Polypeptide, RNA molecule and medical use thereof

Abstract

The present disclosure relates to a polypeptide, an RNA molecule and a medical use thereof, and specifically relates to an ANGPTL polypeptide, an RNA molecule, a polynucleotide, a vector, lipid nanoparticles, a pharmaceutical composition and a medical use thereof.

Description

Polypeptides, RNA molecules and their medicinal uses Technical Field

[0001] This disclosure pertains to the field of biomedicine, specifically relating to ANGPTL peptides, RNA molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and pharmaceutical uses. Background Technology

[0002] Osteoarthritis (OA) is a degenerative joint disease that affects mobility and is accompanied by pain. In OA patients, cartilage degeneration occurs in the joints, with catabolism exceeding anabolism, disrupting cartilage homeostasis. Glycoproteins and collagen fibers, among other extracellular matrix components, continuously degrade, leading to calcification of the cartilage layer. As the disease progresses, the bone structure in the joints also gradually changes, with subchondral bone remodeling, bone sclerosis, and bone spur formation. This cartilage degradation is accompanied by inflammation, with upregulation of pro-inflammatory factors such as IL-1β, TNFα, and IL-6. These pro-inflammatory factors activate collagenases and glycoproteins, including MMP13, further degrading the extracellular matrix, creating a vicious cycle that continuously advances the disease. The main clinical manifestations of OA patients include joint pain and stiffness, a grinding sensation, and a feeling of swelling and bone spurs.

[0003] The causes of osteoarthritis are complex, including age, obesity, joint injury, genetics, and certain metabolic diseases. Current treatments for osteoarthritis are relatively limited. In the early stages, pain relief medications, such as anti-inflammatory drugs (NSAIDs), are the primary approach. Later-stage patients may require surgical procedures such as joint replacement. Clinically, there is a significant lack of medications and treatments that can alleviate or even improve the condition.

[0004] With a deeper understanding of the mechanisms of osteoarthritis, drug development for osteoarthritis has flourished in recent years, targeting various avenues including inflammation suppression, chondrocyte protection, and cartilage repair. Inflammation suppression targets primarily work by inhibiting pro-inflammatory signaling pathways, reducing their activation of collagenases and glycoproteases, and decreasing the degradation of the cartilage extracellular matrix, thus protecting the cartilage. Examples include GNSC-001 and FX201 targeting IL-1β, and ART-I02 targeting IFN-β. Chondrocyte protection targets directly inhibit the activity of collagenases and glycoproteases, reducing the degradation of the cartilage extracellular matrix or inhibiting chondrocyte senescence to reduce cartilage degeneration. Examples include GLPG1972 and M6495 targeting ADAMTS5. Mechanistically, both of these fall under the development strategy of disease-relieving drugs. Cartilage repair targets, on the other hand, promote chondrocyte regeneration, repair degenerated cartilage layers, and reshape the joint environment, belonging to the disease-improving target category. Due to the complexity of the disease, research on these drugs is relatively limited.

[0005] Angiopoietin-like proteins (ANGPTLs) are a family of eight secreted glycoproteins (ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7, and ANGPTL8). They exhibit structural homology with angiopoietin but have different physiological functions, including roles in lipid metabolism, stem cell expansion, inflammation, tissue remodeling, and angiogenesis.

[0006] Members of the ANGPTL family share common structural features (except for ANGPTL8, which exhibits only atypical structural features), and they are similar to, yet differ from, angiopoietins (Ang). Apart from ANGPTL8, the remaining ANGPTL members include an N-terminal signal peptide sequence (SS); a coiled-coil domain (CCD) that mediates homologous oligomer formation; and a C-terminal fibrinogen-like domain (FLD; or fibrinogen-homology domain, FHD) that regulates ligand activity. ANGPTL8 (also known as Lipasin) lacks both the CCD and FLD domains.

[0007] This disclosure relates to the design of proteins with chondrogenic activity and the expression of drug proteins via mRNA-encoded protein sequences for use in cartilage damage, osteoarthritis, and other cartilage degeneration-related diseases. Summary of the Invention

[0008] This disclosure relates to ANGPTL peptides and fusion proteins, RNA molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and pharmaceutical uses.

[0009] Specifically, this disclosure designs and screens modified peptides (ANGPTL peptides) or fusion proteins for the treatment, relief, and / or prevention of osteoarthritis-related diseases. First, by modifying and optimizing the length of ANGPTL family proteins (e.g., ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4), a series of truncated peptide fragments are obtained. Further, by combining sequences from different proteins (ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4) from the same family, the function of the peptides is optimized (e.g., to exert the specific functions of each protein), increasing protein expression levels, enhancing chondrogenic activity, relieving joint pain, increasing half-life, and reducing dosing frequency, etc. This disclosure also further screens a series of amino acid mutations to enhance the above-mentioned optimized functions. This disclosure also provides nucleic acid molecules (e.g., RNA, DNA) capable of expressing the above-mentioned peptides or fusion proteins, as well as nucleic acid construction vectors.

[0010] This disclosure provides a polypeptide comprising at least two polypeptide fragments derived from ANGPTL1, ANGPTL2, ANGPTL3 or ANGPTL4, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with them.

[0011] This disclosure also provides a polypeptide comprising at least one polypeptide fragment derived from ANGPTL3, said ANGPTL3 polypeptide fragment comprising at least one heterologous amino acid sequence.

[0012] This disclosure also provides a polypeptide comprising one or more polypeptide fragments derived from ANGPTL3 having an amino acid mutation selected from any one or more of the following positions: position 259, position 280, position 289, position 304, position 330, position 350, position 384, position 423, position 424, and position 432; wherein the amino acid mutation site is a natural sequence site relative to the amino acid sequence shown in SEQ ID NO: 3.

[0013] This disclosure also provides a polypeptide comprising: one or more ANGPTL polypeptide fragments, wherein...

[0014] The ANGPTL polypeptide fragment is a truncated ANGPTL1, ANGPTL2, or ANGPTL4 polypeptide fragment, or a polypeptide fragment that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0015] This disclosure also provides a polypeptide, characterized in that the amino acid sequence of the polypeptide is any one of SEQ ID NOs: 5-32 and 34-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0016] This disclosure also provides a fusion protein, characterized in that the amino acid sequence of the fusion protein is any one of SEQ ID NOs: 5-10, 26-30, 32, and 35-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0017] This disclosure also provides a polypeptide comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein: (1) the first polypeptide comprises amino acids 269-290 of the amino acid sequence shown in SEQ ID NO: 2; (2) the second polypeptide comprises amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) the third polypeptide comprises amino acids 295-300 of the amino acid sequence shown in SEQ ID NO: 2; (4) the fourth polypeptide comprises amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) the fifth polypeptide comprises amino acids 488-493 of the amino acid sequence shown in SEQ ID NO: 2, wherein (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the polypeptide further comprises a K423Q mutation.

[0018] “ANGPTL1” is a member of the angiopoietin-like protein family, comprising a signal peptide (amino acids 1-23 of SEQ ID NO:1, or a corresponding fragment thereof), an N-terminal coiled-coil domain (CCD, amino acids 80-168 of SEQ ID NO:1, or a corresponding fragment thereof), and a C-terminal fibroinogen-like domain (amino acids 271-491 of SEQ ID NO:1, or a corresponding fragment thereof). The amino acid sequence of ANGPTL1 (GenBank accession number NP_001363692) is shown in SEQ ID NO:1; the corresponding polynucleotide sequence (GenBank accession number NM_001376763) is shown in SEQ ID NO:141. “ANGPTL1 polypeptide” refers to a naturally expressed or modified polypeptide. For the purposes of this disclosure, amino acid numbering is typically determined with reference to the full-length wild-type human ANGPTL1 polypeptide sequence (SEQ ID NO:1). Therefore, in embodiments where the polypeptide of the present invention contains only the C-terminal portion of the full-length ANGPTIL1 and excluding the N-terminal portion, the position is still based on the SEQ ID NO:1 number, even though the length of the peptide is less than 491 amino acids. For example, referring to position 423 of the ANGPTIL1 polypeptide of the present invention refers to position 423 of SEQ ID NO:1, even if the ANGPTIL1 polypeptide itself may be only 200 amino acids long. The amino acid in the target sequence “corresponding” to the position in the reference sequence, such as SEQ ID NO:1, can be determined by optimal sequence alignment, for example, using the default or commonly used CLUSTAL alignment parameters or the default BLAST2 alignment parameters, and by comparing the sequences. For example, position 423 of the target sequence “determined by reference SEQ ID NO:1”, or the amino acid “corresponding to” position 423 of SEQ ID NO:1, refers to the amino acid aligned with position 423 of SEQ ID NO:1 after optimal alignment of the target sequence with SEQ ID NO:1.

[0019] “ANGPTL2” is a member of the angiopoietin-like protein family, comprising a signal peptide (amino acids 1-22 of SEQ ID NO:2, or a corresponding fragment thereof), an N-terminal coiled-coil domain (CCD, amino acids 76-115 and 152-206 of SEQ ID NO:2, or a corresponding fragment thereof), and a C-terminal fibrinogen-like domain (amino acids 269-489 of SEQ ID NO:2, or a corresponding fragment thereof). The amino acid sequence of ANGPTL2 (GenBank accession number NP_036230) is shown in SEQ ID NO:2; the corresponding polynucleotide sequence (GenBank accession number NM_012098) is shown in SEQ ID NO:142. “ANGPTL2 polypeptide” refers to a naturally expressed or modified polypeptide. For the purposes of this disclosure, amino acid numbering is typically determined with reference to the full-length wild-type human ANGPTL2 polypeptide sequence (SEQ ID NO:2). Therefore, in embodiments where the polypeptide of the present invention contains only the C-terminal portion of the full-length ANGPTIL2 and excluding the N-terminal portion, the position is still based on SEQ ID NO:2, even though the length of the peptide is less than 493 amino acids. For example, referring to position 423 of the ANGPTIL2 polypeptide of the present invention refers to position 423 of SEQ ID NO:2, even if the ANGPTIL2 polypeptide itself may be only 200 amino acids long. The amino acid in the target sequence “corresponding” to the position in the reference sequence, such as SEQ ID NO:2, can be determined by optimal sequence alignment, for example, using the default or commonly used CLUSTAL alignment parameters or the default BLAST2 alignment parameters, and by comparing the sequences. For example, position 423 of the target sequence “determined by reference SEQ ID NO:2”, or the amino acid “corresponding” to position 423 of SEQ ID NO:2, refers to the amino acid aligned with position 423 of SEQ ID NO:2 after optimal alignment of the target sequence with SEQ ID NO:2.

[0020] ANGPTL3 is a member of the angiopoietin-like protein family. Primarily expressed in the liver, it possesses the characteristic structure of angiopoietin, comprising a signal peptide (amino acids 1-16 of SEQ ID NO:3, or a corresponding fragment thereof), an N-terminal coiled-coil domain (CCD, amino acids 85-210 of SEQ ID NO:3, or a corresponding fragment thereof), and a C-terminal fibroinogen-like domain (amino acids 237-455 of SEQ ID NO:3, or a corresponding fragment thereof). ANGPTL3 has been shown to bind to αV / β3 integrin, and the FBN-like domain alone is sufficient to induce endothelial cell adhesion and angiogenesis in vivo (Camenisch et al., J. Biol. Chem. 277:17281-17290, 2002). The amino acid sequence of ANGPTL3 (GenBank accession number NP_055310) is shown in SEQ ID NO:3; the corresponding polynucleotide sequence (GenBank accession number NM_014495) is shown in SEQ ID NO:143. "ANGPTL3 polypeptide" refers to a naturally expressed or modified polypeptide. For the purposes of this disclosure, amino acid numbering is typically determined with reference to the full-length wild-type human ANGPTL3 polypeptide sequence (SEQ ID NO:3). Therefore, in embodiments where the polypeptide of the present invention contains only the C-terminal portion of the full-length ANGPTL3 and excluding the N-terminal portion, the position is still based on the SEQ ID NO:3 number, even though the length of the peptide is less than 460 amino acids. For example, reference to position 423 of the ANGPTL3 polypeptide of the present invention refers to position 423 of SEQ ID NO:3, even if the ANGPTL3 polypeptide itself may be only 200 amino acids long. The amino acid in the target sequence that "corresponds" to a position in a reference sequence, such as SEQ ID NO:3, can be determined by optimally aligning the sequence, for example, using the default or common CLUSTAL alignment parameters or the default BLAST2 alignment parameters, and comparing the sequences. For example, position 423 of the target sequence "determined by reference SEQ ID NO:3", or the amino acid "corresponding to" position 423 of SEQ ID NO:3, refers to the amino acid that aligns with position 423 of SEQ ID NO:3 after optimal alignment of the target sequence with SEQ ID NO:3.

[0021] “ANGPTL4” is a member of the angiopoietin-like protein family, comprising a signal peptide (amino acids 1-25 of SEQ ID NO:4, or a corresponding fragment thereof), an N-terminal coiled-coil domain (CCD, amino acids 100-143 of SEQ ID NO:4, or a corresponding fragment thereof), and a C-terminal fibroinogen-like domain (amino acids 179-401 of SEQ ID NO:4, or a corresponding fragment thereof). The amino acid sequence of ANGPTL4 (GenBank accession number NP_647475) is shown in SEQ ID NO:4; the corresponding polynucleotide sequence (GenBank accession number NM_139314) is shown in SEQ ID NO:144. “ANGPTL4 polypeptide” refers to a naturally expressed or modified polypeptide. For the purposes of this disclosure, amino acid numbering is typically determined with reference to the full-length wild-type human ANGPTL4 polypeptide sequence (SEQ ID NO:4). Therefore, in embodiments where the polypeptide of the present invention contains only the C-terminal portion of the full-length ANGPTIL4 and excluding the N-terminal portion, the position is still based on SEQ ID NO:4, even though the length of the peptide is less than 406 amino acids. For example, referring to position 400 of the ANGPTIL4 polypeptide of the present invention, even if the ANGPTIL4 polypeptide itself may only be 200 amino acids long, refers to position 423 of SEQ ID NO:4. The amino acid in the target sequence “corresponding” to the position in the reference sequence, such as SEQ ID NO:4, can be determined by optimal sequence alignment, for example, using the default or commonly used CLUSTAL alignment parameters or the default BLAST2 alignment parameters, and by comparing the sequences. For example, position 400 of the target sequence “determined by reference SEQ ID NO:4”, or the amino acid “corresponding” to position 400 of SEQ ID NO:4, refers to the amino acid aligned with position 400 of SEQ ID NO:4 after optimal alignment of the target sequence with SEQ ID NO:4.

[0022] A schematic diagram of the functional domain divisions of ANGPTL1, ANGPTL2, ANGPTL3, and ANGPTL4 is shown in Figure 11. In some embodiments, the ANGPTL1 peptide described herein contains the amino acid sequence shown in SEQ ID NO:1, the ANGPTL2 peptide contains the amino acid sequence shown in SEQ ID NO:2, the ANGPTL3 peptide contains the amino acid sequence shown in SEQ ID NO:3, and the ANGPTL4 peptide contains the amino acid sequence shown in SEQ ID NO:4.

[0023] As described above and further herein, the present invention contemplates the use of various ANGPTL peptides or fusion proteins that have chondrogenic activity and / or protease resistance. The term "protease resistance" herein refers to a modified peptide that makes it less susceptible to cleavage by trypsin-like proteases compared to the corresponding unmodified wild-type peptide.

[0024] This article provides a polypeptide (e.g., a modified polypeptide) comprising at least two polypeptide fragments derived from ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4.

[0025] The polypeptide described herein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more polypeptide fragments. In some embodiments, the polypeptide comprises 2-10 polypeptide fragments. In some embodiments, the polypeptide comprises 2 polypeptide fragments. In some embodiments, the polypeptide comprises 3 polypeptide fragments. In some embodiments, the polypeptide comprises 4 polypeptide fragments. In some embodiments, the polypeptide comprises 5 polypeptide fragments. In some embodiments, the polypeptide comprises 6 polypeptide fragments.

[0026] "A polypeptide fragment derived from ANGPTL," such as a polypeptide fragment derived from ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4, in this document refers to a polypeptide fragment derived from a member of an angiopoietin-like protein family (e.g., ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4). This polypeptide fragment can be a truncated ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4 (e.g., ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4 polypeptide fragment), a modified ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4 polypeptide fragment (e.g., containing one or more mutations, insertions, deletions, or substitutions compared to its corresponding wild-type sequence), or a polypeptide fragment having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned fragments. In some embodiments, an ANGPTL-derived polypeptide fragment has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with its corresponding wild-type amino acid sequence (e.g., the ANGPTL3 polypeptide fragment of amino acids 424-460 in SEQ ID NO:3).

[0027] In some embodiments, “ANGPTL1 polypeptide, ANGPTL2 polypeptide, ANGPTL3 polypeptide, or ANGPTL4 polypeptide” refers to the wild-type full-length protein sequence (e.g., shown in SEQ ID NO: 1, 2, 3, 4). In some embodiments, “ANGPTL1 polypeptide fragment, ANGPTL2 polypeptide fragment, ANGPTL3 polypeptide fragment, or ANGPTL4 polypeptide fragment” or “polypeptide fragment derived from ANGPTL1, polypeptide fragment derived from ANGPTL2, polypeptide fragment derived from ANGPTL3, or polypeptide fragment derived from ANGPTL4” refers to the wild-type full-length protein sequence (e.g., shown in SEQ ID NO: 1, 2, 3, 4). Segments (as shown in NO:1, 2, 3, 4) containing, for example, truncated segments of length 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or more). In some embodiments, "ANGPTL1 polypeptide fragment, ANGPTL2 polypeptide fragment, ANGPTL3 polypeptide fragment, or ANGPTL4 polypeptide fragment" or "polypeptide fragment derived from ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4" refers to a modified ANGPTL1 polypeptide fragment, ANGPTL2 polypeptide fragment, ANGPTL3 polypeptide fragment, or ANGPTL4 polypeptide fragment. In some embodiments, the modified ANGPTL1 polypeptide fragment, ANGPTL2 polypeptide fragment, ANGPTL3 polypeptide fragment, or ANGPTL4 polypeptide fragment contains one or more mutated, inserted, deleted, or substituted sequences compared to its corresponding wild-type sequence.

[0028] In some embodiments, the “ANGPTL1 peptide fragment, ANGPTL2 peptide fragment, ANGPTL3 peptide fragment, or ANGPTL4 peptide fragment” or “peptide fragment derived from ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4” (e.g., truncated ANGPTL1 peptide, truncated ANGPTL2 peptide, truncated ANGPTL3 peptide, or truncated ANGPTL4 peptide) retains at least 90%, 80%, 70%, 60%, and 50% of the sequence length compared to wild-type ANGPTL peptides (e.g., those shown in SEQ ID NO: 1, 2, 3, 4). In some embodiments, the ANGPTL fragment contains at least one segment of the carboxyl-terminal domain of a human ANGPTL peptide (e.g., at least 200, 250, 300, 350, 400, 450 consecutive amino acids) or a sequence substantially identical to the carboxyl-terminal sequence of the human ANGPTL peptide, wherein the peptide and its variants retain chondrogenic activity.

[0029] In some embodiments, the “ANGPTL1 polypeptide fragment, ANGPTL2 polypeptide fragment, ANGPTL3 polypeptide fragment, or ANGPTL4 polypeptide fragment” or “polypeptide fragment derived from ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4” (e.g., truncated ANGPTL1 polypeptide, truncated ANGPTL2 polypeptide, truncated ANGPTL3 polypeptide, or truncated ANGPTL4 polypeptide, or modified thereof) contains at least one fragment (e.g., at least 100, 150, 200, 215, or 219 consecutive amino acids) of the C-terminal domain of a human ANGPTL polypeptide, or a sequence substantially identical to the C-terminal sequence of the human ANGPTL3 polypeptide, wherein the polypeptide and its variants retain chondrogenic activity. In some embodiments, the ANGPTL-derived polypeptide fragments of this disclosure are missing at least a portion of the C-terminal sequence, for example, missing 5, 6, 7, 8, 9, 10, 15, or 20 amino acids from the C-terminus of SEQ ID NO: 3.

[0030] The "sequence identity percentage" is determined by comparing two optimally aligned sequences in a comparison window, wherein the amino acid or polynucleotide sequence portion in the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence (e.g., the polypeptide of the present invention) that does not contain additions or deletions, to achieve optimal alignment of the two sequences. This percentage can be calculated as follows: determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate a matching position number, dividing this matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0031] The term "identical" or "identity percentage," in the case of two or more nucleic acid or polypeptide sequences, can refer to two or more sequences or subsequences being identical sequences. Two sequences are "substantially identical" if, after comparing and aligning them in a comparison window or designated region using one of the following sequence comparison algorithms or by manual alignment and visual inspection to achieve maximum correspondence, they have a specified percentage of identical amino acid residues or nucleotides (i.e., more than 95%, optionally 96%, 97%, 98%, or 99% identical in a designated region, or, when no region is specified, across the entire sequence), then the two sequences are substantially identical. This disclosure provides polypeptides substantially identical to those exemplified in this disclosure (e.g., any one of SEQ ID NOs: 5-32 and 34-42) and their uses, including but not limited to, uses for treating or preventing arthritis or joint damage. Optionally, for nucleic acids, identity is present in a region of at least about 150 nucleotides in length, or more preferably in a region of 300 to 450 or 600 or more nucleotides in length, or across the entire length of a reference sequence. Optionally, for an amino acid sequence, identity exists in a region of at least about 50 amino acids in length, or more preferably in a region of 100 to 150 or 200 or more amino acids in length, or in the entire length of the reference sequence.

[0032] For sequence comparison, typically, one sequence serves as a reference sequence, to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, subsequence coordinates (if necessary), and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters. As is known in the art, the terms "identity" or "homology" refer to the relationship between sequences of two or more polypeptides or polynucleotides as determined by sequence comparison. In the art, identity also means the degree of sequence correlation between sequences as determined by the number of matches between two or more amino acid residues or nucleic acid residue strings. Identity measures the percentage of consistent matches between two or more sequences that have a smaller gap alignment (if any) proposed by a particular mathematical model or computer program (e.g., an "algorithm"). "Identity %" when applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to residues in the amino acid or nucleic acid sequence of a second sequence after sequence alignment and, where necessary, the introduction of gaps to achieve the maximum identity percentage. Methods and computer programs used for alignment are well known in the art. It should be understood that identity depends on the calculation of the identity percentage, but its value can vary due to gaps and penalties introduced in the calculation. Typically, as determined by sequence alignment procedures and parameters described herein and known to those skilled in the art, a variant of a particular polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with that particular reference polynucleotide or polypeptide. Tools used for alignment include those in BLAST kits (Stephen F. Altschul et al. (1997), “GappedBLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25: 3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF and Waterman, MS (1981), “Identification of common molecular subsequences.” J. Mol. Biol. 147: 195-197).The general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB and Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48: 443-453). Recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is claimed to generate global alignments of nucleotide and protein sequences faster than other optimized global alignment methods (including the Needleman-Wunsch algorithm). Other tools are described in this paper, especially in the definition of "identity" below.

[0033] In some embodiments, the ANGPTL polypeptide is derived from ANGPTL polypeptides of vertebrates (e.g., mammals, such as humans, mice, rabbits, or cattle). For example, human ANGPTL polypeptides, or amino acid sequences containing natural substitutions or amino acid mutations (e.g., insertions, deletions, and / or substitutions).

[0034] In some embodiments, the full-length amino acid sequences of the wild-type ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4 polypeptides are shown in SEQ ID NOs: 1-4, respectively.

[0035] The amino acid sequence of wild-type ANGPTL1:

[0036] SEQ ID NO: 1

[0037] The amino acid sequence of wild-type ANGPTL2:

[0038] SEQ ID NO: 2

[0039] The amino acid sequence of wild-type ANGPTL3:

[0040] SEQ ID NO: 3

[0041] The amino acid sequence of wild-type ANGPTL4:

[0042] SEQ ID NO: 4

[0043] In some embodiments, the polypeptide described herein comprises at least two ANGPTL polypeptide fragments, said polypeptide fragments comprising at least two polypeptides or fragments thereof derived from ANGPTL1, ANGPTL2, ANGPTL3 or ANGPTL4, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0044] In some embodiments, the polypeptide described herein comprises at least one polypeptide fragment derived from ANGPTL3, and (1) at least one polypeptide fragment derived from ANGPTL1; (2) at least one polypeptide fragment derived from ANGPTL2; and / or (3) at least one polypeptide fragment derived from ANGPTL4.

[0045] In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL1 and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL3. In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL2 and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL3. In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL4 and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL3. In some embodiments, the polypeptide described herein comprises polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the above.

[0046] In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL1 and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL2. In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL4 and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL2. In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL1 and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL4. In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL1, one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL2, and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL3. In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL1, one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL2, and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL4. In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL2, one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL3, and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL4. In some embodiments, the polypeptide described herein comprises one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL1, one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL2, one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL3, and one or more (e.g., 1, 2, 3, 4, 5 or more) polypeptide fragments derived from ANGPTL4. In some embodiments, the polypeptide described herein comprises polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the above.

[0047] In some embodiments, the polypeptide described herein comprises one or more truncated fragments derived from ANGPTL1 and one or more truncated fragments derived from ANGPTL3. In some embodiments, the polypeptide described herein comprises one or more truncated fragments derived from ANGPTL2 and one or more truncated fragments derived from ANGPTL3. In some embodiments, the polypeptide described herein comprises one or more truncated fragments derived from ANGPTL4 and one or more truncated fragments derived from ANGPTL3. In some embodiments, the polypeptide described herein comprises a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the above.

[0048] In some embodiments, the polypeptide described in this disclosure comprises a fibrinogen-like domain or a fragment thereof. In some embodiments, the fibrinogen-like domain is an ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4 fibrinogen-like domain. This disclosure provides reference start and end sites for the fibrinogen-like domains of each member of the ANGPTL family, illustrated in Figure 11. In some embodiments, the fibrinogen-like domain fragment is a functional fragment of any of the aforementioned fibrinogen-like domains (e.g., a fragment that can bind αV / β3 integrin, or a fragment sufficient to induce endothelial cell adhesion and in vivo angiogenesis, etc.). In some embodiments, the ANGPTL fibrinogen-like domain comprises amino acids 237-455 of the amino acid sequence shown in SEQ ID NO: 3, or a corresponding polypeptide fragment of ANGPTL1, ANGPTL2, or ANGPTL4.

[0049] In some embodiments, the ANGPTL3 fibrinogen-like domain comprises amino acids 237-455 of the amino acid sequence shown in SEQ ID NO: 3, or a functional fragment thereof (e.g., amino acids 242-460, 269-453, 270-453, 259-415, 259-262, 240-241, or 431-460 of the functional fragment). In some embodiments, the ANGPTL1 fibrinogen-like domain comprises amino acids 271-491 of the amino acid sequence shown in SEQ ID NO: 1, or a fragment thereof (e.g., a functional fragment), or a corresponding polypeptide fragment. In some embodiments, the ANGPTL2 fibrinogen-like domain comprises amino acids 269-489 of the amino acid sequence shown in SEQ ID NO: 2, or a fragment thereof (e.g., a functional fragment), or a corresponding polypeptide fragment. In some embodiments, the ANGPTL4 fibrinogen-like domain comprises amino acids 179-401 of the amino acid sequence shown in SEQ ID NO: 4, or a fragment thereof (e.g., a functional fragment), or a corresponding polypeptide fragment. In some embodiments, the polypeptide comprises the fibrinogen-like domain or a fragment thereof, along with one or more additional amino acid sequences.

[0050] In some embodiments, the polypeptide comprises one of the following fragments, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it: (1) amino acids 242-460 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3 (containing the fibrinogen-like domain functional fragment at positions 242-455); (2) amino acids 269-453 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3 (amino acids 269-453 are the fibrinogen-like domain functional fragment); or (3) amino acids 259-415 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3 (amino acids 259-415 are the fibrinogen-like domain functional fragment).

[0051] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 242-460 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; (2) amino acids 269-453 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 259-415 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; (4) amino acids 259-262 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; (5) amino acids 270-453 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; (6) amino acids 240-241 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; or (7) amino acids 431-460 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3.

[0052] In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, the following fragments, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the following fragments: (1) amino acids 259-262 and 269-453 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; (2) amino acids 259-262 and 270-453 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 259-415 and 431-460 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; (4) amino acids 240-241, 259-262, and 269-453 of the ANGPTL3 amino acid sequence shown in SEQ ID NO: 3; or (5) SEQ ID NO: 3. The amino acids at positions 240-241, 259-415, and 431-460 of the ANGPTL3 amino acid sequence shown in NO:3.

[0053] In some embodiments, the polypeptide further comprises the following polypeptide fragments, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them: (1) amino acids 276-292, 297-303, 490-491, 453-466, 273-292, and / or 273-491 of the ANGPTL1 amino acid sequence shown in SEQ ID NO: 1; (2) amino acids 274-290, 295-300, 488-493, 451-464, and / or 269-290 of the ANGPTL2 amino acid sequence shown in SEQ ID NO: 2; and / or (3) SEQ ID NO: 2. The amino acids 184-200, 205-211, 400-406, 362-376, and / or 179-406 of the ANGPTL4 amino acid sequence shown in NO:4.

[0054] In some embodiments, the polypeptide further contains mutations that improve its thermal stability or protease degradation stability. In some embodiments, the mutations include point mutations, insertions, deletions, truncations, fusions, or any combination thereof.

[0055] In some embodiments, the polypeptide comprises one or more ANGPTL3 polypeptide fragments, and the ANGPTL3 polypeptide fragments contain amino acid mutations selected from any one or more of the following positions: position 259, position 280, position 289, position 304, position 330, position 350, position 384, position 423, position 424, and position 432, wherein the amino acid substitution sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3.

[0056] In some embodiments, the ANGPTL3 polypeptide fragment contains amino acids corresponding to one or more of the following sites: I at position 259, T at position 280, T at position 289, H at position 304, I at position 330, H at position 350, G, T, S, Q, N, H, P or V at position 384, Q at position 423, P at position 424, and I at position 432.

[0057] In some embodiments, the ANGPTL3 polypeptide fragment contains amino acid mutations at one or more of the following positions: position 384; position 424; positions 259 and 423; positions 280 and 423; positions 289 and 423; positions 304 and 423; positions 330 and 423; positions 350 and 423; positions 432 and 423; positions 259, 432, and 423.

[0058] In some embodiments, the ANGPTL3 polypeptide fragment contains one or more amino acid mutations selected from any one of the following groups: (1) W384G, W384T, W384S, W384Q, W384N, W384H, W384P, W384V; (2) S424P; (3) M259I and K423Q; (4) S280T and K423Q; (5) I289T and K423Q; (6) Y304H and K423Q; (7) V330I and K423Q; (8) Y350H and K423Q; (9) L432I and K423Q; (10) M259I, L432I and K423Q, wherein the amino acid substitution sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3.

[0059] In some embodiments, the polypeptide contains a K423Q mutation.

[0060] The amino acid substitution sites described above are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3.

[0061] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 274-290 of the amino acid sequence shown in SEQ ID NO: 2; (2) amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 295-300 of the amino acid sequence shown in SEQ ID NO: 2; (4) amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) amino acids 488-493 of the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 5.

[0062] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 274-290 of the amino acid sequence shown in SEQ ID NO: 2; (2) amino acids 259-415 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 451-464 of the amino acid sequence shown in SEQ ID NO: 2; and (4) amino acids 431-460 of the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, (1)-(4) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 8.

[0063] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 269-290 of the amino acid sequence shown in SEQ ID NO: 2; (2) amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 295-300 of the amino acid sequence shown in SEQ ID NO: 2; (4) amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) amino acids 488-493 of the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the polypeptide contains a K423Q mutation. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 26.

[0064] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 269-290 of the amino acid sequence shown in SEQ ID NO: 2; (2) amino acids 259-415 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 451-464 of the amino acid sequence shown in SEQ ID NO: 2; and (4) amino acids 431-460 of the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, (1)-(4) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 27.

[0065] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 276-292 of the amino acid sequence shown in SEQ ID NO: 1; (2) amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 297-303 of the amino acid sequence shown in SEQ ID NO: 1; (4) amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) amino acids 490-491 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 6.

[0066] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 276-292 of the amino acid sequence shown in SEQ ID NO: 1; (2) amino acids 259-415 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 453-466 of the amino acid sequence shown in SEQ ID NO: 1; and (4) amino acids 431-460 of the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, (1)-(4) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 9.

[0067] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 240-241 of the amino acid sequence shown in SEQ ID NO: 3; (2) amino acids 273-292 of the amino acid sequence shown in SEQ ID NO: 1; (3) amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (4) amino acids 297-303 of the amino acid sequence shown in SEQ ID NO: 1; (5) amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (6) amino acids 490-491 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, (1)-(6) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 28.

[0068] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 240-241 of the amino acid sequence shown in SEQ ID NO: 3; (2) amino acids 273-292 of the amino acid sequence shown in SEQ ID NO: 1; (3) amino acids 259-415 of the amino acid sequence shown in SEQ ID NO: 3; (4) amino acids 453-466 of the amino acid sequence shown in SEQ ID NO: 1; and (5) amino acids 431-460 of the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 29.

[0069] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 184-200 of the amino acid sequence shown in SEQ ID NO: 4; (2) amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 205-211 of the amino acid sequence shown in SEQ ID NO: 4; (4) amino acids 270-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) amino acids 400-406 of the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 7.

[0070] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 184-200 of the amino acid sequence shown in SEQ ID NO: 4; (2) amino acids 259-415 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 362-376 of the amino acid sequence shown in SEQ ID NO: 4; and (4) amino acids 431-460 of the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, (1)-(4) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 10.

[0071] This disclosure also provides a polypeptide comprising at least one polypeptide fragment derived from ANGPTL3, said ANGPTL3 fragment comprising at least one heterologous amino acid sequence.

[0072] "Heterologous amino acid sequence" refers to an amino acid sequence derived from a different protein. For example, a heterologous amino acid sequence relative to ANGPTL3 could be an amino acid sequence derived from ANGPTL1, ANGPTL2, or ANGPTL4. In some embodiments, the heterologous amino acid sequence has 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of identity with ANGPTL3. The heterologous amino acid sequence can be about 1-50, 1-40, 1-30, 1-20, 1-10, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, or other sequences of suitable length. In some embodiments, the heterologous amino acid sequence relative to the ANGPTL3 polypeptide fragment may be an ANGPTL1, ANGPTL2, or ANGPTL4 polypeptide fragment, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0073] In some embodiments, for example, amino acids at positions 242-258 and 416-430 of the ANGPTL3 fragment each comprise a heterologous amino acid sequence, indicating that the heterologous amino acid sequence replaces the corresponding amino acid sequence in the ANGPTL3 fragment. In some embodiments, amino acids at positions 242-258 and 416-430 of the ANGPTL3 fragment each consist of a heterologous amino acid sequence. In some embodiments, amino acids at positions 242-258 and 416-430 of the ANGPTL3 fragment do not contain the original (wild-type) amino acid sequence.

[0074] In some embodiments, the length of the heterologous amino acid sequence and its corresponding amino acid segment are inconsistent. For example, when amino acids 242-258 of the polypeptide are composed of amino acids 273-292 of SEQ ID NO:1, the length of the heterologous amino acid sequence is longer than its corresponding amino acid segment. In this case, the length of the polypeptide is determined by the actual amino acid sequence. Even when the length of the heterologous amino acid sequence and its corresponding amino acid segment are inconsistent, the amino acid site numbering of the polypeptide still follows the natural order of the amino acid sequence relative to SEQ ID NO:3.

[0075] In some embodiments, the heterologous amino acid sequence does not contain a fibrinogen-like domain. In some embodiments, the heterologous amino acid sequence comprises amino acids 273-292 of SEQ ID NO:1, amino acids 453-466 of SEQ ID NO:1, amino acids 269-290 of SEQ ID NO:2, amino acids 295-300 of SEQ ID NO:2, or amino acids 488-493 of SEQ ID NO:2.

[0076] In some embodiments, amino acids at positions 242-258 and 416-430 of the polypeptide each comprise a heterologous amino acid sequence; the amino acid sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, amino acids at positions 242-258 and 416-430 of the polypeptide each consist of a heterologous amino acid sequence.

[0077] In some embodiments, amino acids 242-258 of the polypeptide comprise amino acids 273-292 of SEQ ID NO:1; amino acids 416-430 of the polypeptide comprise amino acids 453-466 of SEQ ID NO:1; the amino acid sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:3. In some embodiments, amino acids 242-258 of the polypeptide are composed of amino acids 273-292 of SEQ ID NO:1; amino acids 416-430 of the polypeptide are composed of amino acids 453-466 of SEQ ID NO:1.

[0078] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 29. In some embodiments, the amino acid sequence of the polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 29.

[0079] In some embodiments, amino acids at positions 242-258, 263-268, 416-430, and / or 454-460 of the polypeptide each comprise a heterologous amino acid sequence; the amino acid sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, amino acids at positions 242-258, 263-268, 416-430, and / or 454-460 of the polypeptide are composed of heterologous amino acid sequences.

[0080] In some embodiments, amino acids at positions 242-258, 263-268, and 454-460 of the polypeptide each comprise a heterologous amino acid sequence; the amino acid sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, amino acids at positions 242-258, 263-268, and 454-460 are composed of heterologous amino acid sequences.

[0081] In some embodiments, amino acids 242-258 of the polypeptide comprise amino acids 269-290 of SEQ ID NO:2, amino acids 263-268 of the polypeptide comprise amino acids 295-300 of SEQ ID NO:2, and amino acids 454-460 of the polypeptide comprise amino acids 488-493 of SEQ ID NO:2; the amino acid sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:3. In some embodiments, amino acids 242-258 of the polypeptide are composed of amino acids 269-290 of SEQ ID NO:2, amino acids 263-268 of the polypeptide are composed of amino acids 295-300 of SEQ ID NO:2, and amino acids 454-460 of the polypeptide are composed of amino acids 488-493 of SEQ ID NO:2.

[0082] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 26. In some embodiments, the amino acid sequence of the polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26.

[0083] The polypeptide regions containing heterologous amino acid sequences described above can be appropriately modified. In some embodiments, the polypeptide regions containing heterologous amino acid sequences may include 1, 2, 3, 4, 5 or more amino acid sites before the start site or after the stop site.

[0084] In some embodiments, the polypeptide comprises one or more ANGPTL3 polypeptide fragments, and the ANGPTL3 polypeptide fragments contain amino acid mutations selected from any one or more of the following positions: position 259, position 280, position 289, position 304, position 330, position 350, position 384, position 423, position 424, and position 432, wherein the amino acid substitution sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3.

[0085] In some embodiments, the ANGPTL3 polypeptide fragment contains amino acids corresponding to one or more of the following sites: I at position 259, T at position 280, T at position 289, H at position 304, I at position 330, H at position 350, G, T, S, Q, N, H, P or V at position 384, Q at position 423, P at position 424, and I at position 432.

[0086] In some embodiments, the ANGPTL3 polypeptide fragment contains amino acid mutations at one or more of the following positions: position 384; position 424; positions 259 and 423; positions 280 and 423; positions 289 and 423; positions 304 and 423; positions 330 and 423; positions 350 and 423; positions 432 and 423; positions 259, 432, and 423.

[0087] In some embodiments, the ANGPTL3 polypeptide fragment contains one or more amino acid mutations selected from any one of the following groups: (1) W384G, W384T, W384S, W384Q, W384N, W384H, W384P, W384V; (2) S424P; (3) M259I and K423Q; (4) S280T and K423Q; (5) I289T and K423Q; (6) Y304H and K423Q; (7) V330I and K423Q; (8) Y350H and K423Q; (9) L432I and K423Q; (10) M259I, L432I and K423Q, wherein the amino acid substitution sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3.

[0088] In some embodiments, the polypeptide contains a K423Q mutation.

[0089] The amino acid substitution sites described above are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3.

[0090] In some embodiments, the amino acid sequence of the polypeptide is as shown in any of SEQ ID NOs: 5-10, 26-30, 32, and 35-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0091] In some embodiments, the ANGPTL3 fragment comprises an ANGPTL3 fibrinogen-like domain or a fragment thereof. The ANGPTL3 fibrinogen-like structure is as described herein.

[0092] This disclosure also provides a polypeptide comprising one or more polypeptide fragments derived from ANGPTL3 having amino acid mutations selected from one or more of the following positions: positions 259, 280, 289, 304, 330, 350, 384, 423, 424, and 432, wherein the amino acid substitution sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3. Any embodiment described herein may be used with the polypeptide.

[0093] In some embodiments, the amino acid sequence of the polypeptide comprises or is as shown in any of SEQ ID NOs: 17-25, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0094] In some embodiments, the polypeptide further comprises at least one polypeptide fragment derived from ANGPTL1, ANGPTL2, or ANGPTL4.

[0095] In some embodiments, the polypeptide comprises: (1) one or more polypeptide fragments derived from ANGPTL1 and one or more polypeptide fragments derived from ANGPTL3; (2) one or more polypeptide fragments derived from ANGPTL2 and one or more polypeptide fragments derived from ANGPTL3; or (3) one or more truncated fragments derived from ANGPTL4 and one or more polypeptide fragments derived from ANGPTL3, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the above.

[0096] In some embodiments, the amino acid sequence of the polypeptide comprises a sequence selected from SEQ ID NOs: 26 and 35-42. In some embodiments, the amino acid sequence of the ANGPTL3 polypeptide comprises a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs: 26 and 35-42.

[0097] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 269-290 of the amino acid sequence shown in SEQ ID NO: 2; (2) amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 295-300 of the amino acid sequence shown in SEQ ID NO: 2; (4) amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) amino acids 488-493 of the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the polypeptide contains a K423Q mutation. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 26. In some embodiments, the amino acid sequence of the polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26.

[0098] In some embodiments, amino acids at positions 242-257, 263-268, and 454-460 of the polypeptide each comprise a heterologous amino acid sequence; the amino acid sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, amino acids at positions 242-257, 263-268, and 454-460 are composed of heterologous amino acid sequences.

[0099] In some embodiments, amino acids 242-257 of the polypeptide comprise amino acids 269-289 of SEQ ID NO:2, amino acids 263-268 of the polypeptide comprise amino acids 295-300 of SEQ ID NO:2, and amino acids 454-460 of the polypeptide comprise amino acids 488-493 of SEQ ID NO:2; the amino acid sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:3. In some embodiments, amino acids 242-257 of the polypeptide are composed of amino acids 269-289 of SEQ ID NO:2, amino acids 263-268 of the polypeptide are composed of amino acids 295-300 of SEQ ID NO:2, and amino acids 454-460 of the polypeptide are composed of amino acids 488-493 of SEQ ID NO:2.

[0100] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 269-289 of the amino acid sequence shown in SEQ ID NO: 2; (2) amino acids 258-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) amino acids 295-300 of the amino acid sequence shown in SEQ ID NO: 2; (4) amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) amino acids 488-493 of the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid mutation selected from one or more of the following positions: positions 259, 280, 289, 304, 330, 350, 384, 423, 424, and 432, wherein the amino acid substitution sites are natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO: 35-42.

[0101] This disclosure also provides a polypeptide comprising: one or more ANGPTL polypeptide fragments, wherein the ANGPTL polypeptide fragments are truncated ANGPTL1, ANGPTL2, ANGPTL3 or ANGPTL4 polypeptide fragments, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0102] This disclosure also provides a polypeptide comprising: one or more ANGPTL polypeptide fragments, wherein the ANGPTL polypeptide fragments are truncated ANGPTL1, ANGPTL2 or ANGPTL4 polypeptide fragments, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0103] In some embodiments, the polypeptide comprises a fibrinogen-like domain (e.g., ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL4 fibrinogen-like domain) or a fragment thereof. The ANGPTL1, ANGPTL2, ANGPTL3, and ANGPTL4 fibrinogen-like domains are as described herein.

[0104] In some embodiments, the truncated ANGPTL1 polypeptide, truncated ANGPTL2 polypeptide, or truncated ANGPTL4 polypeptide retains a sequence length of at least 90%, 80%, 70%, 60%, or 50% compared to the wild-type ANGPTL polypeptide (e.g., shown in SEQ ID NO: 1, 2, or 4). In some embodiments, the ANGPTL fragment contains at least one segment (e.g., at least 100, 150, 200, 219, or 250 consecutive amino acids) of the carboxyl-terminal domain of the human ANGPTL polypeptide, or a sequence substantially identical to the carboxyl-terminal sequence of the human ANGPTL polypeptide, wherein the polypeptide and its variants retain chondrogenic activity. In some embodiments, the polypeptide (e.g., the truncated ANGPTL1 polypeptide, truncated ANGPTL2 polypeptide, or truncated ANGPTL4 polypeptide) also contains a polypeptide fragment derived from ANGPTL3 (e.g., a fragment consisting of two or more amino acids).

[0105] In some embodiments, the polypeptide comprises one of the following fragments, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the following fragments: amino acids 276-491 of the ANGPTL1 amino acid sequence shown in SEQ ID NO: 1; amino acids 261-491 of the ANGPTL1 amino acid sequence shown in SEQ ID NO: 1; or amino acids 273-491 of the ANGPTL1 amino acid sequence shown in SEQ ID NO: 1.

[0106] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 240-241 of the amino acid sequence shown in SEQ ID NO: 3; and (2) amino acids 273-491 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, (1)-(2) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 30.

[0107] In some embodiments, the polypeptide comprises one of the following fragments, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the following fragments: amino acids 274-493 of the ANGPTL2 amino acid sequence shown in SEQ ID NO: 2; amino acids 257-493 of the ANGPTL2 amino acid sequence shown in SEQ ID NO: 2; or amino acids 269-493 of the ANGPTL2 amino acid sequence shown in SEQ ID NO: 2.

[0108] In some embodiments, the polypeptide comprises one of the following fragments, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the following fragments: amino acids 184-406 of the ANGPTL4 amino acid sequence shown in SEQ ID NO: 4; amino acids 166-406 of the ANGPTL4 amino acid sequence shown in SEQ ID NO: 4; or amino acids 179-406 of the ANGPTL4 amino acid sequence shown in SEQ ID NO: 4.

[0109] In some embodiments, the polypeptide comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with, the following fragments: (1) amino acids 240-241 of the amino acid sequence shown in SEQ ID NO: 3; and (2) amino acids 179-406 of the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, (1)-(2) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 32.

[0110] In some embodiments, the amino acid sequence of the polypeptide comprises or is as shown in SEQ ID NOs: 11-16 and 30-32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0111] This disclosure also provides a polypeptide characterized in that the amino acid sequence of the polypeptide comprises or is as shown in SEQ ID NOs: 5-32, and 34-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the polypeptide further comprises one or more mutated, inserted, deleted, or substituted sequences.

[0112] This disclosure also provides a polypeptide comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein: (1) the first polypeptide comprises amino acids 269-290 of the amino acid sequence shown in SEQ ID NO: 2; (2) the second polypeptide comprises amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) the third polypeptide comprises amino acids 295-300 of the amino acid sequence shown in SEQ ID NO: 2; (4) the fourth polypeptide comprises amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) the fifth polypeptide comprises amino acids 488-493 of the amino acid sequence shown in SEQ ID NO: 2, wherein (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the above-described polypeptide is also provided herein. In some embodiments, the polypeptide further comprises one or more mutated, inserted, deleted, or substituted sequences. In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 26.

[0113] This disclosure also provides a fusion protein, characterized in that the amino acid sequence of the fusion protein is any one of SEQ ID NOs: 5-10, 26-30, 32, and 35-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0114] In some embodiments, the ANGPTL peptide fragments are not linked together. In other embodiments, the ANGPTL peptide fragments are directly linked together.

[0115] In some embodiments, the ANGPTL polypeptide fragments are linked together by linkers. In some embodiments, the linkers are peptide linkers. In some embodiments, the linkers are selected from: (EAAAK)3 (SEQ ID NO:55), (EAAAR)3 (SEQ ID NO:56), (EGGGK)3 (SEQ ID NO:57), (EGGGR)3 (SEQ ID NO:58), (DAAAR)3 (SEQ ID NO:59), (DAAAK)3 (SEQ ID NO:60), (DGGGR)3 (SEQ ID NO:61), or (DGGGK)3 (SEQ ID NO:62); or the linker is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS (SEQ ID NO:52) and GGGGS (SEQ ID NO:53); or the linker is GGGGG (SEQ ID NO:54). In some implementations, the connector may be one or more combinations of GS, GGS, GGGS, GGGGS, and GGGGG.

[0116] In some embodiments, the polypeptide described herein may include flanking sequences of a non-natural ANGPTL polypeptide. For example, the chondrogenic active portion of the polypeptide (e.g., a fibrinogen-like domain) may be fused with one or more fusion couplers and / or heterologous amino acids to form a fusion protein. Fusion coupler sequences may include, but are not limited to, amino acid tags, non-L (e.g., D-) amino acids or other amino acid mimics (to prolong in vivo half-life and / or protease resistance), targeting sequences, or other sequences. In some embodiments, the ANGPTL polypeptide is an ANGPTL3 polypeptide.

[0117] In some embodiments, the polypeptides described herein are PEGylated. In some embodiments, the polypeptides of this disclosure are fused with heteropeptides. In some embodiments, the polypeptide is fused with any of the following: human serum albumin (HAS), immunoglobulin heavy chain constant region (Fc), polyhistidine, glutathione S-transferase (GST), thioredoxin, protein A, protein G, mannose-binding protein (MBP), or fragments of any of the aforementioned heteropeptides. Fusion domains or fragments thereof can be selected to impart desired properties. For example, some fusion domains are particularly useful for separating fusion proteins by affinity chromatography. For affinity purification, relevant matrices for affinity chromatography can be used, such as glutathione, α-amylase, and nickel- or cobalt-conjugated resins. Many such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QLAexpress™ system (Qiagen) which can be used with (HIS6) fusion couplers. As another example, fusion domains can be selected to facilitate the detection of ANGPTL polypeptides. Examples of detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags"—typically short peptide sequences against which specific antibodies can be obtained. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some embodiments, the ANGPTL peptide is fused with a domain that can stabilize the ANGPTL peptide in vivo (e.g., a "stabilizer" domain). "Stabilization" refers to an increase in serum half-life, regardless of whether the increase is caused by reduced destruction, reduced renal clearance, or other pharmacokinetic effects. Fusion with the Fc moiety of immunoglobulins is known to confer desired pharmacokinetic properties on a wide range of proteins. Similarly, fusion with human serum albumin can confer desired properties. Other types of fusion domains that can be selected include polymerizing (e.g., dimerizing, tetramerizing) domains and functional domains (to confer additional biological function as desired). Fusions can be constructed by fusing heteropeptides to the amino terminus and / or the carboxyl terminus of the peptide of the present invention. In some embodiments, the heterologous peptide is fused to the amino terminus of the disclosed peptide. In other or alternative embodiments, the heterologous peptide is fused to the carboxyl terminus of the peptide of the present invention.

[0118] The polypeptides or fusion proteins described herein (e.g., the ANGPTL fusion polypeptide) possess chondrogenic activity and relieve joint pain. As defined herein, chondrogenesis or chondrogenic activity refers to the generation of chondrocytes from MSCs. Markers of chondrogenic activity include, but are not limited to, cartilage matrix production. Cartilage matrix production can be measured by the production of various markers, such as Sox9, type II collagen or glycosaminoglycans (GAG), DKK1, etc. In some embodiments, the ANGPTL polypeptide of this disclosure can perform cartilage repair and joint pain relief using the methods described in Example 5 or Example 6.

[0119] In some embodiments, the aforementioned polypeptide has an activity selected from at least one of the following:

[0120] (a) It is capable of binding to integrin α5β1, and the detection method is conventional in the art, such as the method in Embodiment 2 of this disclosure;

[0121] (b) Promote the secretion of DKK1, wherein the method for detecting the amount of secretion is conventional in the art, such as the method in Example 4 of this disclosure;

[0122] (c) Promotes cartilage repair and relieves joint pain.

[0123] In some embodiments, the KD value of the aforementioned peptide binding to integrin α5β1 can be ≤1×10⁻⁷ M, or ≤1×10⁻⁸ M, or ≤1×10⁻⁹ M, or ≤1×10⁻¹⁰ M; for example, it can be between 1-1000 nM, 1-800 nM, 1-600 nM, 1-500 nM, 1-300 nM, 1-200 nM, or 1-100 nM. This disclosure also provides a method for designing, modifying, or screening peptides or fusion proteins, comprising the following steps: using one or more ANGPTL proteins or fragments thereof as templates, modifying them according to at least one modification condition or standard, wherein the modification condition or standard includes: homology with the template, protein stability, cartilage repair function, joint pain relief function, and / or resistance to protease degradation, etc.

[0124] In some embodiments, the ANGPTL protein or a fragment thereof is any of the ANGPTL proteins or fragments described herein. Homology with the template includes, but is not limited to, amino acid sequence identity or evolutionary homology. Protein stability includes, but is not limited to, thermal stability and protease degradation stability. In some embodiments, the principles of peptide optimization include maintaining the chondrogenic activity of the ANGPTL3 C-terminal fibroinogen-like domain and not generating additional angiogenic activity. By comprehensively comparing the structures of the original proteins (e.g., ANGPTL3 and ANGPTL2), analyzing relevant hydrophobic core and angiogenesis-related sequences, the modified peptides or fusion proteins are ultimately designed.

[0125] In some implementations, the peptides or fusion proteins are designed, modified, or screened to be any peptide fragments or fusion proteins described herein.

[0126] Nucleic acid constructs

[0127] This disclosure also provides an RNA molecule comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein comprises any of the polypeptides or fusion proteins described herein.

[0128] This article also provides an RNA molecule comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein is a polypeptide comprising at least two polypeptide fragments derived from ANGPTL1, ANGPTL2, ANGPTL3 or ANGPTL4, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0129] This article also provides an RNA molecule comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein is a polypeptide comprising at least one polypeptide fragment derived from ANGPTL3, the polypeptide fragment derived from ANGPTL3 comprising at least one heterologous amino acid sequence; the amino acid site being a natural sequence site relative to the amino acid sequence shown in SEQ ID NO: 3.

[0130] This document also provides an RNA molecule comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein is a polypeptide comprising one or more polypeptide fragments derived from ANGPTL3 having an amino acid mutation selected from any one or more of the following positions: position 259, position 280, position 289, position 304, position 330, position 350, position 384, position 423, position 424, position 432; the amino acid mutation site is a natural sequence site relative to the amino acid sequence shown in SEQ ID NO: 3.

[0131] This document also provides an RNA molecule comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein is a polypeptide comprising one or more polypeptide fragments derived from ANGPTL, wherein the polypeptide fragments derived from ANGPTL are truncated ANGPTL1, ANGPTL2, ANGPTL3 or ANGPTL4 polypeptide fragments, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0132] This document also provides an RNA molecule comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein is a polypeptide comprising one or more polypeptide fragments derived from ANGPTL, wherein the polypeptide fragments derived from ANGPTL are truncated ANGPTL1, ANGPTL2, or ANGPTL4 polypeptide fragments, or polypeptide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0133] This document also provides an RNA molecule comprising an open reading frame (ORF) encoding a target protein; wherein the target protein is a polypeptide, characterized in that the amino acid sequence of the polypeptide comprises or is as shown in SEQ ID NOs: 5-32, and 34-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0134] This article also provides an RNA molecule comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein is a fusion protein, characterized in that the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NOs: 5-10, 26-30, 32, and 35-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0135] This document also provides an RNA molecule comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein is a polypeptide comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein: (1) the first polypeptide comprises amino acids 269-290 of the amino acid sequence shown in SEQ ID NO: 2; (2) the second polypeptide comprises amino acids 259-262 of the amino acid sequence shown in SEQ ID NO: 3; (3) the third polypeptide comprises amino acids 295-300 of the amino acid sequence shown in SEQ ID NO: 2; (4) the fourth polypeptide comprises amino acids 269-453 of the amino acid sequence shown in SEQ ID NO: 3; and (5) the fifth polypeptide comprises amino acids 488-493 of the amino acid sequence shown in SEQ ID NO: 2, wherein (1)-(5) are arranged in order from the N-terminus to the C-terminus of the polypeptide. In some embodiments, the polypeptide comprises a K423Q mutation. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 26.

[0136] This document also provides an RNA molecule comprising a nucleotide sequence as shown in any one of SEQ ID NOs:48, 51, and 101-138, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the ORF encodes an amino acid sequence as shown in any one of SEQ ID NOs:48, 51, and 101-138, or a sequence having at least 90% sequence identity with it.

[0137] This disclosure also provides a nucleic acid construct comprising (a) an open reading frame (ORF) encoding at least one target protein. In some embodiments, the nucleic acid construct is a DNA molecule. In some embodiments, the nucleic acid construct is an RNA molecule (e.g., mRNA). This disclosure also provides a nucleic acid construct comprising any of the RNA molecules described herein (e.g., mRNA).

[0138] In some embodiments, the nucleic acid construct comprises a nucleotide sequence as shown in SEQ ID NO:48, 50-51, or a nucleotide sequence having at least 90% sequence identity with it. In some embodiments, the nucleic acid construct is an RNA molecule (e.g., mRNA) comprising a nucleotide sequence as shown in SEQ ID NO:48 or 51, or a nucleotide sequence having at least 90% sequence identity with it. In some embodiments, the nucleic acid construct is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO:47 or 50, or a nucleotide sequence having at least 90% sequence identity with it.

[0139] In some implementations, the nucleic acid construct further includes an untranslated region element (UTR). The untranslated region element (UTR) can be a 5' untranslated region element (5'UTR) or a 3' untranslated region element (3'UTR).

[0140] In some embodiments, the target gene is heterologous. In other embodiments, the target gene is endogenous.

[0141] In some embodiments, the nucleic acid construct further includes (b) a 5' untranslated region element (5'UTR). In some embodiments, the 5'UTR in the nucleic acid construct is located upstream of the open reading frame. In some embodiments, the 5'UTR in the nucleic acid construct is located at the 5' end of the open reading frame.

[0142] In some embodiments, the 5'UTR in the nucleic acid construct is selected from the 5'UTR or a derivative sequence of any of the following genes: ACTG1, CTSB, Rho GTPase activator protein (ARHGAP), heat shock 27kDa protein 1 (HSPB1), hemoglobin subunit beta (HBB), C-Cmotif chemokine ligand 13 (CCL13), CHCHD10, or NDUFA11.

[0143] In some implementations, the 5'UTR in the nucleic acid construct is a 5'UTR sequence derived from or a derivative of the ACTG1 gene.

[0144] In some embodiments, the 5'UTR of the ACTG1 gene contains or is a nucleotide sequence as shown in SEQ ID NO: 44 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0145] In some embodiments, the open reading frame described herein originates from a different gene than the 5' UTR and / or the 3' UTR. In some embodiments, the nucleic acid construct of this disclosure comprises at least one open reading frame, at least one 5' UTR, or at least one 3' UTR. In some embodiments, the 5' UTR and 3' UTR in the nucleic acid construct of this disclosure are of the same or different origins, for example, from the same or different genes. In some embodiments, the 5' UTR and 3' UTR in the nucleic acid construct of this disclosure originate from the same or different species.

[0146] In some embodiments, the 3'UTR in the nucleic acid construct of this disclosure is located downstream of the open reading frame. In some embodiments, the 3'UTR in the nucleic acid construct is located at the 3' end of the open reading frame. In some embodiments, the 3'UTR in the nucleic acid construct of this disclosure is selected from or derived from the 3'UTR of any gene such as ACTG1, CTSB, FAM166A, NDUFB9, hemoglobin subunit beta (HBB), ARHGAP15, coronin 1A (CORO1A), hemopexin (HPX), etc.

[0147] In some embodiments, the 3'UTR is a 3'UTR sequence derived from or of the CTSB gene or a derivative thereof.

[0148] In some embodiments, the 3'UTR of the nucleic acid construct disclosed herein contains or is a nucleotide sequence as shown in SEQ ID NO: 45 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0149] In some implementations, the nucleic acid construct includes a 5' UTR and a 3' UTR, wherein:

[0150] The 5'UTR is selected from the 5'UTR or a derivative sequence of any gene such as ACTG1, CHCHD10 or NDUFA11, and the 3'UTR is selected from the 3'UTR or a derivative sequence of any gene such as CTSB, FAM166A or NDUFB9.

[0151] In some embodiments, the nucleic acid construct includes a 5'UTR and a 3'UTR, wherein the 5'UTR and the 3'UTR are selected from any one of the following:

[0152] The 5'UTR is derived from or is a 5'UTR of the ACTG1 gene or a derivative thereof, and the 3'UTR is derived from or is a 3'UTR of the CTSB gene or a derivative thereof;

[0153] In some embodiments, the nucleic acid constructs disclosed herein include a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following:

[0154] The 5'UTR contains or is a nucleotide sequence as shown in SEQ ID NO: 44 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and / or the 3'UTR contains or is a nucleotide sequence as shown in SEQ ID NO: 45 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it;

[0155] In some implementations, the nucleic acid construct disclosed herein further includes: (d) a polyadenylate (poly-A) tail.

[0156] In some embodiments, the poly-A tail in the nucleic acid construct is located downstream of the 3' UTR. In some embodiments, the poly-A tail in the nucleic acid construct is located at the 3' end of the 3' UTR. In some embodiments, the poly-A tail is located at the 3' end of the nucleic acid construct. In some embodiments, the poly-A tail is at least about 50, 100, 120, 125, 130, 150, 200, 300, 400, or 500 nucleotides long.

[0157] In some embodiments, the poly-A tail includes, but is not limited to, tails selected from 120A, 125A, Poly A-3070, HGH polyA, SV40polyA, BGH polyA, rbGlob polyA, or SV40late polyA.

[0158] In some specific embodiments, the poly-A tail is selected from 125A, 120A, or Poly A-3070, wherein 125A (poly(A120)) comprises the sequence shown in SEQ ID NO:46 or has at least 90% identity with it, 120A (poly(A125)) comprises the sequence shown in SEQ ID NO:139 or has at least 90% identity with it, and Poly A-3070 (poly(A30L70)) comprises the sequence shown in SEQ ID NO:140 or has at least 90% identity with it.

[0159] This disclosed nucleic acid construct further includes: (e) a 5' cap structure (5'Cap).

[0160] In some embodiments, the 5' cap structure in the nucleic acid construct is located upstream of the 5' UTR. In some embodiments, the 5' cap structure in the nucleic acid construct is located at the 5' end of the 5' UTR. In some embodiments, the 5' cap structure is a cap structure known to those skilled in the art, such as Cap0 (methylation of the first base, e.g., m7GpppN), Cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap2 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphate thioester modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0161] In some implementations, 5'-cap structures (such as Cap0 or Cap1) are formed using chemical RNA synthesis or in vitro RNA transcription (co-transcriptional capping).

[0162] In some embodiments, a capping enzyme (e.g., a vaccinia virus capping enzyme and / or a cap-dependent 2'-O methyltransferase) is used to form a 5'-cap structure (such as Cap0 or Cap1) via enzymatic capping. In some embodiments, an immobilized capping enzyme is used to add a 5' cap structure (Cap0 or Cap1). The capping methods and means described in WO2016 / 193226 are incorporated herein by reference in their entirety.

[0163] In some implementations, the 5' cap structure includes, but is not limited to, ARCA, 3'OMe-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0164] In some specific embodiments, the 5' cap structure is m7G(5')ppp(5')(2'OMeA)pG. Other 5' cap structures or cap structure analogues may also be used.

[0165] In some implementations, from the 5' to 3' direction, the nucleic acid construct, as described in any of the preceding embodiments, contains any one of i)-v):

[0166] i) 5'UTR, and Open Reading Frame (ORF);

[0167] ii) Open reading frame (ORF), and 3' UTR;

[0168] iii) 5'UTR, Open Reading Frame (ORF), and 3'UTR;

[0169] iv) 5'UTR, open reading frame (ORF), 3'UTR, and poly-A tail;

[0170] v) 5' Cap structure, 5' UTR, Open Reading Frame (ORF), 3' UTR, and poly-A tail;

[0171] The ORF therein originates from a different gene than the 5'UTR and / or the 3'UTR.

[0172] In some implementations, the 5'UTR in i), iii) to v) is selected from the 5'UTR or a derivative sequence of any gene such as ACTG1, CHCHD10 or NDUFA11 (e.g., the 5'UTR of ACTG1 or a derivative sequence thereof).

[0173] In some implementations, the 3'UTR in ii) to v) is selected from the 3'UTR or a derivative sequence thereof of any gene such as CTSB, FAM166A or NDUFB9 (e.g., the 3'UTR of CTSB or a derivative sequence thereof).

[0174] In some embodiments, the poly-A tail in iv) to v) comprises, for example, the nucleotide sequence shown in SEQ ID NO: 46, 139 or 140.

[0175] In some embodiments, the nucleic acid construct is an RNA molecule. In some embodiments, the RNA molecule may be mRNA.

[0176] In some embodiments, the nucleic acid construct is an RNA molecule (e.g., mRNA) containing a nucleotide sequence as shown in or having at least 90% identity with any of SEQ ID NO:48 or 51.

[0177] This disclosure also provides polypeptides or fusion proteins (e.g., ANGPTL polypeptides) encoded by any of the aforementioned nucleic acid constructs. The polypeptides or fusion proteins are generated in vitro or in vivo. In some embodiments, the polypeptides or fusion proteins encoded by any of the aforementioned nucleic acid constructs are intermediates or end products (such as precursors, intermediates, variants, or end products before or after protein modification or cleavage).

[0178] The nucleic acid constructs disclosed herein can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on the nucleotide sequence information disclosed herein, and / or can be isolated from suitable natural sources.

[0179] The mRNA disclosed herein can improve the weight-bearing capacity of bipedalism in a model, has chondrogenic activity and relieves joint pain, can increase half-life and reduce dosing frequency, and provides a drug that can effectively treat osteoarthritis and joint damage.

[0180] Polynucleotides

[0181] This disclosure also provides an isolated polynucleotide. Exemplarily, the polynucleotide includes, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), gamma-hydroxyl nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), including LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereomers of LNA), 2′-amino-LNA having 2′-amino functionalization and 2′-amino-α-LNA having 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

[0182] In some embodiments, the polynucleotide encodes any of the aforementioned modified polypeptides, fusion proteins, or isolated polypeptides, for example, the polynucleotide is RNA (e.g., mRNA) containing an open reading frame sequence encoding the aforementioned modified polypeptide, fusion protein, or isolated polypeptide of ANGPTL. In some embodiments, the polynucleotide encodes the aforementioned RNA molecule, for example, the polynucleotide is template DNA (e.g., cDNA) containing the RNA molecule transcribed from the aforementioned RNA molecule.

[0183] In some embodiments, the polynucleotide is isolated DNA, which is transcribed to obtain the aforementioned RNA molecule. Exemplarily, in vitro transcription of RNA is known in the art and described in WO / 2014 / 152027, which is incorporated herein by reference in its entirety. For example, in some embodiments, an RNA transcript is produced by using a non-amplified, linearized DNA template in an in vitro transcription reaction. In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA transcript is purified by chromatographic methods, for example, using an oligomeric dT substrate. Some embodiments do not include the use of DNases. In some embodiments, RNA transcripts are synthesized from a non-amplified, linear DNA template encoding the gene of interest via an enzymatic in vitro transcription reaction using a T7 phage RNA polymerase of desired chemical properties and a nucleotide triphosphate. Any number of RNA polymerases or variants can be used in the methods of the present invention. The polymerase may be selected from (but is not limited to) phage RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNa polymerase), and / or mutant polymerases, such as (but not limited to) polymerases capable of combining modified nucleic acids and / or modified nucleotides (including chemically modified nucleic acids and / or nucleotides).

[0184] In some embodiments, non-amplified, linearized plasso DNA is used as template DNA for in vitro transcription. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of RNA polynucleotides, such as (but not limited to) HSV RNA, such as HSV mRNA. In some embodiments, cells (e.g., bacterial cells, such as E. coli, such as DH-1 cells) are transfected with the plasso DNA template. In some embodiments, the transfected cells are cultured to replicate the plasso DNA, which is then isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, such as a 5' promoter located at the gene of interest and a T7 promoter operatively linked to the gene of interest.

[0185] In some implementations, the polynucleotide is codon-optimized. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some implementations, optimization algorithms are used to optimize open reading frame (ORF) sequences.

[0186] In some embodiments, the polynucleotide is isolated DNA containing a nucleotide sequence as shown in any one of SEQ ID NOs:47, 50, and 63-100, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the DNA encodes or is transcribed into an RNA sequence as shown in any one of SEQ ID NOs:48, 51, and 101-138, or a sequence having at least 90% sequence identity with it. In some embodiments, the DNA encodes an amino acid sequence as shown in any one of SEQ ID NOs:5-42, or a sequence having at least 90% sequence identity with it.

[0187] Chemical modification

[0188] In some embodiments, the polynucleotides (e.g., RNA) disclosed herein contain at least one chemical modification.

[0189] The terms “chemically modified” and “chemically modified” refer to modifications of at least one of the ribonucleotides or deoxyribonucleotides, namely adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C), in terms of their position, pattern, percentage, or population. Typically, these terms do not refer to modifications of the ribonucleotides in the naturally occurring 5′ end cap of mRNA.

[0190] Modifications to polynucleotides include, but are not limited to, those described herein, and include (but are not explicitly limited to) those involving chemical modifications. Polynucleotides (e.g., RNA, such as mRNA) may contain naturally occurring, non-naturally occurring modifications, or a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides may include any suitable modifications to, for example, inter-sugar, base, or nucleoside linkages (e.g., linkages to phosphate esters, phosphodiester linkages, or the phosphodiester backbone).

[0191] For example, chemical modifications include, but are not limited to, the modification types disclosed in WO2017070601A and WO2017070623A, which are incorporated herein by reference.

[0192] In some embodiments, the polynucleotide (e.g., RNA, such as mRNA) contains multiple (more than one) different modifications. In some embodiments, specific regions of the polynucleotide contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified RNA (e.g., modified mRNA) introduced into cells or organisms exhibits reduced degradation relative to the unmodified polynucleotide in cells or organisms. In some embodiments, the modified RNA (e.g., modified mRNA) introduced into cells or organisms may exhibit reduced immunogenicity (e.g., reduced innate response) in cells or organisms.

[0193] In some implementations, the polynucleotide (e.g., RNA, such as mRNA) comprises a non-naturally modified nucleotide introduced during or after polynucleotide synthesis to achieve the desired function or property. Modifications can be present at internucleotide links, bases such as purines or pyrimidines, or sugars. Modifications can be introduced chemically or via polymerase at the chain terminus or any other location on the chain. Any region of the polynucleotide can be chemically modified.

[0194] This disclosure provides modified nucleosides and nucleotides of polynucleotides (e.g., RNA, such as mRNA). "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof, combined with a base (e.g., purine, pyrimidine, or a derivative thereof). "Nucleotide" refers to a nucleoside, including a phosphate ester group. Modified nucleotides can be synthesized by any suitable method, such as chemical, enzymatic, or recombinant methods, to include one or more modified or non-natural nucleosides. Polynucleotides may contain one or more regions of the linked nucleoside. Such regions may have variable backbone bonds. The bonds may be standard phosphodiester bonds, in which case the polynucleotide will contain the nucleotide region.

[0195] Modified nucleotide base pairings encompass not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides, including non-standard or modified bases, wherein the arrangement of hydrogen bond donors and acceptors allows hydrogen bonding between non-standard bases and standard bases or between two complementary non-standard base structures (for example, such as those polynucleotides having at least one chemical modification). An example of such non-standard base pairings is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker may be incorporated into the polynucleotides of this disclosure.

[0196] In some embodiments, the chemical modification of the nucleotide (e.g., RNA) is a uracil modification. Suitable polynucleotide (e.g., RNA, such as mRNA) modifications (including but not limited to chemical modifications) for this disclosure include, but are not limited to, the following: pseudouridine (ψ), 2-thiouridine (s2U), 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxy Urate, 2′-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4′-thiouridine, 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine, (I), 1-methyl-inosine (m1I), wyoside (imG), methyl wyoside (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl 7-Deza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine, 2-geranylthiouridine, 2-lysicryl, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2′-O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl- 2-Thiouridine, 5-Carboxymethyl-2-thiouridine, 5-Carboxymethylaminomethyl-2-geranylthiouridine, 5-Carboxymethylaminomethyl-2-selenouridine, 5-Cyanomethyluridine, 5-Hydroxycytidine, 5-Methylaminomethyl-2-geranylthiouridine, 7-Aminocarboxypropyl-demethylwyoside, 7-Aminocarboxypropylwyoside, 7-Aminocarboxypropylwyoside methyl ester, 8-Methyladenosine, N4,N4-Dimethylcytidine, N6-Formyladenosine, N6-Hydroxymethyladenosine, agmatidine, Cyclic N6-Threonylcarbamoyladenosine, Glutamyl-quinoline, Incompletely methylated hydroxywayoside, N4,N4,2′-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQ0base, preQ1base, and combinations of two or more thereof. In some embodiments, at least one chemically modified nucleoside is selected from the group consisting of: pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some embodiments, RNA (such as mRNA) comprises at least two (e.g., 2, 3, 4, or more) combinations of the aforementioned modified bases. In some embodiments, RNA (such as mRNA) comprises at least two (e.g., 2, 3, 4, or more) combinations of the aforementioned modified bases.

[0197] In some embodiments, the modified bases in the polynucleotide (e.g., RNA, such as mRNA) are selected from the group consisting of: 1-methyl-pseuuridine (m1ψ), 1-ethyl-pseuuridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, the RNA comprises a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified bases, including but not limited to chemical modifications.

[0198] In some embodiments, the polynucleotide (e.g., RNA, such as mRNA) comprises pseudouridine (ψ) and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA (such as mRNA) comprises 1-ethyl-pseudouridine (e1ψ). In some embodiments, the RNA (such as mRNA) comprises 1-methyl-pseudouridine (m1ψ) and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises 1-ethyl-pseudouridine (e1ψ) and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises 2-thiouridine (s2U). In some embodiments, the RNA (such as mRNA) comprises 2-thiouridine and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises methoxyuridine (mo5U). In some embodiments, the RNA (such as mRNA) comprises 5-methoxyuridine (mo5U) and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises 2′-O-methyluridine. In some embodiments, the RNA (such as mRNA) comprises 2′-O-methyluridine and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises N6-methyladenosine (m6A). In some embodiments, the RNA (such as mRNA) comprises N6-methyladenosine (m6A) and 5-methylcytidine (m5C).

[0199] In some implementations, polynucleotides (e.g., RNA, such as mRNA) are uniformly modified (e.g., completely modified, modified throughout the sequence) to have specific modifications. For example, a polynucleotide may be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a polynucleotide may be uniformly modified to any similar nucleoside residues present in the sequence by replacing modified residues such as those described above.

[0200] host cells

[0201] This disclosure also provides a host cell comprising any of the nucleic acid constructs, RNA, polynucleotides, or vectors described in the preceding embodiments. In some embodiments, the cell is capable of expressing one or more polypeptides of the nucleic acid constructs, RNA, polynucleotides, or vectors disclosed herein. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0202] Bacterial cells include, for example, cells of Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0203] Fungal cells include, for example, cells of species from the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells of species from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichia pastoris* and *Pichia methanolica*), and *Hansenula*.

[0204] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0205] However, this disclosure may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0206] Production or preparation method

[0207] This disclosure provides a method for preparing ANGPTL peptides, polynucleotides, vectors, or RNA. The ANGPTL peptides, polynucleotides, vectors, or RNA may be any of the ANGPTL peptides (e.g., modified peptides), polynucleotides, vectors, or RNA described in this disclosure.

[0208] In some embodiments, the methods used to prepare ANGPTL peptides, polynucleotides, or RNA are known in the art, such as specific suitable vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions. Similarly, protein isolation and purification techniques suitable for the methods of producing the encoded peptides of this disclosure are well known to those skilled in the art.

[0209] In some embodiments, the method for preparing the ANGPTL peptide includes: culturing host cells containing polynucleotides, vectors, or RNA, and recovering the resulting ANGPTL peptide from the culture.

[0210] In some embodiments, the method for preparing the RNA molecule includes: generating the RNA encoding the ANGPTL polypeptide in an in vitro transcription reaction using a linearized DNA template. In some embodiments, the RNA is generated in an in vitro transcription reaction using a non-amplified linearized DNA template. In some specific embodiments, the method further includes adding a 5' cap to the 5' end of the RNA molecule.

[0211] In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of a polynucleotide (RNA, such as, but not limited to, respiratory viral mRNA). In some embodiments, cells, such as bacterial cells, such as *E. coli*, such as DH-1 cells, are transfected with the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA, and then the plasmid DNA is isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, such as the T7 promoter located at the 5' of the target gene and operatively linked thereto.

[0212] In some embodiments, the polynucleotides, vectors, RNAs, or ANGPTL peptides of this disclosure may also be obtained by other production methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0213] lipid nanoparticles

[0214] This disclosure also provides a lipid nanoparticle comprising any of the aforementioned nucleic acid constructs, RNA molecules, polynucleotides, or carriers. In some embodiments, the lipid nanoparticle comprises the RNA molecule described in any of the preceding embodiments.

[0215] In some embodiments, the RNA molecules of this disclosure can be delivered into cells and / or in vivo using nanolipid nanoparticles of any type in the art. Exemplarily, the lipid nanoparticles may comprise ionizable cationic lipids, non-cationic lipids, sterols and / or PEG lipid components, and a target nucleic acid. Exemplarily, the nanolipid nanoparticles include, but are not limited to, the lipid particles disclosed in WO2017075531, WO2018081480A1, WO2017049245A2, WO2017099823A1, WO2022245888A1, WO2022150717A1, CN101291653A, CN102119217A, WO2011000107A1, and CN107028886A, all of which are incorporated herein by reference in their entirety.

[0216] Pharmaceutical Composition

[0217] This disclosure also provides a pharmaceutical composition comprising any of the ANGPTL peptides, polynucleotides, carriers, RNA molecules, nucleic acid constructs, and / or lipid nanoparticles described in any of the preceding claims.

[0218] In some embodiments, the pharmaceutical composition comprises pharmaceutically acceptable excipients, diluents, or excipients.

[0219] Product or reagent kit

[0220] This disclosure provides a product or kit comprising any of the foregoing ANGPTL peptides, nucleic acid constructs, RNA molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions. The kit can be used for related detection or diagnostic purposes.

[0221] Methods of treating and / or preventing diseases and pharmaceutical uses

[0222] In some embodiments, this disclosure provides the use of any of the foregoing peptides or fusion proteins (modified ANGPTL peptides or fusion proteins), nucleic acid constructs, RNA molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions for the prevention, mitigation, and / or treatment of diseases.

[0223] In some embodiments, this disclosure provides the use of any of the foregoing peptides or fusion proteins (modified ANGPTL peptides or fusion proteins), nucleic acid constructs, RNA molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions in the preparation of medicaments for the prevention, relief, and / or treatment of diseases.

[0224] In some embodiments, the disease is a joint-related disease. In some embodiments, the disease includes, but is not limited to, osteoarthritis, rheumatoid arthritis, other autoimmune diseases, or osteochondritis dessicans. In some embodiments, the osteoarthritis includes, but is not limited to, knee osteoarthritis, hand osteoarthritis, hip osteoarthritis, or spinal osteoarthritis.

[0225] In some embodiments, the peptides or fusion proteins (modified ANGPTL peptides or fusion proteins), nucleic acid constructs, RNA molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions of this disclosure may be used to treat, alleviate, or prevent various cartilage disorders and / or related symptoms or effects. Exemplary conditions or diseases using the ANGPTL peptides, nucleic acid constructs, RNA molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions of this disclosure include, but are not limited to, cartilage degeneration-related diseases such as osteoarthritis and joint injuries. In some embodiments, the osteoarthritis is knee osteoarthritis, hand osteoarthritis, hip osteoarthritis, or spinal osteoarthritis.

[0226] In some embodiments, this disclosure provides at least one use of the peptides or fusion proteins (modified ANGPTL peptides or fusion proteins), nucleic acid constructs, RNA molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions described in any of the foregoing:

[0227] (1) Used to induce cartilage formation;

[0228] (2) Used for the prevention, inhibition, relief and / or treatment of joint diseases and / or cartilage-related conditions;

[0229] (3) Used for the prevention, inhibition, relief and / or treatment of osteoarthritis or joint damage;

[0230] (4) Preparation of drugs for inducing cartilage formation;

[0231] (5) To prepare drugs for the prevention, inhibition, relief and / or treatment of joint diseases and / or cartilage-related conditions;

[0232] (6) Prepare drugs for the prevention, inhibition, relief and / or treatment of osteoarthritis or joint damage.

[0233] In some implementations, the ANGPTL (e.g., ANGPTL3) fragment may be a human ANGPTL protein or a fragment thereof, or a protein or peptide derived from a human ANGPTL protein (e.g., a modified human ANGPTL1, ANGPTL2, ANGPTL3, or ANGPTL14 protein, a conserved variant, or a peptide mimic of the protein).

[0234] In some embodiments, this disclosure provides methods for preventing and / or treating diseases, including administering to a subject in need a preventive and / or therapeutically effective amount of the aforementioned ANGPTL peptide, nucleic acid construct, RNA molecule, polynucleotide, carrier, lipid nanoparticle, and / or pharmaceutical composition.

[0235] In some embodiments, the ANGPTL peptides, nucleic acid constructs, RNA molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions of this disclosure are applied, either alone or in combination with a suitable carrier (to prolong protein release), by direct injection into the synovial fluid, systemic administration (oral or intravenous), or direct application to cartilage defects. In some embodiments, the ANGPTL peptides, nucleic acid constructs, RNA molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions of this disclosure are used in combination with other procedures to repair cartilage damage, including but not limited to joint lavage, bone marrow stimulation, dermabrasion, subchondral drilling, or microfractures of the proximal subchondral bone. Attached Figure Description

[0236] Figures 1A and 1B: Design of ANGPTL family protein constructs. The amino acid sites of ANGPTL1, ANGPTL2, ANGPTL3, and ANGPTL4 are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[0237] Figure 2: Evaluation of chondrogenic activity of each protein construct. Figure 2A: Evaluation of the regulatory capacity of each protein construct on the Wnt pathway by stimulating chondrocytes to secrete DKK1, thus determining the chondrogenic activity of candidate molecules. Figure 2B: Evaluation of high-dose toxicity of each protein construct to chondrocytes.

[0238] Figure 3: Evaluation of the bipedal dysfunction repair level of each protein construct using a rat meniscectomy osteoarthritis model.

[0239] Figure 4: Pathological evaluation of the cartilage repair activity of each protein construct in a rat model of meniscus tear osteoarthritis.

[0240] Figure 5: Using a rat meniscectomy osteoarthritis model, the bipedal disability repair level of each protein construct was further screened.

[0241] Figure 6: Pathological evaluation of the cartilage repair activity of protein construct PR0029 in a rat model of meniscus tear osteoarthritis.

[0242] Figure 7: In vitro expression level of the mRNA construct after transfection into 293 cells. The mRNA in the construct encodes PR0029, which is tagged with HIS.

[0243] Figure 8: Evaluation of chondrogenic activity of mRNA nucleic acid constructs expressing target protein in vitro.

[0244] Figure 9: The ameliorative effect of mRNA nucleic acid constructs on bipedal dysfunction in a rat model of osteoarthritis. Figure 9A: Bipedal weight-bearing difference: weight-bearing of the non-affected hind limb - weight-bearing of the affected hind limb. Figure 9B: Bipedal weight-bearing ratio: weight-bearing of the non-affected hind limb / weight-bearing of the affected hind limb.

[0245] Figure 10: The ability of mRNA nucleic acid constructs to repair cartilage in the affected knee joint in a rat osteoarthritis model. Figure 10A: Scoring of cartilage degeneration based on the OARSI scoring system. Figure 10B: Statistical analysis of cartilage degeneration area in each group.

[0246] Figure 11: Schematic diagram of the functional structural domain division of ANGPTL1, ANGPTL2, ANGPTL3 and ANGPTL4. Detailed Implementation

[0247] the term

[0248] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0249] The term "mutation" refers to the deletion, addition, or substitution of amino acid residues in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, substitution of an amino acid at a specific position in the protein sequence is referred to using the annotation "(amino acid residue in wild-type protein)(amino acid position)(amino acid residue in engineered protein)". For example, the annotation Y75A indicates that the tyrosine (Y) residue at position 75 of the amino acid sequence of the reference protein has been replaced by an alanine (A) residue (in a mutant of the reference protein). In cases where variations exist at the same position of amino acid residues between different wild-type sequences, the amino acid code preceding the position number may be omitted from the annotation, such as "75A".

[0250] The terms "fragment" or "truncated form" are understood to mean a fragment lacking one or more amino acids at the N-terminus and / or C-terminus compared to an ANGPTL peptide, but still exhibiting similar biological activity to the ANGPTL peptide of this disclosure. "Similar biological activity to the ANGPTL peptide of this disclosure" means that the ANGPTL peptide fragment has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the biological activity of the ANGPTL peptide. The activity of the ANGPTL peptide and its fragments can be determined by methods known in the art.

[0251] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids, as well as to both natural and non-natural amino acid polymers. Unless otherwise stated, a particular polypeptide sequence also implicitly encompasses variants with conserved modifications. In some embodiments, a polypeptide may also refer to a fusion protein comprising multiple peptide segments.

[0252] The term "amino acid" refers to both natural and synthetic amino acids, as well as amino acid analogs and amino acid simulants that function in a manner similar to natural amino acids. Natural amino acids are those encoded by the genetic code, and subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as natural amino acids—namely, carbon atoms bound to hydrogen, carboxyl groups, amino groups, and R groups—such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. The naturally encoded amino acids are 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine, pyrroline-carboxy-lysine, and selenocysteine.

[0253] The terms “modification” and “alteration” are used interchangeably in this document to refer to substitutions, truncations, amplifications, or chemical modifications to the amino acid or nucleotide sequences of proteins, peptides, or nucleic acid fragments. The “altered” or “modified” peptide or nucleic acid sequences described herein may contain one or more mutations, insertions, deletions, or substitutions.

[0254] The term "recombinant protein" refers to a protein that has a sequence that is not naturally occurring, a sequence modified (such as a chemical modification), or a sequence that is an artificial combination of two otherwise separated sequence segments.

[0255] "Recombinant nucleic acid molecules" are molecules that have sequences that are not naturally occurring, such as those comprising one or more nucleic acid substitutions, deletions (or truncations), or insertions, and / or sequences that are artificially combined by means of two otherwise separated sequence segments. For example, such artificial combinations can be achieved through chemical synthesis or by manipulating the separated nucleic acid segments (e.g., through genetic engineering techniques).

[0256] The term "natural protein, sequence, or disulfide bond" refers to a polypeptide, sequence, or disulfide bond that has not been modified, for example, by selective mutation. For example, selective mutations concentrate the antigenicity of an antigen at a target epitope or introduce a disulfide bond into a protein that is not present in its natural state. A natural protein or natural sequence is also called a wild-type protein or wild-type sequence. A non-natural disulfide bond is a disulfide bond that is not present in a natural protein, for example, a disulfide bond formed in a protein by introducing one or more cysteine ​​residues through genetic engineering.

[0257] “Nucleic acid molecule”: A polymeric form of nucleotides, which may include sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers described above. A nucleotide refers to a modified form of ribonucleotide, deoxynucleotide, or any type of nucleotide. As used herein, the term “nucleic acid molecule” is synonymous with “nucleic acid” and “polynucleotide.” The term includes DNA in both single-stranded and double-stranded forms. Polynucleotides may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. “cDNA” refers to DNA in single-stranded or double-stranded form that is complementary to or identical to mRNA. “Encoding” refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide (e.g., gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological characteristics.

[0258] “Vector” or “expression vector” refers to a replicon, such as a plasmid, rod-like particle, bacteriophage, virus, virion, or granule, that can link another DNA segment, or “insertion,” to enable replication of the linked segment within the cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral in origin and / or final form, such as the PUC57 DNA vector used herein. The term “vector” encompasses any genetic element that, when bound to a suitable control element, is capable of replication and can transfer a gene sequence into a cell. In some embodiments, a vector can be an expression vector or a recombinant vector.

[0259] "Nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule, such as a DNA fragment, that is modified or synthesized to contain nucleic acid segments in a manner not naturally present, said nucleic acid molecule containing one or more control sequences or regulatory elements. In the context of this disclosure, nucleic acid constructs contain recombinant nucleotide sequences that are substantially composed of, optionally, one, two, three, or more separate nucleotide sequences, including a 5' UTR, an open reading frame (ORF), and a 3' UTR. In embodiments involving constructs comprising two or more sequences, the sequences are operatively linked to each other within the construct.

[0260] "Operationally ligated" is defined in this paper as a structure in which a control sequence, i.e. a promoter sequence and / or a 5'UTR sequence, is appropriately positioned relative to the coding DNA sequence such that the control sequence directs the transcription of the coding sequence and the translation of mRNA into a polypeptide sequence encoded by the coding DNA.

[0261] An "open reading frame" (ORF) is a segment or region of an mRNA molecule that encodes a polypeptide. An ORF consists of consecutive, non-overlapping in-frame codons, starting with a start codon and ending with a stop codon, and is translated by the ribosome.

[0262] A "linker" or "connector" refers to a linking unit that connects two polypeptide fragments or their encoding polynucleotides. Linkers are typically flexible, and their use does not cause the loss of the original function of the protein domain. Linkers can be peptide linkers or their encoding polynucleotides, containing one or more amino acids (typically about 1-30, 2-24, or 3-15 amino acids) or their encoding polynucleotides. Linkers used in this disclosure can be the same or different.

[0263] A “substitution” is defined as a change in an amino acid or nucleotide sequence such that, compared to the amino acid or nucleotide sequence of a reference peptide, one or more amino acids or nucleotides are replaced by different ones. If the substitution is conserved, the amino acid substituted into the peptide has similar structural or chemical properties (e.g., charge, polarity, hydrophobicity, etc.) to the amino acid it substituted. In some embodiments, peptide variants may have “non-conserved” variations, where the substituted amino acids differ in structure and / or chemical properties.

[0264] A “deletion” is defined as a change in an amino acid or nucleotide sequence such that one or more amino acid or nucleotide residues are missing compared to the amino acid or nucleotide sequence of a reference polypeptide. In the case of a polypeptide or polynucleotide sequence, the deletion may involve the deletion of 2, 5, 10, up to 20, up to 30, or up to 50 or more amino acid or nucleotide residues, taking into account the length of the modified polypeptide or polynucleotide sequence.

[0265] "Insertion" or "addition" refers to a change in an amino acid or nucleotide sequence such that, compared to the amino acid or nucleotide sequence of a reference polypeptide, this change results in the addition of one or more amino acid or nucleotide residues, respectively. "Insertion" typically refers to the addition of one or more amino acid residues (or nucleotide residues within a polynucleotide) within the amino acid sequence of a polypeptide, while "addition" can refer to an insertion or the addition of an amino acid residue at the N- or C-terminus of a polypeptide (or a nucleotide residue at the 5' or 3' terminus of a polynucleotide). In the case of polypeptide or polynucleotide sequences, insertions or additions can be up to 10, 20, 30, 50, or more amino acids (or nucleotide residues).

[0266] "Codon optimization" refers to replacing codons in a target sequence that are generally rare in genes highly expressed in a given species with codons that are common in genes highly expressed in the same species, while the original and unreplaced codons encode the same amino acid. Different species exhibit specific preferences for certain codons of particular amino acids. Codon preferences (differences in codon use between organisms) are generally related to the translation efficiency of messenger RNA (mRNA), which is considered to be particularly dependent on the characteristics of the codons translated and the utilization of specific transfer RNA (tRNA) molecules. The dominance of the chosen tRNA in the cell is generally a reflection of the most frequently used codons in peptide synthesis. Therefore, based on codon optimization, genes can be modified to target optimal gene expression in a given organism. Thus, the selection of the optimal codon depends on the codon use preferences of the host genome.

[0267] "Cell" or "host cell" includes any cell type that is readily transformed, transfected, transduced, etc., by the nucleic acid constructs or vectors of this disclosure. As a non-limiting example, the host cell can be any of isolated primary cells, pluripotent stem cells, CD34+ cells, induced pluripotent stem cells, or many immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell can be an in situ or in vivo cell in a tissue, organ, or organism.

[0268] An "effective amount" or "pharmacologically effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medically diagnosed disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0269] "Pharmaceutical acceptable" means that these therapeutic agents, materials, compositions and / or dosage forms are suitable for contact with patient tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, within reasonable medical judgment, have a reasonable benefit / risk ratio, and are effective for their intended use.

[0270] Treatment or prevention of disease: For example, in subjects suffering from, for instance, osteoarthritis, suppressing the full development of the disease or condition. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after its onset. Regarding a disease or pathological condition, the term "improvement" refers to any observable beneficial effect of treatment. For example, a beneficial effect can be demonstrated by delaying the onset of clinical symptoms of the disease in susceptible subjects, reducing the severity of some or all of the clinical symptoms of the disease, slowing the progression of the disease, reducing viral load, improving the overall health or well-being of the subject, or by parameters known in the art that are specific to a particular disease. "Preventive" treatment is treatment administered to subjects who do not exhibit signs of disease or only exhibit early signs, with the aim of reducing the risk of pathological development.

[0271] The term "mammal" includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as non-human primates. Therefore, administration to a subject can include administration to human subjects. Non-limiting examples of veterinary subjects include domestic animals (e.g., cats and dogs), livestock (e.g., cattle, horses, pigs, sheep, and goats), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, and non-human primates).

[0272] Example

[0273] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in the Cell Culture Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0274] Example 1: ANGPTL family protein constructs

[0275] Based on ANGPTL family proteins 1-4 (i.e., ANGPTL1, 2, 3, and 4, SEQ ID NOs: 1-4), protein constructs were designed using chimerism, truncation, mutation, and combinations thereof. Figures 1A and 1B show the construction methods of the protein constructs, including the ANGPTL3 C-terminal domain construct (PR0006), the ANGPTL3 C-terminal domain construct containing the K423Q mutation (PR0005), the N-terminal and C-terminal sequence substitutions of ANGPTL1, 2, and 4 with ANGPTL3 as the backbone (PR0008-PP0013 and PR0029-PR0032), the truncated forms of ANGPTL1, 2, and 4 with the backbone (PR0014-PR0019 and PR0033-PR0035), and the trypsin or chymotrypsin resistant mutants with the ANGPTL3 truncated form as the backbone (PR0020-PR0028). All of the above sequences are listed in Table 1 (SEQ ID NOs: 5-34).

[0276] Table 1. Protein construct sequences

[0277] Furthermore, PR0029 was selected for stability modification using an ancestral sequence reconstruction (ASR) strategy. In short, phylogenetic trees were constructed using 82 orthologous proteins from the Prototheca subclass (platypus) or mammals with higher evolutionary relationships. Based on these, ancestral sequence reconstruction was performed, and the reasonableness of the obtained sequences was further evaluated at both the structural model and amino acid property levels. Ultimately, eight single-point or combined mutants were obtained, and all sequences are listed in Table 2 (SEQ ID NOs: 35-42).

[0278] Table 2. Protein sequences based on PR0029 modification.

[0279] All of the above builds were originally designed with a C-end His tag, in HEK293 Freestyle TM Expression was performed in cells, and purification was carried out by Ni-NTA affinity chromatography. The purity of each construct was determined by SDS-PAGE and SEC-HPLC. The proteins of each construct were obtained by detection.

[0280] Example 2: Binding experiment of protein construct with integrin α5β1

[0281] The binding kinetics between the protein construct and integrin α5β1 are mediated by... The results were obtained using the R8 BLI System. In short, before the experiment, biotinylated integrin α5β1 and the protein construct were first transferred to binding buffer (1x PBS, 5mM MgCl2, 1mM CaCl2). Then, integrin α5β1 was immobilized on the streptavidin sensor at a concentration of 5 μg / mL, and the protein construct was diluted in a continuous 3-fold concentration gradient. The last sample was added with buffer as a control. Next, the instrument reaction temperature was set to 25℃, and both binding and dissociation times were set to 300 seconds. After the experiment, the data were obtained using... The analysis was performed using Analysis Studio Software. After removing the reference background, the resulting spectral curves were fitted with a 1:1 combined model to obtain the dynamic parameters.

[0282] Experimental results showed that the protein constructs in Example 1 could all bind to integrin α5β1, with dissociation equilibrium constants (KD) of approximately 1-1000 nM. The detection results for PR0029 and PR0005 are shown in Table 3. The results showed that the dissociation equilibrium constant (KD) between PR0029 and integrin α5β1 was approximately 41 nM, and the dissociation equilibrium constant (KD) between PR0005 and integrin α5β1 was approximately 56 nM.

[0283] Table 3. Binding kinetic parameters of integrin α5β1

[0284] Example 3: Evaluation of the thermal stability of the protein construct

[0285] The thermal stability of the construct PR0029 and its mutants was evaluated using differential scanning calorimetry (DSC), and the data are shown in Table 4. PR0029 exhibits good thermal stability, and the Tm values ​​of the mutants M6, M7, and M8, which were reconstructed from the ancestral sequence, were significantly improved.

[0286] Table 4. Tm values ​​of protein constructs

[0287] Example 4: In vitro activity evaluation of protein constructs

[0288] The chondrogenic mechanism of ANGPTL family proteins is not fully understood. Based on existing knowledge, DKK1 is a WNT-regulated biomarker during the transformation of hMSCs into chondrocytes (Zhong, L. et al. Stem Cells Dev. 2016 Dec. 25, 23). Therefore, we evaluated the chondrogenic activity of the protein construct by inducing chondrocytes to secrete DKK1 using the protein construct, and assessed its safety by evaluating cytotoxicity under high-dose construct administration. Specifically, different concentrations of each protein construct were incubated overnight with chondrocytes in 1% serum-containing medium. The cell supernatant was then used to detect the secretion of DKK1 using an ELISA kit, and cell viability was also assessed.

[0289] Testing revealed that all protein constructs in Example 1 promoted DKK1 secretion. As shown in Figure 2, PR0029 induced DKK1 secretion in chondrocytes significantly better than PR0005; at protein concentrations below 125 μg / mL, PR0029 induced DKK1 secretion in chondrocytes in a dose-dependent manner; and regarding safety, high concentrations of PR0029 protein had minimal impact on cell viability.

[0290] Example 5: In vivo efficacy evaluation of the protein construct

[0291] The chondrogenic activity of each construct was evaluated using a rat meniscus tear model. The difference in weight-bearing between the model leg and the normal leg when the rats were standing on both feet was used to evaluate the improvement in weight-bearing after cartilage repair. At the end of the experiment, the affected joint was fixed and stained with Safranin and Fast Green. The degree of cartilage matrix loss and cartilage degeneration in the experimental animals were analyzed and scored using the OARSI scoring criteria. Specifically, except for the control (sham surgery / Sham) group, all rats underwent osteoarthritis modeling 6 days before grouping (Day 6). The animals were anesthetized by intraperitoneal injection of 2% sodium pentobarbital. The hair in the right hind limb knee joint area was shaved, and the skin was disinfected with iodine. A longitudinal incision of about 2 cm was made in the subcutaneous tissue on the medial side of the knee joint. The subcutaneous fascia was cut open, and the medial collateral ligament was exposed by blunt dissection. The medial collateral ligament was cut below the medial meniscus. The joint cavity was opened to expose the medial meniscus. The medial meniscus was lifted and cut at the narrowest point. The joint capsule was closed by suturing layer by layer to create the MMT (medial meniscal tear) OA model. The wounds and surrounding skin of all surgical animals were disinfected. After the animals were placed on an electric blanket and awakened, they were returned to their animal cages.

[0292] Six days later (Day 0), pain asymmetry in animals was assessed using a bipedal balance analgesia device, and animals were randomly assigned to groups. On Day 1 after grouping, each protein construct was administered via intra-articular injection at 20 μg. Bipedal weight-bearing difference was assessed using the bipedal balance analgesia device on Day 13 and Day 25 after grouping to evaluate bipedal disability status during standing. At the experimental endpoint on Day 26, rat joints were dissected. Routine pre-dissection observations were performed before tissue collection, including opening the joint capsule, observing the joint capsule, exposing the femoral condyle and tibial plateau, and performing tissue damage scoring (Pelletier score). The Pelletier scoring criteria are shown in Table 5. Routine tissue fixation was then performed. The affected knee joint was routinely fixed for 48 hours, followed by decalcification with formic acid decalcification solution for approximately 7 days. The joint was then cut into anterior and posterior parts along the medial collateral ligament, with both cut surfaces facing down, and embedded in an embedding cassette. Sections were prepared, with a thickness of 4–8 μm, and stained with safranin and fast green. The OARSI scoring criteria were used to analyze and score the degree of cartilage matrix loss and cartilage degeneration in the experimental animals. The OARSI scoring criteria are shown in Table 6. The ratio of cartilage degeneration area = matrix loss area / total tibial area * 100%. Statistical and evaluation results are shown in Table 7, Figures 3 and 4.

[0293] The results showed that at day 13, compared with the model group, all protein constructs showed an improving trend in the evaluation of the difference in weight-bearing capacity between the operated and non-operated legs. PR006, PR0029, and PR0032 showed statistically significant improvements in the difference in weight-bearing capacity between the operated and non-operated legs compared to the model group (p<0.05). PR0005, PR0006, and PR0020 showed statistically significant improvements in the ratio of the difference in weight-bearing capacity between the operated and non-operated legs compared to the model group (p<0.05).

[0294] In terms of gross injury score, cartilage degeneration and defect score, PR0005, PR0029 and PR0032 also showed an improvement trend compared with the model group. PR0029 showed statistically significant improvement in all three scores compared with the model group (p<0.05).

[0295] Table 5. Pelletier scoring criteria for joint injuries

[0296] Table 6. OARSI Histopathological Scoring Table 1 - Cartilage Degeneration Note: For each joint, three consecutive slices with a 200 μm interval reflecting the most severe condition were selected. The medial tibial plateau (MTP, which extends only from the medial side of the joint to the cruciate ligament; MTP corresponds to the MFC, i.e., the medial femoral condyle) of each joint slice was divided into three zones: zone 1, zone 2, and zone 3. Zone 1 is the outer 1 / 3 of the MTP, and zone 3 is the 1 / 3 of the MTP closest to the cruciate ligament. Using the standards in the table above, the degree of cartilage degeneration in each slice's three zones was evaluated on a scale of "none" to "severe" (score 0–5). The total cartilage degeneration score was calculated by summing the values ​​obtained from each zone.

[0297] Table 7. In vivo efficacy evaluation of each protein construct.

[0298] Example 6: In vivo efficacy evaluation of protein constructs and heat-stable protein constructs

[0299] Next, we continued to screen for protein constructs with superior chondrogenic activity and further evaluated the in vivo chondrogenic activity of constructs PR0029 and PR0032. Specifically, a rat meniscus tear model was constructed as described above. Six days later (Day 0), animals were randomly assigned to groups based on their bilateral leg disability. On Day 1, Day 8, and Day 15, 20 μg of each protein construct was injected intra-articularly. On Day 12 and Day 24, bilateral leg disability was assessed using a bipedal balance analgesia device. On Day 26, the experimental endpoint, a section of the affected knee joint was fixed and embedded, stained with Safranin-Fix-Green, and analyzed using the OARSI scoring criteria. The statistical and evaluation results are shown in Table 8, Figure 5, and Figure 6.

[0300] In the evaluation of bipedal disability, PR0027, PR0029, and PR0032 all showed a reduction in weight-bearing differences, indicating that each construct possessed the ability to improve weight-bearing in the rat's legs. Among them, the bipedal imbalance repair level of PR0029 was more similar to that of PR0005, and further pathological evaluation was conducted. Based on Pelletier and OARSI scores, compared with the model group, PR0029 showed cartilage repair capacity with significant differences (p<0.01), and the cartilage regeneration activity of PR0029 was superior to that of PR0005. P value indicates comparison with the model group.

[0301] Table 8. In vivo efficacy evaluation of PR0029

[0302] In addition, the thermostable mutants PR0029, PR0029M7 and M8, were also evaluated for in vivo cartilage repair activity using methods largely consistent with those described above. The results are shown in Table 9. Both mutants exhibited pain relief function similar to PR0029. The P-value represents the difference between the mutants and the model group.

[0303] Table 9. In vivo efficacy evaluation of the PR0029 mutant

[0304] Example 7: In vitro expression and activity evaluation of mRNA nucleic acid constructs

[0305] Prepare an mRNA nucleic acid construct encoding the amino acid sequence of PR0029 (SEQ ID NO:26), the construct comprising a CAP cap, 5'UTR, signal peptide (SP), 3'UTR and PolyA tail, the sequence shown in SEQ ID NO:43-46 and modified with pseudouracil (SEQ ID NO:48); or, construct an mRNA nucleic acid construct encoding the amino acid sequence of His-tagged PR0029 (SEQ ID NO:49), the construct comprising a CAP cap, 5'UTR, signal peptide (SP), 3'UTR and PolyA tail, the sequence shown in SEQ ID NO:43-46 and modified with pseudouracil (SEQ ID NO:51).

[0306] The specific process for obtaining mRNA through in vitro transcription is as follows: the plasmid template is digested by restriction endonuclease to linearize the DNA template, and then transcribed in vitro using T7 RNA polymerase. For in vitro transcription, T7 RNA polymerase (Roche), the corresponding reaction buffer, pyrophosphatase, RNase inhibitor, and NTP are used. To effectively cap RNA, an excess of the cap-like compound CleanCap (m7G(5')ppp(5')(2'OMeA)pG, Trilink, N-7113) is added to the reaction. Simultaneously, to reduce the immunogenicity of the mRNA and improve translation efficiency, all mRNAs in this disclosure have undergone nucleic acid modification, replacing all uridine triphosphate (UTP) in the reaction system with pseudouridine triphosphate (ψUTP, purchased from Thermo Fisher). This modification method is referenced in patent US2014 / 0194494 A1. After incubation at 37°C for 2.5 hours, the in vitro transcription system was used to purify RNA using carboxylated magnetic beads (Invitrogen) and resuspend it in nuclease-free water. RNA concentration and quality were assessed by spectrophotometry and analysis on an Agilent 5200 bioanalyzer. The target mRNA was obtained.

[0307] The specific process of mRNA encapsulation in liposomes is as follows: Ionizable lipids, DSPC (Avitar), cholesterol (Avitar), and DMG-PEG (Avitar) were dissolved in ethanol at a molar ratio of 48:10:40.5:1.5 to prepare a lipid solution. mRNA was dissolved in pH 5 acetate buffer to prepare an aqueous mRNA solution. The ethanol lipid solution and the mRNA aqueous solution were mixed using a microfluidic system, with a total lipid to mRNA weight ratio of approximately 20:1 to prepare lipid nanoparticles. Ethanol was removed by dialyzing in 20 mM Tris pH 7.5 solution, and finally, the mixture was transferred to 20 mM Tris pH 7.5, 8% sucrose solution for freezing to obtain mRNA-encapsulated liposome nanoparticles.

[0308] PR0029 was labeled with an mRNA-encoded HIS tag, encapsulated as liposomes, and co-incubated with HEK-293T cells. The expression level of the target protein in the supernatant was then detected. Specifically, the mRNA-encoded liposomes were transfected into 96-well plates at 100-400 ng / well. After 24 h, the supernatant was collected, and the expression level of PR0029 was detected using ELISA. The plates were coated with 2 μg / ml Monoclonal Mouse anti-Human ANGPTL3 Antibody (LSBio, LS-C755396), and incubated with cell supernatant for 90 min. 500 ng / ml HRP-conjugated 6*His His-Tag Monoclonal antibody (Proteintech, HRP-66005) was used as the detection antibody. After color development, the OD values ​​at 450 nm / 570 nm were read using a microplate reader. The results are shown in Figure 7. The protein expression level of PR0029 showed a dose-dependent increase with increasing transfection dose.

[0309] The transfection supernatant was co-incubated with chondrocytes, and the chondrogenic activity of the mRNA construct encoding PR0029 was evaluated by DKK1 secretion. In short, liposome nanoparticles were transfected into 293 cells, and the transfection system was replaced with medium containing 1% serum. After 24 hours, the supernatant was collected, mixed 1:1 with fresh medium containing 1% serum, and incubated with chondrocytes overnight. The cell supernatant was then used to detect the amount of DKK1 secreted using an ELISA kit. The results, shown in Figure 8, indicate that the amount of DKK1 secreted by chondrocytes after co-incubation with the transfection supernatant was positively correlated with the transfection amount.

[0310] Example 8: In vivo activity evaluation of mRNA nucleic acid constructs

[0311] The chondrogenic properties of the mRNA nucleic acid construct were evaluated using the rat meniscus tear model described in Example 6 above. In short, an osteoarthritis model was established in all rats. Seven days later, rats were randomly assigned to groups based on their bipedal incapacity. On days 0, 7, and 14 after grouping, PR0005 20 μg was administered intra-articularly. On days 0 and 14 after grouping, different doses (0.1, 0.4, and 1.6 μg, respectively) of the mRNA nucleic acid construct were administered intra-articularly. On days 31 and 38 after grouping, bipedal weight-bearing capacity was assessed using a bipedal balance analgesia device to evaluate the animals' bipedal incapacity when standing. The results (Figure 9) showed that, compared to the model group, the mRNA nucleic acid construct significantly improved the bipedal weight-bearing capacity of the model rats (Table 10). Furthermore, the recovery level of bipedal weight-bearing capacity was comparable to that of PR0005 (three administrations) with fewer administrations (two administrations). Similarly, after fixing the affected knee joint in the same manner as above, safranin and fixed green staining was performed, and the OARSI system score was used to evaluate the cartilage repair activity of each construct. The results showed (Figure 10) that the mRNA nucleic acid construct showed a cartilage regeneration trend that was comparable to or better than PR0005, and the cartilage repair was basically consistent with the behavior.

[0312] Table 10. In vivo efficacy evaluation of PR0029 mRNA nucleic acid construct

[0313] The sequence of this disclosure is shown below:

[0314] >Signal peptide amino acid sequence

[0315] DNA sequence of a nucleic acid construct containing mRNA encoding the PR0029 protein with the T7 promoter.

[0316] >PR0029 mRNA nucleic acid construct sequence

[0317] >HIS-tagged amino acid sequence of PR0029

[0318] DNA sequence of a nucleic acid construct containing the mRNA encoding the HIS-tagged PR0029 protein with the T7 promoter.

[0319] >The nucleic acid construct sequence of the mRNA encoding the HIS marker PR0029

[0320] >Connecting subsequences

[0321] >Connecting subsequences

[0322] >Connecting subsequences

[0323] >Connecting subsequences

[0324] >Connecting subsequences

[0325] >Connecting subsequences

[0326] >Connecting subsequences

[0327] >Connecting subsequences

[0328] >Connecting subsequences

[0329] >Connecting subsequences

[0330] >Connecting subsequences

[0331] DNA sequence encoding PR0008

[0332] DNA sequence encoding PR0009

[0333] DNA sequence encoding PR0010

[0334] DNA sequence encoding PR0011

[0335] DNA sequence encoding PR0012

[0336] DNA sequence encoding PR0013

[0337] DNA sequence encoding PR0014

[0338] DNA sequence encoding PR0015

[0339] DNA sequence encoding PR0016

[0340] DNA sequence encoding PR0017

[0341] DNA sequence encoding PR0018

[0342] DNA sequence encoding PR0019

[0343] DNA sequence encoding PR0020

[0344] DNA sequence encoding PR0021

[0345] DNA sequence encoding PR0022

[0346] DNA sequence encoding PR0023

[0347] DNA sequence encoding PR0024

[0348] DNA sequence encoding PR0025

[0349] DNA sequence encoding PR0026

[0350] DNA sequence encoding PR0027

[0351] DNA sequence encoding PR0028

[0352] DNA sequence encoding PR0029

[0353] DNA sequence encoding PR0030

[0354] DNA sequence encoding PR0031

[0355] DNA sequence encoding PR0032

[0356] DNA sequence encoding PR0033

[0357] DNA sequence encoding PR0034

[0358] DNA sequence encoding PR0035

[0359] DNA sequence encoding PR0005

[0360] DNA sequence encoding PR0006

[0361] DNA sequence encoding PR0029M1

[0362] DNA sequence encoding PR0029M2

[0363] DNA sequence encoding PR0029M3

[0364] DNA sequence encoding PR0029M4

[0365] DNA sequence encoding PR0029M5

[0366] DNA sequence encoding PR0029M6

[0367] DNA sequence encoding PR0029M7

[0368] DNA sequence encoding PR0029M8

[0369] RNA sequence encoding PR0008

[0370] RNA sequence encoding PR0009

[0371] RNA sequence encoding PR0010

[0372] RNA sequence encoding PR0011

[0373] RNA sequence encoding PR0012

[0374] RNA sequence encoding PR0013

[0375] RNA sequence encoding PR0014

[0376] RNA sequence encoding PR0015

[0377] RNA sequence encoding PR0016

[0378] RNA sequence encoding PR0017

[0379] RNA sequence encoding PR0018

[0380] RNA sequence encoding PR0019

[0381] RNA sequence encoding PR0020

[0382] RNA sequence encoding PR0021

[0383] RNA sequence encoding PR0022

[0384] RNA sequence encoding PR0023

[0385] RNA sequence encoding PR0024

[0386] RNA sequence encoding PR0025

[0387] RNA sequence encoding PR0026

[0388] RNA sequence encoding PR0027

[0389] RNA sequence encoding PR0028

[0390] RNA sequence encoding PR0029

[0391] RNA sequence encoding PR0030

[0392] RNA sequence encoding PR0031

[0393] RNA sequence encoding PR0032

[0394] RNA sequence encoding PR0033

[0395] RNA sequence encoding PR0034

[0396] RNA sequence encoding PR0035

[0397] RNA sequence encoding PR0005

[0398] RNA sequence encoding PR0006

[0399] RNA sequence encoding PR0029M1

[0400] RNA sequence encoding PR0029M2

[0401] RNA sequence encoding PR0029M3

[0402] RNA sequence encoding PR0029M4

[0403] RNA sequence encoding PR0029M5

[0404] RNA sequence encoding PR0029M6

[0405] RNA sequence encoding PR0029M7

[0406] RNA sequence encoding PR0029M8

[0407] ANGPTL1 full-length DNA sequence

[0408] ANGPTL2 full-length DNA sequence

[0409] ANGPTL3 full-length DNA sequence

[0410] ANGPTL4 full-length DNA sequence

Claims

1. A polypeptide comprising at least one polypeptide fragment derived from ANGPTL3, and (1) at least one polypeptide fragment derived from ANGPTL1 ; (2) at least one polypeptide fragment derived from ANGPTL2; and / or (3) at least one polypeptide fragment derived from ANGPTL4; Preferably, the polypeptide comprises 2-10 polypeptide fragments; More preferably, the ANGPTL1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, the ANGPTL2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, the ANGPTL3 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3, and the ANGPTL4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

4.

2. The polypeptide of claim 1, wherein the polypeptide fragment derived from ANGPTL3 is an ANGPTL3 fibrinogen-like domain or a functional fragment thereof; Preferably, the ANGPTL3 fibrinogen-like domain comprises amino acids 237-455 of the amino acid sequence set forth in SEQ ID NO: 3, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

3. The polypeptide of claim 1 or 2, wherein the ANGPTL3 fibrinogen-like domain comprises the following fragment, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto: (1) amino acids 269-453 of the ANGPTL3 amino acid sequence set forth in SEQ ID NO: 3; or (2) amino acids 259-415 of the ANGPTL3 amino acid sequence set forth in SEQ ID NO:

3.

4. The polypeptide of any one of claims 1-3, further comprising a mutation that increases its thermostability, or protease degradation stability; Preferably, the mutation comprises a point mutation, an insertion, a deletion, a truncation, a fusion, or any combination thereof.

5. The polypeptide of any one of claims 1-4, further comprising an amino acid mutation at any one or more positions selected from the group consisting of: position 259, position 280, position 289, position 304, position 330, position 350, position 384, position 423, position 424, and position 432, wherein the amino acid substitution position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 3; Preferably, the ANGPTL3 polypeptide fragment comprises an amino acid at one or more of the following positions: position 259 is I, position 280 is T, position 289 is T, position 304 is H, position 330 is I, position 350 is H, position 384 is G, T, S, Q, N, H, P, or V, position 423 is Q, position 424 is P, position 432 is I; More preferably, the ANGPTL3 polypeptide fragment comprises a K423Q mutation.

6. The polypeptide of any one of claims 1-5, wherein the polypeptide fragments are directly linked or linked by a linker; Preferably, the linker is selected from GGGS (SEQ ID NO: 52), GGGGS (SEQ ID NO: 53), GGGGG (SEQ ID NO: 54), (EAAAK)3(SEQ ID NO: 55), (EAAAR)3(SEQ ID NO: 56), (EGGGK)3(SEQ ID NO: 57), (EGGGR)3(SEQ ID NO: 58), (DAAAR)3(SEQ ID NO: 59), (DAAAK)3(SEQ ID NO: 60), (DGGGR)3(SEQ ID NO: 61), or (DGGGK)3(SEQ ID NO: 62).

7. A polypeptide, characterized in that, The amino acid sequence of the polypeptide comprises or is represented by any one of SEQ ID NOs: 5-32, and 34-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

8. An RNA molecule comprising an open reading frame (ORF) encoding a protein of interest; wherein, The target protein is A polypeptide comprising at least one polypeptide fragment derived from ANGPTL3, and (1) at least one polypeptide fragment derived from ANGPTL1 ; (2) at least one polypeptide fragment derived from ANGPTL2; and / or (3) at least one polypeptide fragment derived from ANGPTL4; Preferably, the polypeptide comprises 2-10 polypeptide fragments; More preferably, the ANGPTL1 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 1, the ANGPTL2 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 2, the ANGPTL3 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 3, and the ANGPTL4 polypeptide comprises the amino acid sequence represented by SEQ ID NO:

4.

9. The RNA molecule of claim 8, wherein the polypeptide fragment derived from ANGPTL3 is an ANGPTL3 fibrinogen-like domain or a functional fragment thereof; Preferably, the ANGPTL3 fibrinogen-like domain comprises amino acids 237-455 of the amino acid sequence represented by SEQ ID NO: 3, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

10. The RNA molecule of claim 8 or 9, wherein the ANGPTL3 fibrinogen-like domain comprises the following fragment, or a polypeptide fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto: (1) amino acids 269-453 of the ANGPTL3 amino acid sequence represented by SEQ ID NO: 3; or (2) amino acids 259-415 of the ANGPTL3 amino acid sequence represented by SEQ ID NO:

3.

11. The RNA molecule according to any one of claims 8-10, wherein the target protein further contains a mutation that improves its thermal stability or protease degradation stability; Preferably, the mutation includes point mutations, insertions, deletions, truncations, fusions, or any combination thereof.

12. The RNA molecule according to any one of claims 8-11, wherein the target protein contains an amino acid mutation selected from one or more of the following positions: position 259, position 280, position 289, position 304, position 330, position 350, position 384, position 423, position 424, and position 432, wherein the amino acid substitution site is a natural sequence site relative to the amino acid sequence shown in SEQ ID NO: 3; Preferably, the ANGPTL3 polypeptide fragment contains amino acids corresponding to one or more of the following sites: The 259th bit is I, the 280th bit is T, the 289th bit is T, the 304th bit is H, the 330th bit is I, the 350th bit is H, the 384th bit is G, T, S, Q, N, H, P or V, the 423rd bit is Q, the 424th bit is P, and the 432nd bit is I; More preferably, the ANGPTL3 polypeptide fragment contains a K423Q mutation.

13. The RNA molecule according to any one of claims 8-12, wherein the polypeptide fragments are directly linked or linked by linkers; Preferably, the connector is selected from GGGS (SEQ ID NO:52), GGGGS (SEQ ID NO:53), GGGGG (SEQ ID NO:54), (EAAAK)3 (SEQ ID NO:55), (EAAAR)3 (SEQ ID NO:56), (EGGGK)3 (SEQ ID NO:57), (EGGGR)3 (SEQ ID NO:58), (DAAAR)3 (SEQ ID NO:59), (DAAAK)3 (SEQ ID NO:60), (DGGGR)3 (SEQ ID NO:61), or (DGGGK)3 (SEQ ID NO:62).

14. An RNA molecule comprising an open reading frame (ORF) encoding a protein of interest; wherein, The target protein is A polypeptide, characterized in that the amino acid sequence of the polypeptide is any one of SEQ ID NOs: 5-32 and 34-42, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

15. An RNA molecule comprising or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the nucleotide sequences shown in any of the SEQ ID NOs:48, 51, and 101-138.

16. The RNA molecule according to any one of claims 8-15, further comprising untranslated region elements (UTRs); preferably, the untranslated region elements (UTRs) comprise 5' untranslated region elements (5'UTRs) and 3' untranslated region elements (3'UTRs); Preferably, the 5'UTR contains the sequence shown in or having at least 90% identity with SEQ ID NO:44, and the 3'UTR contains the sequence shown in or having at least 90% identity with SEQ ID NO:

45.

17. The RNA molecule according to any one of claims 8-16, further comprising a poly-A tail; Preferably, the poly-A tail is selected from 120A, 125A, Poly A-3070, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA, or SV40 late polyA.

18. The RNA molecule according to any one of claims 8-17, further comprising a 5' cap structure; Preferably, the 5'Cap is selected from Cap0, Cap1, Cap2, Cap3, Cap4, ARCA, modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. More preferably, the 5'Cap is selected from ARCA, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5' )(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

19. The RNA molecule according to any one of claims 8-18, comprising at least one chemical modification; preferably, the chemical modification is uracil modification.

20. A polynucleotide encoding a polypeptide as described in any one of claims 1-7.

21. A vector comprising the polynucleotide as described in claim 20.

22. A host cell comprising the polynucleotide of claim 20 or the vector of claim 21.

23. Lipid nanoparticles comprising an RNA molecule as described in any one of claims 8-19, a polynucleotide as described in claim 20, or a carrier as described in claim 21.

24. A pharmaceutical composition comprising any one or any combination of the following: The polypeptide as described in any one of claims 1-7, the RNA molecule as described in any one of claims 8-19, the polynucleotide as described in claim 20, the carrier as described in claim 21, or the lipid nanoparticle as described in claim 23; Optionally, it contains pharmaceutically acceptable excipients, diluents, or excipients.

25. A method for preparing ANGPTL peptides, including: The ANGPTL polypeptide is generated by expressing the RNA molecule as described in any one of claims 8-19, the polynucleotide as described in claim 20, or the vector as described in claim 21 in a host cell.

26. Methods for preparing RNA molecules include: RNA molecules are obtained by transcription using the polynucleotide of claim 20 or the vector of claim 21 as templates. Preferably, the method further includes adding a 5'Cap to the 5' end of the RNA molecule.

27. The use of the polypeptide of any one of claims 1-7, the RNA molecule of any one of claims 8-19, the polynucleotide of claim 20, the carrier of claim 21, or the lipid nanoparticle of claim 23, or the pharmaceutical composition of claim 24 in (1) the preparation of a medicament for inducing cartilage formation, or (2) the preparation of a medicament for the prevention, relief or treatment of joint diseases and / or cartilage-related conditions; Preferably, the joint disease includes osteoarthritis, joint injury, and other cartilage degeneration-related diseases; more preferably, the osteoarthritis is knee osteoarthritis, hand osteoarthritis, hip osteoarthritis, or spinal osteoarthritis.

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