Stable RNA compositions with stem loop, and methods thereof

Abstract

The present invention provides, among other things, linear mRNA compositions comprising a poly A tail and a stem loop structure downstream to the poly A tail. Also provided herein are methods of producing mRNA by ligating a stem loop exonuclease blocker using a double-stranded RNA ligase.

Description

[0001] STABLE RNA COMPOSITIONS WITH STEM LOOP, AND METHODS THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] [1] This application claims priority to, and the benefit of U.S. Provisional Application Serial No. 63 / 561,046 filed on March 4, 2024, the contents of which is incorporated herein by reference in its entirety .

[0004] REFERENCE TO SEQUENCE LISTING

[0005] [2] The instant application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety7. The sequence listing file entitled BEM-030W01_SL.xml, was created on December 31, 2024, which is 16,384 bytes in size.

[0006] BACKGROUND

[0007] [3] Messenger RNA therapy is a promising approach to treat a variety' of diseases via administration of messenger RNA (mRNA) to a patient in need of the therapy. The administered mRNA produces a protein or peptide encoded by the mRNA within the patient’s body. mRNA is typically synthesized using in vitro transcription systems (IVT), resulting in product mRNA with a 5 '-cap and a 3 '-poly A tail.

[0008] [4] The 5’ cap and 3’ poly A tail play a role in mRNA expression and stability in vivo. The cap at the 5’ end prevents degradation and improves translation. The poly A tail at the 3’ end protects the mRNA from exonuclease degradation and improves integrity and stability of mRNA for mRNA therapeutics and can enhance their translation both in vivo and in vitro. The rate limiting step of most mRNA degradation is deadenylation. Methods to increase mRNA stability' include increasing poly A length, using circular RNA, chemically modified messenger-oligonucleotide conjugated RNAs (mocRNA). etc.

[0009] [5] Effective mRNA therapy requires delivery of mRNA to the patient and efficient production of the protein encoded by the mRNA within the patient’s body. There is a need for methods to stabilize mRNA that can be used for any linear polyadenylated mRNA.

[0010] SUMMARY OF THE INVENTION

[0011] [6] The present invention provides, among other things, linear mRNA tail. Without wishing to be bound by any particular theory, the stem loop structure leads to increased mRNA stability, increased resistance to degradation by exonucleases, increasing the half-life of mRNA resulting in enhanced protein production. Any mRNA with a poly A tail can be stabilized using a stem loop structure of the present invention (e g., base editors, transposases. antibodies, chimeric antigen receptors, among others), which comprises contiguous uracil nucleotides (e.g., 4-14), among other nucleotides.

[0012] [7] In some aspects, provided herein is an in vitro synthesized linear messenger RNA comprising a poly A tail and a stem loop structure downstream of the poly A tail.

[0013] [8] In some embodiments, the 5' end lacks a stem loop structure. In some embodiments, the 5' end lacks a hairpin loop structure. In some embodiments, the 5' end lacks a tetraloop structure.

[0014] [9] In some embodiments, the 5' end comprises a 5' triphosphate or 5’ cap structure. In some embodiments, the 5' end comprises a 5' triphosphate structure. In some embodiments, the 5' end comprises a 5' cap structure.

[0015]

[0010] In some embodiments, the stem loop structure comprises adenine, guanine, cytosine and / or uracil nucleotides. In some embodiments, the stem loop structure comprises adenine nucleotides. In some embodiments, the stem loop structure comprises guanine nucleotides. In some embodiments, the stem loop structure comprises cytosine nucleotides. In some embodiments, the stem loop structure comprises uracil nucleotides.

[0016]

[0011] In some embodiments, the stem loop structure comprises a plurality of uracil nucleotides at the 3’ end.

[0017]

[0012] In some embodiments, the plurality of uracil nucleotides form intramolecular base pairs with a portion of the poly A tail.

[0018]

[0013] In some embodiments, the stem loop structure comprises a spacer or loop wherein the adenine, guanine, cytosine and / or uracil nucleotides do not form intramolecular base pairs.

[0019]

[0014] In some embodiments, the stem region of the stem loop structure comprises contiguous uracil nucleotides.

[0020]

[0015] In some embodiments, the stem region of the stem loop structure comprises 4- 14 contiguous uracil nucleotides.

[0016] In some embodiments, the stem region of the stem loop structure comprises 4 contiguous uracil nucleotides.

[0021]

[0017] In some embodiments, the stem region of the stem loop structure comprises 6 contiguous uracil nucleotides.

[0022]

[0018] In some embodiments, the stem region of the stem loop structure comprises 8 contiguous uracil nucleotides.

[0023]

[0019] In some embodiments, the stem region of the stem loop structure comprises 10 contiguous uracil nucleotides.

[0024]

[0020] In some embodiments, the stem region of the stem loop structure comprises 12 contiguous uracil nucleotides.

[0025]

[0021] In some embodiments, the stem region of the stem loop structure comprises 14 contiguous uracil nucleotides.

[0026]

[0022] In some embodiments, the stem region of the stem loop structure comprises greater than 14 contiguous uracil nucleotides.

[0027]

[0023] In some embodiments, one of more contiguous RNA nucleotides is a modified nucleotide.

[0028]

[0024] In some embodiments, the modified nucleotide is a 2'-O-methyl, phosphorothioate and / or N1 -methylpseudouridine. In some embodiments, the modified nucleotide is a 2’-O-methyl. In some embodiments, the modified nucleotide is a phosphorothioate. In some embodiments, the modified nucleotide is aNl- methylpseudouridine.

[0029]

[0025] In some embodiments, the poly A tail is at least 30 nt long.

[0030]

[0026] In some embodiments, the poly A tail is at least 100 nt long.

[0031]

[0027] In some embodiments, provided herein is a composition comprising the mRNA described herein.

[0032]

[0028] In some aspects, provided herein is a composition comprising a population of mRNA molecules, wherein at least 80% of mRNA molecules comprise a poly A tail and a stem loop structure downstream of the poly A tail.

[0033]

[0029] In some aspects, provided herein is a method of producing mRNA, the method exonuclease blocker downstream of the poly A tail of the mRNA, wherein the ligation is carried out by a double-stranded RNA ligase.

[0034]

[0030] In some embodiments, the 5' end does not comprise a stem loop exonuclease blocker.

[0035]

[0031] In some aspects, provided herein is a method of producing protein from mRNA, the method comprising the steps of: (a) providing mRNA comprising a stem loop exonuclease blocker downstream of the poly A tail of mRNA, and (b) translating the mRNA from step (a) into protein.

[0036]

[0032] In some embodiments, the double-stranded RNA ligase is any ligase that is able to hgate double stranded RNA. In some embodiments, the double-stranded RNA ligase is a T4 RNA ligase 2. In some embodiments, the double-stranded RNA ligase is a T4 RNA ligase II. In some embodiments, the double-stranded RNA ligase is a T4 dsRNA ligase.

[0037]

[0033] In some embodiments, the stem loop exonuclease blocker is a hairpin loop or a tetraloop. In some embodiments, the stem loop exonuclease blocker is a hairpin loop. In some embodiments, the stem loop exonuclease blocker is a tetraloop.

[0038]

[0034] In some embodiments, the 5' end of mRNA comprises a 5’ phosphate.

[0039]

[0035] In some embodiments, the ligation is carried out at 37°C.

[0040]

[0036] In some embodiments, the ligation is carried out for between 3h to overnight.

[0041]

[0037] In some embodiments, the ligation is carried out at 20°C.

[0042]

[0038] In some embodiments, the ligation is carried out for between 6h to overnight.

[0043]

[0039] In some embodiments, the ligation is earned out for 6h.

[0044]

[0040] In this application, the use of "or" means "and / or" unless stated otherwise. As used in this disclosure, the term “comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps. As used in this application, the terms “about” and “approximately” are used as equivalents. Both terms are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.

[0045]

[0041] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

[0046] BRIEF DESCRIPTION OF THE DRAWING

[0047]

[0042] The drawings are for illustration purposes only not for limitation.

[0048]

[0043] FIG. 1 is a schematic of a messenger RNA comprising an exemplary stem loop, a tetraloop ligated dow nstream of the poly A tail by a T4 RNA ligase 2 (top), and a schematic of a linear RNA ligated downstream of a poly A tail by a T4 RNA ligase 1 that does not form a stem loop structure (bottom).

[0049]

[0044] FIG. 2A depicts mass spectrometric results of a control emGFP engineered with a PEST sequence. FIG. 2B depicts mass spectra of an emGFP engineered with a PEST sequence having 6 uridines showing successful blocker ligation (emGFP-PEST-6U). FIG. 2C depicts mass spectra of an emGFP engineered with a PEST sequence having 8 uridines showing successful blocker ligation (emGFP-PEST-8U). FIG. 2D depicts deconvoluted mass spectra from emGFP-PEST-H20 control. FIG. 2E depicts deconvoluted mass spectra from an emGFP-PEST-6U sample. FIG. 2F depicts deconvoluted mass spectra from an emGFP- PEST-8U sample.

[0050]

[0045] FIG. 3A depicts immunofluorescence microscopy images of emGFP-PEST expression over time (25 ng). FIG. 3B depicts immunofluorescence microscopy images of emGFP-PEST expression over time (50 ng). FIG. 3C depicts immunofluorescence microscopy images of emGFP-PEST expression over time (100 ng).

[0051]

[0046] FIG. 4A shows mean emGFP expression over time (24, 48 and 72h) when seeded at a concentration of 25 ng / well with emGFP-PEST-H2O (control), emGFP-PEST-6U or emGFP-PEST-8U. FIG. 4B shows fold change of emGFP expression in samples ligated with 6U or 8U relative to control.

[0052]

[0047] FIG. 5A shows mean emGFP expression over time (24, 48 and 72h) when seeded at a concentration of 50 ng / well with emGFP-PEST-H2O (control), emGFP-PEST-6U or emGFP-PEST-8U. FIG. 5B shows fold change of emGFP expression in samples ligated with 6U or 8U relative to control.

[0048] FIG. 6A shows mean emGFP expression over time (24. 48 and 72h) when seeded at a concentration of 100 ng / well with emGFP-PEST-H2O (control), emGFP-PEST- 6U or emGFP-PEST-8U. FIG. 6B shows fold change of emGFP expression in samples ligated with 6U or 8U relative to control.

[0053]

[0049] FIG. 7A and FIG. 7B shows that GFP expression was improved over time by Conjugating 6U or 8U Compared to H2O Ligation. FIG. 7A shows GFP expression over time at 25 ng / well. FIG. 7B shows GFP expression over time at 50 ng / well.

[0054] DEFINITIONS

[0055]

[0050] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0056]

[0051] Approximately or about'. As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherw ise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0057]

[0052] Biologically active'. As used herein, the term “biologically active” refers to a characteristic of any agent that has acti vity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.

[0058]

[0053] Delivery. As used herein, the term “delivery” encompasses both local and systemic deliver}'. For example, deliver}' of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and taken up by other tissues (also referred to as “systemic distribution’7or “systemic delivery). In some embodiments, delivery is pulmonary delivery, e.g., comprising nebulization.

[0059]

[0054] Expression'. As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody) and / or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody). In this application, the terms “expression” and “production.” and their grammatical equivalents, are used interchangeably.

[0060]

[0055] Full-length mRNA: As used herein, “full-length mRNA” is as characterized when using a specific assay, e.g., gel electrophoresis or detection using UV and UV absorption spectroscopy with separation by capillary electrophoresis. The length of an mRNA molecule that encodes a full-length polypeptide and as obtained following any of the purification methods described herein is at least 50% of the length of a full-length mRNA molecule that is transcribed from the target DNA, e.g., at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.01%, 99.05%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the length of a full-length mRNA molecule that is transcribed from the target DNA and prior to purification according to any method described herein.

[0061]

[0056] Functional'. As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0062]

[0057] Contiguous nucleotides or homopolymer or homopolymeric nucleotides'. As used herein, “contiguous nucleotides”, “homopolymer” or homopolymeric nucleotides,” and grammatical equivalents thereof, refers to a sequence of consecutive identical bases. In some embodiments, the nucleotides comprising the homopolymeric nucleotides are selected from A, U, G, or C. In some embodiments, the term “homopolymer” or “homopolymeric nucleotides,” refers to a sequence of substantially identical bases. For example, in some embodiments, the term includes a consecutive series of nucleotides that has one or more nonidentical nucleotides.

[0063]

[0058] Improve, increase, or reduce'. As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[0064]

[0059] In Vitro'. As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0065]

[0060] In Vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cellbased systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0066]

[0061] Isolated: As used herein, the term “isolated” refers to a substance and / or entity that has been (1) separated from at least some of the components with which it w as associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%. or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%. about 90%. about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and / or entities should not include excipients (e.g., buffer, solvent, water. etc.).

[0067]

[0062] messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g.. in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5’ to 3’ direction unless otherwise _ j 163] mRNA Integrity: As used herein, the term “mRNA integrity” generally refers to the quality of mRNA. In some embodiments, mRNA integrity refers to the percentage of mRNA that is not degraded after a purification process (e.g., a method described herein). mRNA integrity may be determined using methods particularly described herein, such as TAE Agarose gel electrophoresis or by SDS-PAGE with silver staining, or by methods well known in the art, for example, by RNA agarose gel electrophoresis (e.g., Ausubel et al., John Wiley & Sons, Inc., 1997, Current Protocols in Molecular Biology).

[0068]

[0064] Nucleic acid: As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, z.e., analogs having other than a phosphodiester backbone. For example, the so- called “peptide nucleic acids,” which are know n in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences that encode proteins and / or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g, in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5 ' to 3’ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g, adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine); nucleoside analogs (e.g, 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine. 2-aminoadenosine, C5- C5-methylcytidine, 2-aminoadenosine, 7-deazaad enosine, 7-deazaguanosine. 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2 -thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2’-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5’-N-phosphoramidite linkages). In some embodiments, the present invention is specifically directed to “unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery. In some embodiments, the nucleotides T and U are used interchangeably in sequence descriptions.

[0069]

[0065] Substantially. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary' skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0070] DETAILED DESCRIPTION

[0071]

[0066] The present invention is based, in part, on providing an in vitro synthesized linear messenger RNA comprising a poly A tail and a stem loop structure downstream of the poly A tail. Without wishing to be bound by any particular theory7, it is contemplated that the stem loop structure at the 3' end provides a linear mRNA with greater stability, having a longer half-life, leading to increased mRNA translation and increased protein product over time.

[0072]

[0067] Various aspects of the invention are further described below.

[0073]

[0068] In some aspects, provided herein is an in vitro synthesized linear messenger RNA comprising a poly A tail and a stem loop structure downstream of the poly A tail. As described in greater detail in the specification below, mRNAs may be synthesized according to any of a variety of known methods, including in vitro transcription (IVT). In some embodiments, the mRNA is capped. In some embodiments, the 5' cap is added post- embodiments, the poly A tail is added either post-transcriptionally or co-transcnptionally. In some embodiments, the poly A tail confers stability to mRNA therapeutic product. In some embodiments, the mRNA encodes an expressed protein (e.g., a base editor, antibody, CAR, transposase, etc.). In some embodiments, the mRNA encodes a base editor. In some embodiments, the mRNA encodes an antibody. In some embodiments, the mRNA encodes a chimeric antigen receptor. In some embodiments, the mRNA encodes a transposase.

[0074]

[0069] In some embodiments, the 5' end lacks a stem loop structure.

[0075]

[0070] In some embodiments, the 5' end comprises a 5' triphosphate or 5?cap structure. In some embodiments, the 5' end comprises a 5' triphosphate. In some embodiments, the 5' end comprises a 5’ cap structure.

[0076] Stem loop structure

[0077]

[0071] A stem loop structure is an intramolecular base pairing in single stranded nucleic acid e.g., DNA or RNA, if sequences of two regions of the same strand are complementary to each other and a double-stranded stem forms which has an unpaired loop at ends. In some embodiments, this structure is also known as a hairpin, and resembles a loop or a U-shape.

[0078] RNA Hairpins

[0079]

[0072] In some embodiments, the stem loop structure is a hairpin. Hairpins are a common type of secondary structure in RNA molecules. In RNA. the secondary structure is the basic shape that the sequence of A, C, U, and G nucleotides form after they are linked in series, such a folding or curling of the nucleic acid strand. mRNA hairpins can be formed when two complementary sequences in a single mRNA molecule meet and bind together, after a folding or wrinkling of the molecule. Hairpin loops can also form in DNA molecules, but are most commonly observed in mRNA.

[0080]

[0073] In some embodiments, a hairpin loop is the termination sequence for transcription, for example, in some prokaryotes. Once a polymerase meets this loop, it falls of and transcription ends. Another more general example is tRNA, a central player in protein synthesis, which is partially formed by hairpin loops. The tRNA molecule actually contains three hairpin loops that form the shape of a three-leafed clover. One of these hairpin loops contains a sequence called the anticodon, which recognizes and decodes the mRNA molecule three nucleotides (one codon) at a time during translation. This clover-leaf structure supports the eventual connection between every codon, anti-codon and amino acid.

[0081] RNA Tetraloops

[0082]

[0074] In some embodiments, the stem loop structure is a tetraloop or miniloop. An RNA tetraloop structure is formed by a Watson-Crick base-paired stem and four loop nucleotides. The first and the fourth nucleotides form a base pair in most of the tetraloops, leaving two unpaired nucleotides in the loop. A sharp turn in the backbone is stabilized by ribose-base, base-phosphate hydrogen bonds and base stacking interactions. Tetraloops help in the folding of RNA by initiating the process. In some embodiments, they provide sites for RNA tertiary contacts and for protein binding, facilitating the assembly of ribonucleoprotein particles. Sequence-specific and structure-specific interactions are involved in the recognition of tetraloops by proteins and RNAs.

[0083]

[0075] RNA tetraloops form compact and stable structures, involving unusual basebase, base-sugar, base-phosphate and sugar-phosphate interactions. In addition, base stacking extends to the loop region. UUCG, GCAA and CUUG tetraloops, for example, contain U-G, G-A and C-G base pairs, respectively, leaving two highly constrained nucleotides in the loop. In some embodiments, the tetraloops comprise one or more of UNCG, GNRA, CUUG, UGAA and CAAC nucleotides.

[0084]

[0076] In some embodiments, the stem loop structure comprises adenine, guanine, cytosine and / or uracil nucleotides. In some embodiments, the stem loop structure comprises adenine nucleotides. In some embodiments, the stem loop structure comprises guanine nucleotides. In some embodiments, the stem loop structure comprises cytosine nucleotides. In some embodiments, the stem loop structure comprises uracil nucleotides. In some embodiments, the stem loop structure comprises adenine, guanine, cytosine and uracil nucleotides.

[0085]

[0077] In some embodiments, the stem loop structure comprises a plurality of uracil nucleotides at the 3’ end.

[0086]

[0078] In some embodiments, the lurality of uracil nucleotides form intramolecular base pairs with a portion of the poly A tail.

[0079] In some embodiments, the stem loop structure comprises a spacer or loop wherein the adenine, guanine, cytosine and / or uracil nucleotides do not form intramolecular base pairs.

[0087]

[0080] In some embodiments, the stem region of the stem loop structure comprises contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 4-14 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 4 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 5 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 6 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 7 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 8 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 9 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 10 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 11 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 12 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 13 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises 14 contiguous uracil nucleotides. In some embodiments, the stem region of the stem loop structure comprises greater than 14 contiguous uracil nucleotides.

[0088] Modified RNA nucleotides

[0089]

[0081] In some embodiments, one of more contiguous RNA nucleotides is a modified nucleotide. In some embodiments, the modified nucleotide is a 2’-O-methyl, phosphorothioate and / or N1 -methylpseudouridine. In some embodiments, the modified nucleotide is a 2'-O-methyl nucleotide. In some embodiments, the modified nucleotide is a phosphorothioate nucleotide. In some embodiments, the modified nucleotide is a Nl- methylpseudouridine nucleotide.

[0090]

[0082] The messenger RNA of any one of the preceding claims, wherein the poly A tail is at least 30 nt long. In some embodiments, the poly A tail is at least 40 nt long. In some embodiments, the poly A tail is at least 50 nt long. In some embodiments, the poly A tail is at

[0091] _ _ _ _ embodiments, the poly A tail is at least 80 nt long. In some embodiments, the poly A tail is at least 90 nt long. In some embodiments, the poly A tail is at least 100 nt long. In some embodiments, the poly A tail is between 40 nt and 100 nt long.

[0092]

[0083] In some aspects, provided herein is a composition comprising a population of mRNA molecules, wherein at least 80% of mRNA molecules comprise a poly A tail and a stem loop structure downstream of the poly A tail. In some embodiments, at least 85% of mRNA molecules comprise a poly A tail and a stem loop structure dow nstream of the poly A tail. In some embodiments, at least 90% of mRNA molecules comprise a poly A tail and a stem loop structure downstream of the poly A tail. In some embodiments, at least 95% of mRNA molecules comprise a poly A tail and a stem loop structure downstream of the poly A tail. In some embodiments, at least 96% of mRNA molecules comprise a poly A tail and a stem loop structure downstream of the poly A tail. In some embodiments, at least 97% of mRNA molecules comprise a poly A tail and a stem loop structure downstream of the poly A tail. In some embodiments, at least 98% of mRNA molecules comprise a poly A tail and a stem loop structure downstream of the poly A tail. In some embodiments, at least 99% of mRNA molecules comprise a poly A tail and a stem loop structure dow nstream of the poly A tail. In some embodiments, 100% of mRNA molecules comprise a poly A tail and a stem loop structure dow nstream of the poly A tail.

[0093] Synthesis of mRNA

[0094]

[0084] mRNAs may be synthesized according to any of a variety of known methods. For example, mRNAs may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions will vary' according to the specific application.

[0095]

[0085] In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.

[0096] Synthesis of mRNA using T3 RNA Polymerase In some embodiments. mRNA is produced using T3 RNA Polymerase. T3 RNA Polymerase is a DNA-dependent RNA polymerase from the T3 bacteriophage that catalyzes the formation of RNA from DNA in the 5'— > 3' direction on either single-stranded DNA or double-stranded DNA, and is able to incorporate modified nucleotide. T3 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T3 promoter. T3 binds to a consensus promoter sequence of 5’-AATTAACCCTCACTAAAGGGAGA-3’.

[0097] Synthesis of mRNA using T7 RNA Polymerase

[0098]

[0086] In some embodiments, mRNA is produced using T7 RNA Polymerase. T7 RNA Polymerase is a DNA-dependent RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'— > 3' direction. T7 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T7 promoter. T7 binds to a consensus promoter sequence of 5'-TAATACGACTCACTATAGGGAGA-3'. The T7 polymerase also requires a double stranded DNA template and Mg2+ion as cofactor for the synthesis of RNA. It has a very low error rate.

[0099] Synthesis of mRNA using SP6 RNA Polymerase

[0100]

[0087] In some embodiments, mRNA is produced using SP6 RNA Polymerase. SP6 RNA Polymerase is a DNA-dependent RNA polymerase with high sequence specificity for SP6 promoter sequences. The SP6 polymerase catalyzes the 5'— >3' in vitro synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter; it incorporates native ribonucleotides and / or modified ribonucleotides and / or labeled ribonucleotides into the polymerized transcript. SP6 binds to a consensus promoter sequence of 5’-ATTTACGACACACTATAGAAGAA-3’. Examples of such labeled ribonucleotides include biotin-, fluorescein-, digoxigenin-, aminoallyl-, and isotope-labeled nucleotides.

[0101] DNA Template

[0102]

[0088] Typically, a DNA template is either entirely double-stranded or mostly singlestranded with a suitable promoter sequence (e g. T3, T7 or SP6 promoter).

[0103]

[0089] Linearized plasmid DNA (linearized via one or more restriction enzymes), linearized genomic DNA fragments (via restriction enzy me and / or physical means), PCR products, and / or synthetic DNA oligonucleotides can be used as templates for in vitro transcription, provided that they contain a double-stranded promoter upstream (and in the 190] In some embodiments, the linearized DNA template has a blunt-end.

[0104]

[0091] In some embodiments, the DNA sequence to be transcribed may be optimized to facilitate more efficient transcription and / or translation. For example, the DNA sequence may be optimized regarding cis-regulatory elements (e.g., TATA box, termination signals, and protein binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements relevant to transcription; the DNA sequence may be optimized regarding cryptic splice sites, mRNA secondary structure, stable free energy of mRNA, repetitive sequences, RNA instability motif, and / or other elements relevant to mRNA processing and stability; the DNA sequence may be optimized regarding codon usage bias, codon adaptability, internal chi sites, ribosomal binding sites (e.g., IRES), premature poly A sites, Shine-Dalgamo (SD) sequences, and / or other elements relevant to translation; and / or the DNA sequence may be optimized regarding codon context, codon-anticodon interaction, translational pause sites, and / or other elements relevant to protein folding. Optimization methods known in the art may be used in the present invention, e.g., GeneOptimizer by ThermoFisher and OptimumGene™, which are described in US 20110081708, the contents of which are incorporated herein by reference in its entirety.

[0105]

[0092] In some embodiments, the DNA template includes a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA' s stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0106]

[0093] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.

[0107]

[0094] Exemplary73' and / or 5' UTR sequences can be derived from mRNA molecules which are stable (e g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1 ) gene, or a fragment thereof to improve the nuclease resistance and / or improve the half-life of the polynucleotide. Also fragment thereof to the 3' end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides’ resistance to in vivo nuclease digestion.

[0108] Large-scale mRNA Synthesis

[0109]

[0095] In some embodiments, the mRNA with poly A tail can be synthesized in a large-scale. In some embodiments, mRNA is synthesized in at least 100 mg. 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more at a single batch. As used herein, the term “batch’’ refers to a quantity or amount of mRNA synthesized at one time, e.g. produced according to a single manufacturing setting. A batch may refer to an amount of mRNA synthesized in one reaction that occurs via a single aliquot of enzy me and / or a single aliquot of DNA template for continuous synthesis under one set of conditions. mRNA synthesized at a single batch would not include mRNA synthesized at different times that are combined to achieve the desired amount.

[0110]

[0096] According to the present invention, 1-100 mg of RNA polymerase is typically used per gram (g) of mRNA produced. In some embodiments, about 1-90 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 10-100 mg, 10-80 mg, 10-60 mg, 10-50 mg of RNA polymerase is used per gram of mRNA produced. In some embodiments, about 5-20 mg of RNA polymerase is used to produce about 1 gram of mRNA. In some embodiments, about 0.5 to 2 grams of RNA polymerase is used to produce about 100 grams of mRNA. In some embodiments, about 5 to 20 grams of RNA polymerase is used to about 1 kilogram of mRNA. In some embodiments, at least 5 mg of RNA polymerase is used to produce at least

[0111] 1 gram of mRNA. In some embodiments, at least 500 mg of RNA polymerase is used to produce at least 100 grams of mRNA. In some embodiments, at least 5 grams of RNA polymerase is used to produce at least 1 kilogram of mRNA. In some embodiments, about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg of plasmid DNA is used per gram of mRNA produced. In some embodiments, about 10-30 mg of plasmid DNA is used to produce about 1 gram of mRNA. In some embodiments, about 1 to 3 grams of plasmid DNA is used to produce about 100 grams of mRNA. In some embodiments, about 1 0 to 30 oranK of nlas id DNA is nsnd tn about 1 kilnaram of mRNA Tn snmn embodiments, at least 10 mg of plasmid DNA is used to produce at least 1 gram of mRNA. In some embodiments, at least 1 gram of plasmid DNA is used to produce at least 100 grams of mRNA. In some embodiments, at least 10 grams of plasmid DNA is used to produce at least 1 kilogram of mRNA.

[0112]

[0097] In some embodiments, the concentration of the RNA polymerase in the reaction mixture may be from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM. 1 to 30 nM, 1 to 20 nM. or about 1 to 10 nM. In certain embodiments, the concentration of the RNA polymerase is from about 10 to 50 nM, 20 to 50 nM, or 30 to 50 nM. A concentration of 100 to 10000 Units / ml of the RNA polymerase may be used, as examples, concentrations of 100 to 9000 Units / ml, 100 to 8000 Units / ml, 100 to 7000 Units / ml. 100 to 6000 Units / ml, 100 to 5000 Units / ml, 100 to 1000 Units / ml. 200 to 2000 Units / ml, 500 to 1000 Units / ml, 500 to 2000 Units / ml, 500 to 3000 Units / ml, 500 to 4000 Units / ml, 500 to 5000 Units / ml, 500 to 6000 Units / ml, 1000 to 7500 Units / ml, and 2500 to 5000 Units / ml may be used.

[0113]

[0098] The concentration of each ribonucleotide (e g., ATP, UTP, GTP, and CTP) in a reaction mixture is between about 0. 1 mM and about 10 mM, e.g., between about 1 mM and about 10 mM, between about 2 mM and about 10 mM, between about 3 mM and about

[0114] 1 mM, betw een about 1 mM and about 8 mM, between about 1 mM and about 6 mM, between about 3 mM and about 10 mM, betw een about 3 mM and about 8 mM, betw een about 3 mM and about 6 mM, between about 4 mM and about 5 mM. In some embodiments, each ribonucleotide is at about 5 mM in a reaction mixture. In some embodiments, the total concentration of rNTPs (for example, ATP, GTP, CTP and UTPs combined) used in the reaction range between 1 mM and 40 mM. In some embodiments, the total concentration of rNTPs (for example, ATP, GTP, CTP and UTPs combined) used in the reaction range between 1 mM and 30 mM, or between 1 mM and 28 mM, or between 1 mM to 25 mM, or between 1 mM and 20 mM. In some embodiments, the total rNTPs concentration is less than 30 mM. In some embodiments, the total rNTPs concentration is less than 25 mM. In some embodiments, the total rNTPs concentration is less than 20 mM. In some embodiments, the total rNTPs concentration is less than 15 mM. In some embodiments, the total rNTPs concentration is less than 10 mM.

[0115]

[0099] The RNA polymerase reaction buffer typically includes a salt / buffering agent, e.g., Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, 1100] The pH of the reaction mixture may be between about 6 to 8.5, from 6.5 to 8.0, from 7.0 to 7.5, and in some embodiments, the pH is 7.5.

[0116]

[0101] Linear or linearized DNA template (e.g., as described above and in an amount / concentration sufficient to provide a desired amount of RNA), the RNA polymerase reaction buffer, and RNA polymerase are combined to form the reaction mixture. The reaction mixture is incubated at between about 37 °C and about 42 °C for thirty’ minutes to six hours, e.g., about sixty to about ninety minutes.

[0117]

[0102] In some embodiments, about 5 mM NTPs. about 0.05 mg / mL RNA polymerase, and about 0.1 mg / ml DNA template in a suitable RNA polymerase reaction buffer (final reaction mixture pH of about 7.5) is incubated at about 37 °C to about 42 °C for sixty to ninety7minutes.

[0118]

[0103] In some embodiments, a reaction mixture contains linearized double stranded DNA template with an RNA polymerase-specific promoter. RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTPs. 10 mM DTT and a reaction buffer (when at lOx is 800 mM HEPES, 20 mM spermidine, 250 mM MgCb, pH 7.7) and quantity sufficient (QS) to a desired reaction volume with RNase-free water; this reaction mixture is then incubated at 37 °C for 60 minutes. The polymerase reaction is then quenched by addition of DNase I and a DNase I buffer (when at lOx is 100 mM Tris-HCl, 5 mM MgCb and 25 mM CaCb, pH 7.6) to facilitate digestion of the double-stranded DNA template in preparation for purification. This embodiment has been shown to be sufficient to produce 100 grams of mRNA.

[0119]

[0104] In some embodiments, a reaction mixture includes NTPs at a concentration ranging from 1 - 10 mM, DNA template at a concentration ranging from 0.01 - 0.5 mg / ml, and RNA polymerase at a concentration ranging from 0.01 - 0. 1 mg / ml, e.g., the reaction mixture comprises NTPs at a concentration of 5 mM, the DNA template at a concentration of 0. 1 mg / ml, and the RNA polymerase at a concentration of 0.05 mg / ml.

[0120] Nucleotides

[0121]

[0105] Various naturally-occurring or modified nucleosides may be used to product mRNA according to the present invention. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine. 2-aminoadenosine, C5- C5-methylcytidine, 2-aminoadenosine, 7-deazaad enosine, 7-deazaguanosine. 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-l-methyl- pseudouridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2’- fluororibose. ribose, 2’-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g, phosphorothioates and 5'-A-phosphoramidite linkages).

[0122]

[0106] In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl- cytidine (“5mC”), pseudouridine (“q / U”), and / or 2-thio-uridine (“2sU”). See, e.g., U.S. Patent No. 8.278,036 or WO2011012316 for a discussion of such residues and their incorporation into mRNA. The mRNA may be RNA, which is defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5 -methylcytidine. Teachings for the use of RNA are disclosed US Patent Publication US20120195936 and international publication WO2011012316, both of which are hereby incorporated by reference in their entirety. The presence of nonstandard nucleotide residues may render an mRNA more stable and / or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In further embodiments, the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine. 5-bromouracil. 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6- aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications. Some embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g, one or more of a 2'-O-alkyl modification, a locked nucleic acid (LNA)). In some embodiments, the RNAs may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNA). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modification may include, but are not limited to a 2’-deoxy-2'-fluoro modification, a 2'-O-methyl modification, a 2'-O- methoxyethyl modification and a 2'-deoxy modification. In some embodiments, any of these modifications may be present in 0-100% of the nucleotides — for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination.

[0123] Post-synthesis processing

[0107] Typically, a 5' cap and / or a 3' tail may be added after the synthesis. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” sen es to protect mRNA from exonuclease degradation.

[0124] 5’ Cap

[0125]

[0108] In some embodiments, a 5' cap is added co-transcriptionally as follows: a cap analog (e.g., CleanCap AG) is added in the in vitro transcription (IVT) reaction so that the mRNA product generated has a cap incorporated at the 5' end of mRNA as it is synthesized during IVT making it a highly efficient process with greater than 95% efficiency.

[0126]

[0109] In some embodiments, an anti-reverse cap analog (ARCA) is used to synthesize cap-0 mRNA first during the IVT reaction, followed by an enzymatic reaction with an enzyme, mRNA cap 2‘-O-methyltransferase to produce cap-1 mRNAs. The capping efficiency of ARCA is about 50-80%.

[0127] [HO] In some embodiments, no cap analog is added in the IVT reaction.

[0128] [Hl] In some embodiments, a 5’ cap is enzymatically added as follows: first, an

[0129] RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’5’5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Additional cap structures are described in published US Application No. US 2016 / 0032356 and U.S. Provisional Application 62 / 464,327, filed February 27, 2017, which are incorporated herein by reference.

[0130] 3 ’-Poly A Tail

[0131]

[0112] The presence of a “tail” at 3’ end serves to protect the mRNA from exonuclease degradation. The 3' tail may be added before, after or at the same time of adding the 5' Cap.

[0132]

[0113] In some embodiments, the poly A tail is added co-transcriptionally. In some embodiments, the poly A tail is added post-transcriptionally. In some embodiments, the stem loop is downstream of a poly A tail.

[0133]

[0114] In some embodiments, the poly A tail is 25-5,000 nucleotides in length. In poly A tail is 30 nucleotides in length. In some embodiments, the poly A tail is 50 nucleotides in length. In some embodiments, the poly A tail is 75 nucleotides in length. In some embodiments, the poly A tail is 100 nucleotides in length. In some embodiments, the poly A tail is 150 nucleotides in length. In some embodiments, the poly A tail is 200 nucleotides in length. In some embodiments, the poly A tail is 250 nucleotides in length. In some embodiments, the poly A tail is 300 nucleotides in length. In some embodiments, the poly A tail is 350 nucleotides in length. In some embodiments, the poly A tail is 400 nucleotides in length. In some embodiments, the poly A tail is 450 nucleotides in length. In some embodiments, the poly A tail is 500 nucleotides in length. In some embodiments, the poly A tail is 550 nucleotides in length. In some embodiments, the poly A tail is 600 nucleotides in length. In some embodiments, the poly A tail is 650 nucleotides in length. In some embodiments, the poly A tail is 700 nucleotides in length. In some embodiments, the poly A tail is 750 nucleotides in length. In some embodiments, the poly A tail is 800 nucleotides in length. In some embodiments, the poly A tail is 850 nucleotides in length. In some embodiments, the poly A tail is 900 nucleotides in length. In some embodiments, the poly A tail is 950 nucleotides in length. In some embodiments, the poly A tail is 1000 nucleotides in length.

[0134]

[0115] In some embodiments, the poly A tail is 1500 nucleotides in length. In some embodiments, the poly A tail is 2000 nucleotides in length. In some embodiments, the poly A tail is 2500 nucleotides in length. In some embodiments, the poly A tail is 3000 nucleotides in length. In some embodiments, the poly A tail is 3500 nucleotides in length. In some embodiments, the poly A tail is 4000 nucleotides in length. In some embodiments, the poly A tail is 4500 nucleotides in length.

[0135]

[0116] Typically, a tail structure includes a poly A tail (A, adenosine). In some embodiments, a poly A tail on the 3' terminus of mRNA includes at least 25 adenosine nucleotides, at least 50 adenosine nucleotides, at least 150 adenosine nucleotides, at least 200 adenosine nucleotides, at least 250 adenosine nucleotides, at least 300 adenosine nucleotides, at least 350 adenosine nucleotides, at least 400 adenosine nucleotides, at least 450 adenosine nucleotides, at least 500 adenosine nucleotides, at least 550 adenosine nucleotides, at least 600 adenosine nucleotides, at least 650 adenosine nucleotides, at least 700 adenosine nucleotides, at least 750 adenosine nucleotides, at least 800 adenosine nucleotides, at least 850 adenosine nucleotides, at least 900 adenosine nucleotides, at least 950 adenosine least 3 kb adenosine nucleotides, at least 4 kb adenosine nucleotides, at least 5 kb adenosine nucleotides, respectively. In some embodiments, a poly A tail may be about 10 to 800 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 300 adenosine nucleotides, about 10 to 400 adenosine nucleotides, about 10 to 500 adenosine nucleotides, about 10 to 550 adenosine nucleotides, about 10 to 600 adenosine nucleotides, about 50 to 600 adenosine nucleotides, about 100 to 600 adenosine nucleotides, about 150 to 600 adenosine nucleotides, about 200 to 600 adenosine nucleotides, about 250 to 600 adenosine nucleotides, about 300 to 600 adenosine nucleotides, about 350 to 600 adenosine nucleotides, about 400 to 600 adenosine nucleotides, about 450 to 600 adenosine nucleotides, about 500 to 600 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides) respectively.

[0136]

[0117] As described herein, the addition of the 5’ cap and / or the 3’ tail facilitates the detection of abortive transcripts generated during in vitro synthesis because without capping and / or tailing, the size of those prematurely aborted mRNA transcripts can be too small to be detected. Thus, in some embodiments, the 5’ cap and / or the 3’ tail are added to the synthesized mRNA before the mRNA is tested for purity (e.g., the level of abortive transcripts present in the mRNA). In some embodiments, the 5’ cap and / or the 3’ tail are added to the synthesized mRNA before the mRNA is purified. In other embodiments, the 5‘ cap and / or the 3’ tail are added to the synthesized mRNA after the mRNA is purified.

[0137]

[0118] In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing.

[0138]

[0119] In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by centrifugation. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by filtration. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by Tangential Flow Filtration (TFF). In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing by chromatography.

[0120] In some embodiments, the nucleotides comprising the contiguous or homopolymeric nucleotides are selected from A, U, G, or C. In some embodiments, the nucleotides comprising the contiguous nucleotides are adenine. In some embodiments, the nucleotides comprising the contiguous nucleotides are uracil. In some embodiments, the nucleotides comprising the contiguous nucleotides are guanine. In some embodiments, the nucleotides comprising the contiguous nucleotides are cytosine.

[0139]

[0121] The contiguous nucleotide length measured according to method of this invention ranges from about 25 nucleotides to greater than about 5000 nucleotides. In some embodiments, the contiguous nucleotide length is about 25 nucleotides, about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides or greater than about 200 nucleotides. In some embodiments, the contiguous nucleotide length is about 25 nucleotides, about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides or greater than about 200 nucleotides.

[0140]

[0122] In some embodiments, the contiguous nucleotide length is between 50 nucleotides and 5,000 nucleotides. In some embodiments, the contiguous nucleotide length is 50 nucleotides. In some embodiments, the contiguous nucleotide length is 100 nucleotides. In some embodiments, the contiguous nucleotide length is 150 nucleotides. In some embodiments, the contiguous nucleotide length is 200 nucleotides. In some embodiments, the contiguous nucleotide length is 250 nucleotides. In some embodiments, the contiguous nucleotide length is 300 nucleotides. In some embodiments, the contiguous nucleotide length is 350 nucleotides. In some embodiments, the contiguous nucleotide length is 400 nucleotides. In some embodiments, the contiguous nucleotide length is 450 nucleotides. In some embodiments, the contiguous nucleotide length is 500 nucleotides. In some embodiments, the contiguous nucleotide length is 550 nucleotides. In some embodiments, the contiguous nucleotide length is 600 nucleotides. In some embodiments, the contiguous nucleotide length is 650 nucleotides. In some embodiments, the contiguous nucleotide length is 700 nucleotides. In some embodiments, the contiguous nucleotide length is 750 nucleotides. In some embodiments, the contiguous nucleotide length is 800 nucleotides. In some embodiments, the contiguous nucleotide length is 850 nucleotides. In some embodiments, the contiguous nucleotide length is 900 nucleotides. In some embodiments, the contiguous nucleotide length is 950 nucleotides. In some embodiments, the contiguous nucleotide length is 1000 nucleotides. In some embodiments, the contiguous nucleotide

[0123] Contiguous or homopolymeric nucleotides serve several functions, for example, protein binding regions, in upstream promoter elements, and in determining DNA location in a nucleosome structure. In some embodiments, contiguous nucleotides or homopolymers contain repeat units (SSRs), microsatellites, minisatellites and macrosatellites. SSRs are composed of 1-5 bp tandemly repeating units. For example, the most abundant SSRs are poly dA-poly dT and poly dG-poly dC, commonly found in non-coding regions and often greater than 9 bp in length. Poly dA-poly dT tracts are common in AT-rich sequences. The SSRs play a role in sequence specific DNA binding.

[0141]

[0124] In some embodiments, contiguous RNA nucleotides or homopolymers such as poly A and poly U are used to make virus-like particles since they provide benefits over RNAs with normal composition comprising a mixture of bases.

[0142]

[0125] In some embodiments, RNA homopolymers are found in tandem repeats, interspersed repeats, transposable elements. DNA transposons, retrotransposons. SINEs (Short Interspersed Nuclear Elements), LINEs (Long Interspersed Nuclear Elements), and CRISPR sequences.

[0143] Methods of Producing Stable mRNA by Ligating a Stem Loop Blocker

[0144]

[0126] In some aspects, provided herein is a method of producing mRNA, the method comprising the steps of: (a) providing in vitro synthesized mRNA, and (b) ligating a stem loop exonuclease blocker downstream of the poly A tail of the mRNA, wherein the ligation is carried out by a double-stranded RNA ligase.

[0145]

[0127] In some embodiments, the 5' end does not comprise a stem loop exonuclease blocker.

[0146]

[0128] In some aspects, provided herein is a method of producing protein from mRNA, the method comprising the steps of: (a) providing mRNA comprising a stem loop exonuclease blocker downstream of the poly A tail of mRNA, and (b) translating the mRNA from step (a) into protein.

[0147]

[0129] In some embodiments, the double-stranded RNA ligase is a T4 RNA ligase 2. In some embodiments, RNA generated by in vitro transcription that comprises a 5' terminal triphosphate is treated with RNA pyrophosphohydrolase (RppH) enzyme which cleaves diphosphate residues yielding mRNA with a terminal 5' monophosphate. In some other monophosphate (GMP) resulting in mRNA product that is a mixture of 5' triphosphorylated mRNA and 5' monophosphorylated mRNA. In some other embodiments, heat annealing is carried out. Messenger RNA comprising 5' monophosphate is then treated with T4 RNA ligase 2.

[0148]

[0130] In some embodiments, the stem loop exonuclease blocker is a hairpin loop or a tetraloop. In some embodiments, the stem loop exonuclease blocker is a hairpin loop. In some embodiments, the stem loop exonuclease blocker is a tetraloop.

[0149]

[0131] In some embodiments, the 5' end of mRNA comprises a 5’ phosphate.

[0150]

[0132] In some embodiments, the ligation is carried out at 37°C. In some embodiments, the ligation is earned out for between 3h to overnight. In some embodiments, the ligation is carried out at 20°C. In some embodiments, the ligation is carried out for between 6h to overnight. In some embodiments, the ligation is carried out for 6h.

[0151] Delivery Systems

[0152]

[0133] The stable mRNA described herein can be delivered to a cell of interest by various delivery systems such as vectors, e.g., plasmids and delivery vectors. In some aspects, mRNA is delivered by said delivery' systems as described herein.

[0153]

[0134] The mRNA described herein can be delivered by nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For instance, organic (e.g. lipid and / or polymer) nanoparticles can be suitable for use as delivery' vehicles in certain embodiments of this disclosure. Exemplary lipids for use in nanoparticle formulations, and / or gene transfer are shown in Table 1 (below).

[0154]

[0135] Table 1

[0155] Lipids Used for Gene Transfer

[0156] Lipid Abbreviation Feature

[0157] 1.2-Dioleoyl-sn-glycero-3-phosphatidylcholine DOPC Helper

[0158] 1.2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE Helper

[0159] Cholesterol Helper Lipids Used for Gene Transfer

[0160] Lipid Abbreviation Feature

[0161] N-[l-(2 3-Dioleyloxy)prophyl]N N,N-trimethylammonium DOTMA Cationic chloride

[0162] 1.2-Dioleoyloxy-3-trimethylammonium-propane DOTAP Cationic

[0163] Dioctadecylamidoglycylspermine DOGS Cationic

[0164] N-(3-Aminopropyl)-N,N-dimethyl-2T3-bis(dodecyloxy)-l- GAP-DLRIE Cationic propanaminium bromide

[0165] Cetyltrimethylammoni urn bromide CTAB Cationic 6-Lauroxyhexyl omithinate LHON Cationic l-(2,3-Dioleoyloxypropyl)-2.4.6-trimethylpyridinium 20c Cationic

[0166] 2.3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N- DOSPA Cationic dimethyl- 1 -propanaminium trifluoroacetate

[0167] 1.2-Dioleyl-3-trimethylammonium-propane DOPA Cationic

[0168] N-(2-Hydroxyethyl)-N.N-dimethyl-2,3-bis(tetradecyloxy)-l- MDRIE Cationic propanaminium bromide

[0169] Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide DMRI Cationic 3p-[N-(N',N'-Dimethylaminoethane)-carbamoyl]cholesterol DC-Chol Cationic Bis-guanidium-tren-cholesterol BGTC Cationic

[0170] L3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide DOSPER Cationic

[0171] Dimethyloctadecylammonium bromide DDAB Cationic Dioctadecylamidoglicylspermidin DSL Cationic rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]- CLIP-1 Cationic dimethylammonium chloride rac-[2(2,3-Dihexadecyloxypropyl- CLIP-6 Cationic oxymethyloxy)ethyl]trimethylammoniun bromide

[0172] Ethyldimyristoylphosphatidylcholine EDMPC Cationic

[0173] 1.2-Distear\’loxy-N,N-dimethyl-3-aminopropane DSDMA Cationic

[0174] 1.2-Dimy ristoy 1-trimethy lammonium propane DMTAP Cationic O,O'-Dimyristyl-N-lysyl aspartate DMKE Cationic

[0175] 1.2-Distearoyl-sn-gly cero-3-ethylpho sphocholine DSEPC Cationic Lipids Used for Gene Transfer

[0176] Lipid Abbreviation Feature

[0177] N-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine diC14-amidine Cationic

[0178] Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl] DOTIM Cationic imidazolinium chloride

[0179] N1 -Cholesteryloxycarbonyl-3.7-diazanonane-1.9-diamine CD AN Cationic

[0180] 2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N- RPR209120 Cationic ditetr adecylcarbamoylme-ethyl-acetamide

[0181] 1.2-dilinoleyloxy-3-dimethylaminopropane DLinDMA Cationic

[0182] 2.2-dilinoleyl-4-dimethylaminoethyl-[ 1 ,3]-dioxolane DLin-KC2- Cationic

[0183] DMA dilinoleyl-methyl-4-dimethylaminobutyrate DLin-MC3- Cationic

[0184] DMA

[0185]

[0136] Table 2 lists exemplary polymers for use in gene transfer and / or nanoparticle formulations.

[0186]

[0137] Table 2

[0187] Polymers Used for Gene Transfer

[0188] Polymer Abbreviation

[0189] Poly(ethylene)glycol PEG

[0190] Polyethylenimine PEI

[0191] Dithiobis (succinimidylpropionate) DSP

[0192] Dimethyl-3,3'-dithiobispropionimidate DTBP

[0193] Poly(ethylene imine)biscarbamate PEIC

[0194] Poly(L-lysine) PLL

[0195] Histidine modified PLL

[0196] Poly(N-vinylpyrrolidone) PVP

[0197] Poly(propylenimine) PPI

[0198] Poly(amidoamine) PAMAM Polymers Used for Gene Transfer

[0199] Polymer Abbreviation

[0200] Poly(amidoethylenimine) SS-PAEI

[0201] Triethylenetetramine TETA

[0202] Poly(P-aminoester)

[0203] Poly(4-hydroxy-L-proline ester) PHP

[0204] Poly(allylamine)

[0205] Poly (a- [4-aminobutyl]-L-gly colic acid) PAGA

[0206] Poly(D,L-lactic-co-glycolic acid) PLGA

[0207] Poly(N-ethyl-4-vinylpyridinium bromide)

[0208] Poly(phosphazene)s PPZ

[0209] Poly(phosphoester)s PPE

[0210] Poly(phosphoramidate)s PPA

[0211] Poly(N-24iydroxy propylmethacrylamide) pHPMA

[0212] Poly (2-(dimethylamino)ethyl methacrylate) pDMAEMA

[0213] Poly(2-aminoethyl propylene phosphate) PPE-EA

[0214] Chitosan

[0215] Galactosylated chitosan

[0216] N-Dodacylated chitosan

[0217] Histone

[0218] Collagen

[0219] Dextran-spermine D-SPM

[0220]

[0138] Table 3 summarizes delivery' methods for a polynucleotide encoding a Cas9 described herein.

[0221] Table 3

[0222] Delivery into Type of

[0223] Non-Dividing Duration of Genome Molecule

[0224] Delivery Vector / Mode Cells Expression Integration Delivered

[0225] Physical e.g., YES Transient NO Nucleic Acids electronoration and Proteins Delivery into Type of

[0226] Non-Dividing Duration of Genome Molecule

[0227] Delivery Vector / Mode Cells Expression Integration Delivered

[0228] Calcium

[0229] Phosphate transfection

[0230] Viral Retrovirus NO Stable YES RNA

[0231] Lentivirus YES Stable YES / NO with RNA modification

[0232] Adenovirus YES Transient NO DNA

[0233] Adeno- YES Stable NO DNA

[0234] Associated

[0235] Virus (AAV)

[0236] Vaccinia Virus YES Very NO DNA

[0237] Transient

[0238] Herpes Simplex YES Stable NO DNA

[0239] Virus

[0240] Non-Viral Cationic YES Transient Depends on Nucleic Acids

[0241] Liposomes what is and Proteins delivered

[0242] Polymeric YES Transient Depends on Nucleic Acids

[0243] Nanoparticles what is and Proteins delivered

[0244] Biological Attenuated YES Transient NO Nucleic Acids

[0245] Non-Viral Bacteria

[0246] Delivery Engineered YES Transient NO Nucleic Acids

[0247] Vehicles Bacteriophages

[0248] Mammalian YES Transient NO Nucleic Acids

[0249] Virus-like Particles

[0250] Biological YES Transient NO Nucleic Acids liposomes:

[0251] Erythrocyte

[0252] Ghosts and

[0253] Exosomes

[0139] In another aspect, the delivery of mRNA described herein may be accomplished by delivering a ribonucleoprotein (RNP) to cells. RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid-mediated methods, for example, as reported by Zuris, J.A. et al., 2015, Nat. Biotechnology, 33(1): 73- 80. RNPs are advantageous for use in CRISPR base editing systems, particularly for cells that are difficult to transfect, such as primary cells. In addition, RNPs can also alleviate difficulties that may occur with protein expression in cells, especially when eukaryotic promoters, e.g., CMV or EFl A, which may be used in CRISPR plasmids, are not well- expressed. Advantageously, the use of RNPs does not require the delivery of foreign DNA into cells. Moreover, because an RNP comprising a nucleic acid binding protein and gRNA complex is degraded over time, the use of RNPs has the potential to limit off-target effects. In a manner similar to that for plasmid based techniques, RNPs can be used to deliver binding protein (e.g., Cas9 variants) and to direct homology directed repair (HDR).

[0254]

[0140] A promoter used to drive a CRISPR system can include AAV ITR. This can be advantageous for eliminating the need for an additional promoter element, which can take up space in the vector. The additional space freed up can be used to drive the expression of additional elements, such as a guide nucleic acid or a selectable marker. ITR activity is relatively w eak, so it can be used to reduce potential toxicity due to over expression of the chosen nuclease.

[0255]

[0141] Any suitable promoter can be used to drive expression of the mRNA. For ubiquitous expression, promoters that can be used include CMV, CAG. CBh. PGK, SV40, Ferritin heavy or light chains, etc. For brain or other CNS cell expression, suitable promoters can include: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For liver cell expression, suitable promoters include the Albumin promoter. For lung cell expression, suitable promoters can include SP- B. For endothelial cells, suitable promoters can include ICAM. For hematopoietic cells, suitable promoters can include IFNbeta or CD45. For osteoblasts, suitable promoters can include OG-2.

[0256]

[0142] In some cases, separate promoters drive expression of the base editor and a compatible guide nucleic acid within the same nucleic acid molecule. For instance, a vector or viral vector can comprise a first promoter operably linked to a nucleic acid encoding the base editor and a second promoter operably linked to the guide nucleic acid. 1143] The promoter used to drive expression of a guide nucleic acid can include Pol III promoters such as U6 or Hl Use of Pol II promoter and intronic cassettes to express gRNA Adeno Associated Virus (AAV).

[0257]

[0144] Messenger RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Patent No. 8,454,972 (formulations, doses for adenovirus), U.S. Patent No. 8,404.658 (formulations, doses for AAV) and U.S. Patent No. 5.846.946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For example, for AAV, the route of administration, formulation and dose can be as in U.S. Patent No. 8,454,972 and as in clinical trials involving AAV. For adenovirus, the route of administration, formulation and dose can be as in U.S. Patent No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery7, the route of administration, formulation and dose can be as in U.S. Patent No. 5,846,946 and as in clinical studies involving plasmids. Doses can be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. The viral vectors can be injected into the tissue of interest. For cell-type specific base editing, the expression of the base editor and optional guide nucleic acid can be driven by a cell-type specific promoter.

[0258]

[0145] For in vivo delivery7, AAV can be advantageous over other viral vectors. In some cases, AAV allows low toxicity7, which can be due to the purification method not requiring ultra-centrifugation of cell particles that can activate the immune response. In some cases, AAV allows low probability of causing insertional mutagenesis because it doesn't integrate into the host genome.

[0259]

[0146] AAV has a packaging limit of 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kb can lead to significantly reduced virus production. An AAV can be AAV1, AAV2, AAV5 or any combination thereof. One can select the type of AAV with regard to the cells to be targeted; e.g.. one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or anv combination thereof for targeting brain or neuronal cells; and one can select certain AAV serotypes as to these cells can be found in Grimm. D. et al, J. Virol. 82: 5887- 5911 (2008)).

[0260]

[0147] Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.

[0261]

[0148] Lentiviruses can be prepared as follows. After cloning pCasESlO (which contains a lentiviral transfer plasmid backbone). HEK293FT at low passage (p=5) were seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and without antibiotics. After 20 hours, media is changed to OptiMEM (serum-free) media and transfection was done 4 hours later. Cells are transfected with 10 pg of lentiviral transfer plasmid (pCasESlO) and the following packaging plasmids: 5 pg of pMD2.G (VSV-g pseudotype), and 7.5 pg of psPAX2 (gag / pol / rev / tat). Transfection can be done in 4 mL OptiMEM with a cationic lipid delivery agent (50 pl Lipofectamine 2000 and 100 ul Plus reagent). After 6 hours, the media is changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods use serum during cell culture, but serum-free methods are preferred.

[0262]

[0149] Lentivirus can be purified as follows. Viral supernatants are harvested after 48 hours. Supernatants are first cleared of debris and filtered through a 0.45 pm low protein binding (PVDF) filter. They are then spun in an ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets are resuspended in 50 pl of DMEM overnight at 4° C. They are then aliquoted and immediately frozen at -80 C.

[0263]

[0150] In another embodiment, minimal non-primate lentiviral vectors based on the equine infectious anemia virus (EIAV) are also contemplated. In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is contemplated to be delivered via a subretinal injection. In another embodiment, use of self-inactivating lentiviral vectors is contemplated.

[0264]

[0151] Any messenger RNA, for example, a Cas9-encoding mRNA, a CAR encoding mRNA, or other mRNA. can be delivered. For example, Cas9 encoding mRNA can be generated using in vitro transcription. For example, Cas9 mRNA can be synthesized using a (GCCACC), nuclease sequence, and 3' UTR such as a 3' UTR from beta globin-polyA tail. The cassette can be used for transcription by T7 polymerase. Guide polynucleotides (e.g, gRNA) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter, followed by the sequence "GG". and guide polynucleotide sequence. To enhance expression and reduce possible toxicity, the Cas9 sequence and / or the guide nucleic acid can be modified to include one or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.

[0265]

[0152] The disclosure in some embodiments comprehends a method of modifying a cell or organism. The cell can be a prokaryotic cell or a eukaryotic cell. The cell can be a mammalian cell. The mammalian cell many be a non-human primate, bovine, porcine, rodent or mouse cell. The modification introduced to the cell by the base editors, compositions and methods of the present disclosure can be such that the cell and progeny of the cell are altered for improved production of biologic products such as an antibody, starch, alcohol or other desired cellular output. The modification introduced to the cell by the methods of the present disclosure can be such that the cell and progeny of the cell include an alteration that changes the biologic product produced.

[0266]

[0153] The system can comprise one or more different vectors. In an aspect, the mRNA is codon optimized for expression the desired cell tvpe. preferentially a eukaryotic cell, preferably a mammalian cell or a human cell.

[0267]

[0154] In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability’ of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp / codon / (visited Jul. 9, 2002), and these tables can be adapted in a number sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10. 15, 20, 25, 50, or more, or all codons) in a sequence encoding an engineered nuclease correspond to the most frequently used codon for a particular amino acid.

[0268]

[0155] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and psi.2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA can be packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line can also be infected with adenovirus as a helper. The helper virus can promote replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid in some cases is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

[0269] Pharmaceutical Compositions

[0270]

[0156] Other aspects of the present disclosure relate to pharmaceutical compositions (e.g., including mRNA). The term “pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, increasing half-life, or other therapeutic compounds).

[0271]

[0157] As used here, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, _ 4 _ _ _ 1,,1 _ : _ +„i _ _ _ 1^ _ _ _ _ stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the deliver}' site) of the body, to another site (e.g, organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).

[0272]

[0158] Some nonlimiting examples of materials which can serve as pharmaceutically- acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository' waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “vehicle,” or the like are used interchangeably herein.

[0273]

[0159] Pharmaceutical compositions can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine. Alternatively, the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions. Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions. The pH buffering compound may be present in 1160] Pharmaceutical compositions can also contain one or more osmotic modulating agents, z.e., a compound that modulates the osmotic properties (e.g, tonicity, osmolality, and / or osmotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals. The osmotic modulating agent can be an agent that does not chelate calcium ions. The osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation. Illustrative examples of suitable types of osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars, such as sucrose, dextrose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and / or types of agents. The osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation.

[0274]

[0161] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e , for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral. intracerebral, and intracerebroventricular administration.

[0275]

[0162] In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site. In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

[0276]

[0163] In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump can be used (See. e.g., Langer, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald etal., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla.. 1974); Controlled New York. 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et ah, 1989, J. Neurosurg. 71: 105.) Other controlled release systems are discussed, for example, in Langer, supra.

[0277]

[0164] In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g.. a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic use as solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade w ater or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0278]

[0165] A pharmaceutical composition for systemic administration can be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in ‘"stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et ah, Gene Ther. 1999, 6: 1438-47). Positively charged lipids such as N-[l-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl- amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g. , U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference. 1166] The pharmaceutical composition described herein can be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; z.e., carrier, or vehicle.

[0279]

[0167] Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g, sterile used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0280]

[0168] In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and can have a sterile access port. For example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture can further comprise a second container comprising a pharmaceutically - acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0281]

[0169] In some embodiments, the pharmaceutical composition comprises any fusion proteins (e.g., including nucleobase editors). In some embodiments, the pharmaceutical composition comprises anv complexes, e.g. a ribonucleoprotein complex comprising an some embodiments, the pharmaceutical composition comprises a gRNA. a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. Pharmaceutical compositions optionally comprise one or more additional therapeutically active substances.

[0282] Kits

[0283]

[0170] In one aspect, the stem loop blocker conjugated mRNA described herein can be provided and or produced by a kit containing any one or more of the elements disclosed in the above methods and compositions. For example, a kit may include a stem loop blocker conjugated mRNA, a ligase, and suitable buffering reagents.

[0284]

[0171] In some embodiments, the kit further comprises a nucleobase editor.

[0285]

[0172] In some aspects, the kit comprises a stable stem loop blocker conjugated mRNA. In some embodiments, the kit comprises a stable mRNA and a gene editing system. For example, a kit may include one or more of a stable mRNA, a gRNA, a ligase, suitable buffering agents and / or a nucleobase editor.

[0286]

[0173] In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide.

[0287]

[0174] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 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. Although methods and materials similar or equivalent to those descnbed herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0288] EXAMPLES

[0289]

[0175] While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.

[0290]

[0176] Example 1. Design of Stem Loop Exonuclease RNA blockers for ligation to exemplary emGFP mRNA

[0291]

[0177] This example illustrates design of stem loop RNA blockers for ligation to mRNA downstream of the poly A tail. An exemplary poly adenylated emGFP mRNA is 5'- GUCUUUGAAUAAAGCCUGAGUAGGAAG-3'-AAAAAAAAAAAAAAAAAAA 3 ‘ (SEQ ID NO: 1). FIG. 1 is a schematic of a messenger RNA comprising an exemplary stem loop, a tetraloop ligated dow nstream of the poly A tail by a T4 RNA ligase 2 (top), and a schematic of a linear RNA ligated downstream of a poly A tail by a T4 RNA ligase 1 that does not form a stem loop structure (bottom).

[0292]

[0178] Exemplary stem loop blockers comprising 4, 6, 8, 10, 12 or 14 uracil nucleotides w ere designed and their theoretical masses were calculated, and the results are shown in Table 4 and Table 5.

[0293]

[0179] Table 4. Exemplary RNA blocker sequence

[0294]

[0180] Table 5. Theoretical masses for conjugated tails of mRNA conjugated with RNA blockers of different lengths.

[0295]

[0181] Overall, this example illustrates design of stem loop RNA blockers for ligation to poly adenylated mRNA downstream of the poly A tail comprising 4-14 uracil nucleotides.

[0296] Example 2. Assessment of Stem Loop RNA Blocker Ligation by Mass Spectrometry

[0297]

[0182] This example illustrates stem loop RNA blocker ligation of mRNA comprising a poly A tail with exemplary stem loop blockers comprising 6 or 8 uracil nucleotides.

[0298]

[0183] Briefly, in vitro transcription was used to generate an exemplary' emGFP- PEST mRNA. A PEST sequence (enriched with Proline, glutamine, serine and threonine) was added to tareel the emGFP protein for nroteosomal degradation due to nhosnhorvlation so that stability measured was due to ligation with the RNA blocker. Following oligodT purification, RNA blocker ligation was carried out with H2O (control) or with stem loop RNA blockers comprising 6 uracils (6U) or 8 uracils (8U). In this example, blocker ligation was carried out at 20 °C overnight. RNA was purified using dynabeads to capture oligo dT and an RNA clean-up column. The sample was then evaluated by mass-spectrometry to confirm ligation of the stem loop blocker.

[0299]

[0184] The mass spectrometric peaks are shown for control RNA (FIG. 2A). RNA ligated with 6U RNA blocker (FIG. 2B) and RNA ligated with 8U RNA blocker (FIG. 2C). Extracted and deconvoluted mass spectra from the emGFP-PEST-H2O ligation showed that the mass spectra peaks resembled the unmodified poly A tail with a mass difference of 329 Da (FIG. 2D). Observed masses were consistent with those observed for emGFP. Further, the peak at retention time of 18.5 minutes did not contain any tail species.

[0300]

[0185] The deconvoluted mass spectra for emGFP-PEST-6U showed that observed masses were comparable to theoretical masses (FIG. 2E and Table 6). No unligated tail species were observed.

[0301]

[0186] Table 6. Theoretical and Observed Masses for emGFP-PEST-6U. 1187] The deconvoluted mass spectra for emGFP-PEST-8U showed that observed masses were comparable to theoretical masses (FIG. 2F and Table 7). No unligated tail species were observed.

[0302]

[0188] Table 7. Theoretical and Observed Masses for emGFP-PEST-8U

[0303]

[0189] The results from mass spectrometry showed that ligated tail species were observed in all samples, processed using dynabeads. The observed masses of the ligated tail species corresponded with theoretical masses. Tails were fully ligated as no unligated tail species were observed. The earlier eluting peak at 18.5 minutes did not contain any tail species.

[0304]

[0190] Overall, the mass spectrometry results confirmed successful ligation of RNA blocker comprising 6 or 8 uracil nucleotides.

[0305] Example 3. Microscopic assessment of stability of polyadenylated RNA ligated with exemplary RNA blockers

[0306]

[0191] This example illustrates stability of an exemplary polyadenylated RNA ligated with 6 or 8 uracil nucleotides.

[0307]

[0192] Briefly, Hela cells were plated in 96 well plates at a density of 8000 cells / well. then imaged 24 hours. 48 hours or 72 hours post-transfection using confocal microscopy (e.g., an Opera Phenix imaging system).

[0308]

[0193] GFP expression of emGFP-PEST mRNA samples treated with concentrations of 25 ng (FIG. 3A), 50 ng (FIG. 3B) or 100 ng mRNA / well (FIG. 3C) was increased or prolonged in samples ligated with 6U and 8U relative to H2O control. Increased or prolonged emGFP expression were detected in 6U and 8U conjugated samples compared to using H2O for ligation. Difference dosing of mRNA showed consistent results, e.g.. when doing with 25 ng (FIG. 3A), 50 ng (FIG. 3B) or 100 ng mRNA / well (FIG. 3C).

[0309]

[0194] FIG. 4A shows mean emGFP expression over time (24, 48 and 72h) when seeded at a concentration of 25 ng / well with emGFP-PEST-H2O (control), emGFP-PEST-6U or emGFP-PEST-8U. FIG. 4B showed fold change of emGFP expression in samples treated with 25 ng emGFP mRNA ligated with 6U or 8U blockers relative to control. FIG. 5A shows mean emGFP expression over time (24. 48 and 72h) when seeded at a concentration of 50 ng / well with emGFP-PEST-H2O (control), emGFP-PEST-6U or emGFP-PEST-8U. FIG. 5B showed fold change of emGFP expression in samples treated with 50 ng emGFP mRNA ligated with 6U or 8U blockers relative to control. FIG. 6A shows mean emGFP expression over time (24, 48 and 72h) when seeded at a concentration of 100 ng / well with emGFP- PEST-H2O (control), emGFP-PEST-6U or emGFP-PEST-8U. FIG. 6B showed fold change of eGFP expression in samples treated with 100 ng eGFP mRNA ligated with 6U or 8U blockers relative to control.

[0310]

[0195] FIG. 7A and FIG. 7B show ed that GFP expression was improved over time by conjugating 6U or 8U Compared to H2O Ligation. FIG. 7A showed GFP expression over time at 25 ng / well. FIG. 7B showed GFP expression over time at 50 ng / well.

[0311]

[0196] Overall, the results showed improved mRNA stability and prolonged expression in mRNA conjugated with exemplary RNA blockers.

[0312]

[0197] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing EQUIVALENTS

[0313]

[0198] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMSWe claim:

1. An in vitro synthesized linear messenger RNA comprising a poly A tail and a stem loop structure downstream of the poly A tail.

2. The messenger RNA of claim 1, wherein the 5' end lacks a stem loop structure.

3. The messenger RNA of claim 1, wherein the 5' end comprises a 5' triphosphate or 5’ cap structure.

4. The messenger RNA of any one of the preceding claims, wherein the stem loop structure comprises adenine, guanine, cytosine and / or uracil nucleotides.

5. The messenger RNA of any one of the preceding claims, wherein the stem loop structure comprises a plurality of uracil nucleotides at the 3’ end.

6. The messenger RNA of claim 5, wherein the plurality of uracil nucleotides form intramolecular base pairs with a portion of the poly A tail.

7. The messenger RNA of any one of the preceding claims, wherein the stem loop structure comprises a spacer or loop wherein the adenine, guanine, cytosine and / or uracil nucleotides do not form intramolecular base pairs.

8. The messenger RNA of any one of the preceding claims, wherein the stem region of the stem loop structure comprises contiguous uracil nucleotides.

9. The messenger RNA of claim 8, wherein the stem region of the stem loop structure comprises 4-14 contiguous uracil nucleotides.

10. The messenger RNA of claim 9, wherein the stem region of the stem loop structure comprises 4 contiguous uracil nucleotides.

11. The messenger RNA of claim 9, wherein the stem region of the stem loop structure comprises 6 contiguous uracil nucleotides.

12. The messenger RNA of claim 9, wherein the stem region of the stem loop structure comprises 8 contiguous uracil nucleotides.

13. The messenger RNA of claim 9, wherein the stem region of the stem loop structure comprises 10 contiguous uracil nucleotides.

14. The messenger RNA of claim 9, wherein the stem region of the stem loop structure comprises 12 contiguous uracil nucleotides.

15. The messenger RNA of claim 9, wherein the stem region of the stem loop structure comprises 14 contiguous uracil nucleotides.

16. The messenger RNA of any one of claims 1-8, wherein the stem region of the stem loop structure comprises greater than 14 contiguous uracil nucleotides.

17. The messenger RNA of any one of the preceding claims, wherein one of more contiguous RNA nucleotides is a modified nucleotide.

18. The messenger RNA of claim 17, wherein the modified nucleotide is a 2'-O-methyl, phosphorothioate and / or N1 -methylpseudouridine.

19. The messenger RNA of any one of the preceding claims, wherein the poly A tail is at least 30 nt long.

20. The messenger RNA of any one of the preceding claims, wherein the poly A tail is at least 100 nt long.

21. A composition comprising the mRNA of any one of the preceding claims.

22. A composition comprising a population of mRNA molecules, wherein at least 80% of mRNA molecules comprise a poly A tail and a stem loop structure downstream of the poly A tail.

23. A method of producing mRNA, the method comprising the steps of(a) providing in vitro synthesized mRNA. and(b) ligating a stem loop exonuclease blocker downstream of the poly A tail of the mRNA, wherein the ligation is carried out by a double-stranded RNA ligase.

24. The method of claim 23, wherein the 5' end does not comprise a stem loop exonuclease blocker.

25. A method of producing protein from mRNA, the method comprising the steps of: (a) providing mRNA comprising a stem loop exonuclease blocker downstream of the poly A tail of mRNA, and (b) translating the mRNA from step (a) into protein.

26. The method of any one of claims 23-25, wherein the double-stranded RNA ligase is a T4 RNA ligase 2.

27. The method of any one of claims 23-26, wherein the stem loop exonuclease blocker is a hairpin loop or a tetraloop.

28. The method of any one of the preceding claims, wherein the 5' end of mRNA comprises a 5' phosphate.

29. The method of any one of the preceding claims, wherein the ligation is carried out at 37°C.

30. The method of claim 29. wherein the ligation is carried out for betw een 3h to overnight.

31. The method of any one of claims 23-28. wherein the ligation is carried out at 20°C.

32. The method of claim 31, wherein the ligation is carried out for between 6h to overnight.

33. The method of claim 32, wherein the ligation is carried out for 6h.

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