Stable nucleic acids encoding messenger ribonucleic acid (mRNA)

Abstract

The present disclosure relates to the field of polyadenylated (poly A) tails. In some embodiments, the DNA encodes a poly A tail located 3'of a nucleotide encoding a protein of interest, where the poly A tail comprises one or more non-adenine nucleotides.

Description

[0001] This application is a divisional application of Chinese application No. 201880066328.X, whose application date is August 14, 2018 and whose invention name is “Stable Nucleic Acid Encoding Messenger Ribonucleic Acid (mRNA)”. Technical Field

[0002] The present disclosure relates to the field of stable messenger ribonucleic acid (mRNA) and DNA encoding stable mRNA. Background Art

[0003] Polyadenylation is the process of adding multiple adenine nucleotides to the 3' end of messenger RNA (mRNA) to form a poly A tail. The poly A tail is composed of multiple repeated adenine nucleotides (e.g., adenosine monophosphate) without other base interruption sequences. The poly A tail is essential for the nuclear export, translation, and stability of mRNA. In nature, when mRNA is produced from DNA, terminal transferase adds adenine nucleotides to the 3' end of mRNA. This enzymatic process can be applied when mRNA is produced in vitro, but the process is difficult to control and produces poly A tails of different lengths. By encoding the poly A tail in a plasmid, it is possible to reduce the heterogeneity in the poly A tail. However, this process does not eliminate heterogeneity and has other disadvantages, such as the potential instability of the plasmid.

[0004] The poly A tail serves as a binding site for poly A binding proteins. Poly A binding proteins facilitate mRNA export from the nucleus, translation, and inhibit mRNA degradation. In the absence of export from the nucleus, mRNA is typically degraded by exosomes. Poly A binding proteins recruit proteins required for translation.

[0005] MRNA is now used as a therapeutic molecule, for example, for treating various diseases and disorders. MRNA is delivered to the subject instead of protein, so that the subject's cell produces the protein encoded by the mRNA in the cell. For these and other purposes, mRNA can be prepared by transcribing from the DNA template usually contained in the plasmid. During mRNA production, the poly-A tail can be enzymatically added to mRNA or encoded on the plasmid itself after transcription from the plasmid. When the poly-A tail is encoded on the plasmid, the poly-A tail may become shorter (that is, lose adenine nucleotides) in the cycle of plasmid DNA replication, which may cause huge changes in the obtained DNA and subsequent mRNA populations. Therefore, this area needs to design the plasmid encoding the poly-A tail, which is stable during DNA replication, and resists the gradual loss of the nucleotides encoding the poly-A adenine nucleotides. Summary of the invention

[0006] Disclosed herein are DNA encoding a polyadenylation (poly A) tail and mRNA comprising a polyadenylation (poly A) tail comprising consecutive adenine nucleotides located 3' to the nucleotides encoding a protein of interest, wherein the poly A tail is stabilized by the insertion of a non-adenine nucleotide "anchor".

[0007] As used herein, the term "poly A tail" refers to a poly A tail on an mRNA molecule, or a sequence encoding a poly A tail in a DNA plasmid. The poly A tail can be encoded by a complementary DNA sequence in a plasmid. A sequence of repeated thymine (T) nucleotides in a DNA sequence (e.g., a homopolymer T sequence) can encode a poly A tail on an mRNA. Two or more consecutive adenosines (e.g., adenosine or deoxyadenosine), thymidines, or other nucleotides are referred to as homopolymers. A natural poly A tail comprises a long, uninterrupted homopolymer A sequence.

[0008] The non-adenine nucleotide anchors disclosed herein interrupt the poly A tail at regularly or irregularly spaced intervals and stabilize the DNA encoding the poly A tail and the mRNA produced from the DNA. Exemplary non-adenine nucleotide anchors are provided in Table 4. For example, the anchor sequence is adjacent to two adenine nucleotide homopolymer sequences within the poly A tail.

[0009] In some embodiments, a DNA composition comprising nucleotides encoding a polyadenylation (poly A) tail located 3' to nucleotides encoding a protein of interest, wherein the poly A tail comprises at least 8 consecutive adenine (A) nucleotides and one or more non-adenine (A) nucleotides is contemplated.

[0010] In some embodiments, the poly A tail comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 consecutive adenine nucleotides.

[0011] In some cases, the one or more non-adenine nucleotides prevent loss of one or more adenine nucleotides during DNA replication compared to the loss that occurs in a DNA comprising a 3' tail of similar or the same length that contains only adenine nucleotides.

[0012] In some embodiments, the one or more non-adenine nucleotides are positioned to interrupt consecutive adenine nucleotides such that a poly(A) binding protein can bind to a stretch of consecutive adenine nucleotides.

[0013] In some embodiments, the poly A tail comprises at least 50 total adenine nucleotides.

[0014] In some embodiments, the poly A tail comprises 40-500 total adenine nucleotides.

[0015] In some cases, the poly A tail comprises 95-100 total adenine nucleotides.

[0016] In some embodiments, the poly A tail comprises or contains a total of 90, 91, 92, 93, 94, 95, 96, or 97 adenine nucleotides.

[0017] In some embodiments, the poly A tail comprises or contains a total of 96 or 97 adenine nucleotides.

[0018] In some embodiments, the poly A tail comprises or contains one non-adenine nucleotide or a continuous stretch of 2-10 non-adenine nucleotides.

[0019] In some embodiments, the non-adenine nucleotide is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides.

[0020] In some cases, the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides.

[0021] In some embodiments, the one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0022] In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or one 2-10 nucleotide continuous segment for every 8-50 continuous adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or one 2-10 nucleotide continuous segment containing at least two non-adenine nucleotides for every 8-50 continuous adenine nucleotides. In some embodiments, the poly A tail has one or more non-adenine nucleotides or one or more 2-10 non-adenine nucleotide continuous segments, which are irregularly spaced anywhere along the length of the poly A tail, wherein at least 8 continuous adenines are present somewhere along the length of the poly A tail. For example, the length of the poly A tail can be 70-1000 nucleotides, and has any number of non-adenines (single or in groups) irregularly spaced along the length, as long as there are one or more segments of at least 8 continuous adenines.

[0023] In some cases, the poly A tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 nucleotides every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0024] In some cases, the poly A tail comprises or contains one non-adenine nucleotide or a 2-10 nucleotide consecutive stretch of at least two non-adenine nucleotides for every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0025] In some embodiments, the poly A tail comprises or contains 1, 2, 3, 4, or 5 consecutive non-adenine nucleotides for every 8-50 consecutive adenine nucleotides.

[0026] In some cases, the poly A tail comprises or contains 1, 2, 3, 4, or 5 consecutive non-adenine nucleotides for every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0027] In some embodiments, the poly A tail comprises or contains one or more non-adenine nucleotides or one or more consecutive stretches of 2-10 nucleotides as irregularly spaced interrupting sequences within the poly A tail.

[0028] In some embodiments, the poly A tail comprises or contains one or more non-adenine nucleotides or one or more consecutive stretches of 2-10 nucleotides comprising at least two non-adenine nucleotides irregularly spaced within the poly A tail.

[0029] In some cases, the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides for every 12 consecutive adenine nucleotides.

[0030] In some embodiments, the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides for every 16 consecutive adenine nucleotides.

[0031] In some embodiments, the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides for every 25 consecutive adenine nucleotides.

[0032] In some cases, the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides for every 30 consecutive adenine nucleotides.

[0033] In some embodiments, the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides for every 39 consecutive adenine nucleotides.

[0034] In some embodiments, the non-adenine nucleotide is guanine, cytosine or thymine. In some cases, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide.

[0035] In some instances where more than one non-adenine nucleotide is present, the non-adenine nucleotides may be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine, and cytosine nucleotides.

[0036] In some embodiments, the non-adenine nucleotide consists of one non-adenine nucleotide selected from guanine, cytosine, and thymine.

[0037] In some cases, the non-adenine nucleotides comprise two non-adenine nucleotides selected from one or more of guanine, cytosine, and thymine.

[0038] In some embodiments, the non-adenine nucleotides comprise three non-adenine nucleotides selected from one or more of guanine, cytosine, and thymine.

[0039] The adenine nucleotide may be adenosine monophosphate.

[0040] In some embodiments, the protein encoded by the mRNA is a therapeutic protein. In some cases, the protein is a cytokine, a chemokine, a growth factor, Cas9, or a modified Cas9.

[0041] In some embodiments, mRNA encoded by any of the DNA described herein is contemplated.

[0042] In some embodiments, the DNA is in a vector. The vector can be in a host cell, including an insect, bacterial, or mammalian (eg, human) cell.

[0043] In some embodiments, the one or more non-adenine nucleotides prevent loss of nucleotides encoding the poly A tail within the vector during growth of the host cell as compared to the loss that occurs in DNA comprising nucleotides encoding a poly A tail of similar or the same length containing only adenine nucleotides.

[0044] Contemplated are methods of producing mRNA from any DNA vector described herein, comprising: linearizing the vector downstream of the poly A tail; denaturing the linearized vector; and contacting the denatured DNA with an RNA polymerase in the presence of guanine, cytosine, uracil, and adenine nucleotides.

[0045] In some embodiments, the present disclosure includes DNA, which comprises nucleotides encoding a polyadenylated (poly A) tail located at 3' of the nucleotides encoding the target protein, wherein the poly A tail comprises a first homopolymer sequence of at least 8 consecutive adenine (A) nucleotides and an interruption sequence comprising one or more non-adenine (A) nucleotides. In some such embodiments, the poly A tail further comprises a second homopolymer sequence of at least consecutive adenine (A) nucleotides. In some embodiments, the poly A tail comprises three or more homopolymer sequences of at least 8 consecutive adenine (A) nucleotides. In some embodiments, the first and / or subsequent homopolymer sequences comprise at least 10, 15, 20, 25, 30, 35 or 40 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides prevent the loss of one or more adenine nucleotides during DNA replication compared to the loss occurring in a 3' tail of similar or identical length containing only adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are positioned to interrupt consecutive adenine nucleotides so that the poly (A) binding protein can bind to a segment of consecutive adenine nucleotides. In some embodiments, the poly A tail comprises at least 50 adenine nucleotides in total. In some embodiments, the poly A tail comprises 40-1000, 40-900, 40-800, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200 or 40-100 adenine nucleotides in total. In some embodiments, the poly A tail comprises 95-100 adenine nucleotides in total. In some embodiments, the poly A tail comprises or contains 90, 91, 92, 93, 94, 95, 96 or 97 adenine nucleotides in total. In some embodiments, the poly A tail comprises or contains 96 or 97 adenine nucleotides in total. In some embodiments, the one or more interruption sequences comprise or contain a non-adenine nucleotide or a 2-10 non-adenine nucleotide continuous section. In some embodiments, the one or more interruption sequences comprise or contain a non-adenine nucleotide or a 2-10 nucleotide continuous section including two or more non-adenine nucleotides. In some embodiments, the non-adenine nucleotide is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0046] In some embodiments, as described in the previous paragraph, the interrupt sequence is a trinucleotide, dinucleotide or mononucleotide interrupt sequence. In some such embodiments, the poly A tail includes or contains one non-adenine nucleotide or a continuous section of 2-10 non-adenine nucleotides for every 8-50 continuous adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or a continuous section of 2-10 non-adenine nucleotides for every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 continuous adenine nucleotides. In some embodiments, the poly A tail comprises or contains 1, 2, 3, 4 or 5 consecutive non-adenine nucleotides for every 8-50 consecutive adenine nucleotides. In some embodiments, the poly A tail comprises or contains 1, 2, 3, 4 or 5 consecutive non-adenine nucleotides for every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive adenine nucleotides. In some embodiments, the poly A tail comprises or contains more than one non-adenine nucleotide or more than one continuous segment of 2-10 non-adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides or more than one continuous section of 2-10 non-adenine nucleotides are irregularly spaced in the poly A tail. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or 2,3,4 or 5 continuous non-adenine nucleotides for every 12 continuous adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or 2,3,4 or 5 continuous non-adenine nucleotides for every 16 continuous adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or 2,3,4 or 5 continuous non-adenine nucleotides for every 25 continuous adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or 2,3,4 or 5 continuous non-adenine nucleotides for every 30 continuous adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or 2,3,4 or 5 continuous non-adenine nucleotides for every 39 continuous adenine nucleotides. In some embodiments, the non-adenine nucleotide is guanine, cytosine or thymine. In some embodiments, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide.In some embodiments, the DNA comprises one or more non-adenine nucleotides selected from the following: (a) guanine and thymine nucleotides; (b) guanine and cytosine nucleotides; (c) thymine and cytosine nucleotides; or (d) guanine, thymine and cytosine nucleotides. In some embodiments described above, the non-adenine nucleotides consist of one non-adenine nucleotide selected from guanine, cytosine and thymine. In some embodiments, the non-adenine nucleotides comprise two non-adenine nucleotides selected from one or more of guanine, cytosine and thymine. In some embodiments, the non-adenine nucleotides comprise three non-adenine nucleotides selected from one or more of guanine, cytosine and thymine. In some embodiments, the adenine nucleotide is adenosine monophosphate. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is a cytokine or chemokine. In some embodiments, the protein is a growth factor. In some embodiments, the protein is Cas9 or modified Cas9.

[0047] The present disclosure also encompasses mRNA encoded by the DNA as described in the previous paragraph.

[0048] In certain embodiments, the DNA described in the previous paragraphs may also be included in a vector. In certain embodiments, the vector is included in a host cell. In certain embodiments where the DNA is in a vector, compared to the loss that occurs in a DNA comprising a poly-A tail of nucleotides encoding only adenine nucleotides or the same length, the one or more non-adenine nucleotides prevent the loss of the nucleotides encoding the poly-A tail in the vector during the growth of the host cell.

[0049] The present disclosure also encompasses a method for producing mRNA from a DNA vector described herein, comprising: (a) linearizing the vector downstream of the poly A tail; (b) denaturing the linearized vector; and (c) contacting the denatured DNA with an RNA polymerase in the presence of guanine, cytosine, uracil, and adenine nucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Shown are sequences encoding a poly A tail containing only adenosine, which decreases in length with the number of growth rounds. Each clone refers to DNA produced by successive rounds of growth / purification of host cells expressing the plasmid encoding the clone.

[0051] Figure 2 The poly A tail containing non-adenine nucleotides is shown to be size-retained over two growth passages.

[0052] Figure 3Shown are secreted embryonic alkaline phosphatase (SEAP) levels measured in a Cas9 mRNA assay using Cas9 mRNA with a poly A tail containing only adenosine or Cas9 mRNA with a poly A tail comprising non-adenine nucleotides and a single guide RNA targeting SEAP (SEQ ID NO: 8).

[0053] Figure 4 Shown is the percent SEAP inhibition measured in a Cas9 mRNA assay using Cas9 mRNA with a poly A tail containing only adenosine or Cas9 mRNA with a poly A tail comprising non-adenine nucleotides and a single guide RNA targeting SEAP (SEQ ID NO: 8) with 24 hours incubation.

[0054] Figure 5 Shown is the percent SEAP inhibition measured in a Cas9 mRNA assay using Cas9 mRNA with a poly A tail containing only adenosine or Cas9 mRNA with a poly A tail comprising non-adenine nucleotides and a single guide RNA targeting SEAP (SEQ ID NO: 8) with 48 hours of incubation.

[0055] Figure 6 Shown are serum transthyretin (TTR) levels in mice 7 days after dosing with a control transformation and storage solution (TSS) buffer or with liquid nanoparticles (LNPs) formulated with a single guide RNA of SEQ ID NO:9 (targeting the mouse TTR gene) and either mRNA encoded by SEQ ID NO:6 (HiCas9 mRNA) or mRNA encoded by SEQ ID NO:7 (interrupting polyAmRNA).

[0056] Figure 7 Shown is the percent SEAP inhibition measured in a Cas9 mRNA assay using Cas9 mRNA with a poly A tail containing only adenosine or Cas9 mRNA with a poly A tail comprising non-adenine nucleotides and a single guide RNA targeting SEAP (SEQ ID NO: 8) with 48 hours of incubation. DETAILED DESCRIPTION

[0057] Disclosed herein is a DNA encoding a polyadenylated tail located at 3' of a nucleotide encoding a target protein, wherein the poly A tail comprises one or more non-adenine nucleotides. During DNA replication, compared to a poly A tail consisting only of adenine nucleotides, a DNA encoding a poly A tail comprising one or more non-adenine nucleotides can show a gradual loss of adenine nucleotides within a less poly A tail. Therefore, a plasmid is provided, the plasmid comprising a DNA encoding a poly A tail comprising one or more non-adenine nucleotides. An mRNA encoded by such a DNA is also contemplated. Compared to similar molecules comprising a non-interrupted poly A tail, the DNA and RNA can exhibit greater stability against the progressive loss of adenine nucleotides.

[0058] The protein of interest may be any natural or non-natural protein. As used herein, "protein" refers to any continuous amino acid sequence. Similarly, a protein may refer to a protein comprising the full amino acid sequence of a natural protein. In addition, a protein may refer to an amino acid sequence comprising a fragment of a full-length protein. A protein may be a natural sequence, a natural sequence with one or more modifications, or an artificial sequence that does not exist in nature.

[0059] The protein of interest may have therapeutic use in a subject, or such a protein may be used in a biochemical reaction. Therapeutic proteins include, for example, growth factors, antigens and enzymes for vaccines or immuno-oncology, etc. Therapeutic proteins may be natural or modified. In some cases, the modified protein may be a fusion protein.

[0060] In some embodiments, the protein is expressed by mRNA for use as a treatment for a disease. In some embodiments, the protein is expressed by mRNA for use as a cancer immunotherapy, for vaccination against infectious diseases, for inducing tolerance to type I allergies, as an alternative therapy, or as a regenerative medicine (see Sergeeva OV et al., Biochemistry (Moscow) 81(7): 709-722 (2016)).

[0061] In some embodiments, autologous dendritic cells are transfected ex vivo with mRNA encoding prostate specific antigen (PSA) to modulate T cell immune responses in subjects with metastatic prostate cancer.

[0062] In some embodiments, mRNA is a preventive vaccine. In some embodiments, mRNA encodes one or more antigenic proteins. In some embodiments, antigenic proteins are viral proteins. In some embodiments, mRNA causes somatic cells to produce and express antigenic proteins. In some embodiments, mRNA causes antigenic proteins to be expressed without danger or disease or transmission between individuals. In some embodiments, the expression of antigenic proteins causes the immune system of the subject to produce antibodies. In some embodiments, these antibodies can neutralize the virus and prevent future infection after exposure to the virus. In some embodiments, mRNA is a preventive vaccine for infectious diseases. In some embodiments, mRNA is a preventive vaccine for influenza, chikungunya, Zika virus, cytomegalovirus, human metapneumovirus (HMPV) or parainfluenza virus type 3 (PIV3). In some embodiments, mRNA is a preventive vaccine for influenza H10 or H7 subtypes.

[0063] In some embodiments, mRNA is a personalized cancer vaccine. In some embodiments, mRNA activates the immune system of a subject with cancer to recognize cancer cells and trigger a response. In some embodiments, this response is adjusted for individual patients' cancers or tumors. In some embodiments, mRNA encodes the patient's specific neoantigens (unique proteins with mutations present in the patient's cancer or tumor). In some embodiments, mRNA expresses the patient's specific neoantigens. In some embodiments, the expression of neoantigens triggers the patient's specific immune response to recognize and destroy cancer cells. In some embodiments, mRNA is used as a personalized cancer vaccine. In some embodiments, mRNA is used as a personalized cancer vaccine together with one or more checkpoint inhibitor antibodies (e.g., anti-PD-1 therapy).

[0064] In some embodiments, mRNA is used for intratumoral immuno-oncology. In some embodiments, injection of mRNA into a tumor reduces off-target effects and / or may be more potent than systemic administration. In some embodiments, mRNA expresses the ligand OX40L (CD252) of CD134. In some embodiments, mRNA expresses cytokines such as interleukin 12 (IL-12).

[0065] In some embodiments, the mRNA is used for protein expression for local therapy. In some embodiments, the mRNA causes more blood vessels to be created in local tissues and improves blood supply. In some embodiments, the mRNA causes vascular endothelial growth factor A (VEGF-A) to be expressed. In some embodiments, the expression of VEGF-A is local and transient. In some embodiments, local and transient VEGF-A expression is used to treat heart failure, or to treat diabetic wound healing or other ischemic vascular diseases after a heart attack.

[0066] In some embodiments, the mRNA expresses a protein for replacement therapy. In some embodiments, the protein is surfactant protein-B.

[0067] In some embodiments, the mRNA expresses an RNA-guided nuclease (e.g., a Class 2 CRISPR-associated Cas endonuclease, such as Cas9 / Csn1 (Cas9)). An exemplary Cas9 sequence is UniProt Q99ZW2. In some embodiments, the protein is a modified Cas9 or a Cas9 protein fused to another functional protein or peptide. A modified form of Cas9 with an inactive catalytic domain (RuvC or HNH) is referred to as a "nickase". In some embodiments, the compositions and methods comprise a nickase. In some embodiments, the compositions and methods comprise a nickase Cas9 that induces a nick instead of a double-strand break on the target DNA.

[0068] In some embodiments, the Cas protein can be modified to contain only one functional nuclease domain. For example, the Cas protein can be modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase Cas with a RuvC domain with reduced activity is used. In some embodiments, a nickase Cas with an inactive RuvC domain is used. In some embodiments, a nickase Cas with an HNH domain with reduced activity is used. In some embodiments, a nickase Cas with an inactive HNH domain is used.

[0069] In some embodiments, the chimeric Cas protein is encoded by a DNA in which a domain or region of the protein is replaced by a portion of a different protein. In some embodiments, the Cas nuclease domain can be replaced by a domain from a different nuclease (e.g., Fok1). In some embodiments, the Cas protein can be a modified nuclease.

[0070] I. DNA encoding a poly(A) tail containing non-adenine nucleotides

[0071] As used herein, "poly A tail" refers to a sequence comprising adenosine or other adenine nucleotides at the 3' end of an mRNA. Although a natural poly A tail may only comprise adenine nucleotides, the "poly A tail" of the present invention is stabilized by one or more non-adenine nucleotide "anchors". In some embodiments, the poly A tail comprises at least 8 consecutive adenine nucleotides and one or more interruption sequences comprising non-adenine nucleotides. In other words, the poly A tail of the present invention comprises at least 8 consecutive adenines, but also comprises one or more non-adenine nucleotides in the interruption or anchor sequence. The interruption sequence disclosed herein interrupts the poly A tail at regular or irregular intervals, and stabilizes the DNA encoding the poly A tail and the mRNA produced from the DNA. Exemplary interruption sequences are provided in Table 4.

[0072] As used herein, "non-adenine nucleotide" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides.

[0073] The natural poly A tail is added during the polyadenylation process that begins after the DNA is transcribed into mRNA. However, in molecular biology methods, the poly A tail is usually encoded by a portion of DNA located in a plasmid encoding the protein of interest. In this case, the size of the poly A tail (i.e., the number of adenine nucleotides contained in the poly A tail) directly depends on the number of DNA nucleotides in the plasmid that encode these consecutive adenine nucleotides.

[0074] The number of DNA nucleotides encoding poly A tails can be during DNA replication, such as plasmids gradually reduce during growth in host cells. When the number of nucleotides encoding continuous adenines in plasmids is reduced, the plasmid yield of encoding full-length poly A tails is reduced, and the resulting mRNA with shorter poly A tails may have reduced stability and / or increased degradation. For example, it can be expected that the stability of mRNA with poly A tails of 40 continuous adenine nucleotides is lower than the mRNA with poly A tails of 90 or more nucleotides. Lower stability refers to that mRNA can be degraded faster, and therefore reduces the expression of target proteins from mRNA with shorter poly A tails. Therefore, in multiple rounds of DNA replication in host cells, it is useful to maintain the length of poly A tails in DNA plasmids. In addition, poly A tails may be important for translation, and maintaining longer poly A tails may improve the expression of proteins from mRNA.

[0075] Inclusion of one or more non-adenine nucleotides in the poly A tail located 3' to the nucleotides encoding the protein of interest can prevent the loss of one or more adenine nucleotides during DNA replication, compared to the loss that occurs in DNA containing a 3' poly A tail of similar or identical length containing only adenine nucleotides. The presence of a longer poly A tail can also improve the efficiency of protein translation from mRNA.

[0076] A. Adenine nucleotide

[0077] The number of consecutive adenine nucleotides in the poly A tail of the present invention is designed to allow poly A binding protein to bind to consecutive adenosines. As used herein, "poly A binding protein (poly-Abinding protein, poly Abinding protein)" or "poly A binding protein" refers to a protein that binds to the poly A tail of mRNA. Poly A binding protein can play a role in regulating translation initiation. By binding to the poly A tail, poly A binding protein can protect them from uridylation of ZCCHC6 / ZCCHC11, and thus contribute to mRNA stability. Poly A binding protein can be located in cytoplasmic messenger ribonucleoprotein (mRNP) particles, which contain untranslated mRNA that shuttles between the cytoplasm and the nucleus. An exemplary poly A binding protein is PABPC1 (Uniprot reference number: P11940). The DNA of the present invention can encode enough consecutive adenine nucleotides so that when transcribed into mRNA, one or more poly A binding proteins retain the ability to bind to the poly A tail. Interrupting non-adenine nucleotide anchors are placed after the functional number of consecutive adenine nucleotides.

[0078] In some embodiments, the one or more non-adenine nucleotides are positioned to interrupt the continuous adenine nucleotides so that the poly A binding protein can bind to a segment of continuous adenine nucleotides (i.e., adenine nucleotide homopolymers or "homopolymer A"). In some embodiments, the poly A tail comprises at least 8 continuous adenine nucleotides. In some embodiments, the at least 8 continuous adenine nucleotides are 8, 9, 10, 11 and / or 12 continuous nucleotides. In some embodiments, the poly A tail comprises at least 10, 15, 20, 25, 30, 35 and / or 40 continuous adenine nucleotides. In some embodiments, the poly A tail comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and / or 90 continuous adenine nucleotides. For example, a homopolymer in a poly A RNA sequence may contain at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, or 40 consecutive adenosine nucleotides. For example, a homopolymer in a plasmid sequence encoding a poly A tail may contain at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, or 40 consecutive thymidine nucleotides. In some embodiments, the poly A tail comprises two or more homopolymer A sequences of different lengths, for example, the interruption sequences in the poly A tail are irregularly spaced. In some embodiments, the poly A tail comprises regularly spaced interruption sequences and two or more homopolymers of the same length.

[0079] In some embodiments, the poly A tail comprises a first homopolymer sequence of at least 8 consecutive adenine nucleotides, a second homopolymer sequence of at least 5 consecutive adenine nucleotides, and an anchor comprising one or more non-adenine nucleotides.

[0080] In some embodiments, the poly A tail comprises one or more groups of 8-50 consecutive adenine nucleotides. In some embodiments, the poly A tail comprises one or more groups of 8-100 consecutive adenine nucleotides. For poly A tails with multiple groups of consecutive adenine nucleotides (i.e., multiple homopolymer sequences), each group of adenine nucleotides does not need to have the same length.

[0081] In addition to the number of consecutive adenine nucleotides, the poly A tail can also be characterized by the total amount of adenine nucleotides. The total amount of adenine nucleotides is simply the sum of all adenine nucleotides in the poly A tail. Therefore, all adenine nucleotides in different groups of consecutive or non-continuous groupings of adenine nucleotides will be included in the total amount of adenine nucleotides in the poly A tail.

[0082] In some embodiments, the poly A tail comprises a total of 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 3 530-540, 540-550, 550-560, 560-570, 570-580, 580-590, or 590-600 adenine nucleotides. In some embodiments, the poly A tail comprises one or more homopolymer A sequences of at least 8, 9, 10, 12, 25, 30, 50 nucleotides in length.

[0083] In some embodiments, the poly A tail comprises 40-1000, 40-900, 40-800, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, or 40-100 total adenine nucleotides.

[0084] In some embodiments, the poly A tail comprises at least a total of 40 adenine nucleotides. In some embodiments, the poly A tail comprises at least a total of 50 adenine nucleotides. In some embodiments, the poly A tail comprises at least 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, or 300 adenine nucleotides.

[0085] In some embodiments, the poly A tail comprises or contains a total of 90, 91, 92, 93, 94, 95, 96, or 97 adenine nucleotides. In some embodiments, the poly A tail comprises or contains a total of 96 or 97 adenine nucleotides.

[0086] In some embodiments, the adenine nucleotide is adenosine monophosphate.The nucleotides may be modified.

[0087] B. Interrupt sequences containing non-adenine nucleotides

[0088] The non-adenine nucleotides of the invention may comprise or consist of natural or non-natural nucleotides (eg, guanine, cytosine or thymine). The nucleotides may be modified.

[0089] In some embodiments, the poly A tail contains a non-adenine nucleotide in the poly A tail that originally consists only of adenine nucleotides. The non-adenine nucleotide can interrupt the sequence of adenine nucleotides. The non-adenine nucleotide can be selected from guanine, cytosine and thymine. In some embodiments, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide. The interruption sequence can be a mononucleotide, a dinucleotide, or a trinucleotide sequence. The interruption sequence can contain 1, 2, 3, 4, 5 or more non-adenine nucleotides, and its length can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0090] In some embodiments, a single non-adenine nucleotide may interrupt a set or group of consecutive adenine nucleotides. The one non-adenine nucleotide may be positioned to interrupt the consecutive adenine nucleotides in a manner such that the poly A binding protein can bind to the stretch of consecutive adenine nucleotides.

[0091] In some embodiments, there is one or more non-adenine nucleotides in the poly A tail. The one or more non-adenine nucleotides can be positioned to interrupt the continuous adenine nucleotides in a manner that allows the poly A binding protein to bind to a continuous adenine nucleotide segment. In some embodiments, the non-adenine nucleotides are interspersed between one or more sets of continuous adenine nucleotides, wherein the number of adenine nucleotides in each series of continuous adenine nucleotides is sufficient to allow the binding of the poly A binding protein.

[0092] The non-adenine nucleotides may be in a segment of more than one non-adenine nucleotide. The non-adenine nucleotides may be in a segment of 2-10 consecutive nucleotides comprising one or more non-adenine nucleotides. The non-adenine nucleotides may be in an interrupted sequence interspersed between one or more consecutive adenine nucleotides (e.g., more than one homopolymer A sequence). In some embodiments, the number of consecutive non-adenine nucleotides may be 1, 2, 3, 4, or 5. In some embodiments, there are 2-10 consecutive segments of non-adenine nucleotides. In some embodiments, there are 2-10 consecutive segments of nucleotides comprising at least two non-adenine nucleotides.

[0093] Continuous non-adenine nucleotides can be more than one identical nucleotide, or continuous non-adenine nucleotides can be different from each other. For example, non-adenine nucleotides can be more than one guanine, cytosine or thymine nucleotides. Non-adenine nucleotides can also be guanine and thymine nucleotides; guanine and cytosine nucleotides; thymine and cytosine nucleotides; or guanine, thymine and cytosine nucleotides. Non-adenine nucleotides can include two non-adenine nucleotides selected from one or more of guanine, cytosine and thymine. Non-adenine nucleotides can include three non-adenine nucleotides selected from one or more of guanine, cytosine and thymine. Non-adenine nucleotides can include more than three non-adenine nucleotides selected from one or more of guanine, cytosine and thymine. The poly A tail can include adenine nucleotides at regular or irregular intervals between non-adenine nucleotides. For example, the poly A tail can be considered to have a certain pattern, wherein the pattern is regular or irregular. The key to the pattern is the presence of one or more non-adenine nucleotides anywhere in the poly A tail, as long as there are at least 8 consecutive adenines anywhere along the length. In some embodiments, poly A may include a segment with at least 8 consecutive adenine nucleotides anywhere along the length, wherein the adenine nucleotide is "interrupted" by one or more non-adenine nucleotides anywhere after 8 or more adenines. The interruption sequence may be a non-adenine nucleotide or 2 to 10 consecutive nucleotides optionally comprising at least two non-adenine nucleotides. Each nucleotide or a continuous segment of nucleotides comprising at least two non-adenine nucleotides may be followed by one or more adenines, optionally followed by one or more non-adenine nucleotides, optionally followed by one or more adenine nucleotides, and so on, until the end of the poly A tail. This pattern of adenine nucleotides / non-adenine nucleotides may be repeated at regular or irregular intervals. Alternatively, there may be no pattern, such as only one or a continuous segment of 2-10 nucleotides, which optionally comprises at least two non-adenine nucleotides along the entire length of poly A.

[0094] II. Exemplary Patterns of Adenine and Non-Adenine Nucleotides in the Poly (A) Tail

[0095] The poly A tail of the present invention may comprise or consist of a plurality of interruption sequences of different patterns, such as consecutive adenine nucleotides and one or more non-adenine nucleotides.

[0096] The poly A tail may start with one or a series of consecutive adenine nucleotides, followed by non-adenine nucleotides. A poly A tail starting with a series of adenine nucleotides means that the 5' end of the poly A tail consists of one or a series of consecutive adenine nucleotides, wherein one or more non-adenine nucleotides are connected after the consecutive adenine nucleotides. "After" means that the non-adenine nucleotide is 3' of the series of consecutive adenine nucleotides.

[0097] In some embodiments, the 5' end of the poly A tail may be composed of a series of continuous adenine nucleotides, followed by one or more non-adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8, 9, 10, 11 or 12 continuous adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8-50 continuous adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8-100 continuous adenine nucleotides. In some embodiments, the non-adenine nucleotides are after 1, 2, 3, 4, 5, 6 or 7 adenine nucleotides, and are followed by at least 8 continuous adenine nucleotides.

[0098] In some embodiments, the 5' end of the poly A tail consists of 1 to 8 adenine nucleotides, followed by one or more non-adenine nucleotides. In such embodiments, the non-adenine nucleotides are followed by more adenine nucleotides. The adenine nucleotides after the one or more non-adenine nucleotides include at least 8 adenine nucleotides before another non-adenine nucleotide.

[0099] The size range of the group of consecutive adenine nucleotides that begin the poly A tail can vary. In some embodiments, the 5' end of the poly A tail consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 99, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100

[0100] In some embodiments, the one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0101] The poly A tail may end with a non-adenine nucleotide segment at the 3' end. The number of non-adenine nucleotides at the 3' end of the poly A tail may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-adenine nucleotides. Alternatively, the 3' end of the poly A tail may consist of one or more adenine nucleotides.

[0102] The poly A tail of the present invention may comprise a continuous adenine nucleotide sequence, followed by one or more non-adenine nucleotides, optionally followed by other adenine nucleotides. The poly A tail of the present invention may also comprise more than one continuous adenine nucleotide sequence interrupted by one or more non-adenine nucleotides. The sequence of continuous adenine nucleotides may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 continuous adenine nucleotides. The number of non-adenine nucleotides in the interrupted sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-adenine nucleotides.

[0103] The poly A tail of the present invention may also include more than one series of continuous adenine nucleotides, which are interrupted by non-adenine nucleotides or interspersed with non-adenine nucleotides. The length of the interruption sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. The length of the interruption sequence may be 1-3, 1-5, 1-10, 2-10, 2-8, 2-6 or 2-5 nucleotides. The poly A tail of the present invention may include more than one group of continuous adenine nucleotides and an interruption sequence comprising one non-adenine nucleotide or more than one 2-10 non-adenine nucleotide continuous segments between each group of continuous adenine nucleotides. The poly A tail of the present invention may include more than one group of continuous adenine nucleotides and one non-adenine nucleotide or more than one 2-10 nucleotide continuous segments comprising at least two non-adenine nucleotides between each group of continuous adenine nucleotides. The poly A tail of the present invention may include more than one group of continuous adenine nucleotides and one or more interruption sequences, each of which includes one or more non-adenine nucleotides. The groups may each include the same or different numbers of adenine nucleotides. In embodiments having multiple groups of consecutive adenine nucleotides, the length of each group of consecutive adenine nucleotides may be sufficient to allow binding of the poly A binding protein.

[0104] In some embodiments, one or more non-adenine nucleotides are interrupting sequences at regular intervals in the poly A tail. Regular intervals mean that a certain number of consecutive adenine nucleotides are followed by non-adenine nucleotides in a repeating manner.

[0105] In some embodiments, one or more non-adenine nucleotides are located at irregular intervals in the poly A tail. Irregular intervals mean that a certain number of consecutive adenine nucleotides are followed by non-adenine nucleotides, followed by another group of consecutive adenine nucleotides, the other group of consecutive adenine nucleotides comprising a different number of adenines than the first group.

[0106] In some embodiments, the poly A tail contains or contains one non-adenine nucleotide or a continuous segment of 2-10 nucleotide non-adenine nucleotides for every 8-50 consecutive adenine nucleotides. In some embodiments, the poly A tail contains or contains one non-adenine nucleotide or a continuous segment of 2-10 non-adenine nucleotides for every 8-50 consecutive adenine nucleotides. In some embodiments, the poly A tail contains or contains one non-adenine nucleotide or a continuous segment of 2-10 nucleotide non-adenine nucleotides for every 8-100 consecutive adenine nucleotides. In some embodiments, the poly A tail contains or contains one non-adenine nucleotide or a continuous segment of 2-10 non-adenine nucleotides for every 8-100 consecutive adenine nucleotides.

[0107] In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or a continuous section of 2-10 nucleotides containing at least two non-adenine nucleotides for every 8-50 consecutive adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or a continuous section of 2-10 nucleotides containing at least two non-adenine nucleotides for every 8-50 consecutive adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or a continuous section of 2-10 nucleotides containing at least two non-adenine nucleotides for every 8-100 consecutive adenine nucleotides. In some embodiments, the poly A tail includes or contains one non-adenine nucleotide or a continuous section of 2-10 nucleotides containing at least two non-adenine nucleotides for every 8-100 consecutive adenine nucleotides.

[0108] In some embodiments, the poly A tail comprises or contains one non-adenine nucleotide or a continuous stretch of 2-10 nucleotides comprising non-adenine nucleotides for every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0109] In some embodiments, the poly A tail comprises or contains one non-adenine nucleotide or a 2-10 nucleotide consecutive stretch of at least two non-adenine nucleotides for every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0110] In some embodiments, the number of non-adenine nucleotides can be 1, 2, 3, 4 or 5 consecutive non-adenine nucleotides. In some embodiments, the number of consecutive adenine nucleotides can be 8-50 adenine nucleotides. In some embodiments, the poly A tail contains or contains 1, 2, 3, 4 or 5 consecutive non-adenine nucleotides every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive adenine nucleotides.

[0111] The number of continuous adenine nucleotides in the poly A tail can be 12, 16, 25, 30 or 39. The number of continuous adenine nucleotides may also be greater than 39. In some embodiments, the poly A tail contains or contains 1, 2, 3, 4 or 5 continuous non-adenine nucleotides for every 12 continuous adenine nucleotides. In some embodiments, the poly A tail contains or contains 1, 2, 3, 4 or 5 continuous non-adenine nucleotides for every 16 continuous adenine nucleotides. In some embodiments, the poly A tail contains or contains 1, 2, 3, 4 or 5 continuous non-adenine nucleotides for every 25 continuous adenine nucleotides. In some embodiments, the poly A tail contains or contains 1, 2, 3, 4 or 5 continuous non-adenine nucleotides for every 30 continuous adenine nucleotides. In some embodiments, the poly A tail contains or contains 1, 2, 3, 4 or 5 continuous non-adenine nucleotides for every 39 continuous adenine nucleotides. The number of continuous non-adenine nucleotides may also be greater than 5.

[0112] Exemplary trinucleotide interruption sequences include GCG, CCG, GTG, TGG, CGG, GGT, TAT, CAT, CGT, CTC, GAT, CCT, TGT, CGC, CAC, TGC, TCG, TCT, CCC, GAC, TAG, GTT, CTG, and TTT. There are 63 possible trinucleotide interruption sequences and 36 trinucleotide interruption sequences without terminal A. In some embodiments, the poly A tail comprises one or more trinucleotide interruption sequences selected from the following: TGG, CGG, GGT, TAT, CAT, CGT, CTC, GAT, CCT, TGT, CGC, CAC, TGC, TCG, TCT, CCC, GAC, TAG, GTT, CTG, and TTT. In some embodiments, the poly A tail comprises multiple interruption sequences designed to minimize hybridization and annealing between 3 or more nucleotides within the sequence encoding the poly A tail or within the poly A tail. In certain embodiments, the interrupt sequence that minimizes annealing between 3 or more nucleotides is selected from 34 trinucleotide interrupt sequences without terminal A. In some embodiments, the interrupt sequence that minimizes annealing between 3 or more nucleotides is selected from TGG, CGG, GGT, TAT, CAT, CGT, CTC, GAT, CCT, TGT, CGC, CAC, TGC, TCG, TCT, CCC, GAC, TAG, GTT, CTG, and TTT. In some embodiments, such as SEQ ID NO: 18, the poly A tail comprises a dinucleotide and / or trinucleotide interrupt sequence selected from the following: TGG, CGG, GGT, TAT, CAT, CGT, CTC, GAT, CCT, TGT, CGC, CAC, TGC, TCG, TCT, CCC, GAC, TAG, GTT, CTG, TTT, and CG. In certain embodiments, the poly A tail comprises a trinucleotide interrupt sequence selected from GCG, CCG, and GTG. Exemplary dinucleotide interrupt sequences include CG, GC, CC, GG, TT, CT, TC, GT, and TG. There are 15 possible dinucleotide interrupt sequences and 9 dinucleotides that do not include a terminal A. Mononucleotide interrupt sequences can be C, G, and T. Note that with respect to any of the above nucleotide sequences, when referring to RNA sequences (e.g., mRNA), in contrast to DNA sequences, T is replaced by U.

[0113] Those skilled in the art will be able to design a variety of different patterns of DNA encoding a poly A tail having consecutive adenine nucleotides and one or more non-adenine nucleotides. Some exemplary poly A tails comprising at least 8 consecutive adenine nucleotides and one or more adenine nucleotides are presented in, for example, SEQ ID Nos: 1-5, 10, 11 and 18.

[0114] III. How to use

[0115] The DNA of the present invention can be used to produce mRNA encoded by the DNA. In some embodiments, mRNA is encoded by the DNA of the present invention.

[0116] In some embodiments, the DNA of the present invention is prepared for the production of mRNA. In some embodiments, the DNA is in a vector. In some embodiments, the vector is in a host cell. In some embodiments, the mRNA encoded by the DNA of the present invention is used to translate the target protein encoded by the DNA.

[0117] In some embodiments, the one or more non-adenine nucleotides prevent the loss of one or more adenine nucleotides during DNA replication compared to the loss that occurs in a DNA containing a 3' tail of similar or identical length containing only adenine nucleotides. DNA replication is a necessary step in plasmid growth for DNA purification. Therefore, compared to the plasmid encoding the poly-A tail consisting only of adenine nucleotides, the plasmid comprising the DNA of the present invention encoding the poly-A tail comprising at least 8 continuous adenine nucleotides and one or more non-adenine nucleotides can show improved stability through one or more rounds of plasmid growth and purification.

[0118] Compared with the plasmid comprising the DNA containing the sequence of the poly A tail consisting only of continuous adenine nucleotides, the plasmid comprising the DNA of the present invention containing the sequence of the poly A tail containing at least 8 continuous adenine nucleotides and one or more non-adenine nucleotides can have greater stability when growing in host cells. During the growth of the host cell expressing the plasmid containing the DNA sequence, compared with the poly A tail consisting only of adenine nucleotides, the DNA sequence encoding the poly A tail comprising continuous adenine nucleotides and one or more non-adenine nucleotides can resist the length reduction of the DNA encoding the poly A tail. In certain embodiments, compared with the plasmid comprising the DNA containing the poly A tail comprising only adenine nucleotides, the plasmid comprising the DNA containing the poly A tail encoding one or more non-adenine nucleotides prevents the loss of adenine during the growth of the host cell.

[0119] Any growth and purification vector known to those skilled in the art can be used for the growth of host cells encoding plasmids. The growth and purification process of the vector can also be referred to as plasmid preparation. The standard steps of plasmid purification include the growth of bacterial culture, the harvesting and lysis of bacteria, and the purification of plasmid DNA. Various kits can be obtained from various manufacturers to purify plasmid DNA. The steps of plasmid preparation can be micropreparation (minipreparation) (expected yield is 20 to 40 μg or 50 to 100 μg plasmid DNA), midipreparation (midipreparation) (expected yield is 100 to 350 μg plasmid DNA), large-scale preparation (maxipreparation) (expected yield is 500-850 μg plasmid DNA), over-preparation (megapreparation) (expected yield is 1.5-2.5 mg plasmid DNA) or super-large-scale preparation (gigapreparation) (expected yield is 7.5-10 mg plasmid DNA). For therapeutic mRNA production, plasmids can be produced on a large scale of 100 mg, 1 g, 10 g or more. Improvements in the stability and replication efficiency of plasmids encoding poly A tails with non-adenine nucleotides as described herein may improve the consistency and efficiency of plasmids produced on this scale.

[0120] In some embodiments, methods for producing mRNA from DNA vectors of the present invention are contemplated. In some embodiments, methods for producing mRNA from DNA vectors include linearizing the vector downstream of the poly A tail; denaturing the linearized vector; and contacting the denatured DNA with an RNA polymerase in the presence of RNA nucleotides, such as guanine, cytosine, uracil, adenine, or chemically modified forms of such nucleotides, such as pseudouridine, N-1-methylpseudouridine, methoxyuridine, etc. Modified residues (e.g., backbone modifications of bases, sugars, and nucleotide residues) can be used in the mRNA, polynucleotides, and methods described herein.

[0121] This description and exemplary embodiments should not be considered as restrictive. For the purpose of this specification and the appended claims, unless otherwise indicated, all numerals and other values ​​used in this specification and the claims indicating quantity, percentage or ratio should be understood to be modified by the term "about" in all cases (if they have not been so modified). Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values, which may vary according to the desired properties sought to be obtained. At least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted at least according to the number of reported significant figures and by applying ordinary rounding techniques.

[0122] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," as well as any singular use of any term, include plural referents unless specifically and unambiguously limited to one referent. As used herein, the term "include" and its grammatical variations are intended to be non-limiting, such that recitation of items in a list does not exclude other like items that can be substituted or added to the listed items.

[0123] Specific implementation plan

[0124] 1. A DNA comprising nucleotides encoding a polyadenylation (poly A) tail located 3' to nucleotides encoding a protein of interest, wherein the poly A tail comprises a first homopolymer sequence of at least 8 consecutive adenine (A) nucleotides and an interrupting sequence comprising one or more non-adenine (A) nucleotides.

[0125] 2. The DNA according to embodiment 1, wherein the poly A tail further comprises at least a second homopolymer sequence of consecutive adenine (A) nucleotides.

[0126] 3. The DNA according to embodiment 1 or 2, wherein the poly A tail comprises three or more homopolymer sequences of at least 8 consecutive adenine (A) nucleotides.

[0127] 4. The DNA according to embodiment 1, wherein the first and / or subsequent homopolymer sequences comprise at least 10, 15, 20, 25, 30, 35 or 40 consecutive adenine nucleotides.

[0128] 5. The DNA according to embodiment 1, wherein the one or more non-adenine nucleotides prevent the loss of one or more adenine nucleotides during DNA replication compared to the loss that occurs in a DNA comprising a 3' tail of similar or the same length containing only adenine nucleotides.

[0129] 6. The DNA according to embodiment 1, wherein the one or more non-adenine nucleotides are positioned to interrupt the consecutive adenine nucleotides so that the poly (A) binding protein can bind to the segment of consecutive adenine nucleotides.

[0130] 7. The DNA according to embodiment 1, wherein the poly A tail comprises at least 50 adenine nucleotides in total.

[0131] 8. The DNA of embodiment 1, wherein the poly A tail comprises a total of 40-1000, 40-900, 40-800, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200 or 40-100 adenine nucleotides.

[0132] 9. The DNA according to embodiment 1, wherein the poly A tail comprises a total of 95-100 adenine nucleotides.

[0133] 10. The DNA according to embodiment 1, wherein the poly A tail comprises or contains a total of 90, 91, 92, 93, 94, 95, 96 or 97 adenine nucleotides.

[0134] 11. The DNA according to embodiment 1, wherein the poly A tail comprises or contains a total of 96 or 97 adenine nucleotides.

[0135] 12. The DNA according to embodiment 1, wherein the one or more interruption sequences comprise or contain a non-adenine nucleotide or a continuous segment of 2-10 non-adenine nucleotides.

[0136] 13. The DNA according to embodiment 1, wherein the one or more interruption sequences comprise or contain a non-adenine nucleotide or a continuous segment of 2-10 nucleotides including two or more non-adenine nucleotides.

[0137] 14. The DNA according to embodiment 12 or 13, wherein the non-adenine nucleotide is located after at least 8, 9, 10, 11 or 12 consecutive adenine nucleotides.

[0138] 15. The DNA according to embodiment 1, wherein the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides.

[0139] 16. The DNA of embodiment 1, wherein the one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0140] 17. The DNA according to any one of embodiments 1 to 16, wherein the interrupt sequence is a trinucleotide, dinucleotide or mononucleotide interrupt sequence.

[0141] 18. The DNA according to any one of embodiments 1 to 16, wherein the poly A tail comprises or contains one non-adenine nucleotide or a continuous segment of 2-10 non-adenine nucleotides for every 8-50 consecutive adenine nucleotides.

[0142] 19. The DNA of any one of embodiments 1 to 16, wherein the poly A tail comprises or contains one non-adenine nucleotide or a continuous segment of 2-10 non-adenine nucleotides for every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive adenine nucleotides.

[0143] 20. The DNA according to any one of the preceding embodiments, wherein the poly A tail comprises or contains 1, 2, 3, 4 or 5 consecutive non-adenine nucleotides for every 8-50 consecutive adenine nucleotides.

[0144] 21. The DNA of any one of embodiments 1 to 20, wherein the poly A tail comprises or contains 1, 2, 3, 4, or 5 consecutive non-adenine nucleotides for every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0145] 22. The DNA according to any one of embodiments 1 to 21, wherein the poly A tail comprises or contains one or more non-adenine nucleotides or one or more consecutive segments of 2-10 non-adenine nucleotides.

[0146] 23. The DNA according to any of the preceding embodiments, wherein the one or more non-adenine nucleotides or the one or more consecutive stretches of 2-10 non-adenine nucleotides are irregularly spaced within the poly A tail.

[0147] 24. The DNA according to any one of the preceding embodiments, wherein the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4 or 5 consecutive non-adenine nucleotides for every 12 consecutive adenine nucleotides.

[0148] 25. The DNA according to any one of the preceding embodiments, wherein the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4 or 5 consecutive non-adenine nucleotides for every 16 consecutive adenine nucleotides.

[0149] 26. The DNA according to any one of the preceding embodiments, wherein the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4 or 5 consecutive non-adenine nucleotides for every 25 consecutive adenine nucleotides.

[0150] 27. The DNA according to any one of the preceding embodiments, wherein the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4 or 5 consecutive non-adenine nucleotides for every 30 consecutive adenine nucleotides.

[0151] 28. The DNA according to any one of the preceding embodiments, wherein the poly A tail comprises or contains one non-adenine nucleotide or 2, 3, 4 or 5 consecutive non-adenine nucleotides for every 39 consecutive adenine nucleotides.

[0152] 29. The DNA according to any one of the preceding embodiments, wherein the non-adenine nucleotide is guanine, cytosine or thymine.

[0153] 30. The DNA of embodiment 29, wherein the non-adenine nucleotide is a guanine nucleotide.

[0154] 31. The DNA of embodiment 29, wherein the non-adenine nucleotide is a cytosine nucleotide.

[0155] 32. The DNA of embodiment 29, wherein the non-adenine nucleotide is a thymine nucleotide.

[0156] 33. The DNA according to embodiment 29, comprising one or more non-adenine nucleotides selected from the group consisting of:

[0157] a. Guanine and thymine nucleotides;

[0158] b. Guanine and cytosine nucleotides;

[0159] c. thymine and cytosine nucleotides; or

[0160] d. Guanine, thymine and cytosine nucleotides.

[0161] 34. The DNA according to any one of the preceding embodiments, wherein the non-adenine nucleotide consists of one non-adenine nucleotide selected from guanine, cytosine and thymine.

[0162] 35. The DNA according to any one of the preceding embodiments, wherein the non-adenine nucleotides comprise two non-adenine nucleotides selected from one or more of guanine, cytosine and thymine.

[0163] 36. The DNA according to any one of the preceding embodiments, wherein the non-adenine nucleotides comprise three non-adenine nucleotides selected from one or more of guanine, cytosine and thymine.

[0164] 37. The DNA according to any one of the preceding embodiments, wherein the adenine nucleotide is adenosine monophosphate.

[0165] 38. The DNA according to any one of the preceding embodiments, wherein the protein is a therapeutic protein.

[0166] 39. The DNA of embodiment 38, wherein the protein is a cytokine or a chemokine.

[0167] 40. The DNA of embodiment 38, wherein the protein is a growth factor.

[0168] 41. The DNA of embodiment 38, wherein the protein is Cas9 or a modified Cas9.

[0169] 42. An mRNA encoded by the DNA according to any one of the preceding embodiments.

[0170] 43. The DNA according to any one of the preceding embodiments, wherein the DNA is in a vector.

[0171] 44. The DNA of embodiment 43, wherein the vector is within a host cell.

[0172] 45. A DNA according to embodiment 43, wherein the one or more non-adenine nucleotides prevent loss of nucleotides encoding the poly (A) tail within the vector during growth of the host cell compared to the loss that occurs in a DNA comprising nucleotides encoding a poly (A) tail of similar or identical length containing only adenine nucleotides.

[0173] 46. ​​A method for producing mRNA from the DNA vector of embodiment 43, comprising:

[0174] a. Linearize the vector downstream of the poly A tail;

[0175] b. denaturing the linearized vector; and

[0176] c. contacting the denatured DNA with RNA polymerase in the presence of guanine, cytosine, uracil and adenine nucleotides.

[0177] Description of the sequence

[0178] This table provides a list of some of the sequences cited herein. Note again that when referring to the RNA form of the DNA sequence in the table below, T is replaced by U. When referring to the DNA form of the RNA sequence in the table below, U is replaced by T.

[0179] Table 1

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238] A phosphorothioate (PS) linkage or bond refers to a bond in which sulfur replaces one of the non-bridging phosphate oxygens in a phosphodiester linkage (e.g., a bond between nucleotide bases). When phosphorothioates are used to produce an oligonucleotide, the modified oligonucleotide may also be referred to as an S-oligonucleotide.

[0239] "*" may be used to depict PS modification. In the present application, the terms A*, C*, U* or G* may be used to indicate a nucleotide that is linked to the next (eg, 3') nucleotide with a PS bond.

[0240] In the present application, the term "mA*", "mC*", "mU*" or "mG*" may be used to indicate a nucleotide that has been substituted with 2'-O-Me and is linked to the next (eg, 3') nucleotide with a PS bond.

[0241] Examples

[0242] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

[0243] Example 1 - Design and stability of stable plasmids for poly A encoding

[0244] Poly A tails containing non-adenine nucleotides were designed. The stability of plasmids encoding these poly A tails with consecutive adenine nucleotides and non-adenine nucleotides (eg, interruption sequences) was compared to a poly A tail composed of adenine nucleotides only.

[0245] Table 2 highlights the problem of the loss of the number of adenosines in the mRNA poly A tail composed only of adenosine. The sequence of the poly A tail containing 96 adenosines was inserted into the pUC57 plasmid (Genscript) and transformed into E. coli. The cells were plated on LB-Amp plates and incubated overnight at 30°C or 37°C. Eight colonies were picked and inoculated into a 96-well plate with LB-Amp medium and grown overnight at 30°C or 37°C (Day 1). Samples from day 1 cultures were added to fresh LB-Amp medium and grown for another two days at 30°C or 37°C (Day 2). DNA was purified from day 1 and day 2 cultures and sequenced to determine the length of the poly A tail in the plasmid. Exemplary results are shown in Tables 2 and 3 below. Figure 1 middle.

[0246] Table 2: Poly A lengths after plasmid growth in E. coli

[0247]

[0248] For multiple colonies, each round of growth is accompanied by a reduction in the number of adenosines in the poly A tail, with only one colony maintaining more than 90 adenosines through two rounds of replication. In addition, the size of the bacterial colony is related to the loss of the poly A tail length in the plasmid (that is, the larger colony corresponds to the loss of the poly A length), which indicates that the sequence encoding the longer poly A tail may inhibit bacterial growth during plasmid production. DNA purified from E. coli colonies represents the DNA colony from individual E. coli containing plasmid DNA. Therefore, the values ​​provided in Table 2 (and similar values ​​described herein) represent the average poly A length of the colony. In addition, during PCR and sequencing of long repeats (such as poly A), polymerase may slip, thereby causing the sequence to look slightly shorter than the actual sequence. Therefore, for the result showing 95 adenosines, it is not certain whether the plasmid has lost an adenosine, or whether it is a PCR artifact. However, during PCR amplification and sequencing, significant loss is not an artifact of polymerase slippage.

[0249] In a separate experiment, E. coli was transformed with the pUC57 plasmid containing the poly A tail of SEQ ID NO: 1 and plated on LB-Amp plates. Eight clones were cultured through two rounds of growth and tested for maintenance of the sequence encoding the poly A tail. Representative data for one clone is shown in Figure 2 In which no change in the tail size of the poly A tail of SEQ ID NO: 1 was seen in 2 rounds of growth of the plasmid encoding it. Miniprep 1 refers to the first round of growth, while miniprep 2 refers to the second round of growth. Miniprep was performed using the Invitrogen Purelink Rapid Plasmid Miniprep Kit.

[0250] Test the ability of plasmids encoding poly A tails (SEQ ID NO:3) with additional non-adenosine patterns to tolerate replication in E. coli. The sequence containing the poly A tail of SEQ ID NO:3 is inserted into pUC19 plasmid (GenScript) and transformed into E. coli. The cells are plated on LB-Kan plates and incubated overnight at 30°C or 37°C. Pick 8 colonies and inoculate them into 96-well plates with LB-Kan culture medium and grow overnight at 30°C or 37°C (Day 1). Samples from day 1 cultures are added to fresh LB-Kan culture medium and grown for two more days (Day 2) at 30°C or 37°C. DNA is purified from day 1 and day 2 cultures and sequenced to determine the length of the poly A tail in the plasmid. Of the eight day 1 cultures sequenced, six maintained segments of 25, 24, 24, and 24 adenosines, and of the twelve day 2 cultures sequenced, nine maintained segments of 25, 24, 24, and 24 adenosines, demonstrating improved poly A retention compared to the adenosine-only sequence.

[0251] These data indicate that DNA encoding a poly A tail comprising non-adenine nucleotides has improved stability over multiple rounds of plasmid growth and purification compared to DNA encoding a poly A tail containing only adenosine.

[0252] Example 2 - Activity of constructs with poly A tails containing non-adenine nucleotides

[0253] Experiments were performed to determine whether there was a difference in the effectiveness of mRNA with poly A tails containing non-adenine nucleotides (interrupt sequences) and those mRNA with poly A tails containing only adenosine. A model system was used in which the mRNA encoding Cas9 protein was transfected into HEK-293 cells by electroporation with a reporter plasmid encoding secreted embryonic alkaline phosphatase (SEAP) and a guide RNA targeting SEAP. The successful expression of Cas9 protein from mRNA caused the SEAP target sequence to crack, thereby causing a color change reflecting the reduction in SEAP production. SEAP HEK-Blue reporter reagent was obtained from Invivogen. The sequence (SEQ ID NO:6) of Cas9mRNA containing T7 promoter and encoding with only adenosine poly A tail (designed with 100 adenosine nucleotides, but shown as having 97 adenosine nucleotides by sequencing) or the sequence (SEQ ID NO:7) of Cas9 mRNA containing T7 promoter and encoding with poly A tails of SEQ ID NO:1 was cloned into pUC57 plasmid (GenSher). mRNAs were generated by in vitro transcription from linearized plasmids encoding each mRNA.

[0254] Figure 3Shown are titrations of Cas9 mRNA with either adenosine poly A alone or poly A of SEQ ID NO: 1 at concentrations of 0.005-50 nM and 1 μM of a single guide RNA targeting SEAP (SEQ ID NO: 8) in a HEK-Blue cell assay.

[0255] The HEK-Blue results show that in the dose-response curve, the effect of mRNA with either poly A tail is similar. Since the baseline blue indicates SEAP expression, as evidenced by the color change to pink, higher concentrations of mRNA lead to a decrease in SEAP reporter gene expression. Therefore, poly A tails containing non-adenine nucleotides do not alter the expression and functional efficacy of the Cas9 construct compared to poly A tails containing only adenosine.

[0256] The editing efficacy conferred by expression of the Cas 9 mRNA of SEQ ID NO: 6 was also compared to the Cas9 mRNA of SEQ ID NO: 7 (i.e., the adenosine poly A tail alone was compared to the poly A tail of SEQ ID NO: 1). In these experiments, HEK-Blue cells were transfected with sgRNA (SEQ ID NO: 8) and the two different mRNAs by electroporation.

[0257] Figure 4 The percentage of SEAP inhibition after 24 h incubation is shown for both constructs. 50 are similar, 0.050 and 0.054 respectively.

[0258] Figure 5 The percentage of SEAP inhibition after 48 h incubation is shown for both constructs. 50 are similar, 0.086 and 0.082 respectively.

[0259] mRNA expression and activity were also confirmed in vivo. Cas9 mRNA of SEQ ID NO:6 (HiCas9 mRNA) and SEQ ID NO:7 (interrupted polyAmRNA) were formulated into lipid nanoparticles (LNPs) with a single guide RNA of SEQ ID NO:9 (targeting the mouse TTR gene) in a 1:1 weight ratio and administered to CD-1 female mice (n=5) by intravenous administration of 1 mg / kg or 0.5 mg / kg of total RNA. Blood was collected from the animals 7 days after administration, and serum levels of TTR protein were measured by ELISA. Briefly, total TTR serum levels were determined using a mouse prealbumin (transthyretin) ELISA kit (Aviva Systems Biology, catalog number OKIA00111). Kit reagents and standards were prepared according to the manufacturer's protocol. The plate was read at an absorbance of 450 nm on a SpectraMax M5 plate reader. Serum TTR levels were calculated using a four-parameter logistic curve fitting standard curve by SoftMax Pro software version 6.4.2. Final serum values ​​were adjusted for assay dilution.

[0260] Figure 6 It was shown that 7 days after dosing, the serum TTR knockdown levels (representing the editing percentage of the TTR gene) of the two poly A constructs were comparable. The serum TTR knockdown results were confirmed by sequencing the TTR locus in the liver of mice harvested on day 7. Mice receiving only adenosine poly A mRNA showed 61.74% and 69.84% editing at 0.5 mg / kg and 1 mg / kg total RNA, respectively, while mice receiving poly A mRNA containing non-adenosine nucleotides showed 63.14% and 70.82% editing at 0.5 mg / kg and 1 mg / kg total RNA.

[0261] Thus, expressing Cas9 mRNA with a poly A tail containing non-adenine nucleotides resulted in similar editing efficacy compared to Cas9 mRNA with a poly A tail containing only adenosine.

[0262] Example 3 - Activity of constructs with poly A tails containing other interruption sequences

[0263] Experiments were performed as in Example 2 to determine the efficacy of mRNAs with poly A tails containing non-adenine nucleotides versus those with poly A tails containing only adenosine nucleotides. Sequences containing a T7 promoter and encoding Cas9 mRNAs having interrupted poly A tails comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11 were prepared by PCR amplification using primers incorporating poly A sequences. mRNAs were generated from these PCR products by in vitro transcription. mRNA of SEQ ID NO: 18 was generated from a linearized plasmid encoding the mRNA by in vitro transcription.

[0264] Figure 7 Titration of Cas9 mRNA with only adenosine poly A [100PA] or poly A of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11 and 1 μM single guide RNA targeting SEAP (SEQ ID NO: 8) at concentrations of 0.02-6 nM is shown in a HEK-Blue cell assay. Specifically, Figure 7 The percent inhibition of SEAP after 48 hours of incubation is shown for the constructs and the EC50 values ​​are provided below in Table 3. All constructs were active.

[0265] Table 3: EC50 values ​​for SEAP inhibition

[0266]

[0267]

[0268] Example 4 - Cloning of long poly A with interruption sequence

[0269] A 300 nucleotide long poly A tail SEQ ID NO: 18 [300pa] was designed that contained 12 interruption sequences from Table 4 (below) and 13 repeats of 12 consecutive adenosines. The anchor sequence of SEQ ID NO: 18 was designed to minimize hybridization and self-annealing between trinucleotide interruption sequences within approximately 300 nt of the poly A tail. Table 4 below provides interruption sequences that minimize annealing between interruption sequences and includes the anchors used in this experiment.

[0270] To clone SEQ ID NO: 18, each of the sequences poly A-1 (SEQ ID NO: 12), poly A-2 (SEQ ID NO: 13), poly A-3 (SEQ ID NO: 14), and poly A-4 (SEQ ID NO: 15) were generated in pUC57 minivectors (GenScript). pA1-2 plasmids were generated by amplifying SEQ ID NO: 12 with Bcl11a primers, digesting the PCR product with restriction enzymes XhoI and AcII, and ligating the restriction fragments into pA2 plasmids containing SEQ ID NO: 13 digested with XhoI and BstBI. pA3-4 plasmids were generated in the same manner, amplifying SEQ ID NO: 14 and ligating it into the same restriction sites on plasmid pA4. The pA1-4 plasmid (comprising SEQ ID NO: 18) was assembled by amplifying the SEQ ID NO: 17 sequence from pA3-4, digesting the PCR fragment with BbsI and XbaI restriction enzymes and cloning the restriction fragment into the polyA1-2 (SEQ ID NO: 16) construct digested with BbsI and XbaI restriction enzymes. The insert fragments in pA1-2 and pA3-4 were evaluated from both directions by Sanger sequencing using [pUC-M seq2 forward primer and pUC-M seq reverse primer] as primers (SEQ ID Nos: 20 and 21).

[0271] The resulting poly A sequence of SEQ ID NO: 18 (300PA) was excised by digesting pA1-4 with XhoI and XbaI for cloning into the same site in the protein encoding vector. All steps were performed under standard conditions.

[0272] Table 4:

[0273]

[0274]

Claims

1. A DNA comprising nucleotides encoding a polyadenylation (poly A) tail located 3' to nucleotides encoding a protein of interest, wherein the poly A tail comprises a first homopolymer sequence of at least 8 consecutive adenine (A) nucleotides and an interrupting sequence comprising one or more non-adenine (A) nucleotides.

2. An mRNA encoded by the DNA according to claim 1.

3. The DNA according to claim 1, wherein the DNA is in a vector.

4. The DNA according to claim 3, wherein the vector is within a host cell.

5. The DNA of claim 3, wherein the one or more non-adenine nucleotides prevent loss of nucleotides encoding the poly A tail within the vector during growth of the host cell, compared to the loss that occurs in a DNA comprising nucleotides encoding a poly A tail of similar or identical length containing only adenine nucleotides.

6. A method for producing mRNA from the DNA vector of claim 3, comprising: a. Linearize the vector downstream of the poly A tail; b. denaturing the linearized vector; as well as c. contacting the denatured DNA with RNA polymerase in the presence of guanine, cytosine, uracil and adenine nucleotides.

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