to achieve robust, penetrant expression of essentially any protein(s) or peptide in a manner that is both temporally, and dose controllable. Described herein are constructs and step-wise protocols for in vitro transcription (IVT) template construction, and synthesis of RNAs, such as non-immunogenic modified-mRNA, and the important parameters and optimization steps involved with their effective in vitro use.

Introduction

[00329] Although once known solely as intermediaries of sequential information transfer from the genetic code to protein, RNA now takes stage as a central regulator of gene expression in eukaryotic cells. Various structurally and functionally diverse RNA molecules have been discovered, and their ability to modulate gene expression in sequence specific manners without leaving any genomic footprint has made them ideal biological tools for interrogating gene function (1-4). Gene knockdown using siRNA/shRNA and miRNA is now routinely used to study transient loss of gene function on genome-wide scales. In contrast, a complementary technique to achieve transient ectopic gene expression with exogenously administered mRNA had lagged in development. Although such a technique has been used historically in overexpression studies involving early vertebrate embryos, protein expression from in vitro transcribed (IVT) mRNA in mature mammalian cells has been hampered by cytotoxicity resulting from activation of innate immune response against foreign RNA (5,6). RNA molecules longer than 21 nucleotides are sensed by Toll-like receptors (e.g., TLR3, TLR7, and TLR8) and foreign RNA sensors (e.g., RIG-I, PKR), which then culminates in translational inhibition as well as apoptosis via interferon and NF-kB dependent pathways (7-15).

[00330] For both experimental and clinical applications, direct administration of modified

RNAs is significantly advantageous over plasmid DNA and viral gene expression vectors due to absent risk of genomic integration, virtually limitless cargo size (both with regard to protein size and the number of co-transfec table mRNAs), ability to achieve highly penetrant delivery as modified RNAs function in the cytoplasm without having to enter the nucleus, and ability to control the level and duration of protein expression by virtue of their transient half-lives (18).

[00331] As described herein, we generated novel DNA templates and constructs and methods of use thereof for producing modified RNAs encoding a protein of interest. In order to facilitate the construction of such DNA templates for in vitro transcription, which is a bottleneck for modRNA production, we cloned the 5' and 3' UTRs in a vector backbone (pZEr02) to generate a construct termed pORFin. The open reading frame (ORF) of a gene of interest can be cloned between two appropriate restriction sites between the UTRs of the construct by blunt-ended cloning, for example. These novel contacts and methods for template construction are straightforward and can be completed in fewer steps compared to other methods, such as splint-mediated ligation (21).

Materials

Reagents

[00332] Reagents used to generate the constructs described herein include:

1. T4 Polynucleotide kinase (New England Biolabs; catalog # M0201s)

2. T4 Polynucleotide kinase (New England Biolabs; catalog # M0201s)

3. 2X KAPA HiFi HotStart ReadyMix (KAPA BIOSYSTEM; catalog # KK2601)

4. T4 DNA ligase (New England Biolabs; catalog # M0202S)

5. ONE SHOT^® TOP10 chemically competent E. coli (Invitrogen; catalog # C4040-03)

6. ONE SHOT® CCDB SURVIVAL™ 2 Tl Phage-Resistant (T1R )(Invitrogen; catalog # A10460 )

7. QIAQUICK^® gel extraction kit (QIAGEN; catalog # 28704)

8. QIAQUICK^® PCR purification kit (QIAGEN; catalog # 28106)

9. QIAPREP^® spin Miniprep kit (QIAGEN; catalog # 27106)

10. MEGASCRIPT^® T7 Kit (Ambion; catalog # AM1334)

11. MEGACLEAR™ (Ambion; catalog # AM1908)

12. 3'-0-Me-m⁷G(5')ppp(5')G RNA Cap analog (New England Biolabs; catalog # S141 IS)

13. 5-Methylcytidine-5' -Triphosphate (Trilink; catalog # N1014) 14. Pseudouridine-5' -Triphosphate (Trilink; catalog # N1019)

15. Antarctic phosphatase (New England Biolabs; catalog # M0289S)

16. OPTI-MEM (Invitrogen; catalog* 31985)

17. LIPOFECTAMINE™ RNAiMax (Invitorgen; catalog#56532)

18. MEGATRAN1.0 (Origene; catalog#TT200002)

19. TRANSIT- mRNA transfection kit (Mirus; catalog#MIR2225)

20. LIPOFECTAMINE™ LTX with plus reagent (Invitrogen; catalog#15338-100)

Primers

[00333] Primers used in the methods described herein include:

Xu-Fl: 5'-TTG GAC CCT CGT ACA GAA GCT AAT ACG-3' (SEQ ID NO: 187)

TTT TTT TTT TCT TCC TAC TCA GGC TTT ATT CAA AGA CCA-3' (SEQ ID NO: 188) Above primers were synthesized at and purchased from Integrated DNA technologies Inc. Xu-T120 was synthesized as Ultramer Oligos (4 nmole scale).

Equipment

[00334] Equipment used in the methods described herein include:

1. Nano-drop

2. PCR Thermocycler (Eppendorf)

3. Microfuge centrifuge (Eppendorf)

4. Vortex

5. Thermomixer (Eppendorf)

Procedures

1. Construction of DNA template for in vitro transcription using pORFin plasmid

[00335] In the original protocol described in Warren et al. 2010, the DNA template for IVT was constructed by ligating 5' and 3' UTRs onto the cloned ORF using oligonucleotide "splints" that span their junctions. As described herein, we cloned 5'- and 3 '-UTRs into pZErO-2, as described below, to generate pORFin, a cloning vector into which any ORF of interest can be inserted between UTRs using standard cloning techniques. To construct pORFin, the 5'- and 3 '-UTRs were de novo synthesized by synthetic oligos, which were annealed together and amplified using forward and reverse primers. Alel and Afel sites were introduced in between 5' and 3' UTRs, which provides entry sites for given open reading frame (ORF). The PCR amplified fragment and pZErO-2 vector were digested with Hindlll and Notl and ligated together to create pORFin. The cloned fragment was verified by sequencing. To generate and insert for gene of interest, the ORF was amplified by using phosphorylated forward and reverse primer pair (FIG. 1, Box 1, 2, 3). The amplified fragment was gel -extracted and cloned in Alel and Afel digested and dephosphorylated pORFin (FIG. 1) by blunt- end ligation. The right-orientation clones were screened by colony PCR (FIG. 1). Sequence-verified clone was used as DNA template for Tail PCR, which appends a polyadenylation template to the pORFin-encoded construct. We used Ml 3 R and Ml 3 F(-21) primers for sequencing our pORFin constructs.

[00336] When using the constructs and methods described herein, the adenine nucleotide (A) of the 1^st codon (ATG) of the open reading frame encoding the protein of interest should be omitted from forward primer sequence as it is provide by Alel site.

[00337] pORFin plasmid and derivatives therefrom should be propagated in bacterial strain resistant to the ccdB gene product such as, for example, ONE SHOT® CCDB SURVIVAL™ 2 Tl Phage-Resistant (T1R ).

2. Addition of poly-(A) tail by PCR

[00338] 2a. Make tail PCR master mix:

KAPA PCR Ready Mix 90 μΐ

XU-F1+XU-T120 (luM) 36 μΐ

Water 36 μΐ

Diluted ORF plasmid ( 1 -5 ng/μΐ) 18 μΐ

[00339] 2b. Aliquot the above mixture into 8 PCR tubes (~20μ1 each)

[00340] An important step for addition of the poly-(A) tail is to carry out the PCR in multiple tubes in a smaller volume. A minimum of eight PCR tubes is required for sufficient yield of tailed product, which is enough for 10-15 IVT reactions.

[00341] 2c. Run Tail PCR (Box 4).

[00342] Conditions for the Run Tail PCR can vary depending upon DNA polymerase used and ORF length, which should be optimized by the user.

[00343] 2d. After PCR, combine all tubes into one eppendorf and add 3μ1 Dpnl.

[00344] 2e. Incubate at 37°C for 1 hour to digest the methylated plasmid DNA.

[00345] 2f. Use PCR purification kit to purify the reaction, adjust final concentration to 100 ng/μΐ. Ideally, purity of tail PCR reaction can be measured by running an aliquot on 1 % agarose gel.

[00346] 2g. Alternatively, vector can be linearized by restriction enzyme that cuts within the vector backbone (see, for example, FIGS. 1A-1C) and the linearized vector can be use as template for tail-PCR reaction. The purpose of this step is to eliminate any circular templates that would generate run-on transcripts. If this step is performed, a user should make sure that selected restriction enzyme is not present within the ORF or UTRs.

3. In vitro Transcription (40μ1 reaction volume)

[00347] 3a. Prepare Custom NTPS' according to following instructions: Add into 1 vial of lyophilized powder of 3'-0-Me-m7G Cap structure analog (25 A₂6o unit) 28.4μ1 of nuclease free water. In addition add to this vial 4.3 μΐ of GTP (75 mM stock) and 21.8 μΐ of ATP (75 mM stock) and 16.4 μΐ of 5-Me-CTP (100 mM) and 16.4 μΐ Pseudo-UTP (100 mM). (Table 1) [00348] 3b. Mix the reagents (in a PCR tube) in the following order:

Custom NTPS ' mix (from Table 1 ) 16 μΐ

Tailed PCR product ( 100 ng/ μΐ ) 16 μΐ

10 x T7 Buffer 4 μΐ

10 x T7 Enzyme mix 4 μΐ

Total 40 μΐ

[00349] 3c. Incubate for 4 hours at 37°C in a PCR machine or dry air incubator. Avoid incubation in a water bath

[00350] 3d. Add 3μ1 Turbo DNAse to each sample. Mix it gently and incubate for 15 minutes at 37°c.

[00351] 3e. Purify DNAse treated reaction using MEGAclear™ (Ambion®) as per manufacturer's instruction and elute the mRNA into a total 100 μΐ elution buffer.

[00352] It is important for these steps to clean the lab bench and pipettors with an RNase decontamination solution (e.g., AMBION® RNASEZAP® Solution) and use RNase-free pipette tips. Frequent change of gloves is also recommended. RNA elution option 2 was followed in the methods described herein as outlined in MEGACLEAR™ manual. The elution buffer should be pre -incubated at 95 °c.

4. Phosphatase treatment of purified RNA

[00353] 4a. To each RNA sample (~100μ1) add ΙΟμΙ of 10 x Antarctic Phosphatase buffer and then add 3μ1 of Antarctic Phosphatase, gently mix samples and incubated for 30 minutes at 37°c. This step is important as phosphatase treatment of uncapped RNA prevents its recognition by RIG-I complex and therefore it is important to incubate RNA with phosphatase for at least 30 minutes.

[00354] 4b. Purify Antarctic Phosphatase treated reaction using MEGACLEAR™

(AMBION®) as described in previous section.

5. Results

[00355] Measure the concentration of RNA after elution. The expected total yield of RNA is

~50μg RNA (500 ng/μΐ in 100 μΐ elution volume). Adjust the concentration to lOOng/μΙ by adding elution buffer (FIGS. 2A-2C). It is important to pay attention to the nano drop reading as a quality control guideline (e.g., FIG. 2A). A yield below 200 ng/μΐ indicates sub-optimal yield.

6. Important considerations for successful transfection

[00356] mRNA transfection efficiency is highly variable and is determined by various factors like cell types, culture conditions, transfection reagents, which should be taken in to consideration for successful transfection. Herein we highlight important issues related with transfection as a guideline and a reader/user should do an optimization at their end for successful outcome of experiment using modRNA. [00357] 6a. Cell types: The most important parameter is target cell type. Transfectibility is a cell autonomous factor as some cell types are easy to transfect (fibroblasts) while others (blood cells) are hard to transfect. We recommend testing of media, various transiection reagents and a dosing regime as pilot experiment for every target cell type to define the transiection condition (FIG. 3A).

[00358] 6b. Transfection reagents: The second most important parameter is transiection reagents. Although majority of them are either cationic lipid or polymer based, for reasons unknown, they behave differently depending upon cell types. User should test a couple of transfection reagents for their target cell. As shown in FIG. 3B, even for easy to transfect fibroblast cell line, 4 tested transfection reagents gave highly variable transfection efficiency. Although MEGATRAN has a very high penetrance, LIPOFECTAMINE RNAIMAX has outstanding performance in our hands for multiple repeated transfection experiment.

[00359] 6c. Cell culture medium condition: Cell culture media and their FCS or protein content has a profound impact on transfectibility. With increase in FCS concentration there is a reduction in transfection efficiency (FIG. 3C) for tested cell. However, this may not be true for some important cell types where serum is an essential requirement for cell growth/proliferation. We recommend use of chemically defined serum free/low serum containing medium for transfection experiment.

[00360] 6d. RNA to transfection reagent ratio: An optimal ratio of RNA to transfection reagent should be determined prior to important experiment as this lowers the toxicity due to vehicle and increases the delivery of RNA to the cells. We recommend use of a matrix as shown in FIG. 3D.

[00361] 6e. Dose and incubation time: Once the condition is defined for a particular cell type dosing and incubation time should be established. In general with increasing dose and incubation time the transfection efficiency as well as RNA delivery per cell increases till saturation point is reached (FIG. 3E and 3D). However with increasing incubation time there might be overt cytotoxic effect due to transfection reagent.

[00362] Troubleshooting Guidelines

Problem Possible cause Suggested solution

Step l.

a) No/few colonies on the plate a) No/inadequate primer phosphorylation a) Optimal primer phosphorylation

b) Inefficient ligation of insert a) Make sure that DNA ligase is b) Increase the amount of insert c) Adequate backbone dephosphorylation b) Low plasmid yield a) Toxicity due to ccdB a) Change bacterial strain b) Increase the culture volume

Step 2. a) No/Low tailed-template DNA a) Failure of tailed PCR a) Set-up multiple reactions

b) Loss during purification b) Run the PCR product over the gel before purification

Step 3.

a) Low yield «200 ng/ul) a) Inadequate ribonucleotides concentration a) Use correct proportion of each

NTPs

b) Loss during purification step a) Avoid delays during purification b) Pre-heat elution buffer to 95 °c c) RNase contamination a) Check the RNA over gel;

Smearing indicates degraded RNA. Work in RNase-free place c) Poor template quality c) Check the quality of tailed- template DNA

b) Lower than expected RNA size a) Inadequate denaturation a) Use denaturing agarose gel b) Denature RNA sufficiently before loading

b) Premature termination a) Verify quality of RNA by

functional test

b) Lower reaction temperature (May reduce the yield) c) RNA product > expected size a) Circular template a) Linearize plasmid template completely (Increase restriction digestion incubation time) b) Persistent secondary structure a) MEGAscript product

occasionally runs as two bands due to persistent secondary structure (see instruction manual)

Box 1.

[00363] Phosphorylation of primers*

10 x Phosphorylation Buffer 5 μΐ

Forward primer (100 μΜ) 3 μΐ

Reverse Primer (100 μΜ) 3 μΐ

100 mM ATP 0.5 μΐ

T4 polynucleotide kinase 10 U

Water q.s. 50 μΐ

[00364] Mix the reagent and incubate at 37 °C for 1 hr. Heat inactivates the enzyme by incubating at 65°C for 20 min. Reaction mix was diluted to 300 μΐ by adding 250 μΐ of water giving final concentration of Oligo mix ΙμΜ. (* As per manufacturer instruction) Box 2.

[00365] Typical PCR reaction set-up and PCR reaction condition for ORF amplification

[00366] A) PCR reaction mix*

HiFi HotStart ready mix (2 x) 25 μΐ

Primer mix (1 μΜ) from above 10 μΐ

Template DNA 1-100 ng

Water q.s. 50 μΐ

[00367] B) PCR reaction condition*

Initial denaturation 9955 °°CC 2-3 min 1 cycle

Denaturation 9988 °°CC 20 sec 25-35 cycles

Primer annealing 6655 °°CC 15 sec

Extension 7722 °°CC 15-60 sec/kb

Final extension 7722 °°CC 1-5 min 1 cycle

( * As per manufacturer instruction)

Box 3.

[00368] Ligation of amplified ORF in to linearized and dephosphorylated pORFin

[00369] A) Digestion of pORFin with Alel and Afel

Plasmid DNA 2 μg

10 x Buffer 4 3 μΐ

Alel 5 U

Afel 5 U

Water q.s. 30 μΐ

[00370] Digestion was carried out at 37 °C for 1 hr. 5 μΐ of reaction mixture was loaded on gel to check for digestion. Digested plasmid was purified through PCR purification column. Final elution was carried out in 30 μΐ of elution buffer.

[00371] B) Dephosphorylation of linearized pORFin

Linearized plasmid from (A) 30 μΐ

10 x antarctic phosphatase buffer 5 μΐ

Antarctic phosphatase 5 U

Water q.s. 50 μΐ

[00372] Dephosphorylation was carried out at 37 °C for 1 hr. Antarctic phosphatase was heat inactivated by incubating at 65 °C for 15 min. The linearized and dephosphorylated plasmid was gel- extracted. The amount of plasmid was quantified by nano-drop. The linearized, dephosphorylated plasmid can be stored at -20 °C for future use.

[00373] C) Ligation of ORF into pORFin Linearized plasmid 50 ng

ORF 3-fold molar excess

10 x T4 DNA ligase buffer 2 μ\

T4 DNA ligase 4 U

Water q.s. 20 μΐ

[00374] Blunt-end ligation was performed over-night on melting ice at room temperature or at

16 °C. We recommend inclusion of a negative control ligation reaction without ORF to monitor the self -ligation of plasmid. 10 μΐ of ligation mix was used for transformation of competent bacteria and transformant were selected over Kanamycin plate.

[00375] D) Screening of positive clones in correct orientation by colony PCR

[00376] The isolation of positive clones in correct orientation was carried out by colony PCR.

Usually we pick 8 colonies for screening. Single cell clones were individually picked using pipette tips. Individual tip was 1^st stabbed in 200 μΐ of Luria Broth and then rinsed several times in 75 μΐ of TE buffer pH 8.0. LB -containing tubes were incubated at 37 °C with constant shaking. TE-containing tubes were boiled for 5 min to lyse the bacteria. Tubes were briefly spined to pellet the debris. 2 μΐ of supernatant was taken for colony PCR. PCR was carried out using Xu-Fl and gene-specific reverse primer. PCR reaction was run over 1 % agarose gel and positive clones in correct orientation were identified (FIGS. 1 A-IC). 200 μΐ of LB from the identified clones were inoculated in large volume of LB for overnight culture for plasmid isolation. Constructs were verified by sequencing.

Box 4.

[00377] Tail PCR reaction condition

[00378] Initial denaturation 95 °C 2-3 min 1 cycle

Denaturation 98 °C 20 sec 25 cycles

Primer annealing 65 °C 15 sec

Extension 72 °C Variable (15-60 sec/kb) Final extension 72 °C 1-5 min 1 cycle

( * As per manufacturer instruction)

Box 5.

[00379] Quality control guideline for RNA synthesis

[00380] a. Yield is usually between 300-500 ng/ μΐ in 100 μΐ elution volume.

[00381] b. The ratio of A260/A280 should be more than 1.7 (usually between 1.8-2.0 or more).

[00382] c. The ratio of A260/A230 should be approaching 2.0. Value closer to 2.0 indicates purity.

[00383] d. We recommend the purified mRNA should be run on the gel for quality control and must be verified by one or other functional test (expression, western blot, Immuno-cytochemistry, other functional assay). [00384] e. We always include one reporter RNA (for example GFP) in every batch of RNA preparation to monitor whole process of RNA synthesis, which is tested at the end for GFP expression after transfection by FACS analysis.

References

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Claims

CLAIMS We claim:

2. The isolated nucleic acid sequence of claim 1, wherein the 5' UTR sequence comprises the sequence of SEQ ID NO: 20.

3. The isolated nucleic acid sequence of any one of the preceding claims, wherein the first blunt- ended restriction enzyme digestion site comprises the sequence of SEQ ID NO: 60.

4. The isolated nucleic acid sequence of any one of the preceding claims, wherein the second blunt-ended restriction enzyme digestion site comprises the sequence of SEQ ID NO: 61.

5. The isolated nucleic acid sequence of any one of the preceding claims, wherein the 3' UTR sequence comprises the sequence of SEQ ID NO: 21.

6. The isolated nucleic acid sequence of any one of the preceding claims, further comprising a vector backbone sequence.

7. The isolated nucleic acid sequence of any one of the preceding claims, wherein the 3' UTR followed by a second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence is not operably linked to a poly-A tail sequence.

8. The isolated nucleic acid sequence of any one of the preceding claims, further comprising a nucleic acid encoding an open reading frame (ORF) sequence between the first and the second blunt-ended restriction enzyme digestion sites.

9. The isolated nucleic acid sequence of claim 8, wherein the ORF sequence excludes the 5' adenine nucleotide prior to inserting it between the first and the second blunt-ended restriction sites.

11. A kit comprising the isolated nucleic acid of any one of claims 1-9, in a suitable container.

12. The kit of claim 11 , further comprising a first and a second blunt-ended restriction enzyme specific for the first and second blunt-ended restriction enzyme digestion sites respectively.

13. The kit of claims 11 or 12, further comprising a first universal primer comprising the forward universal primer sequence and a second universal primer comprising a poly-T sequence.

14. A kit comprising the plurality of nucleic acids of claim 10, in a suitable container.

15. The kit of any of claims 11-14, wherein the 5' UTR sequence comprises the sequence of SEQ ID NO: 20.

16. The kit of any of claims 11-15, wherein the first blunt-ended restriction enzyme digestion site comprises SEQ ID NO: 60.

17. The kit of any of claims 11-16, wherein the second blunt-ended restriction enzyme digestion site comprises SEQ ID NO: 61.

18. The kit of any of claims 11-17, wherein the 3' UTR sequence comprises the sequence of SEQ ID NO: 21.

19. The kit of any of claims 11-18, wherein the isolated nucleic acid further comprises a vector backbone sequence.

20. The kit of any of claims 11-19, wherein the 3' UTR followed by the second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence is not operably linked to a poly- A tail sequence.

21. A method of synthesizing a nucleic acid construct for transcribing a gene of interest in vitro, the method comprising the steps of:

a. amplifying an open reading frame (ORF) sequence of the gene of interest to form an ORF amplification product using a phosphorylated forward primer and a phosphorylated reverse primer, and omitting the 5 'adenosine of the ORF from the ORF amplification product; b. digesting a vector comprising, in the 5' to 3' direction: a first nucleic acid sequence comprising a first universal primer sequence, a promoter sequence operably linked to a 5' UTR sequence, a first blunt-ended restriction enzyme digestion site, a spacer sequence, a second blunt-ended restriction enzyme digestion site operably linked to a 3' UTR sequence, a second nucleic acid sequence comprising a sequence complementary to a second universal primer sequence, and a vector backbone sequence; with a first and a second blunt-ended restriction enzyme specific for the first and second blunt-ended restriction enzyme digestion sites respectively; c. dephosphorylating the digested vector with a phosphatase enzyme; and

22. The method of claim 21, further comprising one or more steps of screening for proper

orientation of the ORF sequence comprising the steps of:

23. The method of claims 21 or 22, further comprising a step of adding a poly-A tail to the ORF sequence by amplifying the nucleic acid construct with a forward primer comprising the first universal primer sequence and a reverse primer sequence comprising a poly-T sequence.