RNA encoding protein

Abstract

The present invention relates to mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide, and a transcription unit, an expression vector or a gene therapy vector comprising a nucleic acid encoding a protein and a signal peptide. Also disclosed herein are therapeutic compositions comprising the mRNA, transcription unit, expression vector or gene therapy vector and uses of the therapeutic compositions in the treatment of a disease or condition.

Description

[0001] This application is a divisional application of PCT application PCT / EP2019 / 086019, filed on December 18, 2019, with the invention name “Protein-encoding RNA”. The date on which the PCT application entered the Chinese national phase is August 16, 2021, and the application number is 201980092197.7. Technical Field

[0002] The present invention relates to an mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0003] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0004] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0005] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0006] The present invention further relates to an mRNA comprising a nucleic acid sequence encoding:

[0007] i) a protein; ii) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase. The present invention relates to a transcription unit or expression vector comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0008] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0009] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0010] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid. The present invention also relates to a transcription unit or expression vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF). The present invention also relates to therapeutic compositions and kits comprising mRNA and / or transcription units or expression vectors. The present invention also relates to mRNA, transcription units or expression vectors, therapeutic compositions and / or kits for use as a medicament, in particular to an mRNA comprising a nucleic acid sequence encoding i) IGF1; and ii) a signal peptide of brain-derived neurotrophic factor (BDNF), for use as a medicament. The present invention also relates to mRNA and therapeutic compositions thereof for use in methods for treating skeletal muscle injuries. Background Art

[0011] In the past, different attempts have been made to increase the expression and secretion yield of the encoded protein, particularly by using improved in vitro and / or in vivo expression systems. The methods for increasing expression and secretion generally described in the prior art are generally based on the use of expression vectors or expression cassettes containing specific promoters and corresponding regulatory elements. Since these expression vectors or expression cassettes are generally limited to specific cell systems, these expression systems must be adjusted to be applicable to different cell systems. The adjusted expression vectors or expression cassettes are then generally transfected into cells and are generally processed according to specific cell lines. Therefore, it is mainly preferred that nucleic acid molecules, such as mRNA, that can express the encoded protein in target cells through the intrinsic system of the cell are not related to the specific promoters and regulatory elements of the specific cell type. In this case, it is possible to distinguish between mRNA stabilizing elements and elements that increase mRNA translation efficiency. For example, WO 02 / 098443 describes mRNA that is generally in a stable form and is optimized for translation in its coding region. WO 02 / 098443 further discloses a method for determining sequence modification. WO 02 / 098443 describes in addition the possibility of replacing adenine and uracil nucleotides to increase the guanine / cytosine (G / C) content of the sequence in the mRNA sequence. In this case, WO 02 / 098443 generally mentions the sequence as the base sequence of this type of modification, wherein the mRNA encoding of the modification is at least one biologically active peptide or polypeptide, and the biologically active peptide or polypeptide is, for example, not translated completely, or insufficiently translated or there is mistranslation in the patient to be treated. In another method for increasing the expression of the encoded protein, application WO 2007 / 036366 describes the long poly (A) sequence (particularly longer than 120bp) of the beta globin gene and the positive effect of the combination of at least two 3 ' non-translated regions on the stability of mRNA and translational activity. Despite all the progress achieved in this area, the effective expression of the encoded protein in a cell-free system, cell or organism, particularly effective secretion (recombinant expression) remains a challenging problem. Summary of the Invention

[0012] The present invention provides an mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein the amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from

[0013] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0014] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0015] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0016] The present invention further provides an mRNA comprising a nucleic acid sequence encoding

[0017] i) protein; and

[0018] ii) a signal peptide heterologous to said protein,

[0019] wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase, in particular the present invention provides an mRNA comprising a nucleic acid sequence encoding

[0020] i) protein; and

[0021] ii) a signal peptide heterologous to said protein,

[0022] wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is selected from the group consisting of carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases and vasoactive proteins.

[0023] The present invention further provides a transcription unit or expression vector comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein the amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0024] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0025] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0026] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0027] The present invention further provides a transcription unit or expression vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase. The present invention further provides a transcription unit or expression vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is selected from the group consisting of carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases and vasoactive proteins. The present invention further provides a therapeutic composition comprising the above-mentioned mRNA and / or transcription unit or expression vector. The present invention further provides a kit comprising the above-mentioned mRNA, transcription unit or expression vector and / or therapeutic composition, as well as instructions, optionally a vector map, optionally a host cell, optionally a culture medium for culturing the host cell, and / or optionally a selective culture medium for selecting and culturing the transfected host cell. The present invention further provides the above-mentioned mRNA, transcription unit or expression vector, therapeutic composition or kit for use as a medicament. The present invention further provides an mRNA comprising a nucleic acid sequence encoding i) IGF1; and ii) a signal peptide of brain-derived neurotrophic factor (BDNF) for use as a medicament. The present invention further provides an mRNA or a therapeutic composition comprising the mRNA for use in a method for treating skeletal muscle injury.

[0028] The present inventors surprisingly found an mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein the amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from

[0029] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0030] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0031] iii) a naturally occurring amino acid sequence which naturally does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid,

[0032] Cells transfected with mRNA encoding a protein and its native, homologous signal peptide provide more efficient protein secretion compared to cells transfected with mRNA encoding the protein and its native, homologous signal peptide. Specifically, the inventors surprisingly discovered that cells transfected with an mRNA comprising a nucleic acid sequence encoding i) a protein; and ii) a BDNF signal peptide heterologous to the protein secrete the protein more efficiently than cells transfected with the protein's native, homologous signal peptide. The amount of protein secreted is up to six times greater than that obtained with an mRNA containing the same protein and its native, homologous signal peptide. This unexpected discovery can be used to efficiently deliver and express mRNA encoding a desired protein in cells, resulting in higher protein secretion than with the protein's native, homologous signal peptide. The higher protein secretion achieved by the present invention with the same amount of mRNA is extremely useful for reducing the therapeutic dose required for local application to tissues, thereby increasing its safety window and combating potential mRNA-related side effects. Furthermore, it makes this application more suitable for controlled-release and device-coated formulations. In addition, it reduces the risk of immunogenicity associated with mRNA and makes this application more suitable for tissues with limited injection volume or previously inaccessible tissues. The inventors have also found that mRNA, particularly mRNA encoding human IGF-1, can be effectively delivered and expressed to skeletal muscle, thereby allowing the expression of the desired polypeptide in skeletal muscle, thereby providing relevant functional benefits to the muscle. The mRNA is preferably present in a liquid composition, preferably in a naked form. The liquid composition can be delivered directly to skeletal muscle, for example, by injection, and does not require any gene transfer vector or carrier for mRNA or methods for enhancing transfer to tissues, such as electrotransfer or ultrasound. Moreover, it has been shown that injecting mRNA into injured skeletal muscle accelerates the recovery process and leads to enhanced skeletal muscle function. Surprisingly, animals treated with mRNA encoding IGF-1 reached functional levels within the healthy range by 16 days. In contrast, control animals treated with vehicle did not achieve complete functional recovery even by day 28. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1The DNA and RNA sequences of Cpd.1 are shown. (A) shows the DNA sequence of human codon-optimized IGF1 (SEQ ID No: 1), which contains its pre-domain, pro-domain, and coding domain. The sequence of the pre-domain (signal peptide) is in italics, and the sequence of the pro-domain is in Underline The IGF-1 coding domain is in bold and the stop codon is in bold (B) shows the RNA sequence of the pre-, pro-, and coding domains of human IGF1 (SEQ ID NO: 2), where uridine is N1-methyl pseudouridine. The pre- and pro-domains are cleaved during secretion.

[0034] Figure 2 The DNA and RNA sequences of Cpd.2 are shown. (A) shows the DNA sequence of human codon-optimized IGF1 (SEQ ID NO: 3), which contains the IGF2 predomain and the IGF1 pro- and coding domains. The sequence of the predomain (signal peptide) is in italics and the sequence of the prodomain is in Underline The IGF1 coding domain is in bold and the stop codon is in bold (B) shows the RNA sequence of the pre- and pro-domains of IGF2 and IGF1 (SEQ ID NO: 4), wherein the uridine is N1-methyl pseudouridine. The pre- and pro-domains are cleaved upon secretion.

[0035] Figure 3 The DNA and RNA sequences of Cpd.3 are shown. (A) shows the DNA sequence of human codon-optimized IGF1 (SEQ ID NO: 5), which contains the ALB predomain and the IGF1 pro- and coding domains. The sequence of the predomain (signal peptide) is in italics and the sequence of the prodomain is in Underline The IGF1 coding domain is in bold and the stop codon is in bold (B) shows the RNA sequence of the pre- and pro-domains of ALB, IGF1 (SEQ ID NO: 6), where uridine is N1-methyl pseudouridine. The pre- and pro-domains are cleaved upon secretion.

[0036] Figure 4 The DNA and RNA sequences of Cpd.4 are shown. (A) shows the DNA sequence of human codon-optimized IGF1 (SEQ ID NO: 7), which contains the BDNF predomain and the IGF1 pro- and coding domains. The sequence of the predomain (signal peptide) is in italics, and the sequence of the prodomain is in Underline The IGF1 coding domain is in bold and the stop codon is in bold(B) shows the RNA sequence of the pre- and pro-domains of BDNF, IGF1, and IGF2 encoding domains (SEQ ID NO: 8), where uridine is N1-methylpseudouridine. The pre- and pro-domains are cleaved upon secretion.

[0037] Figure 5 The DNA and RNA sequences of Cpd.5 are shown. (A) shows the DNA sequence of human codon-optimized IGF1 (SEQ ID NO: 9), which contains the CXCL12 predomain and the IGF1 pro- and coding domains. The sequence of the predomain (signal peptide) is in italics, and the sequence of the prodomain is in Underline The IGF1 coding domain is in bold and the stop codon is in thick body (B) shows the RNA sequence of the pre- and pro-domains encoding CXCL12 and IGF1 (SEQ ID NO: 10), where uridine is N1-methylpseudouridine. The pre- and pro-domains are cleaved upon secretion.

[0038] Figure 6 The DNA and RNA sequences of Cpd.6 are shown. (A) shows the DNA sequence of human codon-optimized IGF1 (SEQ ID No: 11), which contains the synthetic signal peptide 1 pre-domain and the IGF1 pro- and coding domain. The sequence of the pre-domain (signal peptide) is in italics, and the sequence of the pro-domain is in Underline The IGF1 coding domain is in bold and the stop codon is in bold (B) shows the RNA sequence of the synthetic signal peptide 1 pre- and pro-domain encoding IGF1 (SEQ ID NO: 12), where uridine is N1-methyl pseudouridine. The pre- and pro-domains are cleaved during secretion.

[0039] Figure 7 The DNA and RNA sequences of Cpd.7 are shown. (A) shows the DNA sequence of human codon-optimized IGF1 (SEQ ID NO: 13), which contains the synthetic signal peptide 2 pre-domain and the IGF1 pro- and coding domains. The sequence of the pre-domain (signal peptide) is in italics, and the sequence of the pro-domain is in Underline The IGF1 coding domain is in bold and the stop codon is in bold (B) shows the RNA sequence of the synthetic signal peptide 2 pre- and pro-domain encoding IGF1 (SEQ ID NO: 14), where uridine is N1-methyl pseudouridine. The pre- and pro-domains are cleaved during secretion.

[0040] Figure 8 The DNA sequence of the vector pVAX.A120 is shown, in which Cpd.1 is inserted into bold Marker (SEQ ID NO: 15). The ORF of Cpd.1 was digested from its original plasmid and subcloned into the vector.

[0041] Figure 9 The DNA sequence of the vector pMA-T is shown, in which Cpd.2 is inserted into bold Marker (SEQ ID NO: 16). The ORF of Cpd.2 was digested from its original plasmid and subcloned into the vector.

[0042] Figure 10 The DNA sequence of the vector pMA-T is shown, in which Cpd.3 is inserted into bold Marker (SEQ ID NO: 17). The ORF of Cpd.3 was digested from its original plasmid and subcloned into the vector.

[0043] Figure 11 The DNA sequence of the vector pMA-T is shown, in which Cpd.4 is inserted into bold Marker (SEQ ID NO: 18). The ORF of Cpd.4 was digested from its original plasmid and subcloned into the vector.

[0044] Figure 12 The DNA sequence of the vector pMA-T is shown, in which Cpd.5 is inserted into bold Marker (SEQ ID NO: 19). The ORF of Cpd.5 was digested from its original plasmid and subcloned into the vector.

[0045] Figure 13 The DNA sequence of the vector pMA-RQ is shown, in which Cpd.6 is bold Marker (SEQ ID NO: 20). The ORF of Cpd.6 was digested from its original plasmid and subcloned into the vector.

[0046] Figure 14 The DNA sequence of the vector pMA-RQ is shown, in which Cpd.7 is expressed as bold Marker (SEQ ID NO: 21). The ORF of Cpd.7 was digested from its original plasmid and subcloned into the vector.

[0047] Figure 15 The forward (SEQ ID NO: 22) and reverse primer (SEQ ID NO: 23) sequences of the pMA-T and pMA-RQ plasmids used for IVT amplification of mRNA are shown.

[0048] Figure 16The gene names, UniProt numbers, codon-optimized DNA and amino acid sequences, and vectors for the Cpd.1-Cpd.7 signal peptides are shown. 1-Cpd.7 (SEQ ID Nos: 24-37). Note that the signal peptides for Cpd.6 and Cpd.7 are synthetic peptides that do not match known protein sequences in public databases.

[0049] Figure 17 Figure 24: IGF1 secretion induced by human embryonic kidney cells (HEK293T) transfected with mRNA of Cpd.1-Cpd.7. HEK293T cells were transfected with 2 μg of Cp.1-Cpd.7, and secreted IGF1 was measured in the cell culture supernatant using a specific ELISA after 24 hours. The IGF1 secretion induced by Cpd.4 was significantly higher than that of Cpd.1 (3.3 times). The data represent the standard error of the mean ± mean of 4 repetitions. Significance was assessed subsequently by one-way ANOVA (***, <0.001) using Dunnett's multiple comparison test.

[0050] Figure 18 Figure 2 shows the concentration dependence of IGF1 secretion induced in HEK293T cells after mRNA transfection with Cpd.1 or Cpd.4. Cells were transfected with different concentrations (0, 0.02, 0.06, 0.2, 0.6 or 2 μg) of Cpd.1 or Cpd.4, and secreted IGF1 was measured in the cell culture supernatant after 24 hours using a specific ELISA. 50 0.134μg) than Cpd.1(EC 50 0.889 μg) was significantly more effective in inducing IGF1 secretion. Data represent the mean ± standard error of the mean of 2 replicates. Significance was assessed by two-way ANOVA of the two curves (***, <0.001).

[0051] Figure 19 Figure 2 shows that mouse skeletal muscle cells (C2C12) secrete IGF1 by inducing mRNA transfection with Cpd.1-Cpd.7. C2C12 cells were transfected with 2 μg of Cp.1-Cpd.7, and secreted IGF1 was measured in the cell culture supernatant using a specific ELISA after 24 hours. The IGF1 secretion induced by Cpd.4 was significantly higher than that induced by Cpd.1 (6.1 times). The data represent the mean ± standard error of the mean of 4 repetitions. Significance was assessed by one-way ANOVA followed by Dunnett's multiple comparison test (***, <0.001).

[0052] Figure 20Figure 2 shows that human primary skeletal muscle cells (HSkMC) secrete IGF1 by inducing mRNA transfection with Cpd.1 and Cpd.4. HSkMC cells were transfected with 2 μg of Cp.1 or Cpd.4, respectively, and secreted IGF1 was measured in the cell culture supernatant using a specific ELISA after 24 hours. The IGF1 secretion induced by Cpd.4 was significantly higher than that induced by Cpd.1 (3.1 times). The data represent the mean ± standard error of the mean of 3 repetitions. Significance was assessed by one-way ANOVA followed by Dunnett's multiple comparison test (**, P < 0.01).

[0053] Figure 21 Figure 2 shows functional recovery of the tibialis anterior (TA) muscle after notexin injury. After notexin injury was induced by intramuscular injection (day 0), two IGF-I mRNA treatments (Cpd.4 (1 μg)) were applied by intramuscular injection on days 1 and 4 (see arrows). The control group received vehicle solution. Muscle function was assessed on days 1, 4, 7, 10, 14, 21, and 28 after injury. The data represent the mean ± standard error of the mean (SEM) of 5 mice per group and time point. Asterisks indicate significant differences (p < 0.05) between the Cpd.4-treated group and the control group as assessed by Student's t-test.

[0054] Figure 22 Figure 22 shows that IGF1 secretion was induced in human embryonic kidney cells (HEK293T) by mRNA transfection with Cpd.1 (as a control) and Cpd.8-Cpd.26. HEK293T cells were transfected with 0.3 μg Cpd.1 and Cpd.8-Cpd.26, respectively, and secreted IGF1 was measured in cell culture supernatant using specific ELISA after 24 hours. IGF1 secretion was standardized for Cpd.1 IGF1 secretion. Cpd.8, 9, 10, 11, 12 and 13 showed that IGF1 secretion decreased, while the IGF1 secretion induced by Cpd.14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25 and 26 was higher than that by Cpd.1 (up to 2.6 times). Data represent the standard error of the mean ± mean value of 2-11 repetitions of each Cpd. The significance of each Cpd. compared with Cpd.1 was assessed by Student's t-test (*, p<0.05; **, p<0.001; ***, <0.001).

[0055] Figure 23Figure 24 shows that mRNA transfection with Cpd.1 (as a control) and Cpd.4-Cpd.26 induces human hepatocytes (HepG2) to secrete IGF1. HepG2 cells were transfected with 0.3 μg Cp.1 and Cpd.4-Cpd.26, and secreted IGF1 was measured in cell culture supernatant using specific ELISA after 24 hours. IGF1 secretion was normalized for Cpd.1. Cpd.8, 9 and 12 showed that IGF1 secretion decreased, while the IGF1 secretion induced by Cpd.4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26 was higher than that of Cpd.1 (up to 8.3 times). Data represent the standard error of mean ± mean value of 2-4 repetitions of each Cpd. The significance of each Cpd. compared with Cpd.1 was assessed by Student's t-test (**, p<0.01; ***, <0.001).

[0056] Figure 24 Figure 24: IGF1 secretion induced by mRNA transfection of Cpd.1 (as a control) and Cpd.4-Cpd.24 in human neural cells (IMR32). 0.3 μg Cp.1 and Cpd.4-Cpd.24 were used to transfect IMR32 cells, and secreted IGF1 was measured in cell culture supernatant using specific ELISA after 24 hours. IGF1 secretion was standardized for Cpd.1 IGF1 secretion. The IGF1 secretion induced by Cpd.4, 14, 15, 16, 17, 20, 22, 23 and 24 was higher than that by Cpd.1 (up to 2.6 times). The data represent the standard error of the mean ± mean value of 2-6 repetitions of each Cpd. The significance of each Cpd. compared with Cpd.1 was assessed by student's t test (*, p < 0.05; ***, < 0.001).

[0057] Figure 25 Figure 22 shows that human primary chondrocytes were induced to secrete IGF1 by mRNA transfection with Cpd.1 (as a control) and Cpd.4-Cpd.25. Chondrocytes were transfected with 0.6 μg Cp.1 and Cpd.4-Cpd.25, respectively, and secreted IGF1 was measured in cell culture supernatant using specific ELISA after 24 hours. IGF1 secretion was standardized for Cpd.1 IGF1 secretion. The IGF1 secretion induced by Cpd.4, 14, 15, 16, 20, 21, 22, 24 and 25 was higher than that by Cpd.1 (up to 1.9 times). The data represent the mean ± standard error of the mean of 1-2 repetitions of each Cpd. The significance of each Cpd. compared with Cpd.1 was assessed by Student's t test (*, p < 0.05; ***, < 0.001).

[0058] Figure 26 Shows the induction of rat wild-type (A) or SOD1G expression by transfection with mRNAs of Cpd.1 (as a control) and Cpd.4-Cpd.17. S93A (B) IGF1 secretion by primary motor neurons. Rat wild-type primary motor neurons were transfected with 0.3 μg of Cp.1, Cpd.4, Cpd.14, and Cpd.17, respectively, and rat SOD1G was transfected with 0.3 μg of Cp.1, Cpd.14, and Cpd.17, respectively. S93A For primary motor neurons, secreted IGF1 was measured in the cell culture supernatant after 48 hours using a specific ELISA. IGF1 secretion was normalized to Cpd.1 IGF1 secretion. Cpd.4, 14, and 17 induced IGF1 secretion higher than Cpd.1 (up to 4.3-fold in wild type and up to 1.5-fold in SOD1). S93A The data represent the mean ± standard error of the mean of two replicates for each Cpd. The significance of each Cpd. compared with Cpd.1 was evaluated by Student's t-test, revealing no statistical difference.

[0059] Figure 27 Shown by inducing human embryonic kidney cells (HEK293T, A), human liver cells (HepG2, B) and human lung cancer cells (A549, C) to secrete EPO by mRNA transfection with Cpd.27, Cpd.28 or Cpd.29. Cells were transfected with 0.3-0.9 μg Cp.27, Cpd.28 or Cpd.29 respectively, and secreted EPO was measured in cell culture supernatant using specific ELISA after 24 hours. EPO secretion was standardized for Cpd.27. In all three cell types analyzed, the EPO secretion induced by Cpd.28 and 29 was higher than that of Cpd.27 (up to 1.8 times). Data represent the standard error of the mean ± mean value of 3-8 repetitions of each Cpd. The significance of each Cpd. compared with Cpd.27 was assessed by student's t test (*, p < 0.05; ***, < 0.001).

[0060] Figure 28Shown is the induction of INS secretion in human embryonic kidney cells (HEK293T) by transfection with mRNA of Cpd.30, Cpd.31 or Cpd.32. Cells were transfected with 0.6 μg of Cp.30, Cpd.31 or Cpd.32, respectively, and secreted INS was measured in the cell culture supernatant using a specific ELISA after 24 hours. INS secretion was standardized for Cpd.30. INS secretion induced by Cpd.31 and 32 was higher than that of Cpd.30 (up to 3.9 times). The data represent the mean ± standard error of the mean of 3-5 repetitions of each Cpd. The significance of each Cpd. compared to Cpd.30 was assessed by Student's t-test (*, p < 0.05; ***, < 0.001).

[0061] Figure 29 Shown by inducing human embryonic kidney cells (HEK293T, A), human hepatocytes (HepG2, B), human monocytes (THP-1, C) and human lung cancer cells (A549, D) to secrete IL4 with the mRNA transfection of Cpd.33, Cpd.34 or Cpd.35. Cells were transfected with 0.5-0.6 μg Cp.33, Cpd.34 or Cpd.35 respectively, and secreted IL4 was measured in cell culture supernatant using specific ELISA after 24 hours. IL4 secretion was standardized for Cpd.33. In all three cell types analyzed, the IL4 secretion induced by Cpd.34 and 35 was higher than that of Cpd.33 (up to 2.2 times). Data represent the standard error of the mean ± mean value of 3-8 repetitions of each Cpd. The significance of each Cpd. compared with Cpd.33 was assessed by Student's t-test (*, p<0.05; ***, <0.001).

[0062] Figure 30 Shown by inducing human embryonic kidney cells (HEK293T, A), human hepatocytes (HepG2, B) or human monocytes (THP-1, C) to secrete IL10 with the mRNA transfection of Cpd.36, Cpd.37 or Cpd.38. Cells were transfected with 0.3-0.6 μg Cp.36, Cpd.37 or Cpd.38 respectively, and secreted IL10 was measured in cell culture supernatant using specific ELISA after 24 hours. IL10 secretion was standardized for Cpd.36. In all three cell types analyzed, the IL10 secretion induced by Cpd.37 and 38 was higher than that of Cpd.36 (up to 2.2 times). Data represent the standard error of the mean ± mean value of 4-8 repetitions of each Cpd. The significance of each Cpd. compared with Cpd.36 was assessed by Student's t-test (**, p<0.01; ***, <0.001).

[0063] Figure 31 Figure 3 shows that human hepatocytes (HepG2, A) and human primary chondrocytes (B) secrete IGF-1 by inducing mRNA transfection with Cpd.39. Cells were transfected with 0.3-0.6 μg Cp.39 respectively, and secreted IGF1 was measured in cell culture supernatant using specific ELISA after 24 hours. IGF1 secretion was standardized for Cpd.1 IGF1 secretion. In all two cell types analyzed, the IGF1 secretion induced by Cpd.39 was higher than that of Cpd.1 (up to 1.4 times). The data represent the mean ± standard error of the mean of 4-7 repetitions. The significance of each Cpd.1 compared with Cpd.39 was assessed by Student's t test (**, p < 0.01; ***, < 0.001). DETAILED DESCRIPTION

[0064] As used herein, the term "RNA" includes RNA that encodes an amino acid sequence as well as RNA that does not encode an amino acid sequence. Typically, the RNA used herein is coding RNA, i.e., RNA that encodes an amino acid sequence. Such RNA molecules are also referred to as mRNA (messenger RNA) and are single-stranded RNA molecules. Therefore, the term "RNA" used herein preferably refers to mRNA. RNA can be prepared by synthetic chemistry and enzymatic methods known to those of ordinary skill in the art, or by using recombinant technology, or can be isolated from natural sources, or prepared by a combination thereof. RNA may optionally contain non-natural and naturally occurring nucleoside modifications, for example, N 1 -methylpseudouridine, also referred to herein as methylpseudouridine.

[0065] As used herein, the term "mRNA" (i.e., messenger RNA) refers to a polymer composed of nucleoside phosphate building blocks, wherein mainly adenosine, cytidine, uridine and guanosine are used as nucleosides, and it comprises a coding region encoding a protein. In the context of the present invention, mRNA should be understood to mean any polyribonucleotide molecule, if it enters a cell, then is suitable for expressing a protein or its fragment or can be translated into a protein or its fragment. It should be understood that the mRNA of the present invention comprising a nucleic acid sequence encoding a protein and a signal peptide refers to a polyribonucleic acid molecule, if it enters a cell, then is suitable for inducing the expression and secretion of the protein or its fragment. The mRNA of the present invention is an artificial nucleic acid molecule, i.e., an artificial mRNA. Artificial nucleic acid molecules such as artificial mRNA can generally be understood as not being naturally occurring nucleic acid molecules, such as recombinant mRNA. Recombinant mRNA is a preferred mRNA of the present invention. mRNA comprises a ribonucleotide sequence encoding a protein or its fragment, and the function of the protein or its fragment in a cell or near a cell is usually necessary or beneficial, particularly in the case of skeletal muscle injury healing. mRNA can comprise the sequence of a complete protein or its functional variant. Therefore, the nucleic acid sequence of the mRNA of a complete protein generally comprises a nucleic acid sequence encoding a signal peptide and a nucleic acid sequence encoding a protein. The mRNA of the present invention comprises a nucleic acid sequence encoding a protein and a signal peptide. The nucleic acid sequence encoding a protein may optionally comprise a prodomain of the protein, which is generally located at the N-terminus of the protein. Protein and signal peptide are generally encoded by the nucleic acid sequence of the mRNA of the present invention from 5' to 3' in the following order: i) signal peptide and ii) protein, i.e., the last nucleoside of the signal peptide coding region is followed by the first nucleoside of the protein coding region, or when the protein comprises a prodomain, the last nucleoside of the signal peptide coding region is followed by the first nucleoside of the coding region of the proprotein form of the protein. The ribonucleotide sequence can encode a protein or its functional fragment, and the protein plays a role as a factor, inducer, regulator, stimulator or enzyme, wherein the protein is generally a protein necessary for treating a disease (particularly skeletal muscle injury). Here, functional variants are understood to refer to fragments, which can assume the function of a protein of a desired function in a cell. In addition, mRNA can also have other functional regions and / or 3' or 5' non-coding regions. The 3' and / or 5' non-coding regions can be natural flanking regions of protein coding sequences, or can be artificial sequences that help RNA to be stable, such as the cap at the 5' end and / or the polyA tail at the 3' end. Those skilled in the art can determine the sequence that is suitable for this in each case through routine experiments. The mRNA or the DNA that is used to transcribe the mRNA can be codon optimized. Preferably, the DNA that is used to transcribe the mRNA of the present invention and the mRNA of the present invention can be codon optimized among the present invention.Generally, codon optimization refers to the process of modifying a nucleic acid sequence for expression in a target host cell by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the host cell gene, while maintaining the native amino acid sequence. Codon usage tables are readily available, for example, in the "Codon Usage Database," and these tables can be modified in a variety of ways. Computer algorithms are also available for codon optimization of specific sequences for expression in specific host cells, such as Gene. (Aptagen, Pa), and preferably, (ThermoFischer,Ma).

[0066] As used herein, the term "naked RNA" refers to RNA that is not complexed with other compounds of any kind (particularly proteins, peptides, polymers such as cationic polymers, lipids, liposomes, viral vectors, etc.). Thus, "naked RNA" refers to RNA that is present in a free and uncomplexed form, for example, in a liquid composition, which is molecularly dispersed in a solution. For example, "naked RNA" is not considered to be complexed with lipid and / or polymer carrier systems (e.g., lipid nanoparticles and micelles) / transfection reagents, such as DreamFect TM Gold or (branched) PEI complex. Thus, compositions comprising mRNA, such as therapeutic compositions of the present invention, for example, do not comprise lipid and / or polymer carrier system transfection reagents, such as DreamFect TM Gold or (branch) PEI.

[0067] The terms "nucleic acid sequence", "nucleotide sequence" and "nucleotide acid sequence" are used interchangeably herein and have the same meaning herein, preferably referring to DNA or RNA. The terms "nucleic acid sequence", "nucleotide sequence" and "nucleotide sequence" are preferably used synonymously with the term "polynucleotide sequence". Preferably, a nucleic acid sequence is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester bonds of a sugar / phosphate-backbone. The term "nucleic acid sequence" also encompasses modified nucleic acid sequences, such as base-modified, sugar-modified or backbone-modified DNA or RNA.

[0068] As used herein, the term "open reading frame" refers to a sequence of several nucleotide triplets that can be translated into a peptide or protein. An open reading frame (ORF) preferably comprises a start codon (i.e., a combination of three consecutive nucleotides (ATG) that are commonly used to encode amino acid methionine) and a subsequent region (a multiple of 3 nucleotides in length) at its 5' end. ORF is preferably terminated by a terminator codon (e.g., TAA, TAG, TGA). Typically, this is the unique terminator codon of an open reading frame. Therefore, in the context of the present invention, an open reading frame is preferably a nucleic acid sequence, which is composed of a plurality of nucleotides that can be divided by three, and these nucleotides begin with a start codon (e.g., ATG), and preferably terminate with a terminator codon (e.g., TAA, TGA or TAG). An open reading frame can be isolated, or can be incorporated into a longer nucleic acid sequence, for example, incorporated into a vector or mRNA. An open reading frame can also be referred to as a "(protein) coding region," or preferably, as a "coding sequence."

[0069] The term "signal peptide" is also referred to herein as a signaling peptide, predomain, signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide, which is a short peptide (usually 16-40 amino acids long) that is present at the N-terminus of a newly synthesized protein that will enter the secretory pathway. The signal peptide of the present invention is preferably 10-50, more preferably 11-45, even more preferably 12-45, most preferably 13-45, particularly 14-45, more particularly 15-45, even more particularly 16-40 amino acids long. The signal peptide according to the present invention is located at the N-terminus of the target protein or the N-terminus of the preprotein form of the target protein. Using the signal peptide according to the present invention, the amount of secretion of the target protein is at least equal to the amount of the protein secreted using its natural (homologous) signal peptide, preferably, higher than the amount of the protein secreted using its natural (homologous) signal peptide. The signal peptide according to the present invention is typically of eukaryotic origin, such as a signal peptide of a eukaryotic protein, preferably of mammalian origin, such as a signal peptide of a mammalian protein, more preferably of human origin, such as a signal peptide of a mammalian protein. In some embodiments, the heterologous signal peptide and / or the homologous signal peptide to be modified is a naturally occurring signal peptide of a eukaryotic protein, preferably a naturally occurring signal peptide of a mammalian protein, more preferably a naturally occurring signal peptide of a human protein.

[0070] As used herein, the term "protein" refers to a molecule that typically comprises one or more peptides or polypeptides. A peptide or polypeptide is typically a chain of amino acid residues linked by peptide bonds. Peptides typically contain 2 to 50 amino acid residues. Polypeptides typically contain more than 50 amino acid residues. Proteins typically fold into a three-dimensional form, which may be necessary for the protein to exert its biological function. As used herein, the term "protein" includes fragments and fusion proteins of proteins. Preferably, the protein is of mammalian, more preferably human, origin, i.e., a human protein. Preferably, the protein is a protein that is typically secreted from a cell, i.e., a protein that is naturally secreted from a cell. Proteins referred to herein are typically selected from carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; lactoproteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases, and vasoactive proteins.

[0071] Carboxypeptidases are proteins that are proteases that hydrolyze (cleave) peptide bonds at the carboxyl terminus (C-terminus) of proteins; cytokines are secreted proteins that act locally or systemically as regulators of target cell signaling through cell surface receptors, and cytokines are often involved in immune responses; extracellular ligands and transporters are secreted proteins that act by binding to other proteins or carrying other proteins or other molecules to perform certain biological functions; extracellular matrix proteins are a group of proteins secreted by supporting cells that provide structural and biochemical support to surrounding cells; glucosidases are enzymes involved in breaking down complex carbohydrates (such as starch and glycogen) into monomers; glycosyltransferases are enzymes that establish natural glycosidic bonds; growth factors are enzymes that stimulate cell Secretory proteins that regulate cell growth, proliferation, healing, and cell differentiation, acting locally or systemically as regulators of target cell signaling through cell surface receptors. Growth factors are usually involved in nutrient response and survival or cell homeostasis signaling; growth factor binding proteins are secretory proteins that bind to growth factors to regulate their biological activity; heparin binding proteins are secretory proteins that interact with heparin to regulate its biological function, usually binding to growth factors or hormones together with another substance; hormones are members of a class of signaling molecules produced by glands in multicellular organisms, which are secreted and transported by the circulatory system to distant target organs, regulating physiology and behavior by binding to specific receptors on their target cells; hydrolases are a class of enzymes that destroy chemicals by utilizing water Enzymes that biochemically catalyze the cleavage of molecules by chemical bonds, thereby breaking larger molecules into smaller ones; immunoglobulins are large, Y-shaped secreted proteins produced primarily by plasma cells that are used by the immune system to neutralize pathogens, such as disease-causing bacteria and viruses; isomerases are a large class of enzymes that convert molecules from one isomer to another, thereby promoting intramolecular rearrangements in which bonds are broken and formed; kinases are enzymes that catalyze the transfer of a phosphate group from a high-energy, phosphate-donating molecule to a specific substrate; lyases are enzymes that catalyze the breaking of various chemical bonds by means other than hydrolysis and oxidation, typically forming new double bonds or new ring structures; metalloenzyme inhibitors of matrix metalloproteinases (MMPs) are cytostatic inhibitors; metalloproteinases are proteases whose catalytic mechanism involves metal ions. ; Milk proteins are proteins secreted into milk; Neuroactive proteins are secreted proteins that act locally or remotely to support neuronal function, survival, and physiology; Proteases (also called peptidases or proteinases) are enzymes that hydrolyze proteins by hydrolyzing peptide bonds; Protease inhibitors are proteins that inhibit the function of proteases; Protein phosphatases are enzymes that remove phosphate groups from phosphorylated amino acid residues of their substrate proteins; Esterases are enzymes that break down esters at amino acid residues into acids and alcohols in aqueous chemical reactions; Transferases are a class of enzymes that catalyze the transfer of a specific functional group (such as a methyl or sugar group) from one molecule (called a donor) to another (called an acceptor); Vasoactive proteins are secreted proteins that biologically affect vascular function.The carboxypeptidases, cytokines, extracellular ligands and transporters, extracellular matrix proteins, glucosidases, glycosyltransferases, growth factors, growth factor-binding proteins, heparin-binding proteins, hormones, hydrolases, immunoglobulins, isomerases, kinases, lyases, metalloenzyme inhibitors, metalloproteinases, milk proteins, neuroactive proteins, proteases, protease inhibitors, protein phosphatases, esterases, transferases, and vasoactive proteins mentioned in this article can be found in the UniProt database.

[0072] The terms "fragment" or "sequence fragment", which have the same meaning in the present context, are, for example, shorter parts of the full-length sequence of a nucleic acid molecule such as DNA or RNA or a protein. Thus, a fragment typically comprises or consists of a sequence that is identical to a corresponding stretch within the full-length sequence. In the context of the present invention, a preferred sequence fragment comprises or consists of a continuous stretch of an entity, e.g., nucleotides or amino acids corresponding to a continuous stretch of an entity in the molecule from which the fragment is derived, which represents at least 5%, typically at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, most preferably at least 80% of the entire (i.e. full-length) molecule from which the fragment is derived.

[0073] As used herein, the term "signal peptide heterologous to the protein" refers to a naturally occurring signal peptide that is different from the naturally occurring signal peptide of the protein, i.e., the signal peptide is not derived from the same gene as the protein. Typically, a signal peptide heterologous to a given protein is a signal peptide from another protein that is not related to the given protein, i.e., the amino acid sequence of the signal peptide is different from the signal peptide of the given protein, for example, the difference in its amino acid sequence from the signal peptide of the given protein is greater than 50%, preferably greater than 60%, more preferably greater than 70%, even more preferably greater than 80%, most preferably greater than 90%, and in particular greater than 95%. Preferably, a signal peptide heterologous to a given protein has less than 95%, preferably less than 90%, more preferably less than 80%, even more preferably less than 70%, most preferably less than 60%, and in particular less than 50% sequence identity with the amino acid sequence of the naturally occurring (homologous) signal peptide of the given protein. Although heterologous sequences may originate from the same organism, they do not naturally (essentially) occur in the same nucleic acid molecule, for example, in the same mRNA. A signal peptide that is heterologous to a protein and the protein that is heterologous to the signal peptide may be of the same or different origin, and are typically of the same origin, preferably eukaryotic, more preferably eukaryotic from the same eukaryotic organism, even more preferably mammalian, particularly mammalian from the same mammalian organism, and more particularly human. Example 1 discloses an mRNA comprising nucleic acid sequences encoding human BDNF signal peptide and human IGF1, i.e., the signal peptide is heterologous to the protein, wherein the signal peptide and protein are of the same origin, i.e., human.

[0074] As used herein, the term "signal peptide homologous to the protein" refers to a naturally occurring signal peptide of a protein. A signal peptide homologous to a protein is a signal peptide encoded by a gene for the protein, such as one that occurs naturally. A signal peptide homologous to a protein is typically of eukaryotic origin, for example, a naturally occurring signal peptide of a eukaryotic protein, preferably of mammalian origin, for example, a naturally occurring signal peptide of a mammalian protein, more preferably of human origin, for example, a naturally occurring signal peptide of a human protein.

[0075] As used herein, the term "naturally occurring amino acid sequence that does not essentially have a signal peptide function" refers to a naturally occurring amino acid sequence that is different from the amino acid sequence of any naturally occurring signal peptide. As described herein, the naturally occurring amino acid sequence that does not essentially have a signal peptide function is preferably 10-50, more preferably 11-45, even more preferably 12-45, most preferably 13-45, particularly 14-45, more particularly 15-45, even more particularly 16-40 amino acids long. Preferably, the naturally occurring amino acid sequence that does not essentially have a signal peptide function of the present invention is of eukaryotic origin and is different from any signal peptide of eukaryotic origin, more preferably of mammalian origin and is different from any signal peptide of mammalian origin, more preferably of human origin and is different from any signal peptide of human origin occurring in nature. The naturally occurring amino acid sequence that does not essentially have a signal peptide function is typically the amino acid sequence of a protein coding sequence. According to the present invention, the naturally occurring amino acid sequence that does not essentially have a signal peptide function is typically of eukaryotic origin, preferably of mammalian origin, more preferably of human origin.

[0076] As used herein, the terms "naturally occurring," "native," and "essentially" have equivalent meanings.

[0077] As used herein, the term "amino acids 1-9 at the N-terminus of the amino acid sequence of the signal peptide" refers to the first 9 amino acids at the N-terminus of the amino acid sequence of the signal peptide. Similarly, the term "amino acids 1-7 at the N-terminus of the amino acid sequence of the signal peptide" used herein refers to the first seven amino acids at the N-terminus of the amino acid sequence of the signal peptide, and the term "amino acids 1-5 at the N-terminus of the amino acid sequence of the signal peptide" refers to the first five amino acids at the N-terminus of the amino acid sequence of the signal peptide.

[0078] As used herein, the term "an amino acid sequence modified by the insertion, deletion and / or substitution of at least one amino acid" refers to an amino acid sequence comprising an amino acid substitution, insertion and / or deletion of at least one amino acid within the amino acid sequence. As used herein, the term "a signal peptide heterologous to the protein is modified by the insertion, deletion and / or substitution of at least one amino acid" refers to an amino acid sequence of a naturally occurring signal peptide heterologous to the protein, comprising an amino acid substitution, insertion and / or deletion of at least one amino acid in its naturally occurring amino acid sequence. As used herein, the term "a signal peptide homologous to the protein is modified by the insertion, deletion and / or substitution of at least one amino acid" refers to a naturally occurring signal peptide homologous to the protein, comprising an amino acid substitution, insertion and / or deletion of at least one amino acid in its naturally occurring amino acid sequence. As used herein, the term "a naturally occurring amino acid sequence modified by the insertion, deletion and / or substitution of at least one amino acid" refers to a naturally occurring amino acid sequence comprising an amino acid substitution, insertion and / or deletion of at least one amino acid within its naturally occurring amino acid sequence. "Amino acid substitution" or "substitution" herein refers to the replacement of an amino acid at a specific position in the parent protein sequence with another amino acid. For example, the substitution R34K refers to a polypeptide in which the arginine at position 34 is replaced by lysine. For the previous example, 34K indicates that position 34 is replaced by lysine. For the purposes of this document, multiple substitutions are generally separated by slashes. For example, R34K / L78V refers to a double variant comprising the substitutions R34K and L38V. As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid at a specific position in the parent protein sequence. For example, insertion-34 refers to an insertion at position 34. As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid at a specific position in the parent protein sequence. For example, R34- refers to the deletion of the arginine at position 34.

[0079] Preferably, the deleted amino acid is an amino acid with a hydrophobicity score of less than -0.8, preferably less than 1.9. Preferably, the substituted amino acid is an amino acid with a hydrophobicity score higher than the hydrophobicity score of the substituted amino acid, more preferably, the substituted amino acid is an amino acid with a hydrophobicity score of 2.8 and above, more preferably an amino acid with a hydrophobicity score of 3.8 and above. Preferably, the inserted amino acid is an amino acid with a hydrophobicity score of 2.8 and above, more preferably an amino acid with a hydrophobicity score of 3.8 and above.

[0080] Typically, 1 to 15, preferably 1 to 11 amino acids, more preferably 1 to 10 amino acids, even more preferably 1 to 9 amino acids, particularly 1 to 8 amino acids, more particularly 1 to 7 amino acids, even more particularly 1 to 6 amino acids, particularly preferably 1 to 5 amino acids, more particularly preferably 1 to 4 amino acids, even more particularly preferably 1 to 2 amino acids of a given amino acid sequence are inserted, deleted and / or substituted. Typically, 1 to 15, preferably 1 to 11 amino acids, more preferably 1 to 10 amino acids, even more preferably 1 to 9 amino acids, especially 1 to 8 amino acids, more especially 1 to 7 amino acids, even more especially 1 to 6 amino acids, particularly preferably 1 to 5 amino acids, more particularly preferably 1 to 4 amino acids, even more particularly preferably 1 to 2 amino acids of a given amino acid sequence are inserted, deleted and / or substituted within amino acids 1-11 at the N-terminus of the signal peptide amino acid sequence, preferably within amino acids 1-10, more preferably within amino acids 1-9, even more preferably within amino acids 1-8, in particular within amino acids 1-7, more particularly within amino acids 1-6, even more particularly within amino acids 1-5, particularly preferably within amino acids 1-4, more particularly preferably within amino acids 1-3, even more particularly preferably within amino acids 1-2.

[0081] Preferably, the amino acid sequence is optionally modified by deletion and / or substitution of at least one amino acid.

[0082] Preferably, the average hydrophobicity score of the first 9 amino acids at the N-terminus of the amino acid sequence of the modified signal peptide is increased by 1.0 unit or more compared to that of the unmodified signal peptide.

[0083] As used herein, the term "insulin-like growth factor 1", "insulin-like growth factor 1 (IGF1)" or "IGF1" generally refers to the native sequence of the IGF1 protein without a signal peptide, and may contain a propeptide and / or an E-peptide, preferably refers to the native sequence of the IGF1 protein without a signal peptide and without an E-peptide. As used herein, the term "human insulin-like growth factor 1 (IGF1)" refers to the native sequence of human IGF1 (pro-IGF1, which refers to UniProtKB-P05019 in the Uniprot database, and NM_000618.4, NM_001111285.2 and NM_001111283.2 in the Genbank database, or a fragment thereof. The native DNA sequence encoding human insulin-like growth factor 1 can be codon-optimized. The native sequence of human IGF1 comprises a human signal peptide (nucleotides 1-63) having 21 amino acids, a human propeptide (also called the prodomain) (nucleotides 1-63) having 27 amino acids. The C-terminal domain of human IGF1 comprises or consists of a nucleotide sequence of 64-144, a mature human IGF1 having 70 amino acids (nucleotides 145-354), and a so-called E-peptide (or E-domain). The C-terminal domain of human IGF1 (the so-called E-peptide or E-domain) comprises an Ea-, Eb-, or Ec-domain generated by alternative splicing events. The Ea-domain comprises or consists of 35 amino acids (105 nucleotides), the Eb-domain comprises or consists of 77 amino acids (231 nucleotides), and the Ec-domain comprises or consists of 40 amino acids (120 nucleotides) (see, e.g., Wallis et al. M (2009) New insulin-like growth factor (IGF) -precursor sequences from mammalian genomes: the molecular evolution of IGFs and associated peptides in primates. Growth Horm IGF Res 19 (1): 12-23. Doi: 10.1016 / j.ghir.2008.05.001). As used herein, the term "human insulin-like growth factor 1 (IGF)" generally refers to the native sequence of the human IGF1 protein without a signal peptide, and may include a propeptide and / or an E-peptide, preferably refers to the native sequence of the human IGF1 protein without a signal peptide and without an E-peptide. As used herein, the term "human insulin-like growth factor 1 (IGF)" generally includes mature human IGF1. The term "mature protein" refers to a protein that is synthesized in the endoplasmic reticulum and secreted through the Golgi apparatus in cells that express and secrete the protein.The term "mature IGF1" refers to a protein that is synthesized in the endoplasmic reticulum and secreted through the Golgi apparatus in cells that express and secrete IGF1. The term "mature human IGF1" refers to a protein that is synthesized in the endoplasmic reticulum and secreted through the Golgi apparatus in human cells that express and secrete human IGF1, and generally comprises the amino acids encoded by the nucleotide sequence shown in SEQ ID NO: 39.

[0084] As used herein, term " insulin " or " INS " generally refer to the native sequence of insulin without signal peptide.As used herein, term " human insulin " or " people INS " refer to the native sequence of human insulin, it refers to UniProtKB-P01308 in the Uniprot database, and NM_000207.2, NM_001185097.1, NM_001185098.1 and NM_001291897.1 or its fragment in the Genbank database.The natural DNA sequence encoding human insulin can be by codon optimized.The native sequence of human insulin comprises and has 24 amino acid whose people signal peptide (nucleotide 1-72), has 30 amino acid whose human insulin B chains (nucleotide 73-163), has 31 amino acid whose human insulin propeptides (also referred to as connecting peptide; C-peptide) (nucleotide 64-144) and comprises or is made up of the C-terminal domain (nucleotide 64-144) of 21 amino acid whose human insulin A chains are made up, or is made up of it. As used herein, the term "human insulin" generally includes human insulin without a signal peptide.

[0085] As used herein, the term "erythropoietin", "EPO" or "Epo" generally refers to the native sequence of EPO without a signal peptide. The term "human erythropoietin", "human EPO" or "human Epo" used herein refers to the native sequence of human erythropoietin, which refers to UniProtKB-P01588 in the Uniprot database and NM_000799.2 in the Genbank database, or a fragment thereof. The native DNA sequence encoding human erythropoietin can be codon optimized. The native sequence of human erythropoietin comprises a human signal peptide (nucleotides 1-81) with 27 amino acids, a human Epo coding chain (nucleotides 82-579) with 166 amino acids, or consists of it. As used herein, the term "human erythropoietin" generally includes human EPO without a signal peptide.

[0086] As used herein, the term "interleukin-4" or "IL4" generally refers to the native sequence of IL4 without a signal peptide. As used herein, the term "human interleukin-4" or "human IL4" generally refers to the native sequence of human IL4, which refers to UniProtKB-P05112 in the Uniprot database, and NM_000589.3 and NM_172348.2 in the Genbank database, or a fragment thereof. The native DNA sequence encoding human IL4 can be codon optimized. The native sequence of human IL4 comprises a human signal peptide (nucleotides 1-72) with 24 amino acids, a human IL4 coding chain (nucleotides 73-387) with 129 amino acids, or consists of it. As used herein, the term "human IL4" generally includes a human IL4 without a signal peptide.

[0087] As generally used herein, the term "interleukin-10" or "IL10" refers to the native sequence of IL10 without a signal peptide. As used herein, the term "human interleukin-10" or "human IL10" refers to the native sequence of human IL10, which refers to UniProtKB-P22301 in the Uniprot database and NM_000572.2 in the Genbank database, or a fragment thereof. The native DNA sequence encoding human IL10 may be codon-optimized. The native sequence of human IL10 comprises or consists of a human signal peptide (nucleotides 1-54) of 18 amino acids and a human IL10 coding chain (nucleotides 55-534) of 160 amino acids. As used herein, the term "human IL10" generally includes human IL10 without a signal peptide.

[0088] As used herein, the term "signal peptide of insulin growth factor 1 (IGF1)" or "signal peptide of IGF1" refers to the natural signal peptide of IGF1, which refers to P05019 in the Uniprot database, and NM_000618.4, NM_001111284.1 and NM_001111285.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 24, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 25.

[0089] As used herein, the term "signal peptide of insulin growth factor 2 (IGF2)" or "signal peptide of IGF2" refers to the natural signal peptide of IGF2, which refers to P01344 in the Uniprot database, and NM_000612.5, NM_001007139.5, NM_001127598.2, NM_001291861.2 and NM_001291862.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 26, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 27.

[0090] As used herein, the term "signal peptide of serum albumin (ALB)" or "signal peptide of ALB" refers to the natural signal peptide of ALB, which refers to P02768 in the Uniprot database and NM_000477.6 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 28, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 29.

[0091] As used herein, the term "brain-derived neurotrophic factor (BDNF)" or "signal peptide of BDNF" refers to the natural signal peptide of BDNF, which refers to P23560 in the Uniprot database, and NM_001143805.1, NM_170731.4, NM_170734.3, NM_001143810.1 and NM_001143809.1 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 30, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 31.

[0092] As used herein, the term "signal peptide of stromal cell-derived factor-1 (CXCL12)" or "signal peptide of CXCL12" refers to the natural signal peptide of CXCL12, which refers to P48061 in the Uniprot database, and NM_000609.6, NM_001033886.2, NM_001178134.1, NM_001277990.1 and NM_199168.3 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 32, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 33.

[0093] As used herein, the term "synthetic signal peptide 1 signal peptide (synthetic seq1)" or "synthetic signal peptide of seq1" refers to a synthetic signal peptide 1 having an amino acid sequence as shown in SEQ ID NO: 34 and / or encoded by a DNA sequence as shown in SEQ ID NO: 35.

[0094] As used herein, the term "signal peptide of synthetic signal peptide 2 (synthetic seq2)" or "signal peptide of synthetic seq1" refers to synthetic signal peptide 1 having the amino acid sequence shown in SEQ ID NO:36 and / or encoded by the DNA sequence shown in SEQ ID NO:37.

[0095] As used herein, the term "signal peptide of latent transforming growth factor beta binding protein 2 (LTBP2)" or "signal peptide of LTBP2" refers to the native signal peptide of LTBP2, which refers to Q14767 in the Uniprot database and NM_000428.2 in the Genbank database, and preferably has the amino acid sequence shown in SEQ ID NO: 41, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 42.

[0096] As used herein, the term "signal peptide of insulin-like growth factor binding protein complex acid-labile subunit (IGFALS)" or "signal peptide of IGFALS" refers to the natural signal peptide of IGFALS, which refers to P35858 in the Uniprot database, and NM_001146006.1 and NM_004970.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 46, and / or preferably encoded by the DNA sequence shown in SEQ ID NO: 47.

[0097] As used herein, the term "signal peptide of insulin (INS)" or "signal peptide of INS" refers to the natural signal peptide of INS, which refers to P1308 in the Uniprot database, and NM_001185097.1, NM_000207.2, NM_001185098.1 and NM_001291897.1 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 51, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 52.

[0098] As used herein, the term "signal peptide of erythropoietin (Epo)" or "signal peptide of Epo" refers to the natural signal peptide of Epo, which refers to P01588 in the Uniprot database and NM_000799.2 in the Genbank database, preferably having an amino acid sequence as shown in SEQ ID NO: 56, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 57.

[0099] As used herein, the term "signal peptide of granulocyte colony-stimulating factor (CSF3)" or "signal peptide of CSF3" refers to the natural signal peptide of CSF3, which refers to P09919 in the Uniprot database, and NM_000759.3, NM_001178147.1, NM_172219.2 and NM_172220.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 61, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 62.

[0100] As used herein, the term "signal peptide of β-nerve growth factor (NGF)" or "signal peptide of NGF" refers to the natural signal peptide of NGF, which refers to P01138 in the Uniprot database, and NM_002506.2 and XM_006710663.3 in the Genbank database, preferably having an amino acid sequence as shown in SEQ ID NO: 66, and / or preferably encoded by a DNA sequence shown in SEQ ID NO: 67.

[0101] As used herein, the term "signal peptide of interleukin 4 (IL4)" or "signal peptide of IL4" refers to the natural signal peptide of IL4, which refers to P05112 in the Uniprot database, and NM_000589.3 and NM_172348.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 77, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 78.

[0102] As used herein, the term "signal peptide of interleukin 10 (IL10)" or "signal peptide of IL10" refers to the natural signal peptide of IL10, which refers to P22301 in the Uniprot database and NM_000572.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID No: 82, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 83.

[0103] As used herein, the term "signal peptide of fibroblast growth factor 5 (FGF5)" or "signal peptide of FGF5" refers to the natural signal peptide of FGF5, which refers to P12034 in the Uniprot database, and NM_004464.3 and NM_033143.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 87, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 88 or SEQ ID NO: 183.

[0104] As used herein, the term "complement factor H-related protein 2 (FHR2) signal peptide" or "FHR2 signal peptide" refers to the natural signal peptide of FHR2, which refers to P36980 in the Uniprot database, and NM_001312672.1 and NM_005666.3 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 92, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 93.

[0105] As used herein, the term "insulin-like growth factor binding protein 5 (IBP5) signal peptide" or "signal peptide of IBP5" refers to the native signal peptide of IBP5, which refers to P24593 in the Uniprot database and NM_001312672.1 and NM_000599.3 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 97, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 98.

[0106] As used herein, the term "signal peptide of neurotrophin 3 (NTF3)" or "signal peptide of NTF3" refers to the natural signal peptide of NTF3, which refers to P20783 in the Uniprot database, and NM_002527.4, XM_011520963.2 and NM_001102654.1 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 102, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 103.

[0107] As used herein, the term "signal peptide of prostate and testis expressed protein 2 (PATE2)" or "signal peptide of PATE2" refers to the native signal peptide of PATE2, which refers to Q6UY27 in the Uniprot database and NM_212555.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 107, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 108.

[0108] As used herein, the term "signal peptide of extracellular superoxide dismutase (SOD3)" or "signal peptide of SOD3" refers to the natural signal peptide of SOD3, which refers to P08294 in the Uniprot database and NM_003102.2 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID No: 112, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 113.

[0109] As used herein, the term "coding sequence of glucagon receptor (GL-R)" or "coding sequence of GL-R" refers to the coding chain of GL-R, which is a naturally occurring amino acid sequence that essentially does not have a signal peptide function, refers to P47871 in the Uniprot database, and NM_000160.4 and XM_006722277.1 in the Genbank database, preferably having the amino acid sequence shown in SEQ ID NO: 117, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 118.

[0110] As used herein, the terms "modified signal peptide of insulin growth factor 1 (IGF1)", "modified signal peptide of IGF1" or "modified signal peptide of IGF1" refer to a modified signal peptide of IGF1, wherein the natural signal peptide of IGF1 of P05019 in the Uniprot database, and NM_000618.4, NM_001111284.1 and NM_001111285.2 in the Genbank database is modified by G2L / S5L / T9L / Q10L substitutions and K3- and C15-deletions, and preferably has the amino acid sequence shown in SEQ ID NO: 122, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 123.

[0111] As used herein, the terms "modified signal peptide of insulin growth factor 2 (IGF2)", "modified signal peptide of IGF2" or "IGF2-modified signal peptide" refer to a modified signal peptide of IGF2, wherein the natural signal peptide of IGF2 of P01344 in the Uniprot database, NM_000612.5, NM_001007139.5, NM_001127598.2, NM_001291861.2 and NM_001291862.2 in the Genbank database is modified by G2L / G6L / K7L / S8L substitutions and P4-, M5-, I23- and A24-deletions, and preferably has the amino acid sequence shown in SEQ ID NO: 127, and / or is encoded by the DNA sequence shown in SEQ ID NO: 128.

[0112] As used herein, the terms “modified stromal cell-derived factor-1 (CXCL12) signal peptide,” “modified CXCL12 signal peptide,” or “CXCL12 modified signal peptide” refer to a modified CXCL12 signal peptide, wherein the native signal peptide of CXCL12, P48061 in the Uniprot database and NM_000609.6, NM_001033886.2, NM_001178134.1, NM_001277990.1, and NM_199168.3 in the Genbank database, is modified by N3- and K5-deletions, and preferably has the amino acid sequence shown in SEQ ID NO: 132, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 133.

[0113] As used herein, the terms "modified interleukin 4 (IL4) signal peptide", "modified IL4 signal peptide" or "IL4 modified signal peptide" refer to a modified IL4 signal peptide, wherein the natural signal peptide of IL4 of P05112 in the Uniprot database and NM_000589.3 and NM_172348.2 in the Genbank database is modified by G2-, T4-, S5- and Q6-deletions, and preferably has the amino acid sequence shown in SEQ ID NO: 166, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 167.

[0114] As used herein, the terms "modified interleukin 10 (IL10) signal peptide," "modified IL10 signal peptide," or "IL10 modified signal peptide" refer to a modified IL10 signal peptide, wherein the native signal peptide of IL10 of P22301 in the Uniprot database and NM_000572.2 in the Genbank database is modified by H2V / S3L / S4L and S8L substitutions, and preferably has the amino acid sequence shown in SEQ ID NO: 174, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 175.

[0115] As used herein, the terms “modified signal peptide of insulin (INS)”, “modified signal peptide of INS” or “modified signal peptide of INS” refer to the signal peptide of a modified INS, wherein the native signal peptide of INS of P1308 of the Uniprot database and NM_001185097.1, NM_000207.2, NM_001185098.1 and NM_001291897.1 in the Genbank database is modified by M5- and R6-deletion, and preferably has the amino acid sequence shown in SEQ ID NO: 147, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 148 or SEQ ID NO: 182.

[0116] As used herein, the term "modified signal peptide of brain-derived neurotrophic factor (BDNF)", "modified signal peptide of BDNF" or "modified signal peptide of BDNF" refers to a modified signal peptide of BDNF, wherein the native signal peptide of BDNF of P23560 in the Uniprot database and NM_001143805.1, NM_170731.4, NM_170734.3, NM_001143810.1 and NM_001143809.1 in the Genbank database is modified by T2L / T7L and S11L substitutions, and preferably has the amino acid sequence shown in SEQ ID NO: 137, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 138.

[0117] As used herein, the term "modified erythropoietin (Epo) signal peptide", "modified Epo signal peptide" or "Epo modified signal peptide" refers to a modified Epo signal peptide, wherein the natural signal peptide of Epo of P01588 in the Uniprot database and NM_000799.2 in the Genbank database is modified by G2L / P7L / W9L substitution and H4-, E5- and W11-deletion, and preferably has an amino acid sequence as shown in SEQ ID NO: 152, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 153.

[0118] As used herein, the term "modified insulin growth factor 1 (IGF1) prodomain", "modified IGF1 prodomain" or "modified IGF1-Pro" refers to the propeptide of IGF1, which is a naturally occurring amino acid sequence that essentially does not have a signal peptide function (P05019 in the Uniprot database and NM_000618.4, NM_001111284.1 and NM_001111285.2 in the Genbank database), which is modified by deleting the ten amino acid residues flanking 22-31 in the N-terminus of the propeptide (VKMHTMSSSH), and preferably has the amino acid sequence shown in SEQ ID NO: 142, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 143.

[0119] As used herein, the terms "modified intestinal alkaline phosphatase (ALPI) prodomain", "modified ALPI prodomain", "modified ALPI" or "modified ALPI-Pro" refer to the propeptide of ALPI, which is a naturally occurring amino acid sequence that essentially does not have a signal peptide function (P09923 in the Uniprot database, and NM_001631.4 in the Genbank database), modified by A504L / A505L / S511L / G517L / T518L substitutions and H506-, P507-, A509-, A510- and P513-deletions, and preferably has an amino acid sequence as shown in SEQ ID NO: 189, and / or is preferably encoded by the DNA sequence shown in SEQ ID NO: 190.

[0120] As used herein, the term "mRNA comprises a nucleic acid sequence encoding a propeptide of IGF1 and a nucleic acid sequence encoding mature IGF1, and does not comprise a nucleic acid sequence encoding the E-peptide of IGF1", generally refers to an mRNA that comprises a nucleotide sequence encoding a propeptide of human IGF1 having 27 amino acids (also referred to as a prodomain) and a nucleotide sequence encoding mature human IGF1 having 70 amino acids, but does not comprise a nucleotide sequence encoding the E-peptide of human IGF1 (also referred to as an "E domain"), i.e., does not comprise a nucleotide sequence encoding the Ea-, Eb-, or Ec-domain. The nucleotide sequence encoding the propeptide of human IGF1 having 27 amino acids (also referred to as a prodomain) and the nucleotide sequence encoding the mature human IGF1 having 70 amino acids can be codon-optimized.

[0121] As used herein, the term "vector" or "expression vector" refers to a naturally occurring or synthetically produced nucleic acid construct for uptake, propagation, expression or delivery in a cell, such as a plasmid, minicircle, phagemid, cosmid, artificial chromosome / minichromosome, phage, virus (e.g., baculovirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, phage). The vector can be integrated into the genome of the host cell or retained in the host cell as an autonomously replicating construct. Methods for constructing vectors are well known to those skilled in the art and are described in various publications. In particular, techniques for constructing suitable vectors are known to those skilled in the art, including descriptions of functional and regulatory elements, such as promoters, enhancers, termination and polyadenylation signals, selection markers, origins of replication, and splicing signals. Eukaryotic expression vectors will also typically contain prokaryotic sequences that promote the propagation of the vector in bacteria, such as origins of replication and antibiotic resistance genes for selection in bacteria, which can be removed prior to transfection of eukaryotic cells. A variety of eukaryotic expression vectors containing cloning sites into which a polynucleotide can be operably linked are well known in the art, and some are commercially available from companies such as Agilent Technologies, Santa Clara, Calif.; Invitrogen, Carlsbad, Calif.; Promega, Madison, Wis., or Invivogen, San Diego, Calif.

[0122] As used herein, the term "gene therapy vector" refers to any vector used to deliver a nucleic acid sequence, such as a nucleic acid sequence encoding a gene, to a cell. Gene therapy vectors and gene delivery methods are well known in the art. Non-limiting examples of these methods include viral vector delivery systems, including DNA and RNA viruses, which have episomal or integrated genomes after delivery to cells; non-viral vector delivery systems, including DNA plasmids, naked nucleic acids and nucleic acids complexed with delivery vectors, transposon systems (for delivery and integration into the host genome; Moriarity, et al. (2013) Nucleic Acids Res 41(8), e92, Aronovich, et al., (2011) Hum. Mol. Genet. 20(R1), R14-R20), retrovirus-mediated DNA transport (e.g., Moloney murine leukemia virus, spleen necrosis virus, retroviruses (such as Rous sarcoma virus, Harvey sarcoma virus), avian leukemia virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus; for example, see Kay et al. (1993) Science 262, 117-119, Anderson (1992) Science 256, 808-813), and DNA virus-mediated DNA transport, including adenovirus, herpes virus, parvovirus, and adeno-associated virus (e.g., Ali et al. (1994) Gene Therapy 1, 367-384). Viral vectors also include, but are not limited to, adeno-associated virus, adenovirus, lentivirus, retrovirus, and herpes simplex virus vectors. Vectors capable of integrating into the host genome include, but are not limited to, retroviruses or lentiviruses.

[0123] As used herein, the terms "transcription unit," "expression unit," or "expression cassette" refer to a region within a vector, construct, or polynucleotide sequence that contains one or more genes to be transcribed, wherein the genes contained within the segment are operably linked to one another. They are transcribed from a single promoter, and transcription is terminated by at least one polyadenylation signal. As a result, the different genes are linked at least by transcription. More than one protein or product can be transcribed and expressed from each transcription unit (polycistronic transcription unit). Each transcription unit will contain regulatory elements that are essential for the transcription and translation of any selected sequence contained within the unit, and each transcription unit may contain the same or different regulatory elements. For example, each transcription unit may contain the same terminator. IRES elements or introns can be used to functionally link genes within a transcription unit. A vector or polynucleotide sequence may contain more than one transcription unit.

[0124] As used herein, the term "skeletal muscle injury" refers to any injury or breakage of skeletal muscle caused by eccentric muscle contraction, elongation, and muscle overload, preferably a breakage of skeletal muscle. In principle, any skeletal muscle may be affected by such injury or breakage. Preferably, skeletal muscle injury is injury or breakage of skeletal muscle, wherein the skeletal muscle is selected from the muscle groups of the head, neck, chest, back, abdomen, pelvis, arms, legs, and buttocks.

[0125] More preferably, the skeletal muscle injury is an injury or rupture of a skeletal muscle selected from the group consisting of the tibialis, temporalis, papillary, pectoralis major, tibialis posterior, tibialis anterior, gastrocnemius, coracobrachialis, diaphragm, palmaris longus, rectus abdominis, external anal sphincter, internal anal sphincter, subscapularis, biceps, triceps, quadriceps, calf, groin, Achilles tendon, deltoid, teres major, supraspinatus, infraspinatus, teres minor, rotator cuff, subscapularis, and scapula. rectus femoris, rectus abdominis, external obliques, masseter, trapezius, latissimus, pectoralis, erector spinae, iliocostalis, longissimus, spinalis dorsi, transverse spinae, semispinalis dorsi, semispinalis cervicis, semispinalis capitis, multifidus, rotators, interspinalis, intertransverses, splenius capitis, splenius cervicis, intercostals, subcostals, transverse thoracis, levator costae, serratus posterior inferior, serratus posterior superior, transverse abdominis, rectus abdominis, pyramidalis, cremaster, quadratus lumborum, external obliques, internal obliques.

[0126] Even more preferably, the skeletal muscle injury is an injury and rupture wherein the skeletal muscle is selected from the group consisting of the iliac crest muscle, temporalis muscle, papillary muscle, pectoralis major muscle, tibialis posterior muscle, tibialis anterior muscle, gastrocnemius muscle, coracobrachialis muscle, diaphragm muscle, palmaris longus muscle, rectus abdominis muscle, external anal sphincter, internal anal sphincter, subscapularis muscle, biceps muscle, triceps muscle, quadriceps muscle, calf muscle, groin muscle, Achilles tendon, deltoid muscle, teres major muscle, rotator cuff supraspinatus muscle, rotator infraspinatus muscle, teres minor muscle, rotator cuff subscapularis muscle, rectus femoris muscle, rectus abdominis muscle, external oblique muscle, masseter muscle, trapezius muscle, latissimus muscle, and pectoral muscle.

[0127] Preferably, any damage and disruption of skeletal muscle caused by eccentric muscle contraction, elongation or muscle overload is treated by the method of the present invention, preferably disruption of skeletal muscle.

[0128] In a first aspect, the present invention provides an mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0129] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0130] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0131] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0132] In one aspect, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from the group consisting of:

[0133] i) a signal peptide that is heterologous to the protein, provided that the protein is not an oxidoreductase, or a signal peptide that is heterologous to the protein, provided that the protein is not an oxidoreductase, is modified by insertion, deletion and / or substitution of at least one amino acid;

[0134] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0135] iii) a naturally occurring amino acid sequence which does not have a signal peptide function in nature, or a naturally occurring amino acid sequence which does not have a signal peptide function in nature and is modified by insertion, deletion and / or substitution of at least one amino acid.

[0136] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0137] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0138] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0139] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0140] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0141] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0142] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0143] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid.

[0144] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0145] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0146] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0147] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid.

[0148] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0149] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; and

[0150] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid.

[0151] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0152] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; and

[0153] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0154] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0155] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0156] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0157] In another embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that the protein is not an oxidoreductase.

[0158] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid.

[0159] In another embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0160] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is selected from:

[0161] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0162] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0163] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0164] In one aspect, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is selected from the group consisting of:

[0165] i) a signal peptide that is heterologous to the protein, provided that the protein is not an oxidoreductase, or a signal peptide that is heterologous to the protein, provided that the protein is not an oxidoreductase, is modified by insertion, deletion and / or substitution of at least one amino acid;

[0166] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0167] iii) a naturally occurring amino acid sequence which does not have a signal peptide function in nature, or a naturally occurring amino acid sequence which does not have a signal peptide function in nature and is modified by insertion, deletion and / or substitution of at least one amino acid.

[0168] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is selected from:

[0169] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0170] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0171] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0172] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is selected from:

[0173] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0174] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0175] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid.

[0176] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is selected from:

[0177] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0178] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0179] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid.

[0180] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is selected from:

[0181] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; and

[0182] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid.

[0183] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is selected from:

[0184] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; and

[0185] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0186] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is selected from:

[0187] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0188] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0189] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase.

[0190] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid.

[0191] In another embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1 to 7 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.5, wherein the signal peptide is a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0192] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is selected from:

[0193] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0194] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0195] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0196] In one aspect, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is selected from the group consisting of:

[0197] i) a signal peptide that is heterologous to the protein, provided that the protein is not an oxidoreductase, or a signal peptide that is heterologous to the protein, provided that the protein is not an oxidoreductase, is modified by insertion, deletion and / or substitution of at least one amino acid;

[0198] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0199] iii) a naturally occurring amino acid sequence which does not have a signal peptide function in nature, or a naturally occurring amino acid sequence which does not have a signal peptide function in nature and is modified by insertion, deletion and / or substitution of at least one amino acid.

[0200] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is selected from:

[0201] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0202] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0203] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0204] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is selected from:

[0205] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0206] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0207] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid.

[0208] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is selected from:

[0209] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0210] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0211] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid.

[0212] In one aspect, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is selected from the group consisting of:

[0213] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; and

[0214] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid.

[0215] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is selected from:

[0216] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; and

[0217] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0218] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is selected from:

[0219] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0220] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0221] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase.

[0222] In a further embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-5 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid.

[0223] In another embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1 to 5 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, wherein the signal peptide is a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0224] In a preferred embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the signal peptide comprises or consists of an amino acid sequence having a length of 16 to 40 amino acids, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from: i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that the protein is not an oxidoreductase;

[0225] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0226] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0227] In a further preferred embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the signal peptide comprises or consists of an amino acid sequence having a length of 16 to 40 amino acids, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0228] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; and

[0229] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid.

[0230] In a further preferred embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the signal peptide comprises or consists of an amino acid sequence having a length of 16 to 40 amino acids, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0231] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; and

[0232] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0233] In a further preferred embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the signal peptide comprises or consists of an amino acid sequence having a length of 16 to 40 amino acids, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from:

[0234] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0235] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0236] In a further preferred embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the signal peptide comprises or consists of an amino acid sequence having a length of 16 to 40 amino acids, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence have an average hydrophobicity score greater than 2, wherein the signal peptide is a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that the protein is not an oxidoreductase;

[0237] In a further preferred embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the signal peptide comprises or consists of an amino acid sequence having a length of 16 to 40 amino acids, wherein amino acids 1-9 at the N-terminus of the amino acid sequence have an average hydrophobicity score greater than 2, wherein the signal peptide is a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid.

[0238] In a further preferred embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the signal peptide comprises or consists of an amino acid sequence having a length of 16 to 40 amino acids, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence have an average hydrophobicity score greater than 2, wherein the signal peptide is a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0239] In one embodiment, the signal peptide is selected from:

[0240] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0241] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0242] iii) a naturally occurring amino acid sequence, which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid,

[0243] wherein the modification by insertion, deletion and / or substitution of at least one amino acid is carried out within amino acids 1-11 at the N-terminus of the amino acid sequence of the signal peptide, preferably within amino acids 1-10, more preferably within amino acids 1-9, even more preferably within amino acids 1-8, particularly within amino acids 1-7, more particularly within amino acids 1-6, even more particularly within amino acids 1-5, particularly preferably within amino acids 1-4, more particularly preferably within amino acids 1-3, and more particularly preferably within amino acids 1-2.

[0244] The term "hydrophobicity score" or "hydrophobicity score" is used herein synonymously with the term "hydropathy score" and refers to the degree of hydrophobicity of an amino acid as calculated according to the Kyte-Doolittle scale (Kyte J., Doolittle RF; J. Mol. Biol. 157: 105-132 (1982)). The amino acid hydrophobicity scores according to the Kyte-Doolittle scale are as follows:

[0245]

[0246] The "average hydrophobicity score" of an amino acid sequence, e.g., the average hydrophobicity score of the N-terminal amino acids 1-9 of the amino acid sequence of a signal peptide, is calculated by adding the hydrophobicity scores according to the Kyte-Doolittle scale for each amino acid in the amino acid sequence (e.g., the hydrophobicity scores of each of the 9 amino acids from the N-terminal amino acid 1-9), and dividing the sum by the number of amino acids (e.g., by nine).

[0247] In one embodiment of the invention, the amino acids 1 to 9 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 2.05 or more, preferably 2.1 or more, more preferably 2.15 or more, even more preferably 2.2 or more, particularly 2.25 or more, more particularly 2.3 or more, even more particularly 2.35 or more. In a further embodiment, the amino acids 1 to 9 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 2.05 to 4.5, preferably 2.1 to 4.5, more preferably 2.15 to 4.5, even more preferably 2.2 to 4.5, particularly 2.25 to 4.5, more particularly 2.3 to 4.5, even more particularly 2.35 to 4.5. In a further embodiment, the amino acids 1-9 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 2.05 to 4.0, preferably 2.1 to 4.0, more preferably 2.15 to 4.0, even more preferably 2.2 to 4.0, particularly 2.25 to 4.0, more particularly 2.3 to 4.0, even more particularly 2.35 to 4.0.

[0248] In one embodiment of the invention, the average hydrophobicity score of the last 9 amino acids at the C-terminus of the signal peptide amino acid sequence is at least 1.0 unit lower, preferably at least 1.1 units lower, more preferably at least 1.2 units lower, even more preferably at least 1.3 units lower, in particular 1.0 to 4 units lower, more in particular 1.1 to 4 units lower, even more in particular 1.2 to 4 units lower, and most in particular 1.3 to 4 units lower than the average hydrophobicity score of amino acids 1 to 9 at the N-terminus of the signal peptide amino acid sequence.

[0249] In one embodiment of the invention, amino acids 1-9, amino acids 2-10, amino acids 3-11, amino acids 4-12 and amino acids 5-13 at the N-terminus of the signal peptide amino acid sequence each have an average hydrophobicity score greater than 1.5, preferably an average hydrophobicity score greater than 1.6, more preferably an average hydrophobicity score greater than 1.7, even more preferably an average hydrophobicity score greater than 1.8, more preferably an average hydrophobicity score greater than 1.9, particularly an average hydrophobicity score of 1.5 to 4.5, more particularly an average hydrophobicity score of 1.6 to 4.5, even more particularly an average hydrophobicity score of 1.7 to 4.5, more particularly an average hydrophobicity score of 1.8 to 4.5, and most particularly an average hydrophobicity score of 1.9 to 4.5.

[0250] In one embodiment of the present invention, the average hydrophobicity score of amino acids 8-16 at the N-terminus of the amino acid sequence of the signal peptide is at least equal to or lower than the average hydrophobicity score of amino acids 3-11 at the N-terminus of the amino acid sequence of the signal peptide, preferably at least 0.4 units lower than the average hydrophobicity score of amino acids 3-11 at the N-terminus of the amino acid sequence of the signal peptide, more preferably 0.4-2.0 units lower.

[0251] In one embodiment of the invention, the signal peptide comprises or consists of an amino acid sequence having a length of 18 to 40 amino acids, and wherein the average hydrophobicity score of amino acids 10-18 at the N-terminus of the amino acid sequence of the signal peptide is at least 0.5 units lower, preferably 0.5 to 3.0 units lower, than the average hydrophobicity score of amino acids 3-11 at the N-terminus of the amino acid sequence of the signal peptide.

[0252] In one embodiment of the invention, the average hydrophobicity score of the last 9 amino acids at the C-terminus of the amino acid sequence of the signal peptide is at least 1.5 units lower, preferably 1.5-3.5 units lower, than the average hydrophobicity score of amino acids 3-11 at the N-terminus of the amino acid sequence of the signal peptide.

[0253] In one embodiment of the present invention, the average hydrophobicity score of any 9 consecutive amino acids of the amino acid sequence of the signal peptide does not exceed 4.1.

[0254] In one embodiment of the present invention, the last 9 amino acids at the C-terminus of the amino acid sequence of the signal peptide contain at least one amino acid with a negative hydrophobicity score, preferably, the last 9 amino acids at the C-terminus of the amino acid sequence of the signal peptide contain amino acids selected from G, Q, N, T, S, R, K, H, D, E, P, Y and W.

[0255] In one embodiment of the present invention, the second amino acid of amino acids 1-9 at the N-terminus of the amino acid sequence of the signal peptide is selected from P, Y, W, S, T, G, A, M, C, F, L, V and I.

[0256] In a preferred embodiment of the present invention, the second amino acid of amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide is selected from the group consisting of A, L, S, T, V and W.

[0257] In one embodiment of the invention, amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average polarity of 6.1 or less, preferably an average polarity of less than 6.1, more preferably an average polarity of less than 4, even more preferably an average polarity of less than 2, in particular an average polarity of 6.1 to 0, more in particular an average polarity of 4 to 0, even more in particular an average polarity of 2 to 0, and most in particular an average polarity of 1 to 0.2.

[0258] In one embodiment of the invention, amino acids 1 to 7 at the N-terminus of the amino acid sequence of the signal peptide have an average polarity of 6.1 or less, preferably an average polarity of less than 6.1, more preferably an average polarity of less than 4, even more preferably an average polarity of less than 2, in particular an average polarity of 6.1 to 0, more in particular an average polarity of 4 to 0, even more in particular an average polarity of 2 to 0, and most in particular an average polarity of 1 to 0.2.

[0259] In one embodiment of the invention, amino acids 1 to 5 at the N-terminus of the amino acid sequence of the signal peptide have an average polarity of 6.1 or less, preferably an average polarity of less than 6.1, more preferably an average polarity of less than 4, even more preferably an average polarity of less than 2, in particular an average polarity of 6.1 to 0, more in particular an average polarity of 4 to 0, even more in particular an average polarity of 2 to 0, and most in particular an average polarity of 1.1 to 0.2.

[0260] Polarity is calculated according to the Zimmerman Polarity index (Zimmerman JM, Eliezer N., Simha R.; J. Theor. Biol. 21: 170-201 (1968)). The "average polarity" of an amino acid sequence, for example, the average polarity of the N-terminal amino acids 1-9 of the amino acid sequence of a signal peptide, is calculated by adding the polarity values ​​calculated for each amino acid in the amino acid sequence according to the Zimmerman Polarity index (e.g., the average polarity of each of the nine amino acids preceding the N-terminal amino acids 1-9), and then dividing by the number of amino acids (e.g., divided by nine). The polarity of amino acids according to the Zimmerman Polarity index is as follows:

[0261]

[0262] The above average hydrophobicity score or average polarity of the amino acid sequence of the signal peptide of the present invention can be calculated by using the publicly available online database ProtScale (http: / / www.expasy.org / tools / protscale.html), see Gasteiger E. et al. (Gasteiger E., Hoogland C., Gattiker A., ​​Duvaud S., Wilkins MR, Appel RD, Bairoch A.; Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana The hydrophobicity of the Kyte & Doolittle scale ("Hphobicity / Kyte & Doolittle") or the polarity of the Zimmerman scale ("Polarity / Zimmerman") is selected, and the values ​​are set for a specific window size (e.g., a window size of 9 amino acids) of the signal peptide, with the relative weight value at the edge of the window set to 100%, and no scale normalization is performed. The corresponding numerical data can be retrieved by opening the link on the "Numerical format (verbose)" on the result page.

[0263] In one embodiment of the invention, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.7. In one embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1-7 of the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.7, and the signal peptide comprises, or consists of, an amino acid sequence of 14 to 40 amino acids in length.

[0264] In one embodiment of the invention, the amino acids 1 to 7 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 1.6 or more, preferably 1.7 or more, more preferably 1.75 or more, even more preferably 1.8 or more, in particular 2.0 or more, more in particular 2.1 or more, even more in particular 2.2 or more. In a further embodiment, the amino acids 1 to 7 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 1.6 to 4.5, preferably 1.7 to 4.5, more preferably 1.75 to 4.5, even more preferably 1.8 to 4.5, in particular 2.0 to 4.5, more in particular 2.1 to 4.5, even more in particular 2.2 to 4.5. In a further embodiment, the amino acids 1-7 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 1.6 to 4.0, preferably 1.7 to 4.0, more preferably 1.75 to 4.0, even more preferably 1.8 to 4.0, particularly 2.0 to 4.0, more particularly 2.1 to 4.0, even more particularly 2.2 to 4.0.

[0265] In one embodiment of the invention, the average hydrophobicity score of the last 7 amino acids at the C-terminus of the amino acid sequence of the signal peptide is equal to or lower than the average hydrophobicity score of amino acids 1-7 at the N-terminus of the signal peptide amino acid sequence, preferably at least 0.06 units lower, more preferably at least 1.0 unit lower, even more preferably at least 1.1 units lower, in particular at least 1.2 units lower, more in particular 1.0 to 4 units lower, even more in particular 1.0 and 4 units lower, most in particular 1.2 to 4 units lower.

[0266] In one embodiment of the invention, amino acids 1-7, amino acids 2-8, amino acids 3-9, amino acids 4-10 and amino acids 5-11 at the N-terminus of the signal peptide amino acid sequence each have an average hydrophobicity score greater than 1.4, preferably an average hydrophobicity score greater than 1.5, more preferably an average hydrophobicity score greater than 1.6, even more preferably an average hydrophobicity score greater than 1.7, more preferably an average hydrophobicity score greater than 1.75, particularly an average hydrophobicity score of 1.4 to 4.5, more particularly an average hydrophobicity score of 1.5 to 4.5, even more particularly an average hydrophobicity score of 1.6 to 4.5, more particularly an average hydrophobicity score of 1.7 to 4.5, and most particularly an average hydrophobicity score of 1.75 to 4.5.

[0267] In one embodiment of the invention, the average hydrophobicity score of the last 7 amino acids at the C-terminus of the amino acid sequence of the signal peptide is at least 1.0 unit lower, preferably 1.0-3.6 units lower, than the average hydrophobicity score of amino acids 3-9 at the N-terminus of the amino acid sequence of the signal peptide.

[0268] In one embodiment of the present invention, the average hydrophobicity score of any 7 consecutive amino acids of the amino acid sequence of the signal peptide does not exceed 4.1.

[0269] In one embodiment of the present invention, the last 7 amino acids at the C-terminus of the amino acid sequence of the signal peptide contain at least one amino acid with a negative hydrophobicity score, preferably, the last 7 amino acids at the C-terminus of the amino acid sequence of the signal peptide contain an amino acid selected from the group consisting of G, Q, N, T, S, R, K, H, D, E, P, Y and W.

[0270] In one embodiment of the present invention, the second amino acid of amino acids 1-7 at the N-terminus of the signal peptide amino acid sequence is selected from P, Y, W, S, T, G, A, M, C, F, L, V and I.

[0271] In a preferred embodiment of the present invention, the second amino acid of amino acids 1 to 7 at the N-terminus of the signal peptide amino acid sequence is selected from the group consisting of A, L, S, T, V and W.

[0272] In one embodiment of the invention, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the amino acids 1-5 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3 units. In one embodiment, the mRNA comprises a nucleic acid sequence encoding a protein and a signal peptide, wherein the amino acids 1-5 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 1.3, and the signal peptide comprises, or consists of, an amino acid sequence of 12 to 40 amino acids in length.

[0273] In one embodiment of the invention, the amino acids 1 to 5 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 1.0 or greater, preferably 1.1 or greater, more preferably 1.2 or greater, even more preferably 1.25 or greater, in particular 1.3 or greater, more in particular 1.35 or greater, even more in particular 1.38 or greater. In a further embodiment, the amino acids 1 to 5 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 1 to 4.5, preferably 1.1 to 4.5, more preferably 1.2 to 4.5, even more preferably 1.25 to 4.5, in particular 1.3 to 4.5, more in particular 1.35 to 4.5, even more in particular 1.38 to 4.5. In a further embodiment, the amino acids 1-5 at the N-terminus of the amino acid sequence have an average hydrophobicity score of 1.0 to 4.0, preferably 1.1 to 4.0, more preferably 1.2 to 4.0, even more preferably 1.25 to 4.0, particularly 1.3 to 4.0, more particularly 1.35 to 4.0, even more particularly 1.38 to 4.0.

[0274] In one embodiment of the invention, the average hydrophobicity score of the last 5 amino acids at the C-terminus of the signal peptide amino acid sequence is at least 0.2 units lower, preferably at least 0.24 units lower, more preferably at least 1.0 unit lower, even more preferably at least 1.2 units lower, particularly 0.2 to 4 units lower, more particularly 0.24 to 4 units lower, more particularly 1.0 to 4 units lower, and most particularly 1.2 to 4 units lower than the average hydrophobicity score of amino acids 1 to 5 at the N-terminus of the signal peptide amino acid sequence.

[0275] In one embodiment of the invention, amino acids 1-5, amino acids 2-6, amino acids 3-7, amino acids 4-8 and amino acids 5-9 at the N-terminus of the signal peptide amino acid sequence each have an average hydrophobicity score greater than 1.0, preferably an average hydrophobicity score greater than 1.15, more preferably an average hydrophobicity score greater than 1.2, even more preferably an average hydrophobicity score greater than 1.21, more preferably an average hydrophobicity score greater than 1.23, particularly an average hydrophobicity score of 1.0 to 4.5, more particularly an average hydrophobicity score of 1.15 to 4.5, even more particularly an average hydrophobicity score of 1.2 to 4.5, more particularly an average hydrophobicity score of 1.21 to 4.5, and most particularly an average hydrophobicity score of 1.23 to 4.5.

[0276] In one embodiment of the present invention, the average hydrophobicity score of the last 5 amino acids at the C-terminus of the amino acid sequence of the signal peptide is at least 1.2 units lower than the average hydrophobicity score of amino acids 3-7 at the N-terminus of the amino acid sequence of the signal peptide, preferably 1.2 to 3.0 units lower, more preferably 1.2 to 4.3 units lower.

[0277] In one embodiment of the present invention, the average hydrophobicity score of any 5 consecutive amino acids of the amino acid sequence of the signal peptide does not exceed 4.2, preferably does not exceed 4.3.

[0278] In one embodiment of the present invention, the 59 amino acids at the C-terminus of the amino acid sequence of the signal peptide contain at least one amino acid with a negative hydrophobicity score, preferably, the last 5 amino acids at the C-terminus of the amino acid sequence of the signal peptide contain amino acids selected from G, Q, N, T, S, R, K, H, D, E, P, Y and W.

[0279] In one embodiment of the present invention, the second amino acid of amino acids 1-9 at the N-terminus of the signal peptide amino acid sequence is selected from P, Y, W, S, T, G, A, M, C, F, L, V and I.

[0280] In a preferred embodiment of the present invention, the second amino acid of amino acids 1 to 9 at the N-terminus of the signal peptide amino acid sequence is selected from the group consisting of A, L, S, T, V and W.

[0281] In one embodiment of the invention, the signal peptide is i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of amino acids that is less than 50% of the number of amino acids in the amino acid sequence of the signal peptide heterologous to said protein.

[0282] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified signal peptide has a sequence that differs from the amino acid sequence of the unmodified signal peptide heterologous to the protein by one amino acid, preferably two, more preferably three, even more preferably four, most preferably five, particularly six, more particularly seven, even more particularly eight, most particularly nine or ten amino acids.

[0283] In one embodiment, the modified signal peptide has an amino acid sequence that differs from the amino acid sequence of the unmodified signal peptide heterologous to the protein by 1-2 amino acids, preferably 1-3 amino acids, more preferably 1-4 amino acids, even more preferably 1-5 amino acids, most preferably 1-6 amino acids, in particular 1-7 amino acids, more in particular 1-10 amino acids, even more in particular 1-12 amino acids, most in particular 1-15 amino acids.

[0284] In one embodiment, the modified signal peptide has a sequence identity of 95% to 50%, preferably 95% to 60%, more preferably 95% to 70%, even more preferably 95% to 80%, and most preferably 95% to 90% to the amino acid sequence of the unmodified signal peptide heterologous to the protein.

[0285] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, provided that the modified signal peptide has a sequence that differs from the amino acid sequence of the naturally occurring (homologous) signal peptide of the protein by at least one amino acid, preferably at least two, more preferably at least three, even more preferably at least four, most preferably at least five, especially at least six, more especially at least seven, even more especially at least eight, most especially at least nine or ten amino acids. In one embodiment, the modified signal peptide heterologous to the protein has less than 95%, preferably less than 90%, more preferably less than 80%, even more preferably less than 70%, most preferably less than 60%, and especially less than 50% sequence identity with the amino acid sequence of the naturally occurring (homologous) signal peptide of the protein.

[0286] In one embodiment of the present invention, the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the amino acids 1 to 9 at the N-terminus of the amino acid sequence of the unmodified signal peptide homologous to the protein have an average hydrophobicity score of 2 or less, preferably an average hydrophobicity score of less than 2.

[0287] In one embodiment of the present invention, the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by inserting, deleting and / or substituting less than 50% of the number of amino acids in the amino acid sequence of the signal peptide homologous to the protein.

[0288] In one embodiment of the present invention, the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified signal peptide homologous to the protein differs from the amino acid sequence of the unmodified signal peptide homologous to the protein by one, preferably two, more preferably three, even more preferably four, most preferably five, especially six, more especially seven, even more especially 8-12, most especially 9-15 amino acids. In one embodiment, the modified signal peptide homologous to the protein differs from the amino acid sequence of the unmodified signal peptide homologous to the protein by 1-2 amino acids, preferably 1-3 amino acids, more preferably 1-4 amino acids, even more preferably 1-5 amino acids, most preferably 1-6 amino acids, especially 1-7 amino acids, more especially 1-10 amino acids, even more especially 1-12 amino acids, most especially 1-15 amino acids.

[0289] In one embodiment, the modified signal peptide homologous to the protein has a sequence identity of less than 95%, preferably less than 90%, more preferably less than 80%, even more preferably less than 70%, most preferably less than 60%, and in particular less than 50% to the amino acid sequence of the unmodified signal peptide homologous to the protein.

[0290] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of amino acids which is less than 50% of the number of amino acids of the naturally occurring amino acid sequence.

[0291] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence differs from the amino acid sequence of the unmodified naturally occurring amino acid sequence in 1, preferably 2, more preferably 3, even more preferably 4, most preferably 5, particularly 6, more particularly 7, even more particularly 8-12, most particularly 9-15 amino acids.

[0292] In one embodiment, the modified naturally occurring amino acid sequence differs from the amino acid sequence of the unmodified naturally occurring amino acid sequence in 1-2 amino acids, preferably 1-3 amino acids, more preferably 1-4 amino acids, even more preferably 1-5 amino acids, most preferably 1-6 amino acids, particularly 1-7 amino acids, more particularly 1-10 amino acids, even more particularly 1-12 amino acids, most particularly 1-15 amino acids.

[0293] In one embodiment, the modified naturally occurring amino acid sequence has less than 95%, preferably less than 90%, more preferably less than 80%, even more preferably less than 70%, most preferably less than 60%, and in particular less than 50% sequence identity with the amino acid sequence of the unmodified naturally occurring amino acid sequence.

[0294] In one embodiment of the present invention, the modified sequence of a naturally occurring amino acid sequence that essentially does not have a signal peptide function has an amino acid sequence that is more than 50%, preferably more than 60%, more preferably more than 70%, even more preferably more than 80%, most preferably more than 90%, and in particular more than 95% different from the amino acid sequence of a naturally occurring signal peptide. In one embodiment, the modified sequence of a naturally occurring amino acid sequence that essentially does not have a signal peptide function has a sequence identity of less than 100%, preferably less than 95%, more preferably less than 90%, even more preferably less than 80%, most preferably less than 70%, in particular less than 60%, and more in particular less than 50% with the amino acid sequence of a naturally occurring signal peptide.

[0295] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of the signal peptide of brain-derived neurotrophic factor (BDNF), the signal peptide of neurotrophin-3 (NTF-3), the signal peptide of fibroblast growth factor 5 (FGF5), the signal peptide of insulin-like growth factor binding protein 5 (IBP5), the signal peptide of prostate and testis-expressed protein 2 (PATE2), the signal peptide of extracellular superoxide dismutase (SOD3) and the signal peptide of complement factor H-related protein 2 (FHR2), or i) a signal peptide heterologous to the protein modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide heterologous to the protein is selected from the group consisting of the signal peptide of CXC motif chemokine ligand 12 (CXCL12), the signal peptide of insulin growth factor 2 (IGF2), the signal peptide of insulin (INS) and the signal peptide of brain-derived neurotrophic factor (BDNF).

[0296] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of the signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO:30, the signal peptide of neurotrophic factor-3 (NTF-3) as shown in SEQ ID NO:102, the signal peptide of fibroblast growth factor 5 (FGF5) as shown in SEQ ID NO:87, the signal peptide of insulin-like growth factor binding protein 5 (IBP5) as shown in SEQ ID NO:97, the signal peptide of prostate and testis-expressed protein 2 (PATE2) as shown in SEQ ID NO:107, the signal peptide of extracellular superoxide dismutase (SOD3) as shown in SEQ ID NO:112, and the signal peptide of complement factor H-related protein 2 (FHR2) as shown in SEQ ID NO:92, or i) a signal peptide heterologous to the protein modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified signal peptide heterologous to the protein is selected from the group consisting of the signal peptides of SEQ ID NO: A modified signal peptide of CXC motif chemokine ligand 12 (CXCL12) as shown in NO:132, a modified signal peptide of insulin growth factor 2 (IGF2) as shown in SEQ ID NO:127, a modified signal peptide of insulin (INS) as shown in SEQ ID NO:147, and a modified signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO:137.

[0297] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of brain-derived neurotrophic factor (BDNF), a signal peptide of neurotrophin-3 (NTF-3), a signal peptide of fibroblast growth factor 5 (FGF5), a signal peptide of insulin-like growth factor binding protein 5 (IBP5), a signal peptide of prostate and testis-expressed protein 2 (PATE2), a signal peptide of extracellular superoxide dismutase (SOD3) and a signal peptide of complement factor H-related protein 2 (FHR2). Preferably, the signal peptide heterologous to the protein is selected from the signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO:30, the signal peptide of neurotrophic factor-3 (NTF-3) as shown in SEQ ID NO:102, the signal peptide of fibroblast growth factor 5 (FGF5) as shown in SEQ ID NO:87, the signal peptide of insulin-like growth factor binding protein 5 (IBP5) as shown in SEQ ID NO:97, the signal peptide of prostate and testis-expressed protein 2 (PATE2) as shown in SEQ ID NO:107, the signal peptide of extracellular superoxide dismutase (SOD3) as shown in SEQ ID NO:112 and the signal peptide of complement factor H-related protein 2 (FHR2) as shown in SEQ ID NO:92.

[0298] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide heterologous to the protein is selected from the signal peptide of CXC motif chemokine ligand 12 (CXCL12), the signal peptide of insulin growth factor 2 (IGF2), the signal peptide of insulin (INS) and the signal peptide of brain-derived neurotrophic factor (BDNF).

[0299] In a preferred embodiment of the present invention, the signal peptide heterologous to the protein modified by insertion, deletion and / or substitution of at least one amino acid is selected from a modified signal peptide of CXC motif chemokine ligand 12 (CXCL12) as shown in SEQ ID NO: 132, a modified signal peptide of insulin growth factor 2 (IGF2) as shown in SEQ ID NO: 127, a modified signal peptide of insulin (INS) as shown in SEQ ID NO: 147 and a modified signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO: 137.

[0300] In one embodiment of the present invention, the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide homologous to the protein and the protein are selected from the signal peptide of insulin growth factor 1 (IGF1) and IGF1, the signal peptide of insulin and INS, the signal peptide of erythropoietin (EPO) and EPO, the signal peptide of interleukin 4 (IL-4) and IL-4, and the signal peptide of interleukin 10 (IL-10) and IL-10.

[0301] In a preferred embodiment of the present invention, the signal peptide homologous to the protein modified by insertion, deletion and / or substitution of at least one amino acid is selected from a modified signal peptide of insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 122, a modified signal peptide of insulin as shown in SEQ ID No: 147, a modified signal peptide of erythropoietin (EPO) as shown in SEQ ID NO: 152, a modified signal peptide of interleukin 4 (IL-4) as shown in SEQ ID No: 166, and a modified signal peptide of interleukin 10 (IL-10) as shown in SEQ ID No: 174.

[0302] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is selected from the group consisting of the propeptide of insulin growth factor 1 (IGF1), the coding sequence of glucagon receptor (GL-R) and the propeptide of intestinal alkaline phosphatase (ALPI).

[0303] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence, which does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is the coding sequence of the glucagon receptor (GL-R), iii) a naturally occurring amino acid sequence, which does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of insulin growth factor 1 (IGF1), or iii) a naturally occurring amino acid sequence, which does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of intestinal alkaline phosphatase (ALPI).

[0304] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is a coding sequence for glucagon receptor (GL-R).

[0305] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence which does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of insulin growth factor 1 (IGF1).

[0306] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of intestinal alkaline phosphatase (ALPI).

[0307] In a preferred embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is the coding sequence of the glucagon receptor (GL-R) as shown in SEQ ID NO: 117, iii) a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 142, or iii) a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is the propeptide of intestinal alkaline phosphatase (ALPI) as shown in SEQ ID NO: 189.

[0308] In a preferred embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is the coding sequence of the glucagon receptor (GL-R) as shown in SEQ ID NO: 117.

[0309] In a preferred embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 142.

[0310] In a preferred embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of intestinal alkaline phosphatase (ALPI) as shown in SEQ ID No: 189.

[0311] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, wherein the amount of protein secreted using the signal peptide heterologous to the protein is higher than the amount of protein secreted using the signal peptide homologous to the protein. Preferably, the amount of protein secreted using the signal peptide heterologous to the protein is higher than the amount of protein secreted using the signal peptide homologous to the protein, preferably at least 1.4 times higher.

[0312] In one embodiment of the present invention, the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the amount of secretion of the protein using the modified signal peptide homologous to the protein is higher than the amount of secretion of the protein using the unmodified signal peptide homologous to the protein. Preferably, the amount of secretion of the protein using the modified signal peptide homologous to the protein is higher than the amount of secretion of the protein using the unmodified signal peptide homologous to the protein, preferably at least 1.4 times higher.

[0313] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid, and wherein the amount of secreted protein using the naturally occurring amino acid sequence that does not essentially have a signal peptide function is higher than the amount of secreted protein using a signal peptide homologous to the protein. Preferably, the amount of secreted protein using the optionally modified naturally occurring amino acid sequence is higher than the amount of secreted protein using a signal peptide homologous to the protein, preferably at least 1.4 times higher.

[0314] In one embodiment of the invention, wherein the signal peptide is i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not thioredoxin, more particularly, wherein said protein is not rod-derived cone activity factor.

[0315] In one embodiment of the present invention, the signal peptide is selected from: i) a signal peptide heterologous to the protein, and the protein is selected from insulin growth factor 1 (IGF1), insulin (INS), erythropoietin (EPO), interleukin 4 (IL-4) and interleukin 10 (IL-10).

[0316] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, and the protein is IGF1.

[0317] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, and the protein is selected from the group consisting of insulin growth factor 1 (IGF1), insulin (INS), erythropoietin (EPO), interleukin 4 (IL-4) and interleukin 10 (IL-10);

[0318] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is a coding sequence for a glucagon receptor (GL-R) and the protein is insulin growth factor 1 (IGF1); and iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of insulin growth factor 1 (IGF1) and the protein is insulin growth factor 1 (IGF1).

[0319] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, and the protein is selected from the group consisting of insulin growth factor 1 (IGF1), insulin (INS), erythropoietin (EPO), interleukin 4 (IL-4) and interleukin 10 (IL-10).

[0320] In a preferred embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, and the protein is selected from insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 188, insulin (INS) as shown in SEQ ID NO: 185, erythropoietin (EPO) as shown in SEQ ID NO: 184, interleukin-4 (IL-4) as shown in SEQ ID No: 186, and interleukin-10 (IL-10) as shown in SEQ ID No: 187.

[0321] In one embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, and the protein is IGF1.

[0322] In a preferred embodiment of the present invention, the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, and the protein is IGF1 as shown in SEQ ID NO: 188.

[0323] In one embodiment of the present invention, the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, and the protein is selected from insulin growth factor 1 (IGF1), insulin (INS), erythropoietin (EPO), interleukin 4 (IL-4) and interleukin 10 (IL-10).

[0324] In a preferred embodiment of the present invention, the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, and the protein is selected from insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 188, insulin (INS) as shown in SEQ ID NO: 185, erythropoietin (EPO) as shown in SEQ ID NO: 184, interleukin 4 (IL-4) as shown in SEQ ID No: 186, and interleukin 10 (IL-10) as shown in SEQ ID No: 187.

[0325] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is the coding sequence of the glucagon receptor (GL-R) and the protein is insulin growth factor 1 (IGF1).

[0326] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of insulin growth factor 1 (IGF1), and the protein is insulin growth factor 1 (IGF1).

[0327] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of intestinal alkaline phosphatase (ALPI), and the protein is insulin growth factor 1 (IGF1).

[0328] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is the coding sequence of the glucagon receptor (GL-R), and the protein is the insulin growth factor 1 (IGF1) as shown in SEQ ID No: 188.

[0329] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of insulin growth factor 1 (IGF1), and the protein is insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 188.

[0330] In one embodiment of the present invention, the signal peptide is iii) a naturally occurring amino acid sequence that does not naturally have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified natural amino acid sequence is a modified propeptide of intestinal alkaline phosphatase (ALPI), and the protein is insulin growth factor 1 (IGF1) as shown in SEQ ID No: 188.

[0331] In a preferred embodiment of the present invention, the signal peptide is selected from the group consisting of:

[0332] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of brain-derived neurotrophic factor (BDNF), a signal peptide of neurotrophin-3 (NTF-3), a signal peptide of fibroblast growth factor 5 (FGF5), a signal peptide of insulin-like growth factor binding protein 5 (IBP5), a signal peptide of prostate and testis-expressed protein 2 (PATE2), a signal peptide of extracellular superoxide dismutase (SOD3), and a signal peptide of complement factor H-related protein 2 (FHR2), with the proviso that the protein is not an oxidoreductase;

[0333] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide heterologous to the protein is selected from the group consisting of the signal peptide of CXC motif chemokine ligand 12 (CXCL12), the signal peptide of insulin growth factor 2 (IGF2), the signal peptide of insulin (INS) and the signal peptide of brain-derived neurotrophic factor (BDNF), with the proviso that the protein is not an oxidoreductase.

[0334] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide homologous to the protein is selected from the group consisting of: a signal peptide of insulin growth factor 1 (IGF1), a signal peptide of insulin (INS), a signal peptide of erythropoietin (EPO), a signal peptide of interleukin 4 (IL-4) and a signal peptide of interleukin 10 (IL-10);

[0335] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is a coding sequence for a glucagon receptor (GL-R);

[0336] iii) a naturally occurring amino acid sequence, which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of insulin growth factor 1 (IGF1); and

[0337] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of intestinal alkaline phosphatase (ALPI).

[0338] In a preferred embodiment of the present invention, the signal peptide is selected from the group consisting of:

[0339] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of brain-derived neurotrophic factor (BDNF), a signal peptide of neurotrophin-3 (NTF-3), a signal peptide of fibroblast growth factor 5 (FGF5), a signal peptide of insulin-like growth factor binding protein 5 (IBP5), a signal peptide of prostate and testis-expressed protein 2 (PATE2), a signal peptide of extracellular superoxide dismutase (SOD3), and a signal peptide of complement factor H-related protein 2 (FHR2), and the protein is selected from the group consisting of a cytokine, a growth factor, and a hormone;

[0340] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of CXC motif chemokine ligand 12 (CXCL12), a signal peptide of insulin growth factor 2 (IGF2), a signal peptide of insulin (INS) and a signal peptide of brain-derived neurotrophic factor (BDNF), and the protein is selected from the group consisting of a cytokine, a growth factor and a hormone;

[0341] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide homologous to the protein and the protein are selected from the group consisting of the signal peptide of insulin growth factor 1 (IGF1) and IGF1, the signal peptide of insulin and INS, the signal peptide of erythropoietin (EPO) and EPO, the signal peptide of interleukin 4 (IL-4) and IL-4, and the signal peptide of interleukin 10 (IL-10) and IL-10;

[0342] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the signal peptide is a coding sequence for a glucagon receptor (GL-R), and the protein is selected from the group consisting of a cytokine, a growth factor, and a hormone, preferably a growth factor;

[0343] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of insulin growth factor 1 (IGF1), and the protein is selected from the group consisting of a cytokine, a growth factor, and a hormone, preferably a growth factor; and

[0344] iii) a naturally occurring amino acid sequence which essentially has no signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of intestinal alkaline phosphatase (ALPI), and the protein is selected from the group consisting of cytokines, growth factors and hormones, preferably growth factors.

[0345] In a preferred embodiment of the present invention, the signal peptide is selected from the group consisting of:

[0346] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of brain-derived neurotrophic factor (BDNF), a signal peptide of neurotrophin-3 (NTF-3), a signal peptide of fibroblast growth factor 5 (FGF5), a signal peptide of insulin-like growth factor binding protein 5 (IBP5), a signal peptide of prostate and testis-expressed protein 2 (PATE2), a signal peptide of extracellular superoxide dismutase (SOD3), and a signal peptide of complement factor H-related protein 2 (FHR2), and the protein is selected from the group consisting of insulin growth factor 1 (IGF1), insulin (INS), erythropoietin (EPO), interleukin 4 (IL-4), and interleukin 10 (IL-10);

[0347] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide heterologous to the protein is selected from the group consisting of the signal peptide of CXC motif chemokine ligand 12 (CXCL12), the signal peptide of insulin growth factor 2 (IGF2), the signal peptide of insulin (INS) and the signal peptide of brain-derived neurotrophic factor (BDNF), and the protein is insulin growth factor 1 (IGF1);

[0348] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide homologous to the protein and the protein are selected from the group consisting of the signal peptide of insulin growth factor 1 (IGF1) and IGF1, the signal peptide of insulin and INS, the signal peptide of erythropoietin (EPO) and EPO, the signal peptide of interleukin 4 (IL-4) and IL-4, and the signal peptide of interleukin 10 (IL-10) and IL-10;

[0349] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is a coding sequence for a glucagon receptor (GL-R), and the protein is insulin growth factor 1 (IGF1);

[0350] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of insulin growth factor 1 (IGF1), and the protein is insulin growth factor 1 (IGF1); and

[0351] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of intestinal alkaline phosphatase (ALPI), and the protein is insulin growth factor 1 (IGF1).

[0352] In a preferred embodiment of the present invention, the signal peptide is selected from the group consisting of:

[0353] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein and the protein are selected from the group consisting of a signal peptide of brain-derived neurotrophic factor (BDNF) and IGF1, insulin, EPO, or IL-10; a signal peptide of neurotrophin-3 (NTF-3) and IGF1; a signal peptide of fibroblast growth factor 5 (FGF5) and IGF1 or IL4; a signal peptide of insulin-like growth factor binding protein 5 (IBP5) and IGF1; a signal peptide of prostate and testis-expressed protein 2 (PATE2) and IGF1; a signal peptide of extracellular superoxide dismutase (SOD3) and IGF1; and a signal peptide of complement factor H-related protein 2 (FHR2) and IGF1;

[0354] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide heterologous to the protein and the protein are selected from the group consisting of the signal peptide of CXC motif chemokine ligand 12 (CXCL12) and IGF1; the signal peptide of insulin growth factor 2 (IGF2) and IGF1; the signal peptide of insulin (INS) and IGF1; and the signal peptide of brain-derived neurotrophic factor (BDNF) and IGF1;

[0355] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide homologous to the protein and the protein are selected from the group consisting of the signal peptide of insulin growth factor 1 (IGF1) and IGF1, the signal peptide of insulin and INS, the signal peptide of erythropoietin (EPO) and EPO, the signal peptide of interleukin 4 (IL-4) and IL-4, the signal peptide of interleukin 10 (IL-10) and IL-10;

[0356] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is a coding sequence for a glucagon receptor (GL-R), and the protein is insulin growth factor 1 (IGF1);

[0357] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of insulin growth factor 1 (IGF1), and the protein is insulin growth factor 1 (IGF1); and

[0358] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is a propeptide of intestinal alkaline phosphatase (ALPI) and the protein is insulin growth factor 1 (IGF1).

[0359] In a preferred embodiment of the present invention, the signal peptide is selected from the group consisting of:

[0360] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of brain-derived neurotrophic factor (BDNF) as set forth in SEQ ID NO:30, a signal peptide of neurotrophic factor-3 (NTF-3) as set forth in SEQ ID NO:102, a signal peptide of fibroblast growth factor 5 (FGF5) as set forth in SEQ ID NO:87, a signal peptide of insulin-like growth factor binding protein 5 (IBP5) as set forth in SEQ ID NO:97, a signal peptide of prostate and testis-expressed protein 2 (PATE2) as set forth in SEQ ID NO:107, a signal peptide of extracellular superoxide dismutase (SOD3) as set forth in SEQ ID NO:112, and a signal peptide of complement factor H-related protein 2 (FHR2) as set forth in SEQ ID NO:92, with the proviso that the protein is not an oxidoreductase;

[0361] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide heterologous to the protein is selected from the group consisting of the signal peptide of CXC motif chemokine ligand 12 (CXCL12) as shown in SEQ ID NO: 132, the signal peptide of insulin growth factor 2 (IGF2) as shown in SEQ ID NO: 127, the signal peptide of insulin (INS) as shown in SEQ ID NO: 147, and the signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO: 137, with the proviso that the protein is not an oxidoreductase.

[0362] i) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide homologous to the protein is selected from the group consisting of a modified signal peptide of insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 122, a modified signal peptide of insulin (INS) as shown in SEQ ID No: 147, a modified signal peptide of erythropoietin (EPO) as shown in SEQ ID NO: 152, a modified signal peptide of interleukin 4 (IL-4) as shown in SEQ ID NO: 166, and a modified signal peptide of interleukin 10 (IL-10) as shown in SEQ ID NO: 174.

[0363] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is the coding sequence of the glucagon receptor (GL-R) as shown in SEQ ID NO: 117;

[0364] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 142; and

[0365] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of intestinal alkaline phosphatase (ALPI) as shown in SEQ ID NO: 189.

[0366] In a preferred embodiment of the present invention, the signal peptide is selected from the group consisting of:

[0367] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO:30, a signal peptide of neurotrophic factor-3 (NTF-3) as shown in SEQ ID NO:102, a signal peptide of fibroblast growth factor 5 (FGF5) as shown in SEQ ID NO:87, a signal peptide of insulin-like growth factor binding protein 5 (IBP5) as shown in SEQ ID NO:97, a signal peptide of prostate and testis-expressed protein 2 (PATE2) as shown in SEQ ID NO:107, a signal peptide of extracellular superoxide dismutase (SOD3) as shown in SEQ ID NO:112, and a signal peptide of complement factor H-related protein 2 (FHR2) as shown in SEQ ID NO:92, and the protein is selected from the group consisting of a cytokine, a growth factor, and a hormone;

[0368] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified signal peptide heterologous to the protein is selected from the group consisting of a modified signal peptide of CXC motif chemokine ligand 12 (CXCL12) as shown in SEQ ID NO: 132, a modified signal peptide of insulin growth factor 2 (IGF2) as shown in SEQ ID NO: 127, a modified signal peptide of insulin (INS) as shown in SEQ ID NO: 147, and a modified signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO: 137, and the protein is selected from the group consisting of a cytokine, a growth factor, and a hormone;

[0369] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified signal peptide homologous to the protein and the protein are selected from the group consisting of a modified signal peptide of insulin growth factor 1 (IGF1) as set forth in SEQ ID NO: 122 and IGF1 as set forth in SEQ ID NO: 188; a modified signal peptide of insulin as set forth in SEQ ID NO: 147 and insulin (INS) as set forth in SEQ ID NO: 185; a modified signal peptide of erythropoietin (EPO) as set forth in SEQ ID NO: 152 and EPO as set forth in SEQ ID NO: 184; a modified signal peptide of interleukin 4 (IL-4) as set forth in SEQ ID NO: 166 and IL-4 as set forth in SEQ ID NO: 186; a modified signal peptide of interleukin 10 (IL-10) as set forth in SEQ ID NO: 174 and IL-10 as set forth in SEQ ID NO: 187;

[0370] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is the coding sequence of the glucagon receptor (GL-R) as shown in SEQ ID No: 117, and the protein is selected from the group consisting of a cytokine; a growth factor; and a hormone, preferably a growth factor;

[0371] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of insulin growth factor 1 (IGF1) as shown in SEQ ID No: 142, and the protein is selected from the group consisting of cytokines; growth factors; and hormones, preferably growth factors; and

[0372] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of intestinal alkaline phosphatase (ALPI) as shown in SEQ ID No: 189, and the protein is selected from cytokines; growth factors and hormones, preferably growth factors.

[0373] In a preferred embodiment of the present invention, the signal peptide is selected from the group consisting of:

[0374] i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of the signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO: 30, the signal peptide of neurotrophic factor-3 (NTF-3) as shown in SEQ ID NO: 102, the signal peptide of fibroblast growth factor 5 (FGF5) as shown in SEQ ID NO: 87, the signal peptide of insulin-like growth factor binding protein 5 (IBP5) as shown in SEQ ID NO: 97, the signal peptide of prostate and testis-expressed protein 2 (PATE2) as shown in SEQ ID NO: 107, the signal peptide of extracellular superoxide dismutase (SOD3) as shown in SEQ ID NO: 112, and the signal peptide of complement factor H-related protein 2 (FHR2) as shown in SEQ ID NO: 92, and the protein is selected from the group consisting of insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 188, insulin as shown in SEQ ID NO: 185, erythropoietin (EPO) as shown in SEQ ID NO: 184, and leukocyte antigen 2 (LEO) as shown in SEQ ID NO: 190. NO:186, interleukin-4 (IL-4), and interleukin-10 (IL-10) as shown in SEQ ID NO:187; i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified signal peptide heterologous to the protein is selected from the group consisting of a modified signal peptide of CXC motif chemokine ligand 12 (CXCL12) as shown in SEQ ID NO:132, a modified signal peptide of insulin growth factor 2 (IGF2) as shown in SEQ ID NO:127, a modified signal peptide of insulin (INS) as shown in SEQ ID NO:147, and a modified signal peptide of brain-derived neurotrophic factor (BDNF) as shown in SEQ ID NO:137, and the protein is insulin growth factor 1 (IGF1) as shown in SEQ ID No:188;

[0375] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified signal peptide homologous to the protein and the protein are selected from the group consisting of a modified signal peptide of insulin growth factor 1 (IGF1) as shown in SEQ ID NO: 122 and IGF1 as shown in SEQ ID No: 188, a modified signal peptide of insulin as shown in SEQ ID No: 147 and insulin (INS) as shown in SEQ ID No: 185, a modified signal peptide of erythropoietin (EPO) as shown in SEQ ID No: 152 and EPO as shown in SEQ ID No: 184, a modified signal peptide of interleukin 4 (IL-4) as shown in SEQ ID No: 166 and IL-4 as shown in SEQ ID No: 186, a modified signal peptide of interleukin 10 (IL-10) as shown in SEQ ID No: 174 and IL-10 as shown in SEQ ID No: 187;

[0376] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is the coding sequence of the glucagon receptor (GL-R) as shown in SEQ ID No: 117, and the protein is the insulin growth factor 1 (IGF1) as shown in SEQ ID No: 188;

[0377] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of insulin growth factor 1 (IGF1) as shown in SEQ ID No: 142, and the protein is insulin growth factor 1 (IGF1) as shown in SEQ ID No: 188; and

[0378] iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the modified naturally occurring amino acid sequence is a modified propeptide of intestinal alkaline phosphatase (ALPI) as shown in SEQ ID No: 189, and the protein is insulin growth factor 1 (IGF1) as shown in SEQ ID No: 188.

[0379] In a particularly preferred embodiment of the present invention, the mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide is selected from the group consisting of the mRNA sequence shown in SEQ ID NO: 8, the mRNA sequence shown in SEQ ID NO: 105, the mRNA sequence shown in SEQ ID NO: 90, the mRNA sequence shown in SEQ ID NO: 100, the mRNA sequence shown in SEQ ID NO: 110, the mRNA sequence shown in SEQ ID NO: 115, the mRNA sequence shown in SEQ ID NO: 95, the mRNA sequence shown in SEQ ID NO: 135, the mRNA sequence shown in SEQ ID NO: 130, the mRNA sequence shown in SEQ ID NO: 150, the mRNA sequence shown in SEQ ID NO: 140, the mRNA sequence shown in SEQ ID NO: 125, the mRNA sequence shown in SEQ ID NO: 161, the mRNA sequence shown in SEQ ID NO: 155, the mRNA sequence shown in SEQ ID NO: 169, the mRNA sequence shown in SEQ ID NO: 177, the mRNA sequence shown in SEQ ID NO: 120, the mRNA sequence shown in SEQ ID NO: 1 The mRNA sequence shown in NO:145 and the mRNA sequence shown in SEQ ID NO:192.

[0380] In a further aspect, the present invention provides an mRNA comprising a nucleic acid sequence encoding

[0381] i) protein; and

[0382] ii) a signal peptide heterologous to said protein,

[0383] wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase,

[0384] In particular, an mRNA comprising a nucleic acid sequence encoding

[0385] i) protein; and

[0386] ii) a signal peptide heterologous to said protein,

[0387] wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is selected from the group consisting of carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases and vasoactive proteins.

[0388] In one embodiment of the present invention, the protein is a therapeutic protein. In a preferred embodiment of the present invention, the protein is a human protein, i.e., a human protein. In a further preferred embodiment of the present invention, the protein is selected from the group consisting of carboxypeptidases, cytokines, extracellular ligands and transporters, extracellular matrix proteins, glucosidases, glycosyltransferases, growth factors, growth factor binding proteins, heparin binding proteins, hormones, hydrolases, immunoglobulins, isomerases, kinases, lyases, metalloenzyme inhibitors, metalloproteinases, milk proteins, neuroactive proteins, proteases, protease inhibitors, protein phosphatases, esterases, transferases, and vasoactive proteins, all of which are human. In a more preferred embodiment of the present invention, the protein of the present invention is a human protein selected from the group consisting of human carboxypeptidases; human cytokines; human extracellular ligands and transporters; human extracellular matrix proteins; human glucosidases; human glycosyltransferases; human growth factors; human growth factor binding proteins; human heparin binding proteins; human hormones; human hydrolases; human immunoglobulins; human isomerases; human kinases; human lyases; human metalloenzyme inhibitors; human metalloproteinases; human milk proteins; human neuroactive proteins; human proteases; human protease inhibitors; human protein phosphatases; human esterases; human transferases and human vasoactive proteins.

[0389] In one embodiment, the protein is selected from the group consisting of carboxypeptidases, cytokines, extracellular ligands and transporters, extracellular matrix proteins, glucosidases, glycosyltransferases, growth factors, growth factor binding proteins, heparin binding proteins, hormones, hydrolases, immunoglobulins, isomerases, kinases, lyases, metalloenzyme inhibitors, metalloproteinases, milk proteins, neuroactive proteins, proteases, protease inhibitors, protein phosphatases, esterases, transferases, and vasoactive proteins, wherein the carboxypeptidase is selected from the group consisting of ACE, ACE2, CNDP1, CPA1, CPA2, CPA4, CPA5, CPA6, CPB1, CPB2, CPE, CPN1, CPQ, CPXM1, CPZ, and SCPEP1; wherein the cell Factors selected from BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, C1QTNF4, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L, CCL4L2, CCL5, CCL7, CCL8, CD40LG, CER1, CKLF, CLCF 1,CNTF,CSF1,CSF2,CSF3,CTF1,CX3CL1,CXCL1,CXCL10,CXCL11,CXCL12,CXCL13,CXCL14,CXCL16,CXCL17,CXCL2,CXCL3,CXCL5,CXCL8,CXCL9,DKK1,D KK2,DKK3,DKK4,EDA,EBI3,FAM3B,FAM3C,FASLG,FLT3LG,GDF1,GDF10,GDF11,GDF15,GDF2,GDF3,GDF5,GDF6,GDF7,GDF9,GPI,GREM1,GREM2,GRN,IFNA 1,IFNA13,IFNA10,IFNA14,IFNA16,IFNA17,IFNA2,IFNA21,IFNA4,IFNA5,IFNA6,IFNA7,IFNA8,IFNB1,IFNE,IFNG,IFNK,IFNL1,IFNL2,IFNL3,IFNL4, IFNW1,IL10,IL11,IL12A,IL12B,IL13,IL15,IL16,IL17A,IL17B,IL17C,IL17D,IL17F,IL18,IL19,IL1A,IL1B,IL1F10,IL2,IL20,IL21,IL22,IL23A,IL24,IL25,IL26,IL27,IL3,IL31,IL32,IL33,IL34,IL36A,IL36B,IL36G,IL36RN,IL37,IL4,IL5,IL6,IL7,IL9,LEFTY1,LEFTY2,LIF,LTA,MIF,MSTN ,NAMPT,NODAL,OSM,PF4,PF4V1,SCGB3A1, SECTM1,SLURP1,SPP1,THNSL2,THPO,TNF,TNFSF10,TNFSF11,TNFSF12,TNFSF13,TNFSF13B,TNFSF14,TNFSF1 5, TSLP, VSTM1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1 and XCL2; wherein the extracellular ligand and transporter are selected from APCS, CHI3L1, CHI3L2, CLEC3B, DMBT1, DMKN, EDDM3A, EDDM3B, EFNA4, EMC10, ENAM, EPYC, ERVH48-1, F13B, FCN1, FCN2, GLD N,GPLD1,HEG1,ITFG1,KAZALD1,KCP,LACRT,LEG1,METRN,NOTCH2NL,NPNT,OLFM1,OLFML3,PRB2,PSAP,PSAPL1,PSG1,PSG6,PSG9,PTX3,PTX4,RBP4,RNA SE10,RNASE12,RNASE13,RNASE9,RSPRY1,RTBDN,S100A12,S100A13,S100A7,S100A8,SAA2,SAA4,SCG1,SCG2,SCG3,SCGB1C1,SCGB1C2,SCGB1D1,SCGB1 D2,SCGB1D4,SCGB2B2,SCGB3A2,SCGN,SCRG1,SCUBE1,SCUBE2,SCUBE3,SDCBP,SELENOP,SFTA2,SFTA3,SFTPA1,SFTPA2,SFTPC,SFTPD,SHBG,SLURP2,SM OC1,SMOC2,SMR3A,SMR3B,SNCA,SPATA20,SPATA6,SOGA1,SPARC,SPARCL1,SPATA20,SPATA6,SRPX2,SSC4D,STX1A,SUSD4,SVBP,TCN1,TCN2,TCTN1,TF,TULP3, TFF2, TFF3, THSD7A, TINAG, TINAGL1, TMEFF2, TMEM25 and VWC2L; wherein the extracellular matrix protein is selected from ABI3BP, AGRN, CCBE1, CHL1, COL15A1, COL19A1, COLEC11, DMBT1, DRAXIN, EDIL3, ELN, EMID1, EMILIN1, EMILIN2, EMILIN3, EPDR1, FBLN1, FBLN2, FBLN5, FLRT1, FLRT2, FLRT3, FREM1, GLDN, IBSP, KERA, KIAA0100, KIRREL3 , KRT10, LAMB2, MGP, RPTN, SBSPON, SDC1, SDC4, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SIGLEC1, SIGLEC10, SIGLEC6, SLIT1, SLIT2, SLIT3, SLITRK1, SNED1, SNORC, SPACA3, SPACA7, SPON1, SPON2, STATH, SVEP1, TECTA, TECTB, TNC, TNN, TNR and TNXB; wherein the glucosidase is selected from AMY1A, AMY1B, AMY1C, AMY 2A, AMY2B, CEMIP, CHIA, CHIT1, FUCA2, GLB1L, GLB1L2, HPSE, HYAL1, HYAL3, KL, LYG1, LYG2, LYZL1, LYZL2, MAN2B2, SMPD1, SMPDL3B, SPACA5 and SPACA5B; wherein the glycosyltransferase is selected from ART5, B4GALT1, EXTL2, GALNT1, GALNT2, GLT1D1, MGAT4A, ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST6GAL1 and XYLT1; wherein the growth factor is selected from AMH, ARTN, BTC ,CDNF,CFC1,CFC1B,CHRDL1,CHRDL2,CLEC11A,CNMD,EFEMP1,EGF,EGFL6,EGFL7,EGFL8,EPGN,EREG,EYS,FGF1,FGF10,FGF16,FGF17,FGF18,FGF19,FG F2,FGF20,FGF21,FGF22,FGF23,FGF3,FGF4,FGF5,FGF6,FGF7,FGF8,FGF9,FRZB,GDNF,GFER,GKN1,HBEGF,HGF,IGF1,IGF2,INHA,INHBA,INHBB,INHBC,INHBE, INS, KITLG, MANF, MDK, MIA, NGF, NOV, NRG1, NRG2, NRG3, NRG4, NRTN, NTF3, NTF4, OGN, PDGFA, PDGFB, PDGFC, PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD and WISP3; wherein the growth factor binding protein is selected from CHRD, CYR61, ESM1, FGFBP1, FGFBP2, FGFBP3, HTRA1, GHBP, IGFFALS, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, LTBP1, LTBP2, LTBP3, LTBP4, SOSTDC1, NOG, TWSG1 and WIF1; wherein the heparin binding protein is selected from ADA2, ADAMTSL5, ANGPTL3, APOB, APOE, APOH, COL5A1, COMP, CTGF, FBLN7, FN1, FSTL1, HRG, LAMC2, LIPC, LI PG, LIPH, LIPI, LPL, PCOLCE2, POSTN, RSPO1, RSPO2, RSPO3, RSPO4, SAA1, SLIT2, SOST, THBS1 and VTN; wherein the hormone is selected from ADCYAP1, ADIPOQ, ADM, ADM2, ANGPTL8, APELA, APLN, AVP, C1QTNF12, C1QTNF9, CALCA, CALCB, CCK, CGA, CGB1, CGB2, CGB3, CGB5, CGB8, COPA, CORT, CRH, CSH1, CSH2, CSHL1, ENHO, EPO, ERFE, FBN1, FN DC5,FSHB,GAL,GAST,GCG,GH,GH1,GH2,GHRH,GHRL,GIP,GNRH1,GNRH2,GPHA2,GPHB5,IAPP,INS,INSL3,INSL4,INSL5,INSL6,LHB,METRNL,MLN,NPPA,N PPB,NPPC,OSTN,OXT,PMCH,PPY,PRL,PRLH,PTH,PTHLH,PYY,RETN,RETNLB,RLN1,RLN2,RLN3,SCT,SPX,SST,STC1,STC2,TG,TOR2A,TRH,TSHB,TTR,UCN,UCN2, UCN3, UTS2, UTS2B and VIP; wherein the hydrolase is selected from the group consisting of AADACL2, ABHD15, ACP7, ACPP, ADA2, ADAMTSL1, AOAH, ARSF, ARSI, ARSJ, ARSK, BTD, CHI3L2, ENPP1, ENPP2, ENPP3, ENPP5, ENTPD5, ENTPD6, GBP1, GGH, GPLD1, HPSE, LIPC, LIPF, LIPG, LIPH, LIPI, LIPK, LIPM, LIPN, LPL, PGLYRP2, PLA1A, PLA2G10, PLA2G12A, PLA2G1B , PLA2G2A, PLA2G2D, PLA2G2E, PLA2G2F, PLA2G3, PLA2G5, PLA2G7, PNLIP, PNLIPRP2, PNLIPRP3, PON1, PON3, PPT1, SMPDL3A, THEM6, THSD1 and THSD4; wherein the immunoglobulin is selected from IGSF10, IGKV1-12, IGKV1-16, IGKV1-33, IGKV1-6, IGKV1D-12, IGKV1D-39, IGKV1D-8, IGKV2-30, IGKV2D-30, IGKV3-11, IGKV3D-20, IGKV5-2, IGL C1, IGLC2 and IGLC3; wherein the isomerase is selected from NAXE, PPIA and PTGDS; wherein the kinase is selected from ADCK1, ADPGK, FAM20C, ICOS and PKDCC; wherein the lyase is selected from PM20D1, PAM and CA6; wherein the metalloenzyme inhibitor is selected from FETUB, SPOCK3, TIMP2, TIMP3, TIMP4, WFIKKN1 and WFIKKN2; wherein the metalloprotease is selected from ADAM12, ADAM28, ADAM9, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMT S15,ADAMTS16,ADAMTS17,ADAMTS18,ADAMTS19,ADAMTS2,ADAMTS20,ADAMTS3,ADAMTS4,ADAMTS5,ADAMTS6,ADAMTS7,ADAMTS8,ADAMTS9,CLCA1,CLCA2, CLCA4,IDE,MEP1B,MMEL1,MMP1,MMP10,MMP11,MMP12,MMP13,MMP16,MMP17,MMP19,MMP2,MMP20,MMP21,MMP24,MMP25,MMP26,MMP28,MMP3,MMP7,MMP8,MMP9, PAPPA, PAPPA2, TLL1, and TLL2; wherein the milk protein is selected from CSN1S1, CSN2, CSN3 and LALBA; wherein the neuroactive protein is selected from CARTPT, NMS, NMU, NPB, NPFF, NPS, NPVF, NPW, NPY, PCSK1N, PDYN, PENK, PNOC, POMC, PROK2, PTH2, PYY2, PYY3, QRFP, TAC1 and TAC3; wherein the protease is selected from ADAMTS6, C1R, C1RL, C2, CASP4, CELA1, CELA2A, CELA2B, CFB, CFD, CFI, CMA1, CORIN,CTRB1,CTRB2,CTSB,CTSD,DHH,F10,F11,F12,F2,F3,F7,F8,F9,FAP,FURIN,GZMA,GZMK,GZMM,HABP2,HGFAC,HTRA3,HTRA4,IHH,KLK10,KLK11,K LK12,KLK13,KLK14,KLK15,KLK3,KLK4,KLK5,KLK6,KLK7,KLK8,KLK9,KLKB1,MASP1,MASP2,MST1L,NAPSA,OVCH1,OVCH2,PCSK2,PCSK5,PCSK6,PCSK9,P GA3, PGA4, PGA5, PGC, PLAT, PLAU, PLG, PROC, PRSS1, PRSS12, PRSS2, PRSS22, PRSS23, PRSS27, PRSS29P, PRSS3, PRSS33, PRSS36, PRSS38, PRSS3P2, PRSS42, PRSS44, PRSS47, PRSS48, PRSS53, PRSS57, PRSS58, PRSS8, PRTN3, RELN, REN, TMPRSS11D, TMPRSS11E, TMPRSS2, TPSAB1, TPSB2 and TPSD1; wherein the protease inhibitor is selected from A2M,A2ML1,AMBP,ANOS1,COL28A1,COL6A3,COL7A1,CPAMD8,CST1,CST2,CST3,CST4,CST5,CST6,CST7,CST8,CST9,CST9L,CST9LP1,CSTL1,EPPIN,GPC3 ,HMSD,ITIH1,ITIH2,ITIH3,ITIH4,ITIH5,ITIH6,KNG1,OPRPN,OVOS1,OVOS2,PAPLN,PI15,PI16,PI3,PZP,R3HDML,SERPINA1,SERPINA10,SERPINA11,SERPINA12,SERPINA13P,SERPINA3,SERPINA4,SERPINA5,SERPINA7,SERPINA9,SERPINB2,SERPINB5,SERPINC1,SERPINE1,SERPINE2,SERPINE3,SERPINF2,S ERPING1,SERPINI1,SERPINI2,SPINK1,SPINK13,SPINK14,SPINK2,SPINK4,SPINK5,SPINK6,SPINK7,SPINK8,SPINK9,SPINT1,SPINT3,SPINT4,SPOCK1,SPOCK 2, SPP2, SSPO, TFPI, TFPI2, WFDC1, WFDC10A, WFDC13, WFDC2, WFDC3, WFDC5, WFDC6, and WFDC8; wherein the protein phosphatase is selected from ACP7, ACPP, PTEN and PTPRZ1; wherein the esterase is selected from BCHE, CEL, CES4A, CES5A, NOTUM and SIAE; wherein the transferase is selected from METTL24, FKRP, CHSY1, CHST9 and B3GAT1; wherein the vasoactive protein is selected from AGGF1, AGT, ANGPT1, ANGPT2, ANGPTL4, ANGPTL6, EDN1, EDN2, EDN3 and NTS.

[0390] In a preferred embodiment, the protein is selected from the group consisting of cytokines; growth factors; growth factor binding proteins; heparin binding proteins; hormones; neuroactive proteins; and vasoactive proteins.

[0391] In a preferred embodiment, the protein is selected from the group consisting of cytokines; growth factors; growth factor binding proteins; heparin binding proteins; hormones; neuroactive proteins and vasoactive proteins, wherein the cytokines are selected from the group consisting of BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, C1QTNF4, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4 L3L1,CCL3L3,CCL4,CCL4L,CCL4L2,CCL5,CCL7,CCL8,CD40LG,CER1,CKLF,CLCF1,CNTF,CSF1,CSF2,CSF3,CTF1,CX3CL1,CXCL1,CXCL10,CXCL11,CXCL1 2,CXCL13,CXCL14,CXCL16,CXCL17,CXCL2,CXCL3,CXCL5,CXCL8,CXCL9,DKK1,DKK2,DKK3,DKK4,EDA,EBI3,FAM3B,FAM3C,FASLG,FLT3LG,GDF1,GDF10, GDF11,GDF15,GDF2,GDF3,GDF5,GDF6,GDF7,GDF9,GPI,GREM1,GREM2,GRN,IFNA1,IFNA13,IFNA10,IFNA14,IFNA16,IFNA17,IFNA2,IFNA21,IFNA4,IFN A5,IFNA6,IFNA7,IFNA8,IFNB1,IFNE,IFNG,IFNK,IFNL1,IFNL2,IFNL3,IFNL4,IFNW1,IL10,IL11,IL12A,IL12B,IL13,IL15,IL16,IL17A,IL17B,IL17 C,IL17D,IL17F,IL18,IL19,IL1A,IL1B,IL1F10,IL2,IL20,IL21,IL22,IL23A,IL24,IL25,IL26,IL27,IL3,IL31,IL32,IL33,IL34,IL36A,IL36B,IL3 6G,IL36RN,IL37,IL4,IL5,IL6,IL7,IL9,LEFTY1,LEFTY2,LIF,LTA,MIF,MSTN,NAMPT,NODAL,OSM,PF4,PF4V1,SCGB3A1, SECTM1,SLURP1,SPP1,THNSL2,THPO, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TSLP, VSTM1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1 and XCL2; wherein the growth factor is selected from AMH, ARTN, BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2 2, CLEC11A, CNMD, EFEMP1, EGF, EGFL6, EGFL7, EGFL8, EPGN, EREG, EYS, FGF1, FGF10, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF 3,FGF4,FGF5,FGF6,FGF7,FGF8,FGF9,FRZB,GDNF,GFER,GKN1,HBEGF,HGF,IGF1,IGF2,INHA,INHBA,INHBB,INHBC,INHBE,INS,KITLG,MANF,MDK,MIA,N GF, NOV, NRG1, NRG2, NRG3, NRG4, NRTN, NTF3, NTF4, OGN, PDGFA, PDGFB, PDGFC, PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD and WISP3; wherein the growth factor binding protein is selected from CHRD, CYR61, ESM1, FGFBP1, FGFBP2, F GFBP3, HTRA1, GHBP, IGFALS, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, LTBP1, LTBP2, LTBP3, LTBP4, SOSTDC1, NOG, TWSG1 and WIF1; wherein the heparin binding protein is selected from ADA2, ADAMTSL5, ANGPTL3, APOB, APOE, APOH, COL5A1, COMP, CTGF, FBLN7, FN1, FSTL1, HRG, LAMC2, LIPC, LIPG, LIPH, LIPI, LPL, PCOLCE2, POSTN,RSPO1, RSPO2, RSPO3, RSPO4, SAA1, SLIT2, SOST, THBS1 and VTN; wherein the hormone is selected from ADCYAP1, ADIPOQ, ADM, ADM2, ANGPTL8, APELA, APLN, AVP, C1QTNF12, C1QTNF9, CALCA, CALCB, CCK, CGA, CGB1, CGB2, CGB3, CGB5, CGB8, COPA, CORT, CRH, CSH1, CSH2, CSHL1, ENHO, EPO, ERFE, FBN1, FNDC5, FSHB, GAL, GAST, GCG, GH, GH1, GH2, GHRH, GHRL, GIP, GNRH1, GNRH2, GPHA2, GPHB5, IAPP, INS, INSL3, INSL4, INSL5, INSL6, LHB, METRNL, MLN, NPP A, NPPB, NPPC, OSTN, OXT, PMCH, PPY, PRL, PRLH, PTH, PTHLH, PYY, RETN, RETNLB, RLN1, RLN2, RLN3, SCT, SPX, SST, STC1, STC2, TG, TOR2A, TRH, TSHB, TTR, UCN, UCN2, UCN3, UTS2, UTS2B and VIP; wherein the neuroactive protein is selected from CARTPT, NMS, NMU, NPB, NPFF, NPS, NPVF, NPW, NPY, PCSK1N, PDYN, PENK, PNOC, POMC, PROK2, PTH2, PYY2, PYY3, QRFP, TAC1, and TAC3; wherein the vasoactive protein is selected from AGGF1, AGT, ANGPT1, ANGPT2, ANGPTL4, ANGPTL6, EDN1, EDN2, EDN3 and NTS.

[0392] In an even more preferred embodiment, the protein is selected from the group consisting of cytokines; growth factors; hormones and neuroactive proteins.

[0393] In a specific embodiment of the present invention, the protein is selected from the group consisting of cytokines; growth factors; hormones and neuroactive proteins, wherein the cytokines are selected from the group consisting of BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, C1QTNF4, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L3, CCL4, C CL4L,CCL4L2,CCL5,CCL7,CCL8,CD40LG,CER1,CKLF,CLCF1,CNTF,CSF1,CSF2,CSF3,CTF1,CX3CL1,CXCL1,CXCL10,CXCL11,CXCL12,CXCL13,CXCL14,CX CL16,CXCL17,CXCL2,CXCL3,CXCL5,CXCL8,CXCL9,DKK1,DKK2,DKK3,DKK4,EDA,EBI3,FAM3B,FAM3C,FASLG,FLT3LG,GDF1,GDF10,GDF11,GDF15,GDF2,G DF3,GDF5,GDF6,GDF7,GDF9,GPI,GREM1,GREM2,GRN,IFNA1,IFNA13,IFNA10,IFNA14,IFNA16,IFNA17,IFNA2,IFNA21,IFNA4,IFNA5,IFNA6,IFNA7,IFN A8,IFNB1,IFNE,IFNG,IFNK,IFNL1,IFNL2,IFNL3,IFNL4,IFNW1,IL10,IL11,IL12A,IL12B,IL13,IL15,IL16,IL17A,IL17B,IL17C,IL17D,IL17F,IL18 ,IL19,IL1A,IL1B,IL1F10,IL2,IL20,IL21,IL22,IL23A,IL24,IL25,IL26,IL27,IL3,IL31,IL32,IL33,IL34,IL36A,IL36B,IL36G,IL36RN,IL37,IL4 ,IL5,IL6,IL7,IL9,LEFTY1,LEFTY2,LIF,LTA,MIF,MSTN,NAMPT,NODAL,OSM,PF4,PF4V1,SCGB3A1, SECTM1,SLURP1,SPP1,THNSL2,THPO,TNF,TNFSF10,TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TSLP, VSTM1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1 and XCL2; said growth factors are selected from AMH, ARTN, BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2, CLEC11A, CNMD, EFEMP1, EGF, EGFL6, EGFL7, EGFL8, EPGN, EREG, EYS, FGF1, FGF10, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FRZB, GDNF, GFER, GKN1, HBEGF, HGF, IGF1, IGF2, INHA, INHBA, INHBB, INHBC, INHBE, INS, KITLG, MANF, MDK, MIA, NGF, NOV, NRG1, NRG2, NRG3, NRG4, NRTN, NTF3, NTF4, OGN, PDGFA, PDGFB, PDGFC, PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD and WISP3; said hormones are selected from ADCYAP1, ADIPOQ, ADM, ADM2, ANGPTL8, APELA, APLN, AVP, C1QTNF12, C1QTNF9, CALCA, CALCB, CCK, CGA, CGB1, CGB2, CGB3, CGB5, CGB8, COPA, CORT, CRH, CSH1, CSH2, CSHL1, ENHO, EPO, ERFE, FBN1, FNDC5, FSHB, GAL, GAST, GCG, GH, GH1, GH2, GHRH, GHRL, GIP, GNRH1, GNRH2, GPHA2, GPHB5, IAPP, INS, INSL3, INSL4, INSL5, INSL6, LHB, METRNL, MLN, NPPA, NPPB, NPPC, OSTN, OXT, PMCH,PPY, PRL, PRLH, PTH, PTHLH, PYY, RETN, RETNLB, RLN1, RLN2, RLN3, SCT, SPX, SST, STC1, STC2, TG, TOR2A, TRH, TSHB, TTR, UCN, UCN2, UCN3, UTS2, UTS2B and VIP; the neuroactive protein is selected from CARTPT, NMS, NMU, NPB, NPFF, NPS, NPVF, NPW, NPY, PCSK1N, PDYN, PENK, PNOC, POMC, PROK2, PTH2, PYY2, PYY3, QRFP, TAC1 and TAC3.

[0394] In another specific embodiment of the present invention, the protein is selected from cytokines, growth factors, hormones and neuroactive proteins, wherein the cytokines are selected from BMP-2, BMP-4, CNTF, MSTN, IFNG, IL6, SPP1; wherein the growth factors are selected from EGF, FGF1, GDNF, IGF1, IGF2, NTF3, TGFB1; wherein the hormones are selected from EPO, FBN1, GH, GHRH, OSTN, UCN; wherein the neuroactive proteins are selected from NPFF, NPY, PNOC, POMC.

[0395] In another specific embodiment of the present invention, the protein is selected from cytokines, growth factors, hormones and neuroactive proteins, wherein the cytokines are selected from BMP-2, BMP-4, CNTF, MSTN, IFNG, IL4, IL6, IL10, SPP1; wherein the growth factors are selected from EGF, FGF1, GDNF, IGF1, IGF2, NTF3, TGFB1; wherein the hormones are selected from EPO, FBN1, GH, GHRH, OSTN, UCN, INS; wherein the neuroactive proteins are selected from NPFF, NPY, PNOC, POMC.

[0396] In a more specific embodiment of the present invention, the protein is selected from growth factors. In an even more specific embodiment of the present invention, the protein is selected from growth factors, wherein the growth factor is selected from AMH, ARTN, BDNF, BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2, CLEC11A, CNMD, EFEMP1, EGF, EGFL6, EGFL7, EGFL8, EPGN, EREG, EYS, FGF1, FGF10, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FRZB, GDNF, GFER, GKN1, HBEGF, HG F,IGF1,IGF2,INHA,INHBA,INHBB,INHBC,INHBE,INS,KITLG,MANF,MDK,MIA,NGF,NOV,NRG1,NRG2,NRG3,NRG4,NRTN,NTF3,NTF4,OGN,PDGFA,PDGFB,PDGFC,PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD and WISP3.

[0397] In another more specific embodiment of the present invention, the protein is selected from the group consisting of growth factors, wherein the growth factor is selected from the group consisting of EGF, FGF1, GDNF, IGF1, IGF2, NTF3, TGFB1. Most particularly, the protein is IGF1, preferably human IGF1.

[0398] In an even more specific embodiment of the present invention, the protein is selected from cytokines, growth factors and hormones, wherein preferably, the cytokines are selected from BMP-2, BMP-4, CNTF, MSTN, IFNG, IL4, IL6, IL10, SPP1; the growth factors are selected from EGF, FGF1, GDNF, IGF1, IGF2, NTF3, TGFB1; the hormones are selected from EPO, FBN1, GH, GHRH, OSTN, UCN, INS. Most particularly, the protein is selected from insulin growth factor 1 (IGF1), insulin (INS), erythropoietin (EPO), interleukin 4 (IL-4) and interleukin 10 (IL-10).

[0399] In one embodiment of the present invention, the mRNA is naked mRNA. In a preferred embodiment, the mRNA comprises an antireverse CAP analog, such as m7G(5')G, m7GpppG cap, an internal ribosome entry site (IRES) and / or a polyA tail at the 3' end, in particular to improve translation. The mRNA may further comprise regions known to those skilled in the art that promote translation.

[0400] In a preferred embodiment of the present invention, the mRNA comprises a combination of modified and unmodified nucleotides. In a more preferred embodiment, in such a modified mRNA, 1 to 100%, preferably 10 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, and most preferably 100% of the uridine nucleotides are modified. The nucleotides containing adenosine, guanosine, and cytidine may be unmodified or partially modified, and they are preferably present in unmodified form. Preferably, the content of modified uridine nucleotides in the mRNA is in the range of 5 to 25%. In a particularly preferred embodiment of the present invention, the modified uridine nucleotide is N 1 In a more particularly preferred embodiment of the present invention, the mRNA comprises a combination of modified and unmodified nucleotides, wherein 1 to 100%, preferably 10 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, most preferably 100% of the uridine nucleotides in such modified mRNA are N 1 -Methylpseudouridine.

[0401] In a more preferred embodiment of the present invention, the mRNA is a codon-optimized mRNA and contains a combination of modified and unmodified nucleotides. In a more preferred embodiment, in such a modified mRNA, 1 to 100%, preferably 10 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, and most preferably 100% of the uridine nucleotides are modified. The nucleotides containing adenosine, guanosine, and cytidine can be unmodified or partially modified, and they are preferably present in an unmodified form. Preferably, the content of modified uridine nucleotides in the mRNA is in the range of 5 to 25%. In a particularly preferred embodiment of the present invention, the modified uridine nucleotide is N 1 -methyl pseudouridine. In a more particularly preferred embodiment of the present invention, the RNA is an mRNA comprising a combination of modified and unmodified nucleotides, wherein 1 to 100%, preferably 10 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, most preferably 100% of the uridine nucleotides in such modified mRNA are N 1 -Methylpseudouridine.

[0402] In a preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding human insulin-like growth factor 1 (IGF1) as a protein, more preferably, the mRNA is a naked mRNA comprising a nucleic acid sequence encoding human insulin-like growth factor 1 (IGF1) as a protein. In this preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding mature human IGF-1.

[0403] In a more preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding an IGF1 propeptide (preferably a human IGF1 propeptide), and a nucleic acid sequence encoding an IGF1 mature protein (preferably a human IGF1 mature protein), and does not comprise a nucleic acid sequence encoding the E-peptide of IGF1, preferably does not comprise a nucleic acid sequence encoding the E-peptide of human IGF1.

[0404] In another more preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding an IGF1 propeptide (preferably human IGF1 propeptide), a nucleic acid sequence encoding an IGF1 mature protein (preferably human IGF1 mature protein). Preferably, the mRNA does not comprise a nucleic acid sequence encoding the E-peptide of IGF1, more preferably does not comprise a nucleic acid sequence encoding the E-peptide of human IGF1. In another more preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding an IGF1 propeptide (preferably human IGF1 propeptide), a nucleic acid sequence encoding an IGF1 mature protein (preferably human IGF1 mature protein) and a nucleic acid sequence encoding a brain-derived neurotrophic factor (BDNF) signal peptide. Preferably, the mRNA does not comprise a nucleic acid sequence encoding the E-peptide of IGF1, more preferably does not comprise a nucleic acid sequence encoding the E-peptide of human IGF1.

[0405] In a more preferred embodiment of the present invention, the mRNA comprises a nucleotide sequence encoding the propeptide (also called the prodomain) of IGF1 (preferably the propeptide of human IGF1 having 27 amino acids), and a nucleotide sequence encoding mature IGF1 (preferably mature human IGF1 having 70 amino acids), and preferably does not comprise a nucleotide sequence encoding the E-peptide of IGF1, and preferably does not comprise a nucleic acid sequence encoding the E-peptide of human IGF1.

[0406] In another more preferred embodiment of the present invention, the mRNA comprises a nucleotide sequence encoding the propeptide (also referred to as the prodomain) of IGF1 (preferably a propeptide of human IGF1 having 27 amino acids), a nucleotide sequence encoding mature IGF1 (preferably a mature human IGF1 having 70 amino acids), and a nucleic acid sequence encoding the signal peptide of brain-derived neurotrophic factor (BDNF). Preferably, the mRNA does not comprise a nucleotide sequence encoding the E-peptide of IGF1, more preferably does not comprise a nucleic acid sequence encoding the E-peptide of human IGF1.

[0407] In a particularly preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding a propeptide (also called the prodomain) of human IGF1 having 27 amino acids, and a nucleotide sequence encoding a mature human IGF1 having 70 amino acids, and preferably does not comprise a nucleotide sequence encoding the E-peptide (also called the E-domain) of human IGF1, wherein the nucleotide sequence encoding the propeptide (also called the prodomain) of human IGF1 having 27 amino acids, the nucleotide sequence encoding the mature human IGF1 having 70 amino acids and the nucleotide sequence encoding the E-peptide are UniProtKB-P05019 in the Uniprot database, NM_000618.4, NM_001111285.2 and NM_001111283.2 in the Genbank database, respectively.

[0408] In a more particularly preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding a human IGF1 propeptide (also referred to as the prodomain) having 27 amino acids as shown in SEQ ID No: 38, and a nucleotide sequence encoding a mature human IGF1 having 70 amino acids as shown in SEQ ID No: 39, and preferably does not comprise a nucleotide sequence encoding the E-peptide (also referred to as the E-domain) of human IGF1.

[0409] In a more particularly preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding a human IGF1 propeptide (also referred to as the prodomain) having 27 amino acids as shown in SEQ ID No: 38, a nucleotide sequence encoding a mature human IGF1 having 70 amino acids as shown in SEQ ID No: 39, and a nucleic acid sequence encoding a brain-derived neurotrophic factor (BDNF) signal peptide, preferably a nucleotide sequence encoding a brain-derived neurotrophic factor (BDNF) signal peptide as shown in SEQ ID No: 30. Preferably, the mRNA does not comprise a nucleotide sequence encoding the E-peptide (also referred to as the E-domain) of human IGF1.

[0410] In a particularly preferred embodiment of the present invention, the mRNA comprising the nucleic acid sequence encoding the signal peptide of human insulin-like growth factor 1 (IGF1) and brain-derived neurotrophic factor (BDNF) comprises the nucleic acid sequence shown in SEQ ID NO:8.

[0411] In another more particularly preferred embodiment of the present invention, the mRNA comprising a nucleic acid sequence encoding a signal peptide of human insulin-like growth factor 1 (IGF1) and brain-derived neurotrophic factor (BDNF) comprises a nucleic acid sequence transcribed from the DNA sequence shown in SEQ ID No: 7, preferably, the nucleic acid sequence is transcribed in vitro from the DNA sequence shown in SEQ ID NO: 7.

[0412] In a more particularly preferred embodiment of the present invention, the mRNA comprising a nucleic acid sequence encoding a signal peptide of human insulin-like growth factor 1 (IGF1) and brain-derived neurotrophic factor (BDNF) comprises a nucleic acid sequence as shown in SEQ ID NO: 8, wherein preferably 1 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, and most preferably 100% of the uridine nucleotides are N 1 -Methylpseudouridine.

[0413] In another more particularly preferred embodiment of the present invention, the mRNA comprising a nucleic acid sequence encoding a signal peptide of human insulin-like growth factor 1 (IGF1) and brain-derived neurotrophic factor (BDNF) comprises a nucleic acid sequence transcribed from the DNA sequence shown in SEQ ID No: 7, wherein preferably 1 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, and most preferably 100% of the uridine nucleotides are N 1 -methyl pseudouridine. In this embodiment, the nucleotide sequence is preferably transcribed in vitro from the DNA sequence shown in SEQ ID NO: 7, and as the uridine nucleotide, only N 1 -methylpseudouridine-5'-triphosphate (N 1 -methyl pseudo-UTP) i.e., 100% N 1 -Methyl pseudo-UTP was used for transcription from the DNA sequence shown in SEQ ID NO:7.

[0414] In a preferred embodiment of the present invention, the signal peptide of brain-derived neurotrophic factor (BDNF) is the signal peptide of human BDNF, more preferably the signal peptide shown in SEQ ID No: 31, in particular the signal peptide of human BDNF encoded by the nucleic acid sequence shown in SEQ ID NO: 30.

[0415] In a more preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence encoding the following sequence from 5' to 3':

[0416] i) signal peptide of brain-derived neurotrophic factor (BDNF);

[0417] ii) optionally, a prodomain of a protein; and

[0418] iii) Mature protein.

[0419] In an even more preferred embodiment of the present invention, the mRNA comprises a nucleic acid sequence which encodes in the following order from 5' to 3':

[0420] i) signal peptide of brain-derived neurotrophic factor (BDNF);

[0421] ii) optionally, the prodomain of human IGF; and

[0422] iii) Mature human IGF.

[0423] In a preferred embodiment of the present invention, the signal peptide of brain-derived neurotrophic factor (BDNF) replaces the natural signal peptide of the protein.

[0424] In a further aspect, the present invention provides a transcription unit, an expression vector or a gene therapy vector comprising a nucleic acid encoding a protein and a signal peptide, wherein the amino acid sequence of the signal peptide has an average hydrophobicity score of greater than 2 at the N-terminus of amino acids 1-9, wherein the signal peptide is selected from

[0425] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0426] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0427] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0428] In a further aspect, the present invention provides a transcription unit, an expression vector or a gene therapy vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase, preferably, is not a thioredoxin, and more preferably, is not a rod-derived cone activity factor. The signal peptide and protein of brain-derived neurotrophic factor (BDNF) are the same as those described elsewhere herein.

[0429] In a further aspect, the present invention provides a transcription unit, an expression vector or a gene therapy vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is selected from the group consisting of carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; lactoproteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases and vasoactive proteins. The signal peptide and protein of brain-derived neurotrophic factor (BDNF) are the same as those described elsewhere herein.

[0430] In a further aspect, the present invention provides a therapeutic composition comprising mRNA and / or transcription units, expression vectors or gene therapy vectors as described above. Regarding the signal peptide and protein of brain-derived neurotrophic factor (BDNF), the same as has been described elsewhere herein. Typically, the mRNA of the present invention is provided as a therapeutic composition, which is preferably a liquid composition. A liquid composition is any composition in which the mRNA is present in a liquid solution. In one embodiment of the invention, the mRNA is dissolved in water or a buffered or unbuffered aqueous solution. The solution is preferably an aqueous solution. Thus, the liquid can be water, preferably sterile water, more preferably "water for injection" (WFI) or any other buffered or unbuffered aqueous solution. In one embodiment of the invention, the liquid composition is an unbuffered solution, preferably a saline solution, more preferably a saline solution of a pharmaceutically acceptable salt, even more preferably a NaCl solution, i.e., saline. Preferably, the saline solution is isotonic, and even more preferably it exhibits a physiological pH value. In a preferred embodiment of the invention, the solution containing the mRNA is a buffered solution. Preferably, this solution is isotonic with blood. In principle, any buffer that is effective for buffering in the physiological range, in particular in the range of pH 3.0 to 10.5, more preferably in the range of pH 4.0 to 9.0, can be used. Preferred buffers are acetate, phosphate, phosphate-buffered saline (PBS), carbonate, lactate and citrate buffers or Ringer's solution, preferably phosphate-buffered saline (PBS). Therefore, in a more preferred embodiment of the present invention, the solution containing the mRNA is phosphate-buffered saline (PBS).

[0431] The concentration of mRNA in the therapeutic composition is not particularly critical and can be adjusted as needed. Preferably, the concentration is in the range of 0.05 to 20.0 μg / μl, more preferably in the range of 0.1 to 10.0 μg / μl, even more preferably in the range of 0.2 to 5 μg / μl, in particular in the range of 0.4 to 2.0 μg / μl, more particularly in the range of 0.6 to 1.5 μg / μl, even more particularly in the range of 0.80 to 1.20 μg / μl. Particularly preferred is a range of 0.01 μg to 0.1 g, preferably 0.1 μg to 0.01 g, more preferably 0.5 μg to 1 mg, even more preferably 0.5 μg to 10 μg.

[0432] In a further aspect, the present invention provides a kit comprising the above-mentioned mRNA, and / or transcription unit, expression vector, or gene therapy vector, or therapeutic composition, and instructions, optionally a vector map, optionally a host cell, optionally a culture medium for culturing the host cell, and / or optionally a selective culture medium for selecting and culturing the transfected host cell. The kit of the present invention can be provided in a contents kit, (or in the form of) a contents kit. The kit can also comprise one or more components of the therapeutic composition of the present invention, for example, in one or more separate containers. For example, the kit can comprise mRNA (e.g., in a dried form), a solubilizing agent, and an aqueous solution (buffered or unbuffered), for example, in one, two, or three (or more) separate containers, respectively. The kit can also include instructions or an instruction insert.

[0433] In a further aspect, the present invention provides the above mRNA, transcription unit, expression vector or gene therapy vector, therapeutic composition or kit for use as a medicament. Regarding signal peptides, such as signal peptides and proteins of brain-derived neurotrophic factor (BDNF), the same as those described elsewhere herein.

[0434] In a further aspect, the present invention provides mRNA or a therapeutic composition comprising or containing mRNA for use in a method for treating skeletal muscle injury. The present invention also provides the use of mRNA or a therapeutic composition comprising or containing mRNA in the preparation of a medicament for treating skeletal muscle injury in a subject.

[0435] The present invention also provides a method of treating skeletal muscle damage in a subject, the method comprising administering to the subject mRNA or a therapeutic composition comprising or containing mRNA.

[0436] Skeletal muscle injuries, such as muscle ruptures, are among the most common injuries sustained in sports, accounting for 10-55% of all injuries sustained. Muscle injuries can be caused by eccentric muscle contraction, elongation, and muscle overload. Over 90% of all sports-related injuries are caused by eccentric muscle contraction, elongation, or muscle overload. Skeletal muscle injuries occur when a muscle is subjected to sudden, severe stress, such as a direct blow. In a muscle rupture, the muscle is subjected to excessive eccentric tension, resulting in excessive tension on the muscle fibers, causing them to rupture near the myotendinous junction (MTJ). Muscle ruptures are one of the most common complaints treated by physicians, accounting for the majority of all sports-related injuries. Injuries to the hamstring muscle complex (HMC) often affect athletes participating in sports that require rapid acceleration and deceleration during running and eccentric muscle contraction. Mild injuries can be easily managed with conservative treatment, while more devastating injuries are complete hamstring ruptures. Whether hamstring ruptures are treated conservatively or surgically depends on how they are classified. Ruptures can be mild, moderate, or severe. While mild to moderate ruptures can be managed conservatively, severe ruptures are clearly indicated for surgical intervention. Conservative management is determined by clinical presentation and begins immediately with cryotherapy, compression bandaging, immobilization, and nonsteroidal anti-inflammatory drugs, followed by elastic bandaging and physical therapy once the patient is comfortable. Therapeutic ultrasound has been widely discussed as a treatment option but has not been found to have a significant effect on the final regenerative outcome. Within 2 weeks, pain should be significantly reduced so that physical therapy can be added, including active exercises as described above. However, it is recognized in the field that surgical intervention is not without risk and candidates must be selected carefully ( TA, TL, M, V,Vaittinen S,Kalimo H, M (2007) Muscle injuries: optimizing recovery. Best Pract Res Clin Rheumatol 21(2):317-331. DOI:10.1016 / j.berh.2006.12.004; Horst K, Dienstknecht T, Sellei RM, Pape HC (2014) Partial rupture of the hamstring muscle complex: a literature review on treatment options. Eur J Orthop Surg Traumatol 24(3):285-9. DOI:10.1007 / s00590-013-1315-x). Current treatment options offer little benefit beyond the body's own healing processes, and in fact, nonsteroidal anti-inflammatory drugs (NSAIDs) may impair the healing process. As there are currently no effective drug therapies, the unmet medical need is high. In particular, there is a need to provide effective methods for treating skeletal muscle injuries that accelerate the recovery process and lead to increased function of the injured muscle.

[0437] In a preferred embodiment of the present invention, the mRNA used in the method for treating skeletal muscle injury is an mRNA encoding a growth factor, preferably an mRNA encoding human insulin-like growth factor 1 (IGF1). The mRNA encoding the growth factor generally comprises a nucleic acid sequence encoding a signal peptide, optionally comprising a nucleic acid sequence encoding a growth factor propeptide and a nucleic acid sequence encoding a mature growth factor. The mRNA encoding human IGF1 preferably comprises a nucleic acid sequence encoding a signal peptide, optionally comprising a nucleic acid sequence encoding a human IGF1 propeptide and a nucleic acid sequence encoding mature human IGF1, even more preferably comprising a nucleic acid sequence encoding a signal peptide, a nucleic acid sequence encoding a human IGF1 propeptide and a nucleic acid sequence encoding mature human IGF1, and does not comprise a nucleic acid sequence encoding the E-peptide of human IGF1. The signal peptide contained in the mRNA encoding the growth factor may be a signal peptide homologous to the growth factor, i.e., a signal peptide of the growth factor, or may be a signal peptide heterologous to the growth factor, and preferably a signal peptide heterologous to the growth factor, more preferably a signal peptide of brain-derived neurotrophic factor (BDNF), in particular a signal peptide of human BDNF. The signal peptide contained in the mRNA encoding human IGF1 can be a signal peptide homologous to human IGF1, that is, the signal peptide of human IGF1, or can be a signal peptide heterologous to human IGF1, and preferably is a signal peptide heterologous to human IGF1, more preferably is a signal peptide of brain-derived neurotrophic factor (BDNF), in particular the signal peptide of human BDNF.

[0438] Therefore, in a more preferred embodiment of the present invention, the mRNA for use in the method for treating skeletal muscle injury is an mRNA encoding human insulin-like growth factor 1 (IGF1), which comprises a nucleic acid sequence encoding a signal peptide of brain-derived neurotrophic factor (BDNF), in particular a nucleic acid sequence encoding a signal peptide of human BDNF, optionally a nucleic acid sequence encoding a human IGF1 propeptide and a nucleic acid sequence encoding mature human IGF-1. In an even more preferred embodiment of the present invention, the mRNA for use in the method for treating skeletal muscle injury is an mRNA encoding human insulin-like growth factor 1 (IGF1), which comprises a nucleic acid sequence encoding a signal peptide of brain-derived neurotrophic factor (BDNF), in particular a nucleic acid sequence encoding a signal peptide of human BDNF, optionally a nucleic acid sequence encoding a human IGF1 propeptide and a nucleic acid sequence encoding mature human IGF-1, and does not comprise a nucleic acid sequence encoding the E-peptide of human IGF1.

[0439] Therefore, in a further aspect, the present invention provides an mRNA comprising a nucleic acid sequence encoding

[0440] i) IGF1, preferably human IGF1; and

[0441] ii) A signal peptide of brain-derived neurotrophic factor (BDNF), preferably a signal peptide of human BDNF, for use in a method for treating skeletal muscle damage.

[0442] The present invention also provides the use of an mRNA comprising a nucleic acid sequence encoding:

[0443] i) IGF1, preferably human IGF1; and

[0444] ii) a signal peptide of brain-derived neurotrophic factor (BDNF), preferably a signal peptide of human BDNF.

[0445] The present invention also provides a method for treating skeletal muscle damage in a subject, the method comprising administering to the subject an mRNA comprising a nucleic acid sequence encoding:

[0446] i) IGF1, preferably human IGF1; and

[0447] ii) a signal peptide of brain-derived neurotrophic factor (BDNF), preferably a signal peptide of human BDNF.

[0448] Preferably, the present invention provides an mRNA comprising a nucleic acid sequence encoding

[0449] i) mature IGF1, preferably mature human IGF1;

[0450] ii) optionally, the prodomain of IGF1, preferably the prodomain of human IGF1;

[0451] iii) A signal peptide of brain-derived neurotrophic factor (BDNF), preferably a signal peptide of human BDNF, for use in a method for treating skeletal muscle injury.

[0452] The present invention also provides the use of an mRNA comprising a nucleic acid sequence encoding:

[0453] i) mature IGF1, preferably mature human IGF1;

[0454] ii) optionally, the prodomain of IGF1, preferably the prodomain of human IGF1;

[0455] iii) a signal peptide of brain-derived neurotrophic factor (BDNF), preferably a signal peptide of human BDNF.

[0456] The present invention also provides a method for treating skeletal muscle damage in a subject, the method comprising administering to the subject an mRNA comprising a nucleic acid sequence encoding:

[0457] i) mature IGF1, preferably mature human IGF1;

[0458] ii) optionally, the prodomain of IGF1, preferably the prodomain of human IGF1;

[0459] iii) a signal peptide of brain-derived neurotrophic factor (BDNF), preferably a signal peptide of human BDNF.

[0460] Regarding the mRNA containing nucleic acid sequences encoding the signal peptides of human insulin-like growth factor 1 (IGF1) and brain-derived neurotrophic factor (BDNF) for use in methods for treating skeletal muscle damage, the methods are the same as those described elsewhere herein. In a particularly preferred embodiment of the present invention, the mRNA containing nucleic acid sequences encoding the signal peptides of human insulin-like growth factor 1 (IGF1) and brain-derived neurotrophic factor (BDNF) comprises the nucleic acid sequence set forth in SEQ ID NO: 8. In another more particularly preferred embodiment of the present invention, the mRNA containing nucleic acid sequences encoding the signal peptides of human insulin-like growth factor 1 (IGF1) and brain-derived neurotrophic factor (BDNF) comprises a nucleic acid sequence transcribed from the DNA sequence set forth in SEQ ID NO: 7. Preferably, the nucleic acid sequence is transcribed in vitro from the DNA sequence set forth in SEQ ID NO: 7.

[0461] mRNA and / or therapeutic composition can be applied to cells and tissues, such as skeletal muscle, by means well known to those skilled in the art, preferably by injection, more preferably by intramuscular injection, usually by using a syringe with a needle. In principle, any commercially available syringe in combination with a needle can be used for this purpose. Preferably, a hypodermic needle. The diameter of the needle is represented by a needle gauge (G; according to the Stub needle gauge). Typically, a medical needle of 7G (maximum) to 33G (minimum) can be used.

[0462] In some embodiments, mRNA and / or therapeutic compositions can be delivered to cells by direct DNA transfer (Wolff et al. (1990) Science 247, 1465-1468). mRNA and / or therapeutic compositions can be delivered to cells after the cell membrane is slightly mechanically damaged and the cells are temporarily permeabilized. This mild mechanical damage to the membrane can be achieved by gently forcing the cells through a small hole (Sharei et al. PLOS ONE (2015) 10 (4), e0118803). In another embodiment, mRNA and / or therapeutic compositions can be delivered to cells by liposome-mediated DNA transfer (e.g., Gao & Huang (1991) Biochem. Ciophys. Res. Comm. 179, 280-285, Crystal (1995) Nature Med. 1, 15-17, Caplen et al. (1995) Nature Med. 3, 39-46). The term "liposome" can include various unilamellar and multilamellar lipid vehicles formed by the creation of encapsulated lipid bilayers or aggregates. The mRNA can be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, linked to the liposome via a linker molecule that is associated with the liposome and the oligonucleotide, entrapped within the liposome, or complexed with the liposome.

[0463] In one embodiment of the invention, the RNA or therapeutic composition is administered directly into skeletal muscle (preferably by injection) in the form of a therapeutic agent, i.e., a liquid composition comprising the RNA as naked RNA. The mode of administration and the properties of the composition and the RNA contained therein are the same as those described elsewhere herein. In a preferred embodiment, the liquid composition of the invention and the mRNA are each administered directly into skeletal muscle. In this case, the most preferred mode of administration is injection, i.e., intramuscular injection.

[0464] In principle, it is envisaged in the context of the present invention that as early as possible, i.e. at the earliest possible stage of skeletal muscle injury, mRNA and therapeutic composition are administered respectively. For example, this stage is once one or more first symptoms (e.g., pain) have been observed. However, any possible time point after diagnosis is all possible and worthwhile, and therefore, is all conceivable according to the present invention. For example, in the case of surgical intervention (e.g., after muscle rupture), mRNA and therapeutic composition can be administered respectively during the surgical intervention but at least soon after the surgical intervention.

[0465] In one embodiment, mRNA and therapeutic composition are respectively administered during or even before the inflammation period of skeletal muscle regeneration, and in the early proliferation phase. For example, it can be administered between the 0th day and the 10th day after injury, preferably between the 0th day and the 7th day. More specifically, it can be administered on the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th or 7th day after injury. Preferably, it is administered on the 1st day after injury and even more preferably on the 0th day after injury. In a preferred embodiment, therapeutic composition is administered before the inflammatory phase after the skeletal muscle injury. Particularly preferably, it is administered on the 1st day after injury and repeated on the 4th day after injury.

[0466] For example, depending on the progress of the injury to be treated, the administration of the mRNA according to the present invention and the therapeutic composition may be repeated at least once, but preferably multiple times (e.g., 3 to 5 times). Repeated administration may be performed after the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th or 9th day, preferably after the 2nd, 3rd, 4th, 5th, 6th, 7th day, more preferably after the 3rd, 4th or 5th day. Repeated administration may be administered every few weeks (e.g., every 1, 2, 3 or 4 weeks) to every few days (e.g., every 1, 2, 3, 4, 5 or 6 days), preferably every 2 or 3 days.

[0467] The mRNA of the present invention or therapeutic composition can be applied to the patient with a suitable dosage. The dosage regimen can be determined by the attending physician, for example, based on clinical factors. As is well known in the medical field, the dosage of any one patient depends on many factors, including the patient's size, body surface area, age, specific compound to be administered, sex, time of administration and route, overall health status and other drugs administered simultaneously. However, technicians / attending physicians easily derive (therapeutic) effective concentration and / or dosage of the active substance used, for example, (therapeutic) effective concentration and / or dosage of the active substance used in vivo or in vitro. The corresponding sample can be taken from, for example, skeletal muscle (for example, by a suitable probe), in which active compound (naked RNA) and its corresponding concentration can be detected, for example, determined by HPLC.

[0468] Typical dosages of active substances (e.g. mRNA) can be, for example, in the range of 1 ng to a few grams, preferably in the range of 0.1 μg to 1 g, preferably in the range of 1 μg to 0.1 g, more preferably in the range of 10 μg to 1 mg, even more preferably in the range of 15 μg to 0.5 mg, and most preferably in the range of 20 μg to 100 μg. Particularly preferred is a range of 0.01 μg to 0.1 g, preferably 0.1 μg to 0.01 g, more preferably 0.5 μg to 1 mg, even more preferably 0.5 μg to 10 μg. This applies in particular to human patients. When using (m)RNA therapy, the dosage of the (m)RNA used for expression should correspond to this range; however, in principle, dosages below or above this exemplary range are also conceivable, especially taking into account the above-mentioned factors. Typically, the regimen for routine administration as a therapeutic composition should be in the range of 0.1 μg to 10 mg units per kilogram of body weight per day, preferably in the range of 1 μg to 1 mg units, more preferably in the range of 10 μg to 0.1 mg units. Again, this is particularly applicable to human patients. Progress can be monitored by regular assessments. The dosage can vary, but the preferred dosage for (m)RNAs as components of the liquid composition of the present invention, administered by injection, is approximately 10 mg per injection. 5 to 10 15 Again, this applies in particular to human patients.

[0469] In particular, it is contemplated that the therapeutic compositions of the present invention will be administered to a patient, preferably a human patient / human. However, the skeletal muscle injuries described herein may also be treated (or prevented) in non-human animal subjects / patients, such as pets (e.g., dogs, cats, rabbits, rats, and mice), cattle (e.g., cows, pigs, sheep), horses (e.g., racehorses) or ponies, camels (e.g., racing camels), or birds (e.g., chickens, turkeys, parrots).

[0470] In particular, therapeutic compositions comprising mRNA have therapeutic activity in the healing process of injuries, disorders and / or diseases, such as skeletal muscle injuries.

[0471] In a more particularly preferred embodiment of the present invention, the mRNA encoding insulin-like growth factor 1 (IGF1) for use as a medicament comprises a nucleic acid sequence transcribed from the DNA sequence of SEQ ID NO: 7. In an even more particularly preferred embodiment of the present invention, the mRNA encoding insulin-like growth factor 1 (IGF1) for use as a medicament comprises the nucleic acid sequence of SEQ ID NO: 8.

[0472] Any therapeutic combination of the present invention can be provided together with instructions or instructions insert.Instructions / inserts can include guidance for technical personnel / attending physician, instruct how it treats (or prevents) disease as described herein or patient (skeletal muscle injury) according to the present invention.Especially, instructions / inserts can include guidance, and this guidance is respectively about delivery as described herein / mode of administration and delivery / administration scheme (such as delivery / route of administration, dosage regimen, delivery / administration time, delivery / administration frequency).Especially, instructions / inserts can include explanation, and this explanation illustrates injection mRNA and / or preparation for the mRNA that is injected into skeletal muscle respectively.Instructions / inserts can further include explanation, and this explanation illustrates the mRNA that is prepared for use during the inflammation after skeletal muscle injury respectively.In principle, elsewhere herein respectively about the content described in delivery / mode of administration and delivery / administration scheme can be included in instructions / inserts as corresponding explanation.

[0473] Specific implementation plan

[0474] 1. An mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein the amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from

[0475] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase;

[0476] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0477] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0478] 2. The mRNA according to embodiment 1, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average polarity of 6.1 or less.

[0479] 3. The mRNA of embodiment 1 or 2, wherein the average hydrophobicity score of the last 9 amino acids at the C-terminus of the amino acid sequence of the signal peptide is at least 1.0 unit lower than the average hydrophobicity score of amino acids 1-9 at the N-terminus of the amino acid sequence of the signal peptide.

[0480] 4. The mRNA of any one of embodiments 1-3, wherein amino acids 1-9, amino acids 2-10, amino acids 3-11, amino acids 4-12, and amino acids 5-13 at the N-terminus of the amino acid sequence of the signal peptide each have an average hydrophobicity score greater than 1.5.

[0481] 5. The mRNA of any one of embodiments 1-4, wherein the average hydrophobicity score of amino acids 8-16 at the N-terminus of the amino acid sequence of the signal peptide is equal to or lower than the average hydrophobicity score of amino acids 3-11 at the N-terminus of the amino acid sequence of the signal peptide.

[0482] 6. The mRNA of any one of embodiments 1-4, wherein the signal peptide comprises an amino acid sequence of 18 to 40 amino acids in length, and wherein the average hydrophobicity score of amino acids 10-18 at the N-terminus of the amino acid sequence of the signal peptide is at least 0.5 units lower than the average hydrophobicity score of amino acids 3-11 at the N-terminus of the amino acid sequence of the signal peptide.

[0483] 7. The mRNA of any one of embodiments 1-6, wherein the average hydrophobicity score of the last 9 amino acids at the C-terminus of the amino acid sequence of the signal peptide is at least 1.5 units lower than the average hydrophobicity score of amino acids 3-11 at the N-terminus of the amino acid sequence of the signal peptide.

[0484] 8. The mRNA of any one of embodiments 1-7, wherein the average hydrophobicity score of any 9 consecutive amino acids of the amino acid sequence of the signal peptide does not exceed 4.1.

[0485] 9. The mRNA according to any one of embodiments 1-8, wherein the last 9 amino acids at the C-terminus of the amino acid sequence of the signal peptide contain at least one amino acid with a negative hydrophobicity score.

[0486] 10. The mRNA of embodiment 9, wherein the at least one amino acid having a negative hydrophobicity score is selected from the group consisting of G, Q, N, T, S, R, K, H, D, E, P, Y, and W.

[0487] 11. The mRNA of any one of embodiments 1-10, wherein the second amino acid of amino acids 1-9 at the N-terminus of the amino acid sequence of the signal peptide is selected from the group consisting of P, Y, W, S, T, G, A, M, C, F, L, V, and I.

[0488] 12. The mRNA according to any one of embodiments 1-10, wherein the second amino acid of amino acids 1-9 at the N-terminus of the amino acid sequence of the signal peptide is selected from A, L, S, T, V and W.

[0489] 13. The mRNA of any one of embodiments 1-12, wherein the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the average hydrophobicity score of the N-terminal amino acids 1-9 of the amino acid sequence of the modified signal peptide homologous to the protein is at least 1.0 unit higher than the average hydrophobicity score of the N-terminal amino acids 1-9 of the amino acid sequence of the unmodified signal peptide.

[0490] 14. The mRNA of any one of embodiments 1-13 or 2-13, wherein the hydrophobicity score is calculated according to the Kyte-Doolittle scale and the polarity is calculated according to the Zimmerman Polarity index.

[0491] 15. The mRNA of any one of embodiments 1-14, wherein the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of brain-derived neurotrophic factor (BDNF), a signal peptide of neurotrophin-3 (NTF-3), a signal peptide of fibroblast growth factor 5 (FGF5), a signal peptide of insulin-like growth factor binding protein 5 (IBP5), a signal peptide of prostate and testis-expressed protein 2 (PATE2), a signal peptide of extracellular superoxide dismutase (SOD3), and a signal peptide of complement factor H-related protein 2 (FHR2).

[0492] 16. The mRNA of any one of embodiments 1-14, wherein the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide heterologous to the protein is selected from the group consisting of a signal peptide of CXC motif chemokine ligand 12 (CXCL12), a signal peptide of insulin growth factor 2 (IGF2), a signal peptide of insulin (INS), and a signal peptide of brain-derived neurotrophic factor (BDNF).

[0493] 17. The mRNA of any one of embodiments 1-14, wherein the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, wherein the signal peptide homologous to the protein and the protein are selected from the group consisting of the signal peptide of insulin growth factor 1 (IGF1) and IGF1, the signal peptide of insulin and INS, the signal peptide of erythropoietin (EPO) and EPO, the signal peptide of interleukin 4 (IL-4) and IL-4, and the signal peptide of interleukin 10 (IL-10) and IL-10.

[0494] 18. The mRNA of any one of embodiments 1-14, wherein the signal peptide is iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid, wherein the naturally occurring amino acid sequence is selected from the group consisting of the propeptide of insulin growth factor 1 (IGF1), the coding sequence of the glucagon receptor (GL-R) and the propeptide of intestinal alkaline phosphatase (ALPI).

[0495] 19. The mRNA of any one of embodiments 1-18, wherein the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, and wherein the amount of protein secreted using the signal peptide heterologous to the protein is higher than the amount of protein secreted using a signal peptide homologous to the protein.

[0496] 20. The mRNA of any one of embodiments 1-18, wherein the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, and wherein the amount of protein secreted using the modified signal peptide homologous to the protein is higher than the amount of protein secreted using an unmodified signal peptide homologous to the protein.

[0497] 21. The mRNA of any one of embodiments 1-18, wherein the signal peptide is iii) a naturally occurring amino acid sequence that essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid, and wherein the amount of secreted protein using the naturally occurring amino acid sequence that essentially does not have a signal peptide function is higher than the amount of secreted protein using a signal peptide homologous to the protein.

[0498] 22. The mRNA of any one of embodiments 1-21, wherein the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of amino acids less than 50% of the number of amino acids in the amino acid sequence of the signal peptide heterologous to the protein.

[0499] 23. The mRNA of any one of embodiments 1-21, wherein the signal peptide is ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of amino acids less than 50% of the number of amino acids in the amino acid sequence of the signal peptide homologous to the protein.

[0500] 24. The mRNA of any one of embodiments 1-21, wherein the signal peptide is iii) a naturally occurring amino acid sequence that does not essentially function as a signal peptide, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of amino acids that is less than 50% of the number of amino acids in the amino acid sequence of the naturally occurring amino acid sequence.

[0501] 25. The mRNA of any one of embodiments 1-24, wherein the protein is selected from the group consisting of a cytokine, a growth factor, and a hormone.

[0502] 26. The mRNA of any one of embodiments 1-25, wherein the signal peptide is: i) a signal peptide heterologous to the protein, and the protein is selected from insulin growth factor 1 (IGF1), insulin (INS), erythropoietin (EPO), interleukin 4 (IL-4) and interleukin 10 (IL-10).

[0503] 27. The mRNA of any one of embodiments 1-25, wherein the signal peptide is i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid, and the protein is IGF1.

[0504] 28. The mRNA of any one of embodiments 1-27, wherein the signal peptide is iii) a naturally occurring amino acid sequence that does not essentially have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid, and the protein is IGF1.

[0505] 29. The mRNA of any one of embodiments 1-5 and embodiments 7-28, wherein the signal peptide comprises an amino acid sequence of 16 to 40 amino acids in length.

[0506] 30. A transcription unit, expression vector or gene therapy vector comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that the protein is not an oxidoreductase;

[0507] ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and

[0508] iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

[0509] 31. A therapeutic composition comprising the mRNA of any one of embodiments 1-29 and / or the transcription unit, expression vector or gene therapy vector of embodiment 30.

[0510] 32. A kit comprising the mRNA of any one of embodiments 1-29, the transcription unit, expression vector or gene therapy vector of embodiment 30, and / or the therapeutic composition of embodiment 31, and instructions, optionally a vector map, optionally a host cell, optionally a culture medium for culturing the host cell, and / or optionally a selective medium for selecting and culturing the transfected host cell.

[0511] 33. An mRNA comprising a nucleic acid sequence encoding

[0512] i) protein; and

[0513] ii) a signal peptide heterologous to said protein,

[0514] wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase.

[0515] 34. An mRNA comprising a nucleic acid sequence encoding

[0516] i) protein; and

[0517] ii) a signal peptide heterologous to said protein,

[0518] wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is selected from the group consisting of carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases and vasoactive proteins.

[0519] 35. The mRNA of embodiment 33 or 34, wherein the protein is selected from the group consisting of a cytokine; a growth factor; a growth factor binding protein; a heparin binding protein; a hormone; a neuroactive protein; and a vasoactive protein.

[0520] 36. The mRNA of embodiment 33 or 34, wherein the protein is a growth factor.

[0521] 37. The mRNA according to embodiment 36, wherein the growth factor is selected from AMH, ARTN,

[0522] BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2, CLEC11A,

[0523] CNMD,EFEMP1,EGFL6,EGFL7,EGFL8,EPGN,EREG,EYS,FGF1,FGF10,

[0524] FGF16,FGF17,FGF18,FGF19,FGF2,FGF20,FGF21,FGF22,FGF23,FGF3,FGF4,

[0525] FGF5,FGF6,FGF7,FGF8,FGF9,FRZB,GDNF,GFER,GKN1,HBEGF,IGF1,IGF2,

[0526] INHA,INHBA,INHBB,INHBC,INHBE,KITLG,MANF,MDK,MIA,NGF,NOV,

[0527] NRG1,NRG2,NRG3,NRG4,NRTN,NTF3,NTF4,OGN,PDGFA,PDGFB,PDGFC,

[0528] PDGFD,PGF,PROK1,PSPN,PTN,SDF1,SDF2,SFRP1,SFRP2,SFRP3,SFRP4,

[0529] SFRP5,TDGF1,TFF1,TGFA,TGFB1,TGFB2,TGFB3,THBS4,TIMP1,VEGFA,

[0530] VEGFB, VEGFC, VEGFD, and WISP3.

[0531] 38. The mRNA of embodiment 33 or 34, wherein the protein is IGF1.

[0532] 39. The mRNA of any one of embodiments 33-38, wherein the signal peptide of brain-derived neurotrophic factor (BDNF) comprises the amino acid sequence shown in SEQ ID NO: 31.

[0533] 40. The mRNA of any one of embodiments 33-39, wherein the nucleic acid sequence encoding the protein is operably linked to a nucleic acid sequence encoding a signal peptide of a brain-derived neurotrophic factor (BDNF) that is heterologous to the protein.

[0534] 41. The mRNA of any one of embodiments 33-40, wherein the mRNA comprises a nucleic acid sequence encoding the following order from 5' to 3':

[0535] i) signal peptide of brain-derived neurotrophic factor (BDNF);

[0536] ii) optionally, a prodomain of a protein; and

[0537] iii) mature protein;

[0538] The nucleic acid sequence encoding the signal peptide of brain-derived neurotrophic factor (BDNF), optionally the nucleic acid sequence encoding the prodomain of the protein and the nucleic acid sequence encoding the mature protein are operably linked.

[0539] 42. An mRNA according to any one of embodiments 33-41, wherein the mRNA comprises a nucleic acid sequence encoding the propeptide of IGF1, a nucleic acid sequence encoding mature IGF1 and a nucleic acid sequence encoding the signal peptide of brain-derived neurotrophic factor (BDNF), and does not comprise a nucleic acid sequence encoding the E-peptide of IGF1.

[0540] 43. The mRNA of any one of embodiments 33-42, wherein the signal peptide of brain-derived neurotrophic factor (BDNF) replaces the natural signal peptide of the protein.

[0541] 44. A transcription unit, expression vector or gene therapy vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase.

[0542] 45. A transcription unit, expression vector or gene therapy vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is selected from the group consisting of carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases, immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases and vasoactive proteins.

[0543] 46. ​​A therapeutic composition comprising the mRNA of any one of embodiments 33-43 and / or the transcription unit, expression vector or gene therapy vector of embodiment 44 or 45.

[0544] 47. A kit comprising the mRNA of any one of embodiments 33-43, the transcription unit, expression vector or gene therapy vector of embodiment 44 or 45, and / or the therapeutic composition of embodiment 46, and instructions, optionally a vector map, optionally a host cell, optionally a culture medium for culturing the host cell, and / or optionally a selective medium for selecting and culturing the transfected host cell.

[0545] 48. Use of the mRNA according to any one of embodiments 1-28 or 33-43, the transcription unit, expression vector or gene therapy vector according to embodiment 30 or 44-45, the therapeutic composition according to embodiment 31 or 46 or the kit according to embodiment 32 or 47 as a medicament.

[0546] 49. The mRNA of any one of embodiments 1-29 or embodiments 38 or 42, for use in a method of treating skeletal muscle injury.

[0547] 50. An mRNA for use in a method for treating skeletal muscle injury.

[0548] 51. A therapeutic composition comprising mRNA for use in a method for treating skeletal muscle injury.

[0549] 52. The mRNA or composition for use according to embodiment 50 or 51, wherein the mRNA encodes human insulin-like growth factor 1 (IGF1).

[0550] 53. The mRNA or composition for use according to embodiment 52, wherein the mRNA encoding human IGF1 comprises a nucleic acid sequence encoding a signal peptide, optionally a nucleic acid sequence encoding a human IGF1 propeptide and a nucleic acid sequence encoding mature human IGF1.

[0551] 54. The mRNA or composition for use according to embodiment 53, wherein the nucleic acid sequence encoding a signal peptide encodes a signal peptide of brain-derived neurotrophic factor (BDNF).

[0552] Example

[0553] Example 1 Methods and Materials

[0554] Cloning of IGF1 and replacement of signal peptide

[0555] IGF1 is a 70 amino acid polypeptide synthesized in the endoplasmic reticulum and secreted by the Golgi apparatus, which plays a role as an extracellular growth factor in an autocrine and paracrine manner. In order to ensure that the IGF1 induced by mRNA is correctly expressed and secreted from transfected cells, the mRNA sequence includes the natural N-terminal pre-pre-sequence (pre-pro-IGF1) of people IGF1. This sequence is composed of a sequence (nucleotides 1-63) encoding a human IGF1 pre-domain (signal peptide) with 21 amino acids and a sequence (nucleotides 64-144) encoding a human pro-domain with 27 amino acids. In addition, the construct includes a sequence (nucleotides 145-354) encoding a complete coding sequence of a mature human IGF1 with 70 amino acids. In Cpd.2-7, the pre-domain (signal peptide, nucleotides 1-63) is replaced by each pre-domain of IGF2, ALB, BDNF, CXCL12 or a synthetic signal peptide 1 or 2. The C-terminal E-domain is not added to the construct. In summary, the cloning vector contains a copy of the human pre-pro-IGF1 DNA without the E-peptide information and is designated Cpd.1, while Cpds.2-7 contain an alternative pre-domain (signal peptide). Figure 1 The DNA and RNA sequences of IGF1 encoded by its predomain, prodomain and coding domain are shown. Figure 2 The DNA and RNA sequences of IGF1 encoded by the IGF2 predomain, its prodomain and coding domain are shown. Figure 3 The DNA and RNA sequences of IGF1 encoded by the ALB predomain and its prodomain and coding domain are shown. Figure 4 The DNA and RNA sequences of IGF1 encoded by the BDNF predomain and its prodomain and coding domain are shown. Figure 5 The DNA and RNA sequences of IGF1 encoded by the CXCL12 prodomain and its prodomain and coding domain are shown. Figure 6 The DNA and RNA sequences of IGF1 encoded by the synthetic signal peptide 1 predomain and its prodomain and coding domain are shown. Figure 7 The DNA and RNA sequences of IGF1 encoded by the synthetic signal peptide 2 predomain and its prodomain and coding domain are shown. Figure 8 Shown is the pVAX.A120 vector (www.thermofisher.com) with the Cpd.1 insert. Figure 9 Shown is the pMA-T vector (www.thermofisher.com) with the Cpd.2 insert. Figure 10 The pMA-T vector is shown with a Cpd.3 insert. Figure 11 The pMA-T vector is shown with a Cpd.4 insert. Figure 12 The pMA-T vector is shown with a Cpd.5 insert. Figure 13 Shown is the pMA-RQ vector (www.thermofisher.com) with the Cpd.6 insert. Figure 14 Shown is the pMA-RQ vector (www.thermofisher.com) with the Cpd.6 insert. Figure 15 Primers used to amplify Cpd.2-7 are shown. Figure 16 The identification of the different pre-domains is summarized by indicating the gene name, UniProt number, DNA and amino acid sequence of the pre-domain and the vector. For Cpd.6 and Cpd.7, there is no gene name because they are artificial pre-domains. Codon optimization of the DNA and mRNA sequences of Cpds. 1-7 was performed by Fischer-Merck (ThermoFischer, MA).

[0556] The open reading frame of the pre-pro-IGF1 DNA sequence was synthesized by GeneArt (www.thermofisher.com, ThermoFischer, MA) with BamHI and EcoRI restriction sites and subcloned into the pVAX1.A120 vector using the same restriction enzymes. The DNA sequence of the entire vector is shown in Figure 8 The insertion direction and base sequence of the clones were confirmed by Sanger sequencing of several clones. Successful clones were selected as templates for in vitro transcription (IVT) mRNA production. For the alternative pre-domain variants Cpd.2-Cpd.7, pMA-T ( Figure 9-12 ) and pMA-RQ( Figure 13-14 ) vector was used as a template for IVT. All IVT reactions produced mRNA with the same polyA120 tail.

[0557] Replace the signal peptide in Cpd.8-Cpd.39 mRNA.

[0558] In Cpd.8-26 and Cpd.39, the predomain (signal peptide, nucleotides 1-63) of IGF1 (i.e., Cpd.1) was replaced by LTBP2 (Cpd.8; Uniprot ID: Q14767), IGFALS (Cpd.9; Uniprot ID: P35858), INS (Cpd.10; Uniprot ID: P01308), Epo (Cpd.11; Uniprot ID: P01588), CSF3 (Cpd.12; Uniprot ID: P09919), NGF (Cpd.13; Uniprot ID: P01138), FGF5 (Cpd.14; Uniprot ID: P12034), FHR2 (Cpd.15; Uniprot ID: P36980), IBP5 (Cpd.16; Uniprot ID: P36980), and CSF3 (Cpd.17; Uniprot ID: P01588). ID: P24593), NTF3 (Cpd.17; Uniprot ID: P20783), PATE2 (Cpd.18; Uniprot ID: Q6UY27), each predomain of SOD3 (Cpd.19; Uniprot ID: P08294), a partial coding sequence of GLR (Cpd.20; Uniprot ID: P47871), a modified predomain sequence of IGF1 (Cpd.21; Uniprot ID: P05019), a modified predomain sequence of IGF2 (Cpd.22; Uniprot ID: P01344), a modified predomain sequence of CXCL12 (Cpd.23; Uniprot ID: P48061), and a modified predomain sequence of BDNF (Cpd.24; Uniprot ID: P47871). ID: P23560), the modified pre-domain sequence of IGF1 (Cpd.25; Uniprot ID: P05019), the modified pre-domain sequence of ALPI (Cpd.39; Uniprot ID: P09923), and the modified pre-domain sequence of INS (Cpd.26; Uniprot ID: P01308). Similar to Cpd.1, all of the above specific compounds do not have E-peptide. By using Codon optimization of the DNA and mRNA sequences of Cpd.8-26 and Cpd was performed by Fischer-Merck (ThermoFischer, MA).

[0559] Cpd.27 consists of a sequence encoding the pre-domain (signal peptide) of human erythropoietin (Epo; Uniprot ID: P01588) with 27 amino acids (nucleotides 1-81) and a sequence encoding the coding chain of human erythropoietin with 166 amino acids (nucleotides 82-498). In Cpd.28 and Cpd.29, the pre-domain (signal peptide, nucleotides 1-81) of Epo is replaced by a modified pre-domain sequence of Epo (Uniprot ID: P01588) and a pre-domain sequence of BDNF (Uniprot ID: P23560). By using Codon optimization of the DNA and mRNA sequences of Cpds27-29 was performed by Fischer-Merck (ThermoFischer, MA).

[0560] Cpd.30 is composed of a sequence encoding a pre-domain (signal peptide) of human insulin (INS; Uniprot ID: P01308) with 24 amino acids (nucleotides 1-72), a sequence encoding a B-chain domain with 30 amino acids (nucleotides 73-162), a sequence encoding a connecting peptide (C-peptide) domain with 31 amino acids (nucleotides 163-255), and a sequence encoding an A-chain domain with 21 amino acids (nucleotides 256-330). In Cpd.31 and Cpd.32, the pre-domain (signal peptide, nucleotides 1-72) of INS is replaced by a modified pre-domain sequence of INS (Uniprot ID: P01308) and a pre-domain sequence of BDNF (Uniprot ID: P23560). By using Codon optimization of the DNA and mRNA sequences of Cpd.30-32 was performed by Fischer-Merck (ThermoFischer, MA).

[0561] Cpd.33 consists of a sequence encoding a pre-domain (signal peptide) of human interleukin 4 (IL-4; Uniprot ID: P05112) having 24 amino acids (nucleotides 1-72) and a sequence encoding a coding chain domain having 129 amino acids (nucleotides 73-387). In Cpd.34 and Cpd.35, the pre-domain (signal peptide, nucleotides 1-72) of IL-4 is replaced by a modified pre-domain sequence of IL-4 (Uniprot ID: P05112) and a pre-domain sequence of FGF5 (Uniprot ID: P01308). By using Codon optimization of the DNA and mRNA sequences of Cpd.33-35 was performed by Fischer-Merck (ThermoFischer, MA).

[0562] Cpd.36 consists of a sequence encoding a pre-domain (signal peptide) of human interleukin 10 (IL-10; Uniprot ID: P22301) having 24 amino acids (nucleotides 1-54) and a sequence encoding a coding chain domain having 160 amino acids (nucleotides 55-534). In Cpd.37 and Cpd.38, the pre-domain (signal peptide, nucleotides 1-54) of IL-10 is replaced by a modified pre-domain sequence of IL-10 (Uniprot ID: P22301) and a pre-domain sequence of BDNF (Uniprot ID: P23560). By using Codon optimization of the DNA and mRNA sequences of Cpd.36-38 was performed by Fischer-Merck (ThermoFischer, MA).

[0563] Table 1 below shows the amino acid sequence and DNA sequence of the signal peptide of Cpd.1-39, as well as the RNA sequence and DNA sequence of each Cpd.1-39 and the vector.

[0564] Table 1: Amino acid sequence and DNA sequence of the signal peptide of Cpd.1-39, RNA sequence and DNA sequence of Cpd.1-39 and vector

[0565]

[0566]

[0567]

[0568] In vitro transcription (IVT) of Cpd.1 to Cpd.7 mRNA

[0569] The pVAX.A120 vector containing Cpd.1 (SEQ ID No. 15) also has a T7 promoter and a 120 bp poly-A tail. The vector was linearized downstream of the poly-A tail using Xho I enzyme and then in vitro transcribed (IVT) to generate mRNA. For pMA-T and pMA-RQ vectors, IVT-mRNA was generated based on PCR using a homologous primer pair (SEQ ID No: 22 and 23). Figure 15). The reverse primer contained 120 bp of poly-A to include a poly-A tail in the mature mRNA. IVT was performed using T7 RNA polymerase in the MEGAscript T7 kit (www.ambion.com), and the linearized plasmid and PCR amplicons were used as templates for IVT. All mRNAs generated had an anti-reverse CAP analog (ARCA; [m7G(5')G]) at the 5' end and were chemically modified with 100% N1-methylpseudouridine-UTP (www.trilink.com). In vitro transcribed mRNA was purified using the MEGAclear kit (www.ambion.com) and analyzed for quality and concentration using the RNA 6000Nano kit in an Agilent 2100 Bioanalyzer (www.agilent.com).

[0570] In vitro transcription (IVT) of Cpd.1 and Cpd.8 to Cpd.39 mRNA

[0571] For pMA-T and pMA-RQ vectors encoding Cpd.1 (SEQ ID No. 40; before subcloning into pVAX.A120 vector) and Cpd.8 to Cpd.39, PCR-based IVT-mRNA generation was performed using homologous primer pairs (SEQ ID No: 22 and 23) ( Figure 15 ). The reverse primer contained 120 bp of poly-A to include a poly-A tail in the mature mRNA. PCR amplicons were used as templates for IVT using T7 RNA polymerase in the MEGAscript T7 kit (www.ambion.com). All mRNAs generated had an anti-reverse CAP analog (ARCA; [m7G(5')G]) at the 5' end and were chemically modified with 100% N1-methylpseudouridine-UTP (www.trilink.com). In vitro transcribed mRNA was purified using the MEGAclear kit (www.ambion.com) and analyzed for quality and concentration using RNA agarose gel electrophoresis.

[0572] In vitro transfection of HEK293T, C2C12 and HepG2 cells

[0573] Human embryonic kidney 293 cells (HEK293T; ATCC, CRL-1573, Rockville, MD, USA) were maintained in Dulbecco's Modified Eagle's Medium (DMEM, www.biochrom.com) supplemented with 10% (v / v) fetal bovine serum (FBS) and a mixture of penicillin-streptomycin-amphotericin B (882087, Biozym, Oldendorf, Germany). Cells were seeded in 96-well plates at 7,000-20,000 cells / well and incubated for 24 hours at 37°C in a humidified atmosphere containing 5% CO2 before transfection. Cells were grown in DMEM growth medium containing 10% FBS and without antibiotics to a confluence of <60% before transfection.

[0574] The human hepatocellular carcinoma cell line HepG2 (Cat#85011430, ECACC UK) was grown at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum and a mixture of penicillin-streptomycin-amphotericin B (882087, Biozym, Oldendorf, Germany). HepG2 cells were subcultured every 2 days and every 5 days at a split ratio of 1:2 and 1:4, respectively. 24 hours before transfection, cells were plated into 96-well microtiter plates at a density of 20,000-40,000 cells / well. Cells were grown in DMEM growth medium containing 10% FBS and without antibiotics to reach a confluency of 30-40% before transfection.

[0575] Mouse myoblast cell line C2C12 (ATCC, CRL-1772, Rockville, MD, USA) was grown at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum and a mixture of penicillin-streptomycin-amphotericin B (882087, Biozym, Oldendorf, Germany). C2C12 cells were subcultured every 2 days and every 5 days at a split ratio of 1:2 and 1:4, respectively. 24 hours before transfection, cells were plated at a density of 20,000 cells / well into 96-well microtiter plates. Cells were grown in DMEM growth medium containing 2% FBS and without antibiotics to reach a confluence of 80-90% before transfection.

[0576] Afterwards, cells were transfected with 0.3 μg of the different mRNA variants using Lipofectamine 2000 (www.invitrogen.com) according to the manufacturer's instructions. 100 μl of DMEM was removed and replaced with 50 μl of Opti-MEM and 50 μl of Opti-MEM containing the mRNA and Lipofectamine 2000 complex (www.thermofisher.com). After 5 hours, the medium was replaced with fresh medium, and the plates were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 hours.

[0577] In vitro transfection of HSkMC cells

[0578] HSkMC cells were plated in SkM growth medium (PromoCell, Heidelberg, Germany) on microtiter plates at a density of 40,000 cells per 96 wells. Cells were grown for 1 day in a humidified atmosphere at 37°C incubators and 5% CO2 to a confluence of >90%. On the same day of transfection, cells were treated with Lipofectamin 2000 (www.invitrogen.com) and 2 μg of different mRNA variants (Cpd.1 or 4). 100 μl of culture medium was then removed and 1 μl of Lipofectamin / well and 2 μg of mRNA / well were added to OPTIMEM culture medium (www.thermofisher.com). The cells were then incubated at 37°C and 5% CO2 in a humidified atmosphere for 24 hours.

[0579] In vitro transfection of IMR32 cells

[0580] 24 hours before transfection, Caucasian neuroblastoma IMR32 cells (Cat#86041809, ECACC, UK) were plated at a density of 60,000 cells per well in 96 pre-coated BRAND microtiter plates (Cat#782082) in Minimum Essential Medium Eagle (EMEM, Bioconcept Cat#1-31S01-I, www.bioconcept.ch) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS), L-glutamine (2 mM) and non-essential amino acids (NEAA, 1x). Cells were grown overnight at 37°C in a humidified atmosphere containing 5% CO2. Cells were transfected with 0.3 μg of mRNA construct using JetMessenger (www.polyplus-transfection.com) according to the manufacturer's instructions. Briefly, mRNA / JetMessenger complexes were formed by mixing 0.25 μl of JetMessenger reagent per 0.1 μg of mRNA construct. After incubation at room temperature for 15 minutes, 10 μl of JetMessenger complex was added. Five hours after transfection, the medium / mRNA / JetMessenger was removed from the wells and replaced with fresh 100 μl of growth medium, and the plates were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 hours.

[0581] In vitro transfection of A549 cells

[0582] Human lung cancer cell line (Sigma-Aldrich, Buchs Switzerland cat#6012804) was maintained in Dulbecco's Modified Eagle's high glucose medium (DMEM, Sigma-Aldrich, Buchs Switzerland cat#D0822) supplemented with 10% FBS (Thermofischer, Basel, Switzerland cat#10500-064). 24 hours before transfection, A549 cells were plated at a density of 10,000 cells / well in regular growth medium. Thereafter, cells were transfected with different mRNAs (0.3-0.6 μg) using Lipofectamine 2000 (www.invitrogen.com) according to the manufacturer's instructions. 100 μl of DMEM was removed. 50 μl of Opti-MEM (www.thermofisher.com) was added to each well, followed by the addition of 50 μl of Opti-MEM containing the mRNA and Lipofectamine 2000 complex. After 5 hours of culture, the medium was replaced with fresh medium, and the plates were incubated at 37° C. in a humidified atmosphere containing 5% CO 2 for 24 hours.

[0583] In vitro transfection of THP-1 cells

[0584] The human monocytic leukemia cell line THP-1 (Sigma-Aldrich, Buchs, Switzerland, Cat. #88081201) was maintained in growth medium (RPMI 1640) supplemented with 10% FBS and 2 mM glutamine. 72 hours prior to transfection, cells were seeded in 96-well cell culture plates at 30,000 THP-1 cells and activated with 50 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Buchs, Switzerland, Cat. #P8139) diluted in growth medium. Cells were transfected with mRNA (300-600 ng / well) using Lipofectamine 2000 (www.thermofisher.com), and 100 μl of DMEM was removed. 50 μl of Opti-MEM (www.thermofisher.com) was added to each well, followed by 50 μl of Opti-MEM containing mRNA and Lipofectamine 2000 complex. After 5 hours, the medium was replaced with fresh growth medium supplemented with 50 nM PMA, and the plates were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 hours.

[0585] Rat primary spinal cord neurons

[0586] Pregnant female wild-type Wistar rats (Janvier labs, France) or SOD1G93A Sprague Dawley rats (Taconic Bioscience) at 14 days of gestation were sacrificed using deep CO2 anesthesia and cervical dislocation. The fetuses were removed from the uterus and immediately placed in ice-cold Leibovitz medium supplemented with 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). The spinal cords were dissected and treated with 0.05% trypsin-0.02% EDTA for 20 minutes at 37°C. Dissociation was stopped by adding Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g / l glucose, 0.5 mg / mL DNAase I and II, and 10% fetal calf serum (FCS). The cells were mechanically separated by forced passage through a 10 mL pipette tip three times. In addition, the cells were centrifuged at 515 g for 10 minutes at 4°C. The resulting pellet was resuspended in a defined medium consisting of Neurobasal medium containing 2% B27 supplement solution, 2 mmol / l glutamine, 2% PS solution, and 10 ng / ml brain-derived neurotrophic factor (BDNF). Cells were seeded at 20,000 cells per well in 96-well poly-D-lysine pre-coated plates and cultured at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed every two days. After 11-12 days of culture, mRNA constructs (0.3 g) were transfected using JetMessenger (www.polyplus-transfection.com) according to the manufacturer's instructions.

[0587] In vitro transfection of human differentiated chondrocytes.

[0588] Human articular cartilage chondrocytes (Sigma / Cell Applications, Buchs, Switzerland Cat.#402-05A) were maintained in chondrocyte growth medium (Sigma Aldrich, Buchs, Switzerland Cat.#411-500). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Cells were differentiated by growing them on alginate beads in differentiation medium (Sigma Aldrich, Buchs, Switzerland Cat.#A411D-250) for a minimum of 3 weeks. For the preparation of alginate beads, 4x10 6Chondrocytes use 1ml 1.2% sterile alginate solution (1.2% Alginate Sigma Aldrich in 0.9% NaCl, Buchs Switzerland Cat.#A-2033). The cells are resuspended in the 1.2% alginate solution of corresponding volume and dispensed dropwise into the 100mM CaCl solution in 6-well untreated cell culture plates by a 22-gauge needle. After 15 minutes, the polymerized beads are washed 5 times with 0.9% NaCl and washed 2 times with differentiation medium. Chondrocytes / alginate beads are incubated at 37°C in a humid atmosphere containing 5% CO for a minimum of 3 weeks, with differentiation, and the culture medium is changed every two days. 24 hours before transfection, differentiated chondrocytes are released from alginate beads by washing 2 times with 0.9% NaCl and incubating for about 5 minutes in alginate dissolution buffer (55mM sodium citrate, 150mM NaCl, 30mM EDTA pH 6.8). Cells were washed 2 times with 0.9% NaCl. 30,000 cells per well were seeded in 100 μl growth medium in 96-well TPP plates (SigmaAldrich, Buchs, Switzerland Cat.#92096), and grown overnight. According to the manufacturer's instructions, JetMessenger (www.polyplus-transfection.com) was used to transfect cells with 0.6 μg mRNA constructs. 10 μl mRNA / JetMessenger complexes were added in quadruplicate. 0.25 μl JetMessenger reagent was mixed with every 0.1 μg mRNA construct to form mRNA / JetMessenger complexes, and incubated at room temperature for 15 minutes. After 5 hours after transfection, transfection complex (culture medium / mRNA / JetMessenger) was removed from the wells and replaced with 100 μl growth medium. Plates were incubated 24 hours at 37°C in a humid atmosphere containing 5% CO .

[0589] Analysis of protein levels in cell culture supernatants

[0590] 24 hours after transfection, the supernatant of transfected cells was collected, frozen and stored at 20°C until quantitative analysis by ELISA according to the manufacturer's instructions: IGF1 (Cat.#E20, Mediagnost, Reutlingen, Germany), erythropoietin (EPO; Cat.#BMS2035, ThermoFisher, Basel, Switzerland), insulin (INS, Cat.#RAB0327, Sigma-Aldrich, Buchs, Switzerland), interleukin 4 (IL-4, Cat.#88-7046-22, ThermoFisher, Basel, Switzerland) and interleukin 10 (IL-10, Cat.#KIT 10947 SinoBiological, China). Cell supernatants were analyzed after dilution with the corresponding ELISA buffer.

[0591] Data Analysis

[0592] To assess the protein (IGF1, EPO, INS, IL-4, IL-10) levels in the standards or samples, the average absorbance value of the blank was subtracted from the average absorbance of the standards or samples. According to the manufacturer's protocol, a four-parameter nonlinear regression was used to generate and plot a standard curve. To determine the concentration of the protein (IGF1, EPO, INS, IL-4, IL-10) in each sample, the concentration of the different proteins was interpolated from the standard curve. The final protein concentration of the sample was calculated by multiplying by the dilution factor. All calculations were performed using GraphPad Prism8 (San Diego, USA). To represent the increase compared to the endogenous signal peptide construct, the protein level produced by each construct was divided by the protein level produced by the endogenous signal peptide construct at the same concentration.

[0593] result

[0594] Cloning of IGF1

[0595] Successful cloning of all inserts into pVAX.A120 was confirmed by Sanger sequencing. All clones tested resulted in the correct orientation of the IGF1 insert, with 100% sequence accuracy. Positive clones were selected for IVT mRNA production.

[0596] Average hydrophobicity and polarity of Cpd.1-39

[0597] The average hydrophobicity of the N-terminal amino acids 1-9, amino acids 1-7, amino acids 1-5 and the last nine amino acids at the C-terminus of the signal peptide amino acid sequence of Cpd.1-39, as well as the average polarity of the N-terminal amino acids 1-9 of Cpd.1-39 are shown in Tables 2-5 below.

[0598] Table 2: Average hydrophobicity and polarity of the N-terminal amino acids 1-18 and the last 9 amino acids at the C-terminus of the signal peptide amino acid sequence of Cpd.1-39, and average polarity of the N-terminal amino acids 1-9 of the signal peptide of Cpd.1-39

[0599]

[0600]

[0601] Table 3: Average hydrophobicity and polarity of the N-terminal amino acids 1-18 and the last nine amino acids at the C-terminus of the signal peptide amino acid sequence of Cpd.1-39, and average polarity of the N-terminal amino acids 1-9 of the signal peptide of Cpd.1-39 (continued)

[0602]

[0603]

[0604] Table 4: Average hydrophobicity and polarity of the N-terminal amino acids 1-13 and the last nine amino acids at the C-terminus of the signal peptide amino acid sequence of Cpd.1-39, and average polarity of the N-terminal amino acids 1-7 of the signal peptide of Cpd.1-39

[0605]

[0606]

[0607] Table 5: Average hydrophobicity and polarity of the N-terminal amino acids 1-9 and the last 9 amino acids at the C-terminus of the signal peptide amino acid sequence of Cpd.1-39, and the average polarity of the N-terminal amino acids 1-5 of the signal peptide of Cpd.1-39

[0608]

[0609]

[0610] In vitro transcription of mRNA

[0611] IGF1_pVAX.A120 plasmid was linearized with Xho I and IGF1 mRNA (Cpd.1) was generated using the IVT system. Similarly, 50-200 μg of IGF1 mRNA with altered pre-domains (signal peptide, Cpd.2-Cpd.7 encoded in vectors pMA-T and pMA-RQ) was generated using PCR-based IVT. Figure 16 ) for in vitro transfection experiments. Similarly, for Cpd.1 (SEQ ID No. 40) and Cpd.8-26 encoded in the pMA-T and pMA-RQ vectors, PCR-based IVT was used to generate 50-200 μg of IGF1 mRNA with altered signal peptides for in vitro transfection experiments. In addition to IGF1 mRNA, 50-200 μg of mRNA for erythropoietin (EPO, Cpd.27-29), insulin (INS, Cpd.30-32), interleukin 4 (IL4, Cpd.33-35), and interleukin 10 (IL10, Cpd.36-38) with endogenous or altered signal peptides were also generated for in vitro transfection experiments.

[0612] In vitro transfection of HEK293T cells to detect IGF1 secretion

[0613] After HEK293T cells were incubated with Cpd.1-Cpd.7 mRNA for 24 h, the secreted IGF1 levels in the cell culture supernatant were assessed ( Figure 17 ). Cpd.1 was able to induce IGF1 secretion up to 50 ng / ml. Cpd.4 induced significantly higher IGF1 secretion than Cpd.1 (3.3 times, 0.001). To evaluate the concentration dependence of Cpd.1 and Cpd.4, different concentrations of Cpd.1 and Cpd.4 (0.02-2 μg / well) were tested to induce IGF1 secretion into the supernatant ( Figure 18 ). Cpd.1 showed an EC50 of 0.89 μg and Cpd.4 showed an EC50 of 0.13 μg, indicating that Cpd.4 was 6.8 times more potent in inducing IGF1 secretion from HEK293T cells. In summary, Figure 17 and 18 Our data demonstrate that Cpd.4 induces IGF1 secretion in HEK293T cells more potently and efficiently than Cpd.1, suggesting that this signal peptide promotes the exit of IGF1 produced in this cell type from the cells.

[0614] In vitro transfection of C2C12 cells to test IGF1 secretion

[0615] After C2C12 cells were incubated with Cpd.1-Cpd.7 mRNA for 24 h, the secreted IGF1 levels in the cell culture supernatant were assessed ( Figure 19Cpd.1 was able to induce IGF1 secretion up to 60 ng / ml. Cpd.4 induced significantly higher IGF1 secretion than Cpd.1 (6.1-fold, 0.001). The data showed that Cpd.4 induced IGF1 secretion more strongly than Cpd.1 in C2C12 cells, suggesting that this signal peptide also promotes the exit of IGF1 produced in this cell type.

[0616] In vitro transfection of HSkMC cells to test IGF1 secretion

[0617] After HSkMC cells were incubated with Cpd.1 or Cpd.4 mRNA for 24 h, the secreted IGF1 levels in the cell culture supernatant were assessed ( Figure 20 Cpd.1 was able to induce IGF1 secretion up to 30 ng / ml. Cpd.4 induced significantly higher IGF1 secretion than Cpd.1 (3.1-fold, P < 0.05). The data showed that Cpd.4 induced IGF1 secretion more strongly than Cpd.1 in primary HSkMC cells, suggesting that this signal peptide also promotes the exit of IGF1 produced in this cell type.

[0618] In vitro transfection of other mRNAs in HEK293T cells to detect IGF1 secretion

[0619] In another set of tests, the potential of Cpd.8-Cpd.26 to regulate IGF1 secretion from HEK293T cells was analyzed. After 24 hours of incubation with Cpd.1 as a control and Cpd.8-26 as test mRNAs, the levels of secreted IGF1 in the cell culture supernatant were assessed ( Figure 22 ). The Cpd.1 response was normalized to 1, and the data are expressed as fold change of Cpd.1. Cpd.8, Cpd.9, Cpd.10, Cpd.11, Cpd.12, and Cpd13 showed decreased secretion of IGF1, whereas Cpd.14, Cpd.15, Cpd.16, Cpd.17, Cpd.18, Cpd.19, Cpd.20, Cpd.21, Cpd.23, Cpd.24, Cpd.25, and Cpd.26 were able to induce significantly higher IGF1 secretion, up to 2.6-fold, compared to Cpd.1. Among them, Cpd.15 and Cpd.21 showed similar induction to Cpd.4 (see Figure 17 In summary, the data indicate that Cpd.14, Cpd.15, Cpd.16, Cpd.17, Cpd.18, Cpd.19, Cpd.20, Cpd.21, Cpd.23, Cpd.24, Cpd.25, and Cpd.26 induce IGF1 secretion in HEK293T cells more potently and efficiently than Cpd.1, suggesting that these signal peptides promote the exit of IGF1 produced in this cell type from the cells.

[0620] In vitro transfection of HepG2 cells to test IGF1 secretion

[0621] After HepG2 cells were incubated with Cpd.1 as a control and Cpd.4-26 as a test mRNA for 24 h, the secreted IGF1 levels in the cell culture supernatant were assessed ( Figure 23 ). The Cpd.1 response was normalized to 1, and the data are expressed as the fold change of Cpd.1. Among them, Cpd.8, Cpd.9, and Cpd.12 showed reduced secretion of IGF1, while Cpd.4, Cpd.14, Cpd.15, Cpd.16, Cpd.17, Cpd.18, Cpd.19, Cpd.20, Cpd.21, Cpd.22, Cpd.23, Cpd.24, Cpd.25, and Cpd.26 were able to induce significantly higher IGF1 secretion than Cpd.1, up to 8.3-fold. In summary, the data indicate that Cpd.4, Cpd.14, Cpd.15, Cpd.16, Cpd.17, Cpd.18, Cpd.19, Cpd.20, Cpd.21, Cpd.22, Cpd.23, Cpd.24, Cpd.25, and Cpd.26 induce IGF1 secretion in HepG2 cells more potently than Cpd.1, suggesting that these signal peptides promote the exit of IGF1 produced in this cell type from the cells.

[0622] In vitro transfection of IMR32 neural cells to test IGF1 secretion

[0623] After IMR324 neural cells were incubated with Cpd.1 as a control and Cpd.4-24 as a test mRNA for 24 hours, the secreted IGF1 level in the cell culture supernatant was evaluated ( Figure 24 ). Cpd.1 responses were normalized to 1, and data are expressed as fold change of Cpd.1. Cpd.4, Cpd.14, Cpd.15, Cpd.16, Cpd.17, Cpd.20, Cpd.22, Cpd.23, and Cpd.24 were able to induce significantly higher IGF1 secretion, up to 2.6-fold, than Cpd.1. In summary, the data indicate that Cpd.4, Cpd.14, Cpd.15, Cpd.16, Cpd.17, Cpd.20, Cpd.22, Cpd.23, and Cpd.24 induce IGF1 secretion more potently than Cpd.1 in IMR32 neural cells, suggesting that these signal peptides promote the exit of IGF1 produced in this cell type from the cell.

[0624] In vitro transfection of human chondrocytes to test IGF1 secretion

[0625] After chondrocytes were incubated with Cpd.1 as a control and Cpd....

Claims

1. An mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein the amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that said protein is not an oxidoreductase; ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

2. A transcription unit, expression vector or gene therapy vector comprising a nucleic acid sequence encoding a protein and a signal peptide, wherein amino acids 1 to 9 at the N-terminus of the amino acid sequence of the signal peptide have an average hydrophobicity score greater than 2, wherein the signal peptide is selected from i) a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, with the proviso that the protein is not an oxidoreductase; ii) a signal peptide homologous to the protein, wherein the signal peptide homologous to the protein is modified by insertion, deletion and / or substitution of at least one amino acid; and iii) a naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion and / or substitution of at least one amino acid.

3. A therapeutic composition comprising the mRNA of claim 1 and / or the transcription unit, expression vector or gene therapy vector of claim 2.

4. A kit comprising the mRNA of claim 1, the transcription unit, expression vector or gene therapy vector of claim 2, and / or the therapeutic composition of claim 3, and instructions, optionally a vector map, optionally a host cell, optionally a culture medium for culturing the host cell, and / or optionally a selective medium for selecting and culturing the transfected host cell.

5. An mRNA comprising a nucleic acid sequence encoding i) protein; and ii) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to the protein is a signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase.

6. An mRNA comprising a nucleic acid sequence encoding i) protein; and ii) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is selected from the group consisting of carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases and vasoactive proteins.

7. A transcription unit, expression vector or gene therapy vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is not an oxidoreductase.

8. A transcription unit, expression vector or gene therapy vector comprising a nucleic acid sequence encoding a protein and a signal peptide heterologous to the protein, wherein the signal peptide heterologous to the protein is the signal peptide of brain-derived neurotrophic factor (BDNF), and wherein the protein is selected from the group consisting of carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases, immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteinases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases and vasoactive proteins.

9. A therapeutic composition comprising the mRNA of claim 6 and / or the transcription unit, expression vector or gene therapy vector of claim 7 or 8.

10. A kit comprising the mRNA of claim 6, the transcription unit, expression vector or gene therapy vector of claim 7 or 8, and / or the therapeutic composition of claim 9, and instructions, optionally a vector map, optionally a host cell, optionally a culture medium for culturing the host cell, and / or optionally a selective medium for selecting and culturing the transfected host cell.

11. An mRNA for use in a method for treating skeletal muscle damage.

12. A therapeutic composition comprising mRNA for use in a method for treating skeletal muscle injury.

Images