Compositions and methods for producing active proteins from inactive proproteins

Abstract

The present disclosure relates to the nucleic acids encoding a proprotein and at least one enzyme for processing the proprotein into a mature protein. In particular, the present disclosure relates to nucleic acids encoding an inactive proprotein and at least one enzyme for cleavage of the proprotein to produce an active protein.

Description

Docket No.: 69944-20020.40 COMPOSITIONS AND METHODS FOR PRODUCING ACTIVE PROTEINS FROM INACTIVE PROPROTEINS CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims priority benefit of U.S. Provisional Patent Application No. 63 / 637,805 filed on April 23, 2024, which is hereby incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (699442002040SEQLIST.xml; Size: 22,088 bytes; and Date of Creation: April 21, 2025) is herein incorporated by reference in its entirety. FIELD

[0003] The present disclosure relates to the nucleic acids encoding a proprotein and at least one enzyme for processing the proprotein into a mature protein. In particular, the present disclosure relates to nucleic acids encoding an inactive proprotein and at least one enzyme for cleavage of the proprotein to produce an active protein. BACKGROUND

[0004] Many proteins are produced in cells in inactive forms (proproteins) that must be processed by enzymes such as proprotein convertases into active forms (mature proteins). For example, insulin is initially produced in cells as preproinsulin, and is subsequently enzymatically processed into an active, mature form of insulin. However, not all cell types express the appropriate enzymes for production of an active protein from an exogenous nucleic acid encoding an inactive proprotein. This restrict the types of cells that can be used to produce a protein requiring post-translational processing.

[0005] As such, there is a need in the art for tools for expressing an exogenous proprotein of interest and properly processing the proprotein into an active protein in cells where the active protein is not typically produced. 1sf-6697944Docket No.: 69944-20020.40 BRIEF SUMMARY

[0006] The present disclosure relates to the nucleic acids encoding a proprotein and at least one enzyme for processing the proprotein into a mature protein. In particular, the present disclosure relates to nucleic acids encoding an inactive proprotein and at least one enzyme for cleavage of the proprotein to produce an active protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A-1F show schematics of nucleic acids suitable for expression of proteins requiring post-translational processing. FIG. 1A shows an exemplary nucleic acid of the present disclosure, which encodes (from ATG to STOP codon) a proprotein, a self-cleaving 2A (2A) peptide, and an enzyme that processes the proprotein into an active protein. FIG. 1B shows an exemplary nucleic acid, which comprises a coding region (from ATG to STOP codon) of a proprotein, an internal ribosome entry site (IRES), and a coding region (from ATG to STOP codon) of an enzyme that processes the proprotein into an active protein. FIG. 1C shows an exemplary nucleic acid, which encodes (from ATG to STOP codon) a first enzyme, a first 2A peptide, a second enzyme, a second 2A peptide, and a proprotein. FIG. 1D shows an exemplary nucleic acid, which comprises a coding region of (from ATG to STOP codon) a proprotein, an IRES, a coding region (from ATG to STOP codon) of a first enzyme, a 2A peptide, and a second enzyme. FIG. 1E shows an exemplary nucleic acid encoding (from ATG to STOP codon) a first enzyme, a first 2A peptide, a second enzyme, a second 2A peptide, a third enzyme, a third 2A peptide, and a proprotein. FIG. 1F shows an exemplary nucleic acid, which comprises a coding region (from ATG to STOP codon) of a proprotein, an IRES, and a coding region (from ATG to STOP codon) of a first enzyme, a first 2A peptide, a second enzyme, a second 2A peptide, and a third enzyme.

[0008] FIG. 2A shows a schematic diagram of the processing that occurs in pancreatic beta cells to produce insulin from preproinsulin. Signal peptidases are present in all cells, and thus, an exogenous signal peptidase does not have to be provided. In contrast, the enzymes carboxypeptidase (CPE), proprotein convertase 1 / 3 (PCSK1), and proprotein convertase 2 (PCSK2) are present only in specialized cells, such as pancreatic beta cells. FIG. 2B shows a schematic diagram of a nucleic acid encoding (from ATG to STOP codon) carboxypeptidase (CPE), a first 2A peptide, proprotein convertase 1 / 3 (PCSK1), a second 2A peptide, proprotein 2sf-6697944Docket No.: 69944-20020.40 convertase 2 (PCSK2), a third 2A peptide, secretogranin V (SCG5), a fourth 2A peptide, and preproinsulin. The amino acid sequence of this polyprotein is set forth as SEQ ID NO:1. Introduction of the coding regions of CPE, PCSK1, PCSK2, SCG5 and preproinsulin into virtually any type of cell enables that cell to produce insulin in an active form (e.g., mature insulin).

[0009] FIG. 3A shows a schematic diagram of the processing that occurs in intestine cells and brain cells to produce Glicentin, GRPP, Oxyntomodulin, GLP-1, GLP-2, and IP-2 from proglucagon. Proglucagon is processed in this manner only in the specialized cell types, where PCSK1 and CPE are expressed. FIG. 3B shows a schematic diagram of a nucleic acid, which comprises a coding region (from ATG to STOP codon) of carboxypeptidase (CPE), a first 2A peptide, proprotein convertase 1 / 3 (PCSK1), a second 2A peptide, and proglucagon. The amino acid sequence of this polyprotein is set forth as SEQ ID NO:2. FIG. 3C shows schematic diagram of the processing that occurs in pancreatic alpha cells to produce MPGF, GRPP, Glucagon, and IP-1 from proglucagon. Proglucagon is processed in this manner only in specialized cell types where PCSK2 and CPE are expressed. FIG. 3D shows a schematic diagram of a nucleic acid, which comprises a coding region (from ATG to STOP codon) of carboxypeptidase (CPE), a first 2A peptide, proprotein convertase 2 (PCSK2), a second 2A peptide, secretogranin V (SCG5), a third 2A peptide, and proglucagon. The amino acid sequence of this polyprotein is set forth as SEQ ID NO:3.

[0010] FIG. 4A shows a schematic diagram of the processing that occurs in anterior pituitary cells to produce Pro-ACTH, -LPH, N-POMC, and ACTH from proopiomelanocortin (POMC). POMC is processed in this manner only in the specialized cell types, where PCSK1 and CPE are expressed. FIG. 4B shows a schematic diagram of a nucleic acid, which comprises a coding region (from ATG to STOP codon) of carboxypeptidase (CPE), a first 2A peptide, proprotein convertase 1 / 3 (PCSK1), a second 2A peptide, and POMC. The amino acid sequence of this polyprotein is set forth as SEQ ID NO:4. FIG. 4C shows schematic diagram of the processing that occurs in cells of hypothalamus and the intermediate lobe of the pituitary to produce -MSH, CLIP, -LPH, -MSH, -EP, and 3-MSH from POMC. POMC is processed in this manner only in specialized cell types where PCSK1, PCSK2 and CPE are expressed. FIG. 4D shows a schematic diagram of a nucleic acid, which comprises a coding region (from 3sf-6697944Docket No.: 69944-20020.40 ATG to STOP codon) of carboxypeptidase (CPE), a first 2A peptide, proprotein convertase 1 / 3 (PCSK1), a second 2A peptide, proprotein convertase 2 (PCSK2), a third 2A peptide, secretogranin V (SCG5), a fourth 2A peptide, and POMC. The amino acid sequence of this polyprotein is set forth as SEQ ID NO:5.

[0011] FIG. 5A shows a schematic diagram of the processing that occurs in specific cells, such as neurons, to produce brain derived neurotrophic factor (BDNF) from proBDNF. FIG. 5B shows a schematic diagram of a nucleic acid, which comprises a coding region (from ATG to STOP codon) of carboxypeptidase (CPE), a first 2A peptide, proprotein convertase 1 / 3 (PCSK1), a second 2A peptide, and proBDNF. The amino acid sequence of this polyprotein is set forth as SEQ ID NO:6.

[0012] FIG. 6A-C show an alternative design for expression of three proteins. Instead of including coding regions of multiple proteins in a single synthetic mRNA (synRNA), coding regions of each protein can be included in three separate synRNA molecules, all of which typically have a 5’-Cap, 5’-UTR, 3’-UTR, and poly(A) sequence. FIG. 6A shows a schematic diagram of a nucleic acid, which comprises a coding region (from ATG to STOP codon) of proglucagon (NCBI ID: NM_002054.5; NP_002045.1). FIG. 6B shows a schematic diagram of a nucleic acid, which comprises a coding region (from ATG to STOP codon) of proprotein convertase 1 / 3 (PCSK1) (NCBI ID: NM_000439.5; NP_000430.3). FIG. 6C shows a schematic diagram of a nucleic acid, which comprises a coding region (from ATG to STOP codon) of carboxypeptidase (CPE) (NCBI ID: NM_001873.4; NP_001864.1).

[0013] FIG. 7 shows that intradermal injection of RNAs can reduce the weight of diet- induced obese mice. Male C57BL / 6 DIO mice received the following injections into their skin: no RNA control, c-srRNA-CPE-PCSK1-GCG, c-srRNA-FGF21, and a mixture of synRNA- CPE, synRNA-PCSK1, and synRNA-GCG. No RNA control and a mixture of synRNA-CPE, synRNA-PCSK1, and synRNA-GCG were injected on day 0, 1, 2, 7, 8, 9, 10, 11, 14, and 15. c- srRNA-CPE-PCSK1-GCG and c-srRNA-FGF21 were injected on day 0. The body weight was measured every day for 25 days. Average % weight gain / loss of each group (n=4) is shown. 4sf-6697944Docket No.: 69944-20020.40 DETAILED DESCRIPTION

[0014] The present disclosure relates to the artificial production of both proprotein and proprotein convertase in cells. Any vector, such as synthetic mRNA (synRNA) and viral vectors, can be delivered to any cells where a synthesized proprotein is cleaved by proprotein convertase(s) into functional proteins or peptides in these cells. These cells are not necessarily specialized cell types that naturally cleave a proprotein into peptides and secrete them to a circulation to deliver to target organs / tissues / cells. By expressing both proprotein and proprotein convertase(s) in the same cell, the proper cleavage of proprotein can occur in any cells.

[0015] A variety of delivery methods can be used. For example, viral vectors such as adeno-associated virus, adenovirus, herpes simplex virus, retrovirus, lentivirus, and Sendai virus can be used. Non-viral vectors such as DNA vector and synthetic mRNA can be used. In these transcription or translation units, proprotein and proprotein convertase can be linked by the standard methods such as self-cleavage peptides, IRES, and a fusion protein. These transcription or translation units, such as synthetic mRNA, can be delivered to any cells where a synthesized proprotein is cleaved by proprotein convertase(s) into functional proteins or peptides in these cells. These cells are not necessarily specialized cell types that naturally cleave a proprotein into peptides and secrete them to a circulation to deliver to target organs / tissues / cells. By expressing both proprotein and proprotein convertase(s) in the same cell, the proper cleavage of proprotein can occur in any cells. General Techniques and Definitions

[0016] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0017] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients.

[0018] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed, indicating that such embodiments do not include additional elements (except for trace 5sf-6697944Docket No.: 69944-20020.40 impurities). The phrase “consisting essentially of” is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.

[0019] The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value (e.g., about 500 nucleotides in length when used in reference to an RNA molecule refers to an RNA molecule that is from 450 to 550 nucleotides in length).

[0020] As used herein, the term “synthetic mRNA”, abbreviated as “synRNA” refers to a mRNA molecule comprising at least a 5’-UTR (5’-untranslated region), a CDS (coding sequence), and a 3’-UTR (3’-untranslated region), in which the CDS is heterologous to at least the 3’-UTR. As such, synRNAs are not naturally-occurring molecules.

[0021] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a certain length unless otherwise specified. Polypeptides may include natural amino acid residues or a combination of natural and non-natural amino acid residues. The terms also include post-translational modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity (e.g., antigenicity).

[0022] As used herein in reference to a protein of interest, the terms “coding sequence,” “CDS”, “open reading frame” and “ORF” refer to the nucleotide sequences that encode the protein of interest. Due to the degeneracy of the genetic code, multiple distinct nucleotide sequences can encode the same amino acid sequence.

[0023] The terms “isolated” and “purified” as used herein refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). The term “isolated,” when used in reference to a recombinant protein, refers to a protein that has been removed from the culture medium of the host cell that produced the protein. In some embodiments, an isolated protein is at least 75%, 90%, 95%, 96%, 97%, 98% or 99% pure as determined by HPLC. 6sf-6697944Docket No.: 69944-20020.40

[0024] An “effective amount” or a “sufficient amount” of a substance is that amount sufficient to affect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied.

[0025] In the present disclosure, the terms “individual” and “subject” refer to mammals. “Mammals” include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats) and pets (e.g., dogs and cats). In some preferred embodiments, the subject is a human subject.

[0026] The term “dose” as used herein in reference to a composition comprising a mRNA encoding a protein of interest refers to a measured portion of the mRNA taken by (administered to or received by) a subject at any one time.

[0027] The relative terms “higher” and “lower” refer to a measurable increase or decrease, respectively, in a response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition. For instance, the phrases “higher level of protein expression” and “stronger protein expression” refer to a level of protein expression as a consequence of contacting a cell with a composition of the present disclosure comprising a mRNA encoding the protein that is greater than 1, preferably greater than 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold above a level of protein expression as a consequence of a control condition (e.g., administration of a comparator composition that either does not comprise the mRNA or comprises a control mRNA that does not encode the protein). The phrases “lower level of protein expression” and “weaker protein expression” refer to a level of protein expression as a consequence of a control condition (e.g., administration of a comparator composition that either does not comprise the mRNA or comprises a control mRNA that does not encode the protein) that is less than 1, preferably less than 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold below a level of protein expression as a consequence of administration of a composition of the present disclosure comprising an mRNA encoding the protein.

[0028] As used herein, “percent (%) amino acid sequence identity” and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antigen) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, 7sf-6697944Docket No.: 69944-20020.40 to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0029] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Amino acid substitutions may be introduced into an antigen of interest and the products screened for a desired activity, e.g., increased stability and / or immunogenicity.

[0030] Amino acids generally can be grouped according to the following common side- chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0031] Conservative amino acid substitutions will involve exchanging a member of one of these classes with another member of the same class. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes with a member of another class.

[0032] As used herein, the term “excipient” refers to a compound present in a composition comprising an active ingredient (e.g., mRNA encoding a protein of interest). Pharmaceutically acceptable excipients are inert pharmaceutical compounds, and may include for instance, solvents, bulking agents, buffering agents, tonicity adjusting agents, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments the compositions of the present disclosure comprise an excipient that functions as one or more of a 8sf-6697944Docket No.: 69944-20020.40 solvent, a bulking agent, a buffering agent, and a tonicity adjusting agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent). I. POLYPROTEIN ENGINEERING

[0033] Protein, peptide, and their analog have been delivered to human and domestic animals as therapeutic products. In addition to direct delivery of these protein, peptide, and their analog, they can be encoded in nucleic acids and delivered in the form of viral vectors such as adeno-associated virus, adenovirus, herpes simplex virus, retrovirus, lentivirus, and Sendai virus as well as non-viral vectors such as DNA vectors and synthetic mRNAs (synRNAs), including a typical linear mRNAs, self-replicating or self-amplifying RNA, and circular RNA.

[0034] Many proteins are produced in cells in inactive forms (called proprotein) that require to be processed by proprotein convertase into active peptides or proteins. Often, a proprotein is cleaved into multiple peptides that coordinately regulate metabolism and biological function. The same proprotein may be cleaved into different sets of peptides depending on cell types. Therefore, the proprotein-expressing vectors must be delivered only to specific and suitable cell types. However, it is difficult or impossible to target proteins and peptides, viral vectors, and non-viral vector to specific cell types in vivo.

[0035] To address this problem, tools and methods were developed for effectively expressing an exogenous proprotein of interest and properly cleaving them into functional proteins and / or peptides in cells where the proprotein is not necessarily expressed and processed in nature.

[0036] A key idea is to deliver a proprotein and enzymes that cleaves the proprotein in the same cell. By selecting one or a combination of enzymes that function in specific cell types in nature, this method allows any cells to convert a delivered proprotein to a set of peptides that are naturally produced in these specific cell types.

[0037] Enzymes that can be employed here are selected from any enzymes that are known to be involved in the proprotein processing (Thomas 2002, Seidah and Prat 2012). Proprotein convertases are such examples and are listed in Table I. For example, PCSK1 is involved in the processing of hormone and other proproteins. PCSK1 substrates include proglucagon, proopiomelanocortin (POMC), renin, enkephalin, dynorphin, somatostatin, insulin 9sf-6697944Docket No.: 69944-20020.40 and agouti related neuropeptide (AGRP). PCSK1 gene mutations are associated with susceptibility to obesity. PCSK2 is involved in the processing of hormone and other proproteins. PCSK2 is involved in the release of glucagon from proglucagon in pancreatic alpha cells. PCSK2 gene mutations increase susceptibility to myocardial infarction and type 2 diabetes. PCSK2 requires secretogranin V (SCG5), a molecular chaperone, for its function (Zhu, Muller et al. 1998). Table I. Proprotein Convertases10sf-6697944Docket No.: 69944-20020.40

[0038] Carboxypeptidase E (CPE, also known as CPH; BDVS; IDDHH) also plays an important role in proprotein processing (McGirr, Guizzetti and Dhanvantari 2013, Ji, Wu et al. 2017, Liu, Jiang et al. 2023). This protein cleaves C-terminal amino acid residues and is involved in the biosynthesis of peptide hormones and neurotransmitters, including insulin. Mutations in this gene are implicated in type 2 diabetes. Other enzymes such as peptidyl -amidating monooxygenase (PAM), N-acetyltransferase, prolylcarboxypeptidase (PRCP) are also indicated for processing of proopiomelanocortin (POMC).

[0039] Any methods of placing more than two proteins in single transcription or translation unit can be used here. For example, self-cleaving 2A peptides are commonly used to connect two proteins, which produces two separate proteins (Kim, Lee et al. 2011, Liu, Chen et al. 2017). Different 2A peptides are known: P2A, T2A, E2A, and F2A (Kim, Lee et al. 2011, Liu, Chen et al. 2017). Any 2A peptides can be used here. Alternatively, two CDS (ATG-STOP codon) can be connected via internal ribosomal entry site (IRES) (Mokrejs, Vopalensky et al. 2006). Different IRES sequences are known and any IRES sequence can be used here (Mokrejs, Vopalensky et al. 2006). Typical designs of the transcription or translation unit are shown in FIG. 1A-1F. Although the Figures show the design containing only up to three enzymes, more enzymes can be added. Also, any additional protein that is required for the function of enzymes can be included, as shown below. For example, SCG5 can be included when PCSK2 is used. Also, two or more than two proprotein are included in the design, as shown below. Also, order or locations of each protein can be changed. Also, to enhance the production of one protein, the same protein can be included in duplicates, triplicates, and so on. II. PRE-PRO-INSULIN

[0040] Insulin is initially produced as preproinsulin, which is first processed to proinsulin by the removal of a N-terminal signal peptide by a signal peptidase (FIG. 2A). Proinsulin is subsequently cleaved by PCSK1 and PCSK2 (Weiss, Steiner and Philipson 2000, Infante 2022). Compared to PCSK1, PCSK2 plays a minor role and requires SCG5 for its function. Then, CPE removes C-terminal basic amino acids from the peptide chains, leading to the formation of mature insulin. All these processes occur only in pancreatic beta cells, where preproinsulin, signal peptidase, PCSK1, PCSK2, SCG5, and CPE are present. 11sf-6697944Docket No.: 69944-20020.40

[0041] These components that are required to produce mature insulin are introduced into cells. FIG. 2B shows a schematic diagram of an artificial protein (ATG-Stop codon), which can be encoded in any viral and non-viral delivery system, including a synthetic mRNA. A signal peptidase is ubiquitously present, and thus, is not included in this design. However, a signal peptidase can also be included. The diagram shows the design that uses self-cleaving 2A peptides, but IRES can also be used. Preproinsulin, three enzymes (CPE, PCSK1, and PCSK2), and a chaperone (SCG5) are included (FIG. 2B). Alternatively, considering the minor role of PCSK2, it is possible to include only CPE, PCSK1, and preproinsulin. Order or locations of each protein can be changed; however, preproinsulin is preferably located at the C-terminus of the polyprotein, as self-cleaving 2A peptides, after cleavage, leave residual residues at the C- terminus, which is undesirable for insulin. III. PRO-GLUCAGON

[0042] Proglucagon is processed into different sets of peptides depending on cell types where proglucagon is expressed, for example, in pancreatic alpha cells, intestinal cells, and brain neuronal cells (Muller, Finan et al. 2019). When proglucagon is produced in intestine and brain, where PCSK1 is typically expressed, proglucagon is processed into GLP-1, GLP-2, IP-2, Glicentin, GRPP, and Oxyntomodulin (FIG. 3A). This process also requires CPE (McGirr, Guizzetti and Dhanvantari 2013). On the other hand, when proglucagon is produced in pancreatic alpha cells, where PCSK2 is typically expressed, proglucagon is processed into Glucagon, GRPP, IP-1, and MPGF (FIG. 3D). This process also requires CPE and SCG5, a chaperone for PCSK2 (Zhu, Muller et al. 1998, McGirr, Guizzetti and Dhanvantari 2013).

[0043] To recapitulate the processing of intestine and brain proglucagon in cells, proglucagon, PCSK1, and CPE are introduced into cells. FIG. 3B shows a schematic diagram of an artificial protein (ATG-Stop codon), which can be encoded in any viral and non-viral delivery system, including a synthetic mRNA. The diagram shows the design that uses self-cleaving 2A peptides, but IRES can also be used. Order or locations of each protein can be changed; however, proglucagon is preferably located at the C-terminus of the polyprotein, as self-cleaving 2A peptides, after cleavage, leave residual residues at the C-terminus, which is undesirable.

[0044] To recapitulate processing of pancreatic alpha cell proglucagon in cells, proglucagon, PCSK2, SCG5, and CPE are introduced into cells. FIG. 3D shows a schematic 12sf-6697944Docket No.: 69944-20020.40 diagram of an artificial protein (ATG-Stop codon), which can be encoded in any viral and non- viral delivery system, including a synthetic mRNA. The diagram shows the design that uses self- cleaving 2A peptides, but IRES can also be used. Order or locations of each protein can be changed; however, proglucagon is preferably located at the C-terminus, as self-cleaving 2A peptides, after cleavage, leave residual residues at the C-terminus, which is undesirable. IV. PRO-OPIOMELANOCORTIN (POMC)

[0045] POMC is processed into different sets of peptides depending on cell types where POMC is expressed (Wardlaw 2011, Harno, Gali Ramamoorthy et al. 2018). In the anterior pituitary, where PCSK1 is present, POMC is initially cleaved by PCSK1 to yield pro-adrenocorticotropic hormone (pro-ACTH) and -lipotropin ( -LPH). Pro-ACTH is then cleavedby PCSK1 to ACTH and N-terminal POMC (FIG. 4A). On the other hand, in the hypothalamus and in the intermediate lobe of the pituitary, where both PCSK1 and PCSK2 are present, POMC is more extensively processed to produce -MSH, corticotropin-like-intermediate lobe peptide(CLIP), -LPH, -MSH, -endorphin ( -EP), 3-MSH (FIG. 4C). It is also known that CPE isrequired to properly process the POMC (Fricker, Tashima et al. 2021). Other enzymes such as peptidyl -amidating monooxygenase (PAM), N-acetyltransferase, prolylcarboxypeptidase (PRCP) are also involved in processing of proopiomelanocortin (POMC).

[0046] To recapitulate the POMC processing in the anterior pituitary, POMC, PCSK1, and CPE are introduced into cells. FIG. 4B shows a schematic diagram of an artificial protein (ATG-STOP codon), which can be encoded in any viral and non-viral delivery system, including a synthetic mRNA. The diagram shows the design that uses self-cleaving 2A peptides, but IRES can also be used. Order or locations of each protein can be changed; however, POMC is preferably located at the C-terminus, as self-cleaving 2A peptides, after cleavage, leave residual residues at the C-terminus, which is undesirable.

[0047] To recapitulate the POMC processing in the hypothalamus and in the intermediate lobe of the pituitary, POMC, PCSK2, SCG5, and CPE are introduced into cells. FIG. 4D shows a schematic diagram of an artificial protein (ATG-Stop codon), which can be encoded in any viral and non-viral delivery system, including a synthetic mRNA. The diagram shows the design that uses self-cleaving 2A peptides, but IRES can also be used (FIG. 1). Order or locations of 13sf-6697944Docket No.: 69944-20020.40 each protein can be changed; however, POMC can preferably be at C-terminus, as self-cleaving 2A peptides, after cleavage, leave residual residues at the C-terminus, which is undesirable. V. PRO-BDNF

[0048] Brain-derived neurotrophic factor (BDNF) is synthesized in the brain as a precursor proBDNF, which is subsequently processed by PCSK1 and CPE to form mature BDNF (Liu, Jiang et al. 2023) (FIG. 5A).

[0049] To recapitulate the ProBDNF processing in the brain, the design of an artificial protein (ATG-STOP codon) shown in FIG. 5B includes proBDNF, CPE, and PCSK1. Order or locations of each protein can be changed; however, proBDNF is preferably located at the C- terminus, as self-cleaving 2A peptides, after cleavage, leave residual residues at the C-terminus, which is undesirable for BDNF. VI. TARGET PROPROTEINS OF PCSK1 AND PCSK2

[0050] Many proproteins form mature protein after being processed by PCSK1 or PCSK1 and CPE (Stijnen, Ramos-Molina et al. 2016, Burnett, LeDuc et al. 2017). These proproteins include, but not limited to (in names of their proprotein form or mature form) agouti- related peptide (AGRP), arginine vasopressin (AVP), brain-derived neurotrophic factor (BDNF), cocaine- and amphetamine-regulated transcript (CART), cholecystokinin (CCK), corticotropin- releasing hormone (CRH), ghrelin (GHRL), gastric inhibitory peptide (GIP), gonadotropin- releasing hormone (GnRH), orexin (HCRT), melanin-concentrating hormone (MCH), neuropeptide Y (NPY), oxytocin (OXT), proenkephalin (PENK), peptide YY (PYY), renin (REN), somatostatin (SST), thyrotropin-releasing hormone (TRH).

[0051] To recapitulate proper processing of these proproteins in their native cells / tissues, the design of an artificial protein (ATG-STOP codon) shown in FIG. 3B^is used by replacing proglucagon with a proprotein of interest.

[0052] Some of these proproteins are also processed by PCSK2. In such a case, the design of an artificial protein (ATG-STOP codon) shown in FIG. 3D^is used by replacing proglucagon with a proprotein of interest. 14sf-6697944Docket No.: 69944-20020.40

[0053] Some of these proproteins are also processed by the combination of PCSK1 and PCSK2. In such a case, the design of an artificial protein (ATG-STOP codon) shown in FIG. 2B is used by replacing preproinsulin with a proprotein of interest. VII. INTRODUCTION INTO CELLS OF MULTIPLE NUCLEIC ACIDS EACH ENCODING A SEPARATE PROPROTEIN OR ENZYME

[0054] The procedures described thus far are for expression of a proprotein and an enzyme that cleaves the proprotein from a single nucleic acid to produce mature protein. As an alternative procedure, the proprotein and the enzyme can be encoded by separate nucleic acids (Example 7), such as an mRNA, including for instance a synthetic mRNA (synRNA), a self- replicating RNA (srRNA), a controllable self-replicating RNA (c-srRNA), or a DNA, which may be contained within a viral vector.

[0055] For example, proglucagon (GCG), proprotein convertase 1 / 3 (PCSK1), and carboxypeptidase (CPE) can be encoded by three different synthetic mRNAs (synRNAs) as shown in FIG. 6A-C. These proteins can be encoded (ATG-Stop codon) not only by synRNAs, but also by nucleic acids present in a viral or non-viral delivery system. The three proteins can also be encoded by different combinations of two nucleic acids. For example, GCG can be encoded by a first synRNA, and PCSK1 and CPE can be encoded by a second synRNA in which the coding regions of PCSK1 and CPE are separated by an intervening sequence, such as a sequence encoding a self-cleaving 2A peptide.

[0056] The two or more synRNAs can be co-introduced into cells in vitro and in vivo. When the proteins of GCG, PCSK1, and CPE are produced in cells, GRG is cleaved to form glicentin, glicentin-related pancreatic polypeptide (GRPP), oxyntomodulin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and intervening peptide-2 (IP-2) by PCSK1 and CPE, as described in Section III and shown in FIG. 3A. VIII. RNA-INDUCED WEIGHT REDUCTION OF OBESE MICE

[0057] When proglucagon is (GCG) produced and processed by PCSK1 and CPE in intestine and brain, peptides such as GLP-1, GLP-2, IP-2, glicentin, GRPP, and oxyntomodulin are produced. Naturally-produced polypeptides such as GLP-1, glicentin, and oxyntomodulin, are known to have physiological functions that lead to the reduction of body weight. GLP-1 15sf-6697944Docket No.: 69944-20020.40 analogs are currently used as drugs to reduce the body weight. However, the combinations of polypeptides that are naturally processed and produced from the proglucagon may be more potent weight loss agents having reduced side effects.

[0058] Fibroblast growth factor-21 (FGF21) has also been shown to reduce body weight of diet-induced obese mice, when mRNA encoding FGF21 encapsulated in lipid nanoparticle (LNP) is delivered to skin (Bartesaghi et al., 2022).

[0059] Now, as described in Example 7, c-srRNA encoding FGF21, c-srRNA encoding a fusion protein of CPE, PCSK1, and proglucagon (FIG. 3B), or a mixture of three synRNAs, which encode CPE, PCSK1, and proglucagon separately (FIG. 6A-C) are injected into skin of diet-induced obese mice. Unlike the commonly used method (e.g., Bartesaghi et al., 2022), LNPs were not used for RNA delivery. Instead, the RNAs were mixed with chitosan oligosaccharide. As a control, diet-induced obese mice received the same injection without RNAs. Strikingly, compared to obese mice treated with a control injection (no RNA), all the RNA / chitosan-treated groups lost weight over the 25 days post-initial injection period (FIG. 7). IX. ENUMERATED EMBODIMENTS 1. An isolated nucleic acid comprising: a coding sequence (CDS) of a proprotein; and at least one CDS of at least one enzyme capable of cleaving the proprotein to form a mature protein. 2. The nucleic acid of embodiment 1, wherein the at least one CDS of the at least one enzyme comprises a CDS of a first enzyme and a CDS of a second enzyme. 3. The nucleic acid of embodiment 1, wherein the at least one CDS of the at least one enzyme comprises a CDS of a first enzyme, a CDS of a second enzyme, and a CDS of a third enzyme. 4. The nucleic acid of any one of embodiments 1-3, further comprising a CDS of a chaperone. 16sf-6697944Docket No.: 69944-20020.40 5. The nucleic acid of embodiment 4, wherein the chaperone comprises secretogranin V (SCG5). 6. The nucleic acid of any one of embodiments 1-5, wherein the proprotein is a preprotein comprising a signal peptide. 7. The nucleic acid of any one of embodiments 1-6, wherein the proprotein is inactive and the mature protein is active. 8. The nucleic acid of any one of embodiments 1-7, wherein the CDS of the proprotein and the at least one CDS of the at least one enzyme are separated from each other by a linking sequence. 9. The nucleic acid of embodiment 8, wherein the linking sequence comprises an internal ribosome entry site (IRES), a CDS of a flexible linker, a CDS of a 2A self-cleaving peptide, or any combination thereof. 10. The nucleic acid of any one of embodiments 1-9, wherein the proprotein is preproinsulin, the first enzyme is carboxypeptidase E (CPE), the second enzyme is proprotein convertase 1 / 3 (PCSK1), the third enzyme is proprotein convertase 2 (PCSK2). 11. The nucleic acid of embodiment 10, comprising the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1. 12. The nucleic acid of any one of embodiments 1-9, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 17sf-6697944Docket No.: 69944-20020.40 13. The nucleic acid of embodiment 12, comprising the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2. 14. The nucleic acid of any one of embodiments 1-9, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2). 15. The nucleic acid of embodiment 14, comprising the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3. 16. The nucleic acid of any one of embodiments 1-9, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 17. The nucleic acid of embodiment 16, comprising the nucleotide sequence of SEQ ID NO:4, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4. 18. The nucleic acid of any one of embodiments 1-9, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2). 19. The nucleic acid of embodiment 18, comprising the nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5. 20. The nucleic acid of any one of embodiments 1-9, wherein the proprotein is proBDNF, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 18sf-6697944Docket No.: 69944-20020.40 21. The nucleic acid of embodiment 20, comprising the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6. 22. The nucleic acid of any one of embodiments 1-21, wherein the nucleic acid is a DNA molecule. 23. A plasmid comprising the DNA molecule of embodiment 22 in operable combination with a promoter. 24. The nucleic acid of any one of embodiments 1-21, wherein the nucleic acid is an RNA molecule. 25. A recombinant virus comprising the RNA molecule of embodiment 24. 26. A method for expressing a mature protein, comprising contacting a mammalian cell with the nucleic acid, plasmid or recombinant virus of any one of embodiments 1-25 under conditions suitable for expression of the mature protein. 27. The method of embodiment 26, wherein the contacting is in vitro. 28. The method of embodiment 26, wherein the contacting is in vivo. 29. The method of embodiment 28, wherein the contacting comprises injection. 30. The method of embodiment 29, wherein the injection is intradermal injection, subcutaneous injection, intramuscular injection, intraocular injection, intrathecal injection, or intravenous injection, optionally wherein the injection is intradermal injection or subcutaneous injection. 19sf-6697944Docket No.: 69944-20020.40 31. A method for producing a mature protein, comprising: contacting a mammalian cell with a first nucleic acid comprising a coding sequence (CDS) of a proprotein; and contacting the mammalian cell with a second nucleic acid comprising a CDS of a first enzyme capable of cleaving the proprotein, wherein the contacting is done under conditions suitable for production of the mature protein. 32. The method of embodiment 31, further comprising contacting the mammalian cell with a third nucleic acid comprising a CDS of a second enzyme capable of cleaving the proprotein, wherein the contacting is done under conditions suitable for production of the mature protein. 33. The method of embodiment 32, further comprising contacting the mammalian cell with a fourth nucleic acid comprising a CDS of a third enzyme capable of cleaving the proprotein, wherein the contacting is done under conditions suitable for production of the mature protein. 34. The method of any one of embodiments 31-33, further comprising contacting the mammalian cell with a further nucleic acid comprising a CDS of a chaperone. 35. The method of embodiment 34, wherein the chaperone comprises secretogranin V (SCG5). 36. The method of any one of embodiments 31-35, wherein the proprotein is a preprotein comprising a signal peptide. 37. The method of any one of embodiments 31-36, wherein the proprotein is inactive and the mature protein is active. 38. The method of any one of embodiments 31-37, wherein the proprotein is preproinsulin, the first enzyme is carboxypeptidase E (CPE), the second enzyme is proprotein convertase 1 / 3 (PCSK1), the third enzyme is proprotein convertase 2 (PCSK2). 20sf-6697944Docket No.: 69944-20020.40 39. The method of any one of embodiments 31-37, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 40. The method of any one of embodiments 31-37, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2). 41. The method of any one of embodiments 31-37, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 42. The method of any one of embodiments 31-37, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2). 43. The method of any one of embodiments 31-37, wherein the proprotein is proBDNF, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 44. The method of any one of embodiments 31-43, wherein the nucleic acid is a DNA molecule. 45. The method of any one of embodiments 31-43, wherein the nucleic acid is an RNA molecule. 46. The method of embodiment 45, wherein the RNA is part of a recombinant virous vector. 47. The method of any one of embodiments 31-46, wherein the contacting for each nucleic acid is in vitro. 21sf-6697944Docket No.: 69944-20020.40 48. The method of any one of embodiments 31-46, wherein the contacting for each nucleic acid is in vivo. 49. The method of embodiment 48, wherein each contacting comprises injection. 50. The method of embodiment 49, wherein the injection is intradermal injection, subcutaneous injection, intramuscular injection, intraocular injection, intrathecal injection, or intravenous injection, optionally wherein the injection is intradermal injection or subcutaneous injection. 51. The method of any one of embodiments 31-50, wherein each contacting step occurs concurrently. 52. The method of any one of embodiments 31-50, wherein at least one contacting step occurs sequentially with another contacting step. 53. The method of any one of embodiments 31-52, wherein the mammalian cell does not produce at least one of the first enzyme, the second enzyme and the third enzyme in the absence of one or more of the contacting steps. 54. The method of any one of embodiments 31-52, wherein the mammalian cell produces none of the first enzyme, the second enzyme and the third enzyme in the absence of one or more of the contacting steps. 55. The method of embodiment 51, wherein each of the nucleic acids is part of a single nucleic acid molecule. 56. The method of embodiment 51, wherein at least two of the nucleic acids are part of a single nucleic acid molecule. 22sf-6697944Docket No.: 69944-20020.40 57. The method of embodiment 51, wherein each of the nucleic acids is part of a separate nucleic acid molecule. 58. The method of embodiment 56 or embodiment 57, wherein all of the nucleic acid molecules is present in a single composition. 59. A composition comprising a first nucleic acid comprising a coding sequence (CDS) of a proprotein, a second nucleic acid comprising a CDS of a first enzyme capable of cleaving the proprotein to form a mature protein. 60. The composition of embodiment 59, further comprising a third nucleic acid comprising a CDS of a second enzyme capable of cleaving the proprotein to form the mature protein. 61. The composition of embodiment 60, further comprising a fourth nucleic acid comprising a CDS of a third enzyme capable of cleaving the proprotein to form the mature protein. 62. The composition of any one of embodiments 59-61, further comprising a further nucleic acid comprising a CDS of a chaperone. 63. The composition of any one of embodiments 59-62, wherein each of the nucleic acids is part of a separate nucleic acid molecule. 64. The composition of any one of embodiments 61-63, wherein the proprotein is preproinsulin, the first enzyme is carboxypeptidase E (CPE), the second enzyme is proprotein convertase 1 / 3 (PCSK1), and the third enzyme is proprotein convertase 2 (PCSK2). 65. The composition of any one of embodiments 60-63, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 23sf-6697944Docket No.: 69944-20020.40 66. The composition of any one of embodiments 60-63, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2). 67. The composition of any one of embodiments 60-63, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 68. The composition of any one of embodiments 60-63, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2). 69. The composition of any one of embodiments 60-63, wherein the proprotein is proBDNF, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1). 70. The composition of any one of embodiments 59-69, wherein the composition is a pharmaceutical formulation further comprising a pharmaceutically acceptable excipient. 71. The composition of 70, further comprising chitosan. 24sf-6697944Docket No.: 69944-20020.40 EXAMPLES

[0060] Abbreviations: 2A peptide (self-cleaving 2A peptide); BDNF (brain-derived neurotrophic factor); CDS (coding sequence); CPE (carboxypeptidase); FGF21 (fibroblast growth factor 21); GCG (proglucagon); GLP (glucagon-like peptide); GRPP (glicentin-related pancreatic polypeptide); IP-1 (intervening peptide-1); IP-2 (intervening peptide-2); IRES (internal ribosome entry site); IVT (in vitro transcription); kb (kilobase); LNP (lipid nanoparticle); MPGF (major proglucagon fragment); ORF (open reading frame); PCSK1 (proprotein convertase 1 / 3); PCSK2 (proprotein convertase 2); POMC (proopiomelanocortin); srRNA (self-replicating RNA); c-srRNA (controllable self-replicating RNA); synRNA (synthetic mRNA); and secretogranin V (SCG5) Example 1. Production of Insulin

[0061] This example describes ectopic production of insulin by expression of a polyprotein from an exogenous nucleic acid encoding preproinsulin and enzymes that process preproinsulin to insulin. In nature, insulin is produced in pancreatic beta cells, but this technology makes it possible to produce insulin in other cell types such as skin (e.g., keratinocytes) and / or subcutaneous tissue (e.g., fibroblast cells). Materials and Methods

[0062] Polyprotein Engineering: A nucleic acid was engineered to include a coding sequence (CDS) comprising the following open reading frames (ORFs): CPE-2A-PCSK1-2A- PCSK2-2A-SCG5-2A-Preproinsulin (FIG. 2B, SEQ ID NO:1). CDS of the following proteins were used: CPE (NCBI: NM_001873.4), PCSK1 (NCBI: NM_000439.5), PCSK2 (NCBI: NM_002594.5), SCG5 (NCBI: NM_001144757.3), Preproinsulin (NCBI: NM_000207.3). Self- cleaving 2A peptides were used to link each protein: F2A, P2A, T2A, and E2A (Kim et al., 2011 and Liu et al., 2017). Synthetic mRNA (synRNA) is produced by in vitro transcription. Results and Conclusions

[0063] After delivering a synRNA encoding the preproinsulin polyprotein to mouse skin, ELISA is used to detect human insulin in mouse serum. Detection of human insulin in the serum 25sf-6697944Docket No.: 69944-20020.40 indicates that insulin was produced from mouse cells into which the synRNA was introduced, and subsequently secreted into the circulation. Example 2. Production of GLP-1, Glicentin, and Oxyntomodulin

[0064] This example describes ectopic production of Glicentin, GRPP, Oxyntomodulin, GLP-1, GLP-2, IP-2 by expression of an exogenous nucleic acid encoding proglucagon and enzymes that process proglucagon into active proteins. In nature, these active proteins are produced in cells of the intestine and brain, but this technology makes it possible to produce these active proteins in other cell types such as skin (e.g., keratinocytes) and / or subcutaneous tissue (e.g., fibroblast cells). Materials and Methods

[0065] Polyprotein Engineering: A nucleic acid was engineered to include a CDS comprising the following ORFs: CPE-2A-PCSK1-2A-Proglucagon (FIG. 3B, SEQ ID NO:2). CDS of the following proteins were used: CPE (NCBI: NM_001873.4), PCSK1 (NCBI: NM_000439.5), Proglucagon (NCBI: NM_002054.5). Self-cleaving 2A peptides were used to link each protein: F2A and E2A (Kim et al., 2011 and Liu et al., 2017). Synthetic mRNA (synRNA) is produced by in vitro transcription. Results and Conclusions

[0066] After delivering synRNA encoding the proglucagon polyprotein to mouse skin, ELISA is used to detect human Glicentin, Oxyntomodulin, and GLP-1 in mouse serum. Detection of human Glicentin, Oxyntomodulin, and GLP-1 in the serum indicates that these mature proteins were produced from mouse cells into which the synRNA was introduced, and subsequently secreted into the circulation. Example 3. Production of Glucagon and MGFP

[0067] This example describes ectopic production of MPGF, GRPP, Glucagon, and IP-1 by expression of an exogenous nucleic acid encoding proglucagon and enzymes that process proglucagon into active proteins. In nature, these active proteins are produced in pancreatic alpha 26sf-6697944Docket No.: 69944-20020.40 cells, but this technology makes it possible to produce these active proteins in other cell types such as skin (e.g., keratinocytes) and / or subcutaneous tissue (e.g., fibroblast cells). Materials and Methods

[0068] Polyprotein Engineering: A nucleic acid was engineered to include a CDS comprising the following ORFs: CPE-2A-PCSK2-2A-SCG5-2A-Proglucagon (FIG. 3D, SEQ ID NO:3). CDS of the following proteins were used: CPE (NCBI: NM_001873.4), PCSK2 (NCBI: NM_002594.5), SCG5 (NCBI: NM_001144757.3), Proglucagon (NCBI: NM_002054.5). Self-cleaving 2A peptides were used to link each protein: F2A, T2A, and E2A (Kim et al., 2011 and Liu et al., 2017). Synthetic mRNA (synRNA) is produced by in vitro transcription. Results and Conclusions.

[0069] After delivering synRNA encoding the proglucagon polyprotein to mouse skin, ELISA is used to detect human Glucagon and MGFP in mouse serum. Detection of human Glucagon and MGFP in the serum indicates that Glucagon and MGFP were produced from mouse cells into which the synRNA was introduced, and subsequently secreted into the circulation. Example 4. Production of ACTH and -LPH.

[0070] This example describes ectopic production of Pro-ACTH, -LPH, N-POMC, ACTH by expression of an exogenous nucleic acid encoding POMC and enzymes that process POMC into active proteins. In nature, these active proteins are produced in cells of the anterior pituitary, but this technology makes it possible to produce these active proteins in other cell types such as skin (e.g., keratinocytes) and / or subcutaneous tissue (e.g., fibroblast cells). Materials and Methods

[0071] Polyprotein Engineering: A nucleic acid was engineered to include a CDS comprising the following ORFs: CPE-2A-PCSK1-2A-POMC (FIG. 4B, SEQ ID NO:4). CDS of the following proteins were used: CPE (NCBI: NM_001873.4), PCSK1 (NCBI: NM_000439.5), POMC (NCBI: NM_001319204.2). Self-cleaving 2A peptides were used to link each protein: 27sf-6697944Docket No.: 69944-20020.40 F2A and E2A (Kim et al., 2011 and Liu et al., 2017). Synthetic mRNA (synRNA) is produced by in vitro transcription. Results and Conclusions

[0072] After delivering synRNA encoding the POMC polyprotein to mouse skin, ELISA is used to detect human ACTH and -LPH in mouse serum. Detection of human ACTH and - LPH in the serum indicates that these mature proteins were produced from mouse cells into which the synRNA was introduced, and subsequently secreted into the circulation. Example 5. Production of -MSH, CLIP, -MSH, -EP, 3-MSH.

[0073] This example describes ectopic production of -MSH, CLIP, -LPH, -MSH, - EP, and 3-MSH by expression of an exogenous nucleic acid encoding POMC and enzymes that process POMC into active proteins. In nature, these active proteins are produced in cells of hypothalamus and the intermediate lobe of the pituitary, but this technology makes it possible to produce these active proteins in other cell types such as skin (e.g., keratinocytes) and / or subcutaneous tissue (e.g., fibroblast cells). Materials and Methods

[0074] Polyprotein Engineering: A nucleic acid was engineered to include a coding sequence (CDS) comprising the following open reading frames (ORFs): CPE-2A-PCSK1-2A- PCSK2-2A-SCG5-2A-POMC (FIG. 4D, SEQ ID NO:5). CDS of the following proteins were used: CPE (NCBI: NM_001873.4), PCSK1 (NCBI: NM_000439.5), PCSK2 (NCBI: NM_002594.5), SCG5 (NCBI: NM_001144757.3), POMC (NCBI: NM_001319204.2). Self- cleaving 2A peptides were used to link each protein: F2A, P2A, T2A, and E2A (Kim et al., 2011 and Liu et al., 2017). Synthetic mRNA (synRNA) is produced by in vitro transcription. Results and Conclusion

[0075] After delivering synRNA encoding the POMC polyprotein to mouse skin, ELISA is used to detect human -MSH, CLIP, -MSH, -EP, and 3-MSH in mouse serum. Detection of human -MSH, CLIP, -MSH, -EP, and 3-MSH in the serum indicates that these mature proteins were produced from mouse cells into which the synRNA was introduced, and subsequently secreted into the circulation. 28sf-6697944Docket No.: 69944-20020.40 Example 6. Production of BDNF

[0076] This example describes ectopic production of BDNF by expression of an exogenous nucleic acid encoding proBDNF and enzymes that process proBDNF into mature BDNF. In nature, BDNF is produced in brain cells, but this technology makes it possible to produce BDNF in other cell types such as skin (e.g., keratinocytes) and / or subcutaneous tissue (e.g., fibroblast cells). Materials and Methods

[0077] Polyprotein Engineering: A nucleic acid was engineered to include a CDS comprising the following ORFs: CPE-2A-PCSK1-2A-proBDNF (FIG. 5B, SEQ ID NO:6). CDS of the following proteins were used: CPE (NCBI: NM_001873.4), PCSK1 (NCBI: NM_000439.5), and proBDNF (NCBI: NM_170735.6). Self-cleaving 2A peptides were used to link each protein: F2A and E2A (Kim et al., 2011 and Liu et al., 2017). Synthetic mRNA (synRNA) is produced by in vitro transcription. Results and Conclusions

[0078] After delivering synRNA encoding the proBDNF polyprotein to mouse skin, ELISA is used to detect human BDNF in mouse serum. Detection of human BDNF in the serum indicates that BDNF was produced from mouse cells into which the synRNA was introduced, which subsequently secreted into the circulation. Example 7. Weight Reduction by Delivering synthetic mRNAs to Skin

[0079] This example describes the weight reduction of diet-induced obese mice by delivering synRNAs to their skin. Materials and Methods

[0080] Polyprotein engineering and RNA production: Three different pharmaceutical compositions comprising RNA and a control composition lacking RNA were produced.

[0081] (1) c-srRNA-CPE-PCSK1-proglucagon (GCG): A nucleic acid was engineered to include a CDS comprising the following ORFs: CPE-2A-PCSK1-2A-proglucagon (FIG. 3B, 29sf-6697944Docket No.: 69944-20020.40 SEQ ID NO:2). CDS of the following proteins were used: CPE (NCBI: NM_001873.4), PCSK1 (NCBI: NM_000439.5), Proglucagon (NCBI: NM_002054.5). Self-cleaving 2A peptides were used to link each protein: F2A and E2A (Kim et al., 2011 and Liu et al., 2017). As a backbone, c- srRNA was used (see, WO 2021 / 138447 and WO 2022 / 266511 of Elixirgen Therapeutics, Inc.). RNA was produced by in vitro transcription with CleanCapAU (TriLink), without modified nucleotides. To prepare RNA for injection, 80 μg of c-srRNA-CPE-PCSK1-GCG was mixed with 1.2 μg of chitosan oligosaccharide, 288 μg of trehalose, and 60 μL of lactated Ringer’s solution (5% dextrose), lyophilized, and reconstituted with 60 μL of water.

[0082] (2) c-srRNA-FGF21: A nucleic acid was engineered to include a CDS of fibroblast growth factor 21 (FGF21) gene (NCBI: NM_019113.4, NP_061986.1). As a backbone, c-srRNA was used (see, WO 2021 / 138447 and WO 2022 / 266511 of Elixirgen Therapeutics, Inc.). RNA was produced by in vitro transcription with CleanCapAU (TriLink), without modified nucleotides. To prepare RNA for injection, 80 μg of c-srRNA-FGF21 was mixed with 1.2 μg of chitosan oligosaccharide, 288 μg of trehalose, and 60 μL of lactated Ringer’s solution (5% dextrose), lyophilized, and reconstituted with 60 μL of water.

[0083] (3) synRNA-CPE, synRNA-PCSK1, synRNA-GCG (proglucagon): Three synthetic mRNA (synRNA) molecules were designed to encode (from ATG to STOP codon) proglucagon (GCG) (NCBI ID: NM_002054.5; NP_002045.1), proprotein convertase 1 / 3 (PCSK1) (NCBI ID: NM_000439.5; NP_000430.3), and carboxypeptidase (CPE) (NCBI ID: NM_001873.4; NP_001864.1), respectively. As a backbone, 5’-UTR and 3’-UTR of Nodamura virus, which contains a 50 residue poly(A) sequence at its 3’-end was used (see, WO 2024 / 173609 of Elixirgen Therapeutics, Inc.). RNA was produced by in vitro transcription with CleanCapAG (TriLink), with a N1-methyl-pseudouridine modification. To prepare synRNA for injection, 20 μg of synRNA-CPE, 20 μg of synRNA-PCSK1, and 40 μg of synRNA-GCG were mixed with 1.2 μg of chitosan oligosaccharide, 288 μg of trehalose, and 60 μL of lactated Ringer’s solution (5% dextrose), lyophilized, and reconstituted with 60 μL of water.

[0084] (4) Control sample (no RNA): To prepare control samples for injection, 1.2 μg of chitosan oligosaccharide, 288 μg of trehalose, and 60 μL of lactated Ringer’s solution (5% dextrose) were mixed, lyophilized, and reconstituted with 60 μL of water. 30sf-6697944Docket No.: 69944-20020.40

[0085] Diet-induced obese mice: Male diet-induced obese mice (C57BL / 6 DIO) were purchased from the Jackson Laboratory. The mice were fed with a high-fat diet and became obese.

[0086] Intradermal injection of RNA into diet-induced obese mice: Mice (n=4 for each group) received 60 μL solution by intradermal injection of: No RNA control, c-srRNA-CPE- PCSK1-GCG, c-srRNA-FGF21, and a mixture of synRNA-CPE, synRNA-PCSK1, and synRNA-GCG. No RNA control and a mixture of synRNA-CPE, synRNA-PCSK1, and synRNA-GCG were injected on day 0, 1, 2, 7, 8, 9, 10, 11, 14, and 15. c-srRNA-CPE-PCSK1- GCG and c-srRNA-FGF21 were injected on day 0. The body weight was measured every day for 25 days. Average % weight gain / loss of each group (n=4) are shown. Results and Conclusions

[0087] As shown in FIG. 7, obese mice that received a control injection (no RNA) maintained their body weight during the observation period – 25 days. In contrast, obese mice that received one c-srRNA-CPE-PCSK1-GCG or c-srRNA-FGF21 lost body weight over time. Notably, obese mice that received a mixture of synRNA-CPE, synRNA-PCSK1, and synRNA- GCG, lost more weight than any other groups. References Bartesaghi et al. (2022). “Subcutaneous delivery of FGF21 mRNA therapy reverses obesity, insulin resistance, and hepatic steatosis in diet-induced obese mice.” Molecular Therapy: Nucleic Acids 28: 500-513. Burnettet al. (2017). "Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome." J Clin Invest 127(1): 293-305. Fricker et al. (2021). "Neuropeptidomic Analysis of a Genetically Defined Cell Type in Mouse Brain and Pituitary." Cell Chem Biol 28(1): 105-112 e104. Germanos, et al. (2021). "Inside the Insulin Secretory Granule." Metabolites 11(8). 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"Structural elements of PC2 required for interaction with its helper protein 7B2." J Biol Chem 273(2): 1158-1164. Sequences SEQ ID NO:1 >CPE-2A-PCSK1-2A-PCSK2-2A-SCG5-2A-Preproinsulin [artificial] MAGRGGSALLALCGALAACGWLLGAEAQEPGAPAAGMRRRRRLQQEDGISFEYHRYPELREALV SVWLQCTAISRIYTVGRSFEGRELLVIELSDNPGVHEPGEPEFKYIGNMHGNEAVGRELLIFLA QYLCNEYQKGNETIVNLIHSTRIHIMPSLNPDGFEKAASQPGELKDWFVGRSNAQGIDLNRNFP DLDRIVYVNEKEGGPNNHLLKNMKKIVDQNTKLAPETKAVIHWIMDIPFVLSANLHGGDLVANY PYDETRSGSAHEYSSSPDDAIFQSLARAYSSFNPAMSDPNRPPCRKNDDDSSFVDGTTNGGAWY SVPGGMQDFNYLSSNCFEITVELSCEKFPPEETLKTYWEDNKNSLISYLEQIHRGVKGFVRDLQ GNPIANATISVEGIDHDVTSAKDGDYWRLLIPGNYKLTASAPGYLAITKKVAVPYSPAAGVDFE LESFSERKEEEKEELMEWWKMMSETLNFGSGVKQTLNFDLLKLAGDVESNPGPMERRAWSLQCT AFVLFCAWCALNSAKAKRQFVNEWAAEIPGGPEAASAIAEELGYDLLGQIGSLENHYLFKHKNH PRRSRRSAFHITKRLSDDDRVIWAEQQYEKERSKRSALRDSALNLFNDPMWNQQWYLQDTRMTA ALPKLDLHVIPVWQKGITGKGVVITVLDDGLEWNHTDIYANYDPEASYDFNDNDHDPFPRYDPT NENKHGTRCAGEIAMQANNHKCGVGVAYNSKVGGIRMLDGIVTDAIEASSIGFNPGHVDIYSAS WGPNDDGKTVEGPGRLAQKAFEYGVKQGRQGKGSIFVWASGNGGRQGDNCDCDGYTDSIYTISI SSASQQGLSPWYAEKCSSTLATSYSSGDYTDQRITSADLHNDCTETHTGTSASAPLAAGIFALA LEANPNLTWRDMQHLVVWTSEYDPLANNPGWKKNGAGLMVNSRFGFGLLNAKALVDLADPRTWR SVPEKKECVVKDNDFEPRALKANGEVIIEIPTRACEGQENAIKSLEHVQFEATIEYSRRGDLHV TLTSAAGTSTVLLAERERDTSPNGFKNWDFMSVHTWGENPIGTWTLRITDMSGRIQNEGRIVNW KLILHGTSSQPEHMKQPRVYTSYNTVQNDRRGVEKMVDPGEEQPTQENPKENTLVSKSPSSSSV GGRRDELEEGAPSQAMLRLLQSAFSKNSPPKQSPKKSPSAKLNIPYENFYEALEKLNKPSQLKD SEDSLYNDYVDVFYNTKPYKHRDDRLLQALVDILNEENGSGATNFSLLKQAGDVEENPGPMKGG CVSQWKAAAGFLFCVMVFASAERPVFTNHFLVELHKGGEDKARQVAAEHGFGVRKLPFAEGLYH FYHNGLAKAKRRRSLHHKQQLERDPRVKMALQQEGFDRKKRGYRDINEIDINMNDPLFTKQWYL INTGQADGTPGLDLNVAEAWELGYTGKGVTIGIMDDGIDYLHPDLASNYNAEASYDFSSNDPYP YPRYTDDWFNSHGTRCAGEVSAAANNNICGVGVAYNSKVAGIRMLDQPFMTDIIEASSISHMPQ LIDIYSASWGPTDNGKTVDGPRELTLQAMADGVNKGRGGKGSIYVWASGDGGSYDDCNCDGYAS SMWTISINSAINDGRTALYDESCSSTLASTFSNGRKRNPEAGVATTDLYGNCTLRHSGTSAAAP EAAGVFALALEANLGLTWRDMQHLTVLTSKRNQLHDEVHQWRRNGVGLEFNHLFGYGVLDAGAM VKMAKDWKTVPERFHCVGGSVQDPEKIPSTGKLVLTLTTDACEGKENFVRYLEHVQAVITVNAT RRGDLNINMTSPMGTKSILLSRRPRDDDSKVGFDKWPFMTTHTWGEDARGTWTLELGFVGSAPQ KGVLKEWTLMLHGTQSAPYIDQVVRDYQSKLAMSKKEELEEELDEAVERSLKSILNKNGSGEGR GSLLTCGDVEENPGPMVSRMVSTMLSGLLFWLASGWTPAFAYSPRTPDRVSEADIQRLLHGVME QLGIARPRVEYPAHQAMNLVGPQSIEGGAHEGLQHLGPFGNIPNIVAELTGDNIPKDFSEDQGY PDPPNPCPVGKTADDGCLENTPDTAEFSREFQLHQHLFDPEHDYPGLGKWNKKLLYEKMKGGER RKRRSVNPYLQGQRLDNVVAKKSVPHFSDEDKDPEGSGQCTNYALLKLAGDVESNPGPMALWMR LLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGG GPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN SEQ ID NO:2 33sf-6697944Docket No.: 69944-20020.40 >CPE-2A-PCSK1-2A-Proglucagon [artificial] MAGRGGSALLALCGALAACGWLLGAEAQEPGAPAAGMRRRRRLQQEDGISFEYHRYPELREALV SVWLQCTAISRIYTVGRSFEGRELLVIELSDNPGVHEPGEPEFKYIGNMHGNEAVGRELLIFLA QYLCNEYQKGNETIVNLIHSTRIHIMPSLNPDGFEKAASQPGELKDWFVGRSNAQGIDLNRNFP DLDRIVYVNEKEGGPNNHLLKNMKKIVDQNTKLAPETKAVIHWIMDIPFVLSANLHGGDLVANY PYDETRSGSAHEYSSSPDDAIFQSLARAYSSFNPAMSDPNRPPCRKNDDDSSFVDGTTNGGAWY SVPGGMQDFNYLSSNCFEITVELSCEKFPPEETLKTYWEDNKNSLISYLEQIHRGVKGFVRDLQ GNPIANATISVEGIDHDVTSAKDGDYWRLLIPGNYKLTASAPGYLAITKKVAVPYSPAAGVDFE LESFSERKEEEKEELMEWWKMMSETLNFGSGVKQTLNFDLLKLAGDVESNPGPMERRAWSLQCT AFVLFCAWCALNSAKAKRQFVNEWAAEIPGGPEAASAIAEELGYDLLGQIGSLENHYLFKHKNH PRRSRRSAFHITKRLSDDDRVIWAEQQYEKERSKRSALRDSALNLFNDPMWNQQWYLQDTRMTA ALPKLDLHVIPVWQKGITGKGVVITVLDDGLEWNHTDIYANYDPEASYDFNDNDHDPFPRYDPT NENKHGTRCAGEIAMQANNHKCGVGVAYNSKVGGIRMLDGIVTDAIEASSIGFNPGHVDIYSAS WGPNDDGKTVEGPGRLAQKAFEYGVKQGRQGKGSIFVWASGNGGRQGDNCDCDGYTDSIYTISI SSASQQGLSPWYAEKCSSTLATSYSSGDYTDQRITSADLHNDCTETHTGTSASAPLAAGIFALA LEANPNLTWRDMQHLVVWTSEYDPLANNPGWKKNGAGLMVNSRFGFGLLNAKALVDLADPRTWR SVPEKKECVVKDNDFEPRALKANGEVIIEIPTRACEGQENAIKSLEHVQFEATIEYSRRGDLHV TLTSAAGTSTVLLAERERDTSPNGFKNWDFMSVHTWGENPIGTWTLRITDMSGRIQNEGRIVNW KLILHGTSSQPEHMKQPRVYTSYNTVQNDRRGVEKMVDPGEEQPTQENPKENTLVSKSPSSSSV GGRRDELEEGAPSQAMLRLLQSAFSKNSPPKQSPKKSPSAKLNIPYENFYEALEKLNKPSQLKD SEDSLYNDYVDVFYNTKPYKHRDDRLLQALVDILNEENGSGQCTNYALLKLAGDVESNPGPMKS IYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLD SRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRD FPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK SEQ ID NO:3 >CPE-2A-PCSK2-2A-SCG5-2A-Proglucagon [artificial] MAGRGGSALLALCGALAACGWLLGAEAQEPGAPAAGMRRRRRLQQEDGISFEYHRYPELREALV SVWLQCTAISRIYTVGRSFEGRELLVIELSDNPGVHEPGEPEFKYIGNMHGNEAVGRELLIFLA QYLCNEYQKGNETIVNLIHSTRIHIMPSLNPDGFEKAASQPGELKDWFVGRSNAQGIDLNRNFP DLDRIVYVNEKEGGPNNHLLKNMKKIVDQNTKLAPETKAVIHWIMDIPFVLSANLHGGDLVANY PYDETRSGSAHEYSSSPDDAIFQSLARAYSSFNPAMSDPNRPPCRKNDDDSSFVDGTTNGGAWY SVPGGMQDFNYLSSNCFEITVELSCEKFPPEETLKTYWEDNKNSLISYLEQIHRGVKGFVRDLQ GNPIANATISVEGIDHDVTSAKDGDYWRLLIPGNYKLTASAPGYLAITKKVAVPYSPAAGVDFE LESFSERKEEEKEELMEWWKMMSETLNFGSGVKQTLNFDLLKLAGDVESNPGPMKGGCVSQWKA AAGFLFCVMVFASAERPVFTNHFLVELHKGGEDKARQVAAEHGFGVRKLPFAEGLYHFYHNGLA KAKRRRSLHHKQQLERDPRVKMALQQEGFDRKKRGYRDINEIDINMNDPLFTKQWYLINTGQAD GTPGLDLNVAEAWELGYTGKGVTIGIMDDGIDYLHPDLASNYNAEASYDFSSNDPYPYPRYTDD WFNSHGTRCAGEVSAAANNNICGVGVAYNSKVAGIRMLDQPFMTDIIEASSISHMPQLIDIYSA SWGPTDNGKTVDGPRELTLQAMADGVNKGRGGKGSIYVWASGDGGSYDDCNCDGYASSMWTISI NSAINDGRTALYDESCSSTLASTFSNGRKRNPEAGVATTDLYGNCTLRHSGTSAAAPEAAGVFA LALEANLGLTWRDMQHLTVLTSKRNQLHDEVHQWRRNGVGLEFNHLFGYGVLDAGAMVKMAKDW KTVPERFHCVGGSVQDPEKIPSTGKLVLTLTTDACEGKENFVRYLEHVQAVITVNATRRGDLNI NMTSPMGTKSILLSRRPRDDDSKVGFDKWPFMTTHTWGEDARGTWTLELGFVGSAPQKGVLKEW TLMLHGTQSAPYIDQVVRDYQSKLAMSKKEELEEELDEAVERSLKSILNKNGSGEGRGSLLTCG DVEENPGPMVSRMVSTMLSGLLFWLASGWTPAFAYSPRTPDRVSEADIQRLLHGVMEQLGIARP RVEYPAHQAMNLVGPQSIEGGAHEGLQHLGPFGNIPNIVAELTGDNIPKDFSEDQGYPDPPNPC 34sf-6697944Docket No.: 69944-20020.40 PVGKTADDGCLENTPDTAEFSREFQLHQHLFDPEHDYPGLGKWNKKLLYEKMKGGERRKRRSVN PYLQGQRLDNVVAKKSVPHFSDEDKDPEGSGQCTNYALLKLAGDVESNPGPMKSIYFVAGLFVM LVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQDFVQW LMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEE LGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK SEQ ID NO:4 >CPE-2A-PCSK1-2A-POMC [artificial] MAGRGGSALLALCGALAACGWLLGAEAQEPGAPAAGMRRRRRLQQEDGISFEYHRYPELREALV SVWLQCTAISRIYTVGRSFEGRELLVIELSDNPGVHEPGEPEFKYIGNMHGNEAVGRELLIFLA QYLCNEYQKGNETIVNLIHSTRIHIMPSLNPDGFEKAASQPGELKDWFVGRSNAQGIDLNRNFP DLDRIVYVNEKEGGPNNHLLKNMKKIVDQNTKLAPETKAVIHWIMDIPFVLSANLHGGDLVANY PYDETRSGSAHEYSSSPDDAIFQSLARAYSSFNPAMSDPNRPPCRKNDDDSSFVDGTTNGGAWY SVPGGMQDFNYLSSNCFEITVELSCEKFPPEETLKTYWEDNKNSLISYLEQIHRGVKGFVRDLQ GNPIANATISVEGIDHDVTSAKDGDYWRLLIPGNYKLTASAPGYLAITKKVAVPYSPAAGVDFE LESFSERKEEEKEELMEWWKMMSETLNFGSGVKQTLNFDLLKLAGDVESNPGPMERRAWSLQCT AFVLFCAWCALNSAKAKRQFVNEWAAEIPGGPEAASAIAEELGYDLLGQIGSLENHYLFKHKNH PRRSRRSAFHITKRLSDDDRVIWAEQQYEKERSKRSALRDSALNLFNDPMWNQQWYLQDTRMTA ALPKLDLHVIPVWQKGITGKGVVITVLDDGLEWNHTDIYANYDPEASYDFNDNDHDPFPRYDPT NENKHGTRCAGEIAMQANNHKCGVGVAYNSKVGGIRMLDGIVTDAIEASSIGFNPGHVDIYSAS WGPNDDGKTVEGPGRLAQKAFEYGVKQGRQGKGSIFVWASGNGGRQGDNCDCDGYTDSIYTISI SSASQQGLSPWYAEKCSSTLATSYSSGDYTDQRITSADLHNDCTETHTGTSASAPLAAGIFALA LEANPNLTWRDMQHLVVWTSEYDPLANNPGWKKNGAGLMVNSRFGFGLLNAKALVDLADPRTWR SVPEKKECVVKDNDFEPRALKANGEVIIEIPTRACEGQENAIKSLEHVQFEATIEYSRRGDLHV TLTSAAGTSTVLLAERERDTSPNGFKNWDFMSVHTWGENPIGTWTLRITDMSGRIQNEGRIVNW KLILHGTSSQPEHMKQPRVYTSYNTVQNDRRGVEKMVDPGEEQPTQENPKENTLVSKSPSSSSV GGRRDELEEGAPSQAMLRLLQSAFSKNSPPKQSPKKSPSAKLNIPYENFYEALEKLNKPSQLKD SEDSLYNDYVDVFYNTKPYKHRDDRLLQALVDILNEENGSGQCTNYALLKLAGDVESNPGPMPR SCCSRSGALLLALLLQASMEVRGWCLESSQCQDLTTESNLLECIRACKPDLSAETPMFPGNGDE QPLTENPRKYVMGHFRWDRFGRRNSSSSGSSGAGQKREDVSAGEDCGPLPEGGPEPRSDGAKPG PREGKRSYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEFKRELTGQRLREGDGPDGPA DDGAGAQADLEHSLLVAAEKKDEGPYRMEHFRWGSPPKDKRYGGFMTSEKSQTPLVTLFKNAII KNAYKKGE SEQ ID NO:5 >CPE-2A-PCSK1-2A-PCSK2-2A-SCG5-2A-POMC [artificial] MAGRGGSALLALCGALAACGWLLGAEAQEPGAPAAGMRRRRRLQQEDGISFEYHRYPELREALV SVWLQCTAISRIYTVGRSFEGRELLVIELSDNPGVHEPGEPEFKYIGNMHGNEAVGRELLIFLA QYLCNEYQKGNETIVNLIHSTRIHIMPSLNPDGFEKAASQPGELKDWFVGRSNAQGIDLNRNFP DLDRIVYVNEKEGGPNNHLLKNMKKIVDQNTKLAPETKAVIHWIMDIPFVLSANLHGGDLVANY PYDETRSGSAHEYSSSPDDAIFQSLARAYSSFNPAMSDPNRPPCRKNDDDSSFVDGTTNGGAWY SVPGGMQDFNYLSSNCFEITVELSCEKFPPEETLKTYWEDNKNSLISYLEQIHRGVKGFVRDLQ GNPIANATISVEGIDHDVTSAKDGDYWRLLIPGNYKLTASAPGYLAITKKVAVPYSPAAGVDFE LESFSERKEEEKEELMEWWKMMSETLNFGSGVKQTLNFDLLKLAGDVESNPGPMERRAWSLQCT AFVLFCAWCALNSAKAKRQFVNEWAAEIPGGPEAASAIAEELGYDLLGQIGSLENHYLFKHKNH PRRSRRSAFHITKRLSDDDRVIWAEQQYEKERSKRSALRDSALNLFNDPMWNQQWYLQDTRMTA ALPKLDLHVIPVWQKGITGKGVVITVLDDGLEWNHTDIYANYDPEASYDFNDNDHDPFPRYDPT 35sf-6697944Docket No.: 69944-20020.40 NENKHGTRCAGEIAMQANNHKCGVGVAYNSKVGGIRMLDGIVTDAIEASSIGFNPGHVDIYSAS WGPNDDGKTVEGPGRLAQKAFEYGVKQGRQGKGSIFVWASGNGGRQGDNCDCDGYTDSIYTISI SSASQQGLSPWYAEKCSSTLATSYSSGDYTDQRITSADLHNDCTETHTGTSASAPLAAGIFALA LEANPNLTWRDMQHLVVWTSEYDPLANNPGWKKNGAGLMVNSRFGFGLLNAKALVDLADPRTWR SVPEKKECVVKDNDFEPRALKANGEVIIEIPTRACEGQENAIKSLEHVQFEATIEYSRRGDLHV TLTSAAGTSTVLLAERERDTSPNGFKNWDFMSVHTWGENPIGTWTLRITDMSGRIQNEGRIVNW KLILHGTSSQPEHMKQPRVYTSYNTVQNDRRGVEKMVDPGEEQPTQENPKENTLVSKSPSSSSV GGRRDELEEGAPSQAMLRLLQSAFSKNSPPKQSPKKSPSAKLNIPYENFYEALEKLNKPSQLKD SEDSLYNDYVDVFYNTKPYKHRDDRLLQALVDILNEENGSGATNFSLLKQAGDVEENPGPMKGG CVSQWKAAAGFLFCVMVFASAERPVFTNHFLVELHKGGEDKARQVAAEHGFGVRKLPFAEGLYH FYHNGLAKAKRRRSLHHKQQLERDPRVKMALQQEGFDRKKRGYRDINEIDINMNDPLFTKQWYL INTGQADGTPGLDLNVAEAWELGYTGKGVTIGIMDDGIDYLHPDLASNYNAEASYDFSSNDPYP YPRYTDDWFNSHGTRCAGEVSAAANNNICGVGVAYNSKVAGIRMLDQPFMTDIIEASSISHMPQ LIDIYSASWGPTDNGKTVDGPRELTLQAMADGVNKGRGGKGSIYVWASGDGGSYDDCNCDGYAS SMWTISINSAINDGRTALYDESCSSTLASTFSNGRKRNPEAGVATTDLYGNCTLRHSGTSAAAP EAAGVFALALEANLGLTWRDMQHLTVLTSKRNQLHDEVHQWRRNGVGLEFNHLFGYGVLDAGAM VKMAKDWKTVPERFHCVGGSVQDPEKIPSTGKLVLTLTTDACEGKENFVRYLEHVQAVITVNAT RRGDLNINMTSPMGTKSILLSRRPRDDDSKVGFDKWPFMTTHTWGEDARGTWTLELGFVGSAPQ KGVLKEWTLMLHGTQSAPYIDQVVRDYQSKLAMSKKEELEEELDEAVERSLKSILNKNGSGEGR GSLLTCGDVEENPGPMVSRMVSTMLSGLLFWLASGWTPAFAYSPRTPDRVSEADIQRLLHGVME QLGIARPRVEYPAHQAMNLVGPQSIEGGAHEGLQHLGPFGNIPNIVAELTGDNIPKDFSEDQGY PDPPNPCPVGKTADDGCLENTPDTAEFSREFQLHQHLFDPEHDYPGLGKWNKKLLYEKMKGGER RKRRSVNPYLQGQRLDNVVAKKSVPHFSDEDKDPEGSGQCTNYALLKLAGDVESNPGPMPRSCC SRSGALLLALLLQASMEVRGWCLESSQCQDLTTESNLLECIRACKPDLSAETPMFPGNGDEQPL TENPRKYVMGHFRWDRFGRRNSSSSGSSGAGQKREDVSAGEDCGPLPEGGPEPRSDGAKPGPRE GKRSYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEFKRELTGQRLREGDGPDGPADDG AGAQADLEHSLLVAAEKKDEGPYRMEHFRWGSPPKDKRYGGFMTSEKSQTPLVTLFKNAIIKNA YKKGE SEQ ID NO:6 >CPE-2A-PCSK1-2A-ProBDNF [artificial] MAGRGGSALLALCGALAACGWLLGAEAQEPGAPAAGMRRRRRLQQEDGISFEYHRYPELREALV SVWLQCTAISRIYTVGRSFEGRELLVIELSDNPGVHEPGEPEFKYIGNMHGNEAVGRELLIFLA QYLCNEYQKGNETIVNLIHSTRIHIMPSLNPDGFEKAASQPGELKDWFVGRSNAQGIDLNRNFP DLDRIVYVNEKEGGPNNHLLKNMKKIVDQNTKLAPETKAVIHWIMDIPFVLSANLHGGDLVANY PYDETRSGSAHEYSSSPDDAIFQSLARAYSSFNPAMSDPNRPPCRKNDDDSSFVDGTTNGGAWY SVPGGMQDFNYLSSNCFEITVELSCEKFPPEETLKTYWEDNKNSLISYLEQIHRGVKGFVRDLQ GNPIANATISVEGIDHDVTSAKDGDYWRLLIPGNYKLTASAPGYLAITKKVAVPYSPAAGVDFE LESFSERKEEEKEELMEWWKMMSETLNFGSGVKQTLNFDLLKLAGDVESNPGPMERRAWSLQCT AFVLFCAWCALNSAKAKRQFVNEWAAEIPGGPEAASAIAEELGYDLLGQIGSLENHYLFKHKNH PRRSRRSAFHITKRLSDDDRVIWAEQQYEKERSKRSALRDSALNLFNDPMWNQQWYLQDTRMTA ALPKLDLHVIPVWQKGITGKGVVITVLDDGLEWNHTDIYANYDPEASYDFNDNDHDPFPRYDPT NENKHGTRCAGEIAMQANNHKCGVGVAYNSKVGGIRMLDGIVTDAIEASSIGFNPGHVDIYSAS WGPNDDGKTVEGPGRLAQKAFEYGVKQGRQGKGSIFVWASGNGGRQGDNCDCDGYTDSIYTISI SSASQQGLSPWYAEKCSSTLATSYSSGDYTDQRITSADLHNDCTETHTGTSASAPLAAGIFALA LEANPNLTWRDMQHLVVWTSEYDPLANNPGWKKNGAGLMVNSRFGFGLLNAKALVDLADPRTWR SVPEKKECVVKDNDFEPRALKANGEVIIEIPTRACEGQENAIKSLEHVQFEATIEYSRRGDLHV 36sf-6697944Docket No.: 69944-20020.40 TLTSAAGTSTVLLAERERDTSPNGFKNWDFMSVHTWGENPIGTWTLRITDMSGRIQNEGRIVNW KLILHGTSSQPEHMKQPRVYTSYNTVQNDRRGVEKMVDPGEEQPTQENPKENTLVSKSPSSSSV GGRRDELEEGAPSQAMLRLLQSAFSKNSPPKQSPKKSPSAKLNIPYENFYEALEKLNKPSQLKD SEDSLYNDYVDVFYNTKPYKHRDDRLLQALVDILNEENGSGQCTNYALLKLAGDVESNPGPMTI LFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADTFEHVI EELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSD PARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEG CRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR SEQ ID NO:7 PROGLUCAGON (GCG) [HUMAN] MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSK YLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG RRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK SEQ ID NO:8 PCSK1 [HUMAN] MERRAWSLQCTAFVLFCAWCALNSAKAKRQFVNEWAAEIPGGPEAASAIAEELGYDLLGQIGSL ENHYLFKHKNHPRRSRRSAFHITKRLSDDDRVIWAEQQYEKERSKRSALRDSALNLFNDPMWNQ QWYLQDTRMTAALPKLDLHVIPVWQKGITGKGVVITVLDDGLEWNHTDIYANYDPEASYDFNDN DHDPFPRYDPTNENKHGTRCAGEIAMQANNHKCGVGVAYNSKVGGIRMLDGIVTDAIEASSIGF NPGHVDIYSASWGPNDDGKTVEGPGRLAQKAFEYGVKQGRQGKGSIFVWASGNGGRQGDNCDCD GYTDSIYTISISSASQQGLSPWYAEKCSSTLATSYSSGDYTDQRITSADLHNDCTETHTGTSAS APLAAGIFALALEANPNLTWRDMQHLVVWTSEYDPLANNPGWKKNGAGLMVNSRFGFGLLNAKA LVDLADPRTWRSVPEKKECVVKDNDFEPRALKANGEVIIEIPTRACEGQENAIKSLEHVQFEAT IEYSRRGDLHVTLTSAAGTSTVLLAERERDTSPNGFKNWDFMSVHTWGENPIGTWTLRITDMSG RIQNEGRIVNWKLILHGTSSQPEHMKQPRVYTSYNTVQNDRRGVEKMVDPGEEQPTQENPKENT LVSKSPSSSSVGGRRDELEEGAPSQAMLRLLQSAFSKNSPPKQSPKKSPSAKLNIPYENFYEAL EKLNKPSQLKDSEDSLYNDYVDVFYNTKPYKHRDDRLLQALVDILNEEN SEQ ID NO:9 CPE [HUMAN] MAGRGGSALLALCGALAACGWLLGAEAQEPGAPAAGMRRRRRLQQEDGISFEYHRYPELREALV SVWLQCTAISRIYTVGRSFEGRELLVIELSDNPGVHEPGEPEFKYIGNMHGNEAVGRELLIFLA QYLCNEYQKGNETIVNLIHSTRIHIMPSLNPDGFEKAASQPGELKDWFVGRSNAQGIDLNRNFP DLDRIVYVNEKEGGPNNHLLKNMKKIVDQNTKLAPETKAVIHWIMDIPFVLSANLHGGDLVANY PYDETRSGSAHEYSSSPDDAIFQSLARAYSSFNPAMSDPNRPPCRKNDDDSSFVDGTTNGGAWY SVPGGMQDFNYLSSNCFEITVELSCEKFPPEETLKTYWEDNKNSLISYLEQIHRGVKGFVRDLQ GNPIANATISVEGIDHDVTSAKDGDYWRLLIPGNYKLTASAPGYLAITKKVAVPYSPAAGVDFE LESFSERKEEEKEELMEWWKMMSETLNF SEQ ID NO:10 FGF21 [HUMAN] MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIR EDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLL EDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSD PLSMVGPSQGRSPSYAS 37sf-6697944

Claims

Docket No.: 69944-20020.40 CLAIMS We claim:

1. An isolated nucleic acid comprising: a coding sequence (CDS) of a proprotein; and at least one CDS of at least one enzyme capable of cleaving the proprotein to form a mature protein.

2. The nucleic acid of claim 1, wherein the at least one CDS of the at least one enzyme comprises a CDS of a first enzyme and a CDS of a second enzyme.

3. The nucleic acid of claim 1, wherein the at least one CDS of the at least one enzyme comprises a CDS of a first enzyme, a CDS of a second enzyme, and a CDS of a third enzyme.

4. The nucleic acid of any one of claims 1-3, further comprising a CDS of a chaperone.

5. The nucleic acid of claim 4, wherein the chaperone comprises secretogranin V (SCG5).

6. The nucleic acid of any one of claims 1-5, wherein the proprotein is a preprotein comprising a signal peptide.

7. The nucleic acid of any one of claims 1-6, wherein the proprotein is inactive and the mature protein is active.

8. The nucleic acid of any one of claims 1-7, wherein the CDS of the proprotein and the at least one CDS of the at least one enzyme are separated from each other by a linking sequence.

9. The nucleic acid of claim 8, wherein the linking sequence comprises an internal ribosome entry site (IRES), a CDS of a flexible linker, a CDS of a 2A self-cleaving peptide, or any combination thereof. 38sf-6697944Docket No.: 69944-20020.40 10. The nucleic acid of any one of claims 1-9, wherein the proprotein is preproinsulin, the first enzyme is carboxypeptidase E (CPE), the second enzyme is proprotein convertase 1 / 3 (PCSK1), the third enzyme is proprotein convertase 2 (PCSK2).

11. The nucleic acid of claim 10, comprising the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

1.

12. The nucleic acid of any one of claims 1-9, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

13. The nucleic acid of claim 12, comprising the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

2.

14. The nucleic acid of any one of claims 1-9, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2).

15. The nucleic acid of claim 14, comprising the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

3.

16. The nucleic acid of any one of claims 1-9, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

17. The nucleic acid of claim 16, comprising the nucleotide sequence of SEQ ID NO:4, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

4. 39sf-6697944Docket No.: 69944-20020.40 18. The nucleic acid of any one of claims 1-9, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2).

19. The nucleic acid of claim 18, comprising the nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

5.

20. The nucleic acid of any one of claims 1-9, wherein the proprotein is proBDNF, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

21. The nucleic acid of claim 20, comprising the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

6.

22. The nucleic acid of any one of claims 1-21, wherein the nucleic acid is a DNA molecule.

23. A plasmid comprising the DNA molecule of claim 22 in operable combination with a promoter.

24. The nucleic acid of any one of claims 1-21, wherein the nucleic acid is an RNA molecule.

25. A recombinant virus comprising the RNA molecule of claim 24.

26. A method for expressing a mature protein, comprising contacting a mammalian cell with the nucleic acid, plasmid or recombinant virus of any one of claims 1-25 under conditions suitable for expression of the mature protein. 40sf-6697944Docket No.: 69944-20020.40 27. The method of claim 26, wherein the contacting is in vitro.

28. The method of claim 26, wherein the contacting is in vivo.

29. The method of claim 28, wherein the contacting comprises injection.

30. The method of claim 29, wherein the injection is intradermal injection, subcutaneous injection, intramuscular injection, intraocular injection, intrathecal injection, or intravenous injection, optionally wherein the injection is intradermal injection or subcutaneous injection.

31. A method for producing a mature protein, comprising: contacting a mammalian cell with a first nucleic acid comprising a coding sequence (CDS) of a proprotein; and contacting the mammalian cell with a second nucleic acid comprising a CDS of a first enzyme capable of cleaving the proprotein, wherein the contacting is done under conditions suitable for production of the mature protein.

32. The method of claim 31, further comprising contacting the mammalian cell with a third nucleic acid comprising a CDS of a second enzyme capable of cleaving the proprotein, wherein the contacting is done under conditions suitable for production of the mature protein.

33. The method of claim 32, further comprising contacting the mammalian cell with a fourth nucleic acid comprising a CDS of a third enzyme capable of cleaving the proprotein, wherein the contacting is done under conditions suitable for production of the mature protein.

34. The method of any one of claims 31-33, further comprising contacting the mammalian cell with a further nucleic acid comprising a CDS of a chaperone.

35. The method of claim 34, wherein the chaperone comprises secretogranin V (SCG5). 41sf-6697944Docket No.: 69944-20020.40 36. The method of any one of claims 31-35, wherein the proprotein is a preprotein comprising a signal peptide.

37. The method of any one of claims 31-36, wherein the proprotein is inactive and the mature protein is active.

38. The method of any one of claims 31-37, wherein the proprotein is preproinsulin, the first enzyme is carboxypeptidase E (CPE), the second enzyme is proprotein convertase 1 / 3 (PCSK1), the third enzyme is proprotein convertase 2 (PCSK2).

39. The method of any one of claims 31-37, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

40. The method of any one of claims 31-37, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2).

41. The method of any one of claims 31-37, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

42. The method of any one of claims 31-37, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2).

43. The method of any one of claims 31-37, wherein the proprotein is proBDNF, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

44. The method of any one of claims 31-43, wherein the nucleic acid is a DNA molecule. 42sf-6697944Docket No.: 69944-20020.40 45. The method of any one of claims 31-43, wherein the nucleic acid is an RNA molecule.

46. The method of claim 45, wherein the RNA is part of a recombinant virous vector.

47. The method of any one of claims 31-46, wherein the contacting for each nucleic acid is in vitro.

48. The method of any one of claims 31-46, wherein the contacting for each nucleic acid is in vivo.

49. The method of claim 48, wherein each contacting comprises injection.

50. The method of claim 49, wherein the injection is intradermal injection, subcutaneous injection, intramuscular injection, intraocular injection, intrathecal injection, or intravenous injection, optionally wherein the injection is intradermal injection or subcutaneous injection.

51. The method of any one of claims 31-50, wherein each contacting step occurs concurrently.

52. The method of any one of claims 31-50, wherein at least one contacting step occurs sequentially with another contacting step.

53. The method of any one of claim 31-52, wherein the mammalian cell does not produce at least one of the first enzyme, the second enzyme and the third enzyme in the absence of one or more of the contacting steps.

54. The method of any one of claim 31-52, wherein the mammalian cell produces none of the first enzyme, the second enzyme and the third enzyme in the absence of one or more of the contacting steps. 43sf-6697944Docket No.: 69944-20020.40 55. The method of claim 51, wherein each of the nucleic acids is part of a single nucleic acid molecule.

56. The method of claim 51, wherein at least two of the nucleic acids are part of a single nucleic acid molecule.

57. The method of claim 51, wherein each of the nucleic acids is part of a separate nucleic acid molecule.

58. The method of claim 56 or claim 57, wherein all of the nucleic acid molecules is present in a single composition.

59. A composition comprising a first nucleic acid comprising a coding sequence (CDS) of a proprotein, a second nucleic acid comprising a CDS of a first enzyme capable of cleaving the proprotein to form a mature protein.

60. The composition of claim 59, further comprising a third nucleic acid comprising a CDS of a second enzyme capable of cleaving the proprotein to form the mature protein.

61. The composition of claim 60, further comprising a fourth nucleic acid comprising a CDS of a third enzyme capable of cleaving the proprotein to form the mature protein.

62. The composition of any one of claims 59-61, further comprising a further nucleic acid comprising a CDS of a chaperone.

63. The composition of any one of claims 59-62, wherein each of the nucleic acids is part of a separate nucleic acid molecule.

64. The composition of any one of claims 61-63, wherein the proprotein is preproinsulin, the first enzyme is carboxypeptidase E (CPE), the second enzyme is proprotein convertase 1 / 3 (PCSK1), and the third enzyme is proprotein convertase 2 (PCSK2). 44sf-6697944Docket No.: 69944-20020.40 65. The composition of any one of claims 60-63, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

66. The composition of any one of claims 60-63, wherein the proprotein is proglucagon, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2).

67. The composition of any one of claims 60-63, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

68. The composition of any one of claims 60-63, wherein the proprotein is proopiomelanocortin (POMC), the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 2 (PCSK2).

69. The composition of any one of claims 60-63, wherein the proprotein is proBDNF, the first enzyme is carboxypeptidase E (CPE), and the second enzyme is proprotein convertase 1 / 3 (PCSK1).

70. The composition of any one of claims 59-69, wherein the composition is a pharmaceutical formulation further comprising a pharmaceutically acceptable excipient.

71. The composition of 70, further comprising chitosan. 45sf-6697944

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