Sequences for optimal processability of secreted therapeutic proteins delivered via vectors administered as a subcutaneous injection

Abstract

The present invention, at least in part, relates to nucleic acid sequences that can be used in therapeutic gene therapies. More specifically, the present invention provides compositions and methods related to the use of nucleic acid sequences that can promote optimal production and / or cellular export of therapeutic proteins from subcutaneous adipocytes.

Description

[0001] Attorney Docket No. R0872.70005WO00

[0002] SEQUENCES FOR OPTIMAL PROCESSABILITY OF SECRETED THERAPEUTIC PROTEINS DELIVERED VIA VECTORS ADMINISTERED AS A SUBCUTANEOUS INJECTION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 748,391, filed January 22, 2025, entitled “NUCLEIC ACID SEQUENCES FOR OPTIMAL PROCESSABILITY OF SECRETED THERAPEUTIC PROTEINS DELIVERED VIA VECTORS ADMINISTERED AS A SUBCUTANEOUS INJECTION IN WHITE SUBCUTANEOUS ADIPOCYTES”, the entire disclosure of which is hereby incorporated by reference in its entirety.

[0004] FIELD

[0005] The present invention, at least in part, relates to nucleic acid sequences that can be used in therapeutic gene therapies. More specifically, the present invention provides compositions and methods related to the use of nucleic acid sequences that can promote optimal production and / or cellular export of therapeutic proteins from subcutaneous adipocytes.

[0006] BACKGROUND

[0007] Traditionally, adipocytes have been a neglected cell type, and little is known about optimal genetic sequences to enable protein production, secretion, export, and signal peptide cleavage. These elements, however, can be important to obtain optimal productivity levels from therapeutic transgenes delivered to treat disease.

[0008] SUMMARY

[0009] Using subcutaneous adipocytes to produce therapeutic proteins in vivo has a number of advantages including ease of access, abundance of resident cells, and their non-life / nonfunction sustaining nature. However, despite the advantages, adipocytes are not generally targeted by gene therapy since gene therapy has been thought of as a gene-corrective approachaimed at cell types that require a given gene to function properly. Since gene delivery to adipocytes is uncommon, little is known about optimal genetic sequences to enable protein production, secretion, export, and signal peptide cleavage - factors important for producing therapeutic proteins in adipocytes in vivo. The aforementioned elements can be important to obtain optimal productivity levels from therapeutic transgenes delivered to treat disease, particularly for proteins that need to biodistributed to the general circulation or target specific organs that are not optimally served by the general circulation.

[0010] The present invention provides nucleic acid sequences for maximizing the efficiency of production, processability, and / or secretion of therapeutic proteins delivered as a nucleic acid therapy to subcutaneous adipocytes. Thus, provided herein, at least in part, are compositions and methods related to the nucleic acid sequences, motifs, forms, and structures that can provide optimal translation, secretion, cleavage, and / or or generally processing of proteins intended for export from subcutaneous adipocytes.

[0011] In one aspect, a nucleic acid construct for vector-mediated delivery via a subcutaneous injection to white subcutaneous adipocytes intended to express secreted therapeutic proteins for eventual systemic or predominantly systemic biodistribution is provided. The nucleic construct may be comprised of: one or more sequences encoding at least one efficiently processable secretion peptide which contains an adipocyte-processable motif; optionally, one or more sequences encoding at least one amino acid spacer sequence, which optionally contains a cleavage-enhancing sequence; one or more sequences encoding a therapeutic peptide, protein, or fusion protein, and one or more spanning sequences comprised of a nucleic acid sequence that encodes an amino acid sequence which spans the secretion peptide sequence and the therapeutic protein sequence and enables efficient adipocyte-expressed endopeptidase processing.

[0012] In one embodiment of any one of the compositions or methods provided herein, the one or more sequences encoding at least one efficiently processable secretion peptide contains a stretch of 2-4 leucines in the N-region, with a downstream motif of LLLXL in the H-region, and a C-region motif of AXX, where A is the n=-3 amino acid preceding the cleavage site. In one embodiment of any one of the compositions or methods provided herein, the one or more sequences encoding at least one efficiently processable secretion peptide contains the motif LL-(5 to 8 X)-LLLXL-(4 to 6 X)-AXX. In one embodiment of any one of the compositions or methods provided herein, the one or more sequences encoding at least one efficientlyprocessable secretion peptide contains 4 characteristic regions starting with an M(P / E)XXXXXX containing at least two leucines, followed by two leucine-rich regions containing at least two leucines in a row, followed by an LLL(A / L)L stretch and an AXX terminal three amino acid motif. In one embodiment of any one of the compositions or methods provided herein, the one or more sequences encoding at least one efficiently processable secretion peptide contains at least 3 characteristic regions starting with an M(P / E) motif, which is later followed by at least one leucine- alanine stretch of either LLLAL, LLAL, LLALL, LALL and an GX(L / V / A)AXX terminal motif. In one embodiment of any one of the compositions or methods provided herein, the one or more sequences encoding at least one efficiently processable secretion peptide contains at least 3 characteristic regions starting with an M(P / E) motif, which is later followed by at least one leucine- alanine stretch containing at least one dileucine and one trileucine flanked by an alanine or glycine and an GX(L / V / A)AXX terminal motif. In one embodiment of any one of the compositions or methods provided herein, the one or more sequences encoding at least one efficiently processable secretion peptide, contains the GX(L / V / A)AXX terminal motif derived at least 50% from an adipocyte secreted protein.

[0013] In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct encodes a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is the same as the last amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is the same as the first amino acid of the therapeutic protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct encodes a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are the same as the last two amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is the same as the first amino acid of the therapeutic protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct encodes a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is the same as the last amino acid of the natural secretionsignal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are the same as the first two amino acid of the therapeutic protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct encodes a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are the same as the last two amino acids of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are the same as the first two amino acid of the therapeutic protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct encodes a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is of a similar charge or hydrophobicity as the last amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is of a similar charge or hydrophobicity as the first amino acid of the therapeutic protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct encodes a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are of a similar charge or hydrophobicity as the last two amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is of a similar charge or hydrophobicity as the first amino acid of the therapeutic protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct encodes a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is of a similar charge or hydrophobicity as the last amino acid of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are of a similar charge or hydrophobicity as the first two amino acid of the therapeutic protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct encodes a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretionsignal are of a similar charge or hydrophobicity as the last two amino acids of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are of a similar charge or hydrophobicity as the first two amino acid of the therapeutic protein.

[0014] In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first three amino acids of said protein, which follows the secretion signal is of approximately the same charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first two amino acids of said protein, which follows the secretion signal is of approximately the same charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first amino acid of said protein, which follows the secretion signal is an amino acid of a similar charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first three amino acids of said protein, which follows the secretion signal is the same amino acid as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first two amino acids of said protein, which follows the secretion signal is the same amino acid as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semisynthetic sequence derived from said protein, with the last 12 base pairs of the secretion signalencoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second postcleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted proteinexpressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, thenucleic acid construct is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the firstor second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V7T]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encodingnucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second postcleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, where the second X is the last pre-clevage or first post-cleavage site amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytesor a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V7G]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, which contains at least a stretch encoding 5 amino acids with the motif containing at least two Methionines and at least an Alanine, Glycine, Lysine, or Arginine, followed by a downstream stretch encoding a sequence of at least 5 amino acids with at least two Lysines and at least an Alanine, Valine, or Isoleucine, followed by a sequence encoding the motif [A / V / S / T / G]X[A / G / S / T]X where the last X amino acid is the last pre-cleavage or first postcleavage amino acid. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, which contains at least the following motif [A / V / S / T / G]X[A / G / S / T]Z where Z is the first amino acid of the therapeutic protein or peptide and is the same as the first amino acid of the secreted adipocyte expressed protein, or Z is the last amino acid of the secretion signal and is the same as the last amino acid of the therapeutic protein’s natural secretion signal. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, which contains at least the following motif [A / V / S / T / G]X[A / G / S / T]XZ where the second X is the first amino acid of the therapeutic protein or peptide and is the same as the first amino acid of the secreted adipocyte expressed protein and Z is the second amino acid of the therapeutic protein or peptide and is of a similar charge or polarity to the second amino acid of the secreted adipocyte expressed protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding asecretion signal from a different secreted protein expressed in adipocytes or a semi-synthetic sequence derived from said protein, which contains at least the following motif [A / V7S / T / G]X[A / G / S / T]XZ where the second X is the first amino acid of the therapeutic protein or peptide and is the same as the first amino acid of the secreted adipocyte expressed protein and Z is the second amino acid of the expressed adipocyte secreted protein and is of a similar charge or polarity to the second amino acid of the therapeutic peptide or protein. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first amino acid of said protein, which follows the secretion signal is the same amino acid as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal.

[0015] In one embodiment of any one of the compositions or methods provided herein, the sequence encoding an efficiently processable secretion peptide, encodes in addition to the therapeutic protein or peptide of interest, at least the first three amino acids, and another 5 amino acid stretch, and the last 4 amino acids of at least one of the following sequences: MPPWGAALALILAVLALLGLLGP, MLLLGAVLLLLALPGHDQ, MDPPRPALLALLALPALLLLLLAGARA, MVRMVPVLLSLLLLLGPAVP, MAHAHIQGGRRAKSRFVVCIMSGARSKLALFLCGCYVVALG, MLLLVTSLLLCELPHPAFLLIP, MKFLLLVLAALGFLTQVIPASA, MELDRAVGVLGAATLLLSFLGMAWA, MDLWQLLLTLALAGSSDA, MARLLQASCLLSLLLAGFVS, MRPFFLLCFALPGLLHA, MHWGTLCGFLWLWPYLFYVQA, MESKALLVLTLAVWLQSLTASRGGVAA, MECLYYFLGFLLLAARLPLDAA, MQLKIMPKKKRLSAGRVPLILFLC, MRGANAWAPLCLLLAAATQLSRQ, MVPGAAGWCCLVLWLPACVAA, MAPTWGPGMVSVVGPMGLLVVLLVGGCAA, MAKRSRGPGRRCLLALVLFCAWGTLA, MAPSAWAICWLLGGLLLHGGSS, MRLSPAPLKLSRTPALLALALPLAAALA, MHKEAEMLIGPQLDEKRWGWRLGDGSAAPPFLPQALSFLLLLPLASA, METPAWPRVPRPETAVARTLLLGWVFAQVAGA, MPGSPRPAPSWVLLLRLLALLRPPGLGEA, MPWPLLLLLAVSGAQT, MSPLLRRLLLAALLQLAPAQA, MRSGEPACTMDQARGLDDAAA, MELWGAYLLLCLFSLLTQVTT, MLAATVLTLALLGNAHA, MHSWERLAVLVLLGAAACAA, MKFVPCLLLVTLSCLGTLG,MGSRGQGLLLAYCLLLAFASGLVLS, MNQLSFLLFLIATTRGWS, MSGAPTAGAALMLCAATAVLLSAQG, MKGLRSLAATTLALFLVFVFLGNSSC, MKSLPILLLLCVAVCSA, MHLLAILFCALWSAVLA, MKTLLLLAVIMIFGLLQAHG, MKASSLAFSLLSAAFYLLWTPSTG, MKLVSVALMYLGSLAFLGADT, MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAG, MDYLLMIFSLLFVACQG, MNSFSTSAFGPVAFSLGLLLVLPAAFPAP, MHSSALLCCLVLLTGVRA, MLLLLLLLLLLALALA, MWWRLWWLLLLLLLLWPMVWA, MDMRVPAQLLGLLLLWLRGARC, MPLLLLLPLLWAGALA, MDAMKRGLCCVLLLCGAVFVSPS, MLLLLLLLGLRLQLSLG. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding an efficiently processable secretion peptide contains an adipocyte-processable motif [A / G / S]X[A / G / S / L]R immediately preceding the signal peptide cleavage site. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding an efficiently processable secretion peptide contains an adipocyte-processable motif [A / G / S]X[A / G / S / L]R and at least 60% of a secretion signal from a protein expressed in adipocytes and is followed by the first amino acid of the therapeutic peptide or spacer sequence, which is the same as the first amino acid of the aforementioned adipocyte expressed protein. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding an efficiently processable secretion peptide contains an adipocyte-processable motif [A / G / S]X[A / G / S / L]R and at least 60% of a secretion signal from a protein expressed in adipocytes and is followed by the first amino acid of the therapeutic peptide or spacer sequence, which has approximately the same charge, polarity, or hydrophobicity as the first amino acid of the aforementioned adipocyte expressed protein. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding an efficiently processable secretion peptide contains the following motif [LAV]L[X]nL[L / F / V][L / A]L[L / A][X]mpreceding the last 4 amino acids of the secretion signals such that n is between 2 and 6 and m is between 2 and 7. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding an efficiently processable secretion peptide contains the following motif [L / F / V][L / A]L[L / A][X]npreceding the last 4 amino acids of the secretion signals such that n is between 1 and 8.

[0016] In one embodiment of any one of the compositions or methods provided herein, the nucleic acid sequence encodes at least the leading two amino acids, followed by at least 4 consecutive amino acids from the non-leading core, and the last 4 amino acids from the C-region from one or more of the following amino acid sequences MLLLGILTLAFAGRTAG,MSQTGSHPGRGLAGRWLWGAQPCLLLPIVPLSWLVWLLLLLLASLLPSARL, MQFVSWATLLTLLVRDLA, MKRLPLLVVFSTLLNCSYT, MLLLGAVLLLLALPGHDQ, MKLVSVALMYLGSLAFLGADT, MLSQLAMLQGSLLLVVATMSVAQQ, MSGAPTAGAALMLCAATAVLLSAQG, MLKKPLSAVTWLCIFIVAFVSHPAWL, MGSRGQGLLLAYCLLLAFASGLVLS, MIIDSSRIPSFTQLHSTMTRAPLLLLCVALVLLGHVNG, MEKKCTLYFLVLLPFFMILVTA, MRGTPKTHLLAFSLLCLLSKVRT, MLAATVLTLALLGNAHA, MHSWERLAVLVLLGAAACAA, MELWGAYLLLCLFSLLTQVTT, MVPDTACVLLLTLAALGASG, MDFPCLWLGLLLPLVAA, MGASSPRSPEPVGPPAPGLPFCCGGSLLAVVVLLALPVAWG, MRPQGPAASPQRLRGLLLLLLLQLPAPSSA, MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAG, MKIAVLFCFFLLIIFQTDFG, MDYLLMIFSLLFVACQG, MWGRLLLWPLVLGFSLS, MRRGRLLEIALGFTVLLASYTSHG, MKFVPCLLLVTLSCLGTLG, MDLWQLLLTLALAGSSDA, MHSSALLCCLVLLTGVRA, MKASSLAFSLLSAAFYLLWTPSTG, MDSYLLMWGLLTFIMVPGCQA, MNSFSTSAFGPVAFSLGLLLVLPAAFPAP, MDGLPGRALGAACLLLLAAGWLGPEAWG, MLLLLGLCLGLSLCVG, MNQLSFLLFLIATTRGWS, MHWGTLCGFLWLWPYLFYVQA, MTLTLSVLICLGLSVGPRTCVQA, MRFAWTVLLLGPLQLCALVHC, MESKALLVLTLAVWLQSLTASRGGVAA, MLTPPLLLLLPLLSALVAA, MARCFSLVLLLTSIWTTRL, MNCQQLWLGFLLPMTVSG, MKSLPILLLLCVAVCSA, MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRT, MARRAGGARMFGSLLLFALLAAGV, MRGANAWAPLCLLLAAATQLSRQ, MKTLLLLAVIMIFGLLQAHG, MAWKTLPIYLLLLLSVFVIQQVSS, MLTTLLPILLLSGWAFC, MHLLAILFCALWSAVLA, MAPSAWAICWLLGGLLLHGGSS, MKGLRSLAATTLALFLVFVFLGNSSC, MGSPAHRPALLLLLPPLLLLLLLRVPPSRS, MASQLTQRGALFLLFFLTPAVTP, MLAPRGAAVLLLHLVLQRWLAAGAQA, MPGSPRPAPSWVLLLRLLALLRPPGLGEA, MRVLLAALGLLFLGALRA, MSLQEMFRFPMGLLLGSVLLVASAPATL, MRTVVLTMKASVIEMFLVLLVTGVHS, MRARPQVCEALLFALALQTGVCYG, MLLLLLLLLLLALALA, MWWRLWWLLLLLLLLWPMVWA, MDMRVPAQLLGLLLLWLRGARC,MPLLLLLPLLWAGALA, MDAMKRGLCCVLLLCGAVFVSPS, MLLLLLLLGLRLQLSLG, MGPTSGPSLLLLLLTHLPLALG, MSSFSTTTVSFLLLLAFQLLGQTRA, MLLFLLSALVLLTQPLGYLE, MARRSSFQSCQIISLFTFAVGVNICLG, MRRLLIPLALWLGAVGVGVA, MFTIKLLLFIVPLVIS, MPPWGAALALILAVLALLGLLGP, MDPPRPALLALLALPALLLLLLAGARA, MVRMVPVLLSLLLLLGPAVP, MAHAHIQGGRRAKSRFVVCIMSGARSKLALFLCGCYVVALG, MLLLVTSLLLCELPHPAFLLIP, MKFLLLVLAALGFLTQVIPASA, MELDRAVGVLGAATLLLSFLGMAWA, MARLLQASCLLSLLLAGFVS, MRPFFLLCFALPGLLHA, MECLYYFLGFLLLAARLPLDAA, MQLKIMPKKKRLSAGRVPLILFLC, MVPGAAGWCCLVLWLPACVAA, MAPTWGPGMVSVVGPMGLLVVLLVGGCAA, MAKRSRGPGRRCLLALVLFCAWGTLA, MRLSPAPLKLSRTPALLALALPLAAALA, MHKEAEMLIGPQLDEKRWGWRLGDGSAAPPFLPQALSFLLLLPLASA, METPAWPRVPRPETAVARTLLLGWVFAQVAGA, MPWPLLLLLAVSGAQT, MSPLLRRLLLAALLQLAPAQA, MRSGEPACTMDQARGLDDAAA, MRKRAPQSEMAPAGVSLRATILCLLAWAGLAAG, MTSKLAVALLAAFLISAALC, MKIILWLCVFGLFLATLFPISWQ, MPVESGLSSEDSASSESFA, METPAQLLFLLLLWLPDTTG, MKSIYFVAGLFVMLVQGSWQ, MALWMRLLPLLALLALWGPDPAAA, MKWVTFISLLFLFSSAYSRGVFRRD, MKWVTFISLLFSSAYS, MKALCLLLLPVLGLLVSS, MYSAPSACTCLCLHFLLLCFQVQVLVA, MKCLLYLAFLFIGVNC, MPGRAPLRTVPGALGAWLLGGLWAWTLCGLCSLGAVG MKIILWLCVFGLFLATLFPISWQMPVESGLSSEDSASSESFA. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid sequence encodes at least 5 consecutive amino acids from the secretion signals of the following human proteins ADIPOQ, ADM, ANGPTL4, AOC3, APOB, AZGP1, BTD, C1QTNF1, CETP, CFD, CLEC3B, CSF2RA, CXCL3, DEFB132, EDN1, ENPP1, FCN2, FGFBP2, GHR, GPX3, IL10, IL20, IL6, ITLN1, LAMB3, LEP, LPL, MAPT, MMP3, NMB, NRCAM, PCOLCE2, PLA2G2A, PRADC1, PRXL2A, PTPRS, PTX3, PXDN, RBP4, SEMA3G, SPON1, SPX, SSC4D, TF, TIMP4, TSKU, VEGFB, or ZBED3.

[0017] In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes a flexible amino acid spacer sequence that follows the signalpeptide cleavage site and is comprised of an amino acid sequence of 2-55 amino acids in length and does not form a structured alpha helix, beta strand, hairpin loop, or other structured motif when followed by the therapeutic protein or peptide sequence. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes a flexible amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of at least a 4 amino acid stretch of one or more of GGGGSLVPRGSGGGGS, GSGSGS, GSGSGSGSGSGSGSGS, GGGGSLVPRGSGGGG, GGSGGHMGSGG, GGSGGSGGSGG, GGSGG, GGSGGGGG, GSGSGSGS, GGGSEGGGSEGGGSEGGG, AAGAATAA, GGGGG, GGSSG, GSGGGTGGGSG, GT, GSGSGSGSGGSG, GSGGSGGSGGSGGS, GSGGSGSGGSGGSG, SPNSASHSGSAPQTSSAPGSQ, GGGSGGGSGGGSGGGS, GGSG, GGSGGSG, GGSGGSGGSG, GGSGGSGGSGGSGSG, QPELQKPFKYTTVTKRSRRIRPTHPA, GGGGS, SGSG, APSVAPEPDGC, AAAAA, PAAAA, GEAAEGPAAA, AAGVGGERSS, GGPSGAGAGDE, VRTHGTLESVNGPKA, DQKVRPNEENNKDADL, GVKDTD, LPVQNGCPESAMEMN, (GGGGS)n, GGGG, or GGGGGG. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes a semi-flexible amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of an amino acid sequence of 2-55 amino acids in length and features a semi-structured motif enriched with a combination of proline, alanine, and serine. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes a semi-flexible amino acids spacer sequence that follows the signal peptide cleavage site and is comprised of at least a 4 amino acid stretch of one or more of GSPAG, GGASPAGG, or GGASPAAPAPAG. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes a rigid amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of an amino acid sequence of 2-55 amino acids in length that can form a structured alpha helix, beta strand, hairpin loop, or other structured motif when followed by the therapeutic protein or peptide sequence. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes a rigid amino acid spacer sequence that follows the signal peptide cleavage site and features poly-proline, poly-proline-threonine, poly-alanine, and may be stabilized by glutamic acid / lysine salt bridges. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes a rigid amino acid spacer sequence that follows the signal peptide cleavage site and features the following motif (XP)n where n is between 2 and 28 inclusively and X is an Alanine, Lysine, orGlycine. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes a rigid amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of at least a 4 amino acid stretch of one or more of GPPPG, GPPPPPPPG, GTPTPTPTPTG, or GGAEAAAKEAAAKAGG. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes an amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of at least one endopeptidase cleavage site. In one embodiment of any one of the compositions or methods provided herein, the optional spacer sequence encodes an amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of PPTIFFRL, KPIEFFRL, R[S / K][R / S / K][R / K]XXX[G / E], XXX[L / F]VXXX, PFHL[L / V / K][V / I / Y][Y / H / G][S / N], DEVD[G / S]XXX, X[E / D]XDXXXX, QTGGKXEX, PQGIAGQ, DXXD, XRXKRRX, KRIKR, RGRR, RGKR, LRGGKXXX, or [D / L]ETD[G / S / A]XXX.

[0018] In one embodiment of any one of the compositions or methods provided herein, the amino acid sequence which is part of the optional spacer sequence or near or at the end of the signal peptide and is processable by one or more of trypsin, chymotrypsin, caspases, elastase, thermolysin, pepsin, glutamyl endopeptidase, plasmin, MMPs, calpain, furin, renin, neprilysin or related or other mammalian endopeptidase. In one embodiment of any one of the compositions or methods provided herein, the amino acid sequence which is part of the optional spacer sequence and is specifically processable by mammalian extracellular endopeptidases, following secretion to expose one or more active sites at the N-terminal of the therapeutic protein. In one embodiment of any one of the compositions or methods provided herein, the amino acid sequence which is part of the optional spacer sequence and is specifically processable by Neprilysin, kallikreins, cathepsins, MMPs, ADAMTS, or plasmin following secretion from the cell.

[0019] In one embodiment of any one of the compositions or methods provided herein, the sequence encoding a therapeutic peptide encodes glucagon-like peptide 1, dipeptidyl peptidase-4-resistant glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, exendin-4, or derivatives, or dual- or triple-agonists, at least one of the antagonistic targets of which is the GLP-1 receptor. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding a therapeutic peptide encodes insulin, including furin-cleavable insulin, enhanced-stability insulin, seleno-insulin, four-disulfide insulin analog, or derivatives. In one embodiment of any one of the compositions or methods provided herein, the sequenceencoding a therapeutic peptide encodes human growth hormone, human growth hormone antagonist, human growth hormone agonist, insulin receptor agonist or antagonist, Thyroid Stimulating Hormone (TSH), Adrenocorticotropic Hormone (ACTH), Vasopressin (Antidiuretic Hormone - ADH), Oxytocin, Parathyroid Hormone (PTH), Calcitonin, Glucagon, Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), somatostatin receptor agonist or antagonist, Corticotropin-Releasing Hormone (CRH), Amylin, Prolactin, Natriuretic Peptides (ANP and BNP), pancreatic polypeptide, Insulin-like Growth Factor 1 (IGF-1), Leptin, Peptide YY, Ghrelin, Cholecystokinin (CCK), Adiponectin, peptide from the Relaxin family, Secretin, Gastrin, GIP agonist, GIP-GLP dual agonist or other peptide hormone, polypeptide hormone, dual or triple agonist, or a fusion thereof with at least one fragment of another functional protein. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding a therapeutic peptide encodes a peptide hormone or peptide fragment of a natural protein that has potential therapeutic activity.

[0020] In one embodiment of any one of the compositions or methods provided herein, the sequence encoding a therapeutic protein encodes at least a fraction of one or more of Adalimumab, Rituximab, Trastuzumab, Bevacizumab, Infliximab, Pembrolizumab, Atezolizumab, Etanercept, Cetuximab, Ranibizumab, Omalizumab, Natalizumab, Golimumab, Tocilizumab, Ustekinumab, Daratumumab, Ixekizumab, Secukinumab, Alemtuzumab, Eculizumab, Palivizumab, Abciximab, Basiliximab, Tositumomab, Panitumumab, Ofatumumab, Trastuzumab emtansine, Mogamulizumab, Obinutuzumab, Sarilumab, Brentuximab vedotin, Dinutuximab, Efgartigimod or other monoclonal, bispecific, or multispecific antibody, antibody fragment, nanobody, or fusion thereof with at least one other functional fragment of another protein. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding a therapeutic protein encodes at least IL-2, alpha-L-iduronidase, iduronate-2- sulfatase, alpha-glucosidase, alpha-galactosidase, glucocerebrosidase, phenylalanine hydroxylase, Factor VIII, Factor IX, Factor XI, or a secreted protein or enzyme the function of which is lost in a human genetic disorder, or their analog, homolog, ortholog, or their derivative. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding a therapeutic protein encodes a protein or a functional protein fragment or subunit with potential therapeutic activity.

[0021] In one embodiment of any one of the compositions or methods provided herein, the sequence encoding a fusion protein encodes a fusion of a fragment of IL-12 and IL-23, an anti-CD30 antibody and monomethyl auristatin E, alpha-L-iduronidase enzyme and IGF-1, TNF-alpha receptor and an Fc fragment of an antibody, VEGFR1 and VEGFR2 or other fusion protein between two or more fragments of a protein at least one of which is functional. In one embodiment of any one of the compositions or methods provided herein, the sequence encoding a fusion protein encodes a fusion between at least one protein or functional protein fragment and another protein or protein fragment, either of which has potential therapeutic activity.

[0022] In one embodiment of any one of the compositions or methods provided herein, the spanning sequence encodes one or more of the following amino acid sequences, AXAXYXXX, AXAXXXEG, AXAXXXQG, AXAX[P / K]P[T / I][I / E], AXAXR[S / K][R / S / K][R / K], AXAXXXX[L / F], AXAX[P / D][F / E][H / V][L / D], AXAXX[E / D]XD, AXAX[Q / P][T / Q]G[G / I], AXAXDXXD, AXAXXRXK, [A / V / S / T / G]X[A / G / S / T]XYXXX, | A / V / S / T / G|X| A / G / S / T|XXXEG, [A / V / S / T / G]X[A / G / S / T]XXXQG, [A / V / S / T / G]X[A / G / S / T]X[P / K]P[T / I][I / E], [A / V / S / T / G]X[A / G / S / T]XR[S / K][R / S / K][R / K], [A / V / S / T / G]X[A / G / S / T]XXXX[L / F] , [A / V / S / T / G]X[A / G / S / T]X[P / D] [F / E] [H / V] [L / D] , | A / V / S / T / G |X| A / G / S / T| XX| E / D |XD, [A / V / S / T / G]X[A / G / S / T]X[Q / P] [T / Q]G[G / I] , [A / V / S / T / G]X[A / G / S / T]XDXXD, or [A / V / S / T / G]X[A / G / S / T]XXRXK, which span the secretion signal cleavage site starting at the n-4, n-3, n-2, or n-1 position, where n-1 is the amino acid immediately preceding the cleavage site. In one embodiment of any one of the compositions or methods provided herein, the spanning sequence encodes an amino acid sequence, which spans 1 to 4 amino acids of the therapeutic protein and 1 to 4 amino acids of the secretion peptide.

[0023] In one embodiment of any one of the compositions or methods provided herein, the white subcutaneous adipocytes are part of human, canine, feline, equine, or other mammalian tissues.

[0024] In one embodiment of any one of the compositions or methods provided herein, the nucleic acid construct is an RNA, DNA, or RNA-DNA hybrid construct, with or without chemical modifications.

[0025] In one embodiment of any one of the compositions or methods provided herein, the vector is a non-antigenic or minimally antigenic vector that does not elicit a neutralizing immune response following subcutaneous administration at levels anticipated to be clinically effective.In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered as part of a 0.1 to 50mL subcutaneous injection. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered by physical, chemical, or biochemical methods. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered by viral vectors. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered by virus like particles. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered by polymeric nanoparticles. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered by lipid nanoparticles. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered by liposomes. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered by transfection. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are delivered by electroporation or particle mediated transfer. In one embodiment of any one of the compositions or methods provided herein, the sequences and / or constructs are as described in any one of the examples provided herein.

[0026] In one embodiment of any one of the compositions or methods provided herein, the sequences have been codon optimized. In one embodiment of any one of the compositions or methods provided herein, the sequences have been codon optimized for a given species using codon frequency usage algorithms for said species. In one embodiment of any one of the compositions or methods provided herein, the sequences are used in tandem with other sequences promoting increased gene expression including one or more of promoters, terminators, insulators, 3’UTRs, 5’UTRs, enhancers, or combinations thereof. In one embodiment of any one of the compositions or methods provided herein, the sequences are used as part of therapies for human or veterinary pathologies.

[0027] In one embodiment of any one of the compositions or methods provided herein, the nucleic acid sequences encode more than one natural or in silico optimized secretion signal. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid sequences encode at least a fragment of one secretion signal and at least a fragment of one other secretion signal.In one embodiment of any one of the compositions or methods provided herein, the nucleic acid, amino acid sequences, or combinations thereof are as described in any one of the examples provided herein.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the amount of Exenatide construct by dose (Example 1).

[0029] FIG. 2 provides a table of secretion signals as discussed in Example 2.

[0030] FIG. 3 provides the example construct of Example 5.

[0031] FIG. 4 provides results from the alignment discussed in Example 6.

[0032] FIG. 5 shows the example construct of Example 6.

[0033] FIG. 6 shows the Signal P 6.0 results described in Example 8.

[0034] FIG. 7 shows alignment of Example 9.

[0035] FIG. 8 illustrates the alignment pattern described in Example 9.

[0036] FIG. 9 shows the Signal P 6.0 results described in Example 9.

[0037] FIG. 10 shows the signal sequence rankings and strength described in Example 11. FIG. 11 shows in vivo results described in Example 11.

[0038] FIG. 12 shows the titer over time described in Example 11.

[0039] DETAILED DESCRIPTION

[0040] The use of subcutaneous adipocytes to produce therapeutic proteins in vivo has a number of advantages over repeat subcutaneous protein administration via injection. Some such advantages include reduced pharmacokinetic spikes and therefore an optimized pharmacokinetic profile of the treatment, reduced injection burden, reduced cost, and / or increased treatment efficacy due to eliminated potential for missed dose or non-compliance. Adipocytes are also beneficial targets for nucleic acid therapy to facilitate protein expression due to their simple accessibility via a subcutaneous injection, abundance of resident cells in the subcutis, and their non-life / non-function sustaining nature. While other cell types are alsopresent in the subcutaneous tissues, adipocytes are a major cell type in the subcutaneous adipose tissues, and the white subcutaneous adipocytes are at least locally a non-function and non-life sustaining organ.

[0041] The present invention generally pertains to nucleic acid sequences at least a part of which encode amino acid sequences of therapeutic peptides or proteins. The nucleic acid sequences described in the present invention can be for vector-mediated delivery using either viral or non-viral vectors specifically via a subcutaneous injection or injection into adipose tissues. The present invention generally describes delivery of genetic sequences to adipocytes, at least a part of which encode therapeutic peptides or proteins, such as delivery of nucleic acid sequences to white subcutaneous adipocytes, preferably in some embodiments with adipocyte specificity or localization of the transgene. The present invention provides for the delivery of nucleic acid sequences to adipocytes, with a goal in some embodimentes of enabling eventual systemic or predominantly systemic biodistribution of the secreted therapeutic protein, peptide, fusion protein, or polypeptide. To enable effective delivery, transcription, translation, secretion, and / or, importantly, systemic efficacy, the therapeutic protein or peptide of interest may be at least initially attached to a secretion peptide. The secretion peptide can be encoded in the delivered nucleic acid and should preferably be processable by adipocytes to enable secretion of the therapeutic protein or peptide of interest and cleavage or exposure of the active N-terminal site, if one is located at the N-terminal. Thus, preferably, in some embodiments the nucleic acid sequences provided herein may contain one or more sequences encoding at least one efficiently processable secretion peptide which contains an adipocyte-processable motif, and one or more optional sequences encoding at least one amino acid spacer sequence, which optionally contains a cleavage-enhancing sequence, and one or more sequences encoding a therapeutic peptide, protein, or fusion protein, and one or more spanning sequences comprised of a nucleic acid sequence that encodes an amino acid sequence which spans the secretion peptide sequence and the therapeutic protein sequence and enables efficient adipocyte-expressed endopeptidase processing.

[0042] In general, the present invention describes nucleic acids, RNA or DNA, but preferably in some embodiments DNA if long term expression is required for vector-mediated delivery via one or more subcutaneous injections. The said vector or generally subcutaneous injection may be preferentially targeting adipocytes or may be passively transfecting cells in a tissue predominantly populated by adipocytes to obtain preferential transfection. The vector may be viral or non-viral, physical or chemical, or a combination thereof, with the goal of deliveringDNA to the adipocytes for durable expression at least for weeks, but preferably in some embodiments months, and more preferably in some embodiments years. Expression may be used for protein replacement therapies that are produced locally in the subcutis but eventually biodistributed systemically and may be expressed under the regulation of constitutive, circadian, inducible, or other promoters. As a result, the nucleic acids delivered can remain predominantly locally, in preferably in some embodiments mostly white subcutaneous adipocytes, but be expressed and secreted efficiently to enable beneficial systemic biodistribution. In general, in some embodiments, the sequences described encode at least one efficiently processable secretion peptide which contain at least one adipocyte-processable amino acid motif. The sequences delivered may also encode at least one amino acid spacer sequence, which optionally contains a cleavage-enhancing sequence and one or more sequences encoding a therapeutic peptide, protein, or fusion protein. In some embodiments of the present invention the delivered nucleic acid sequences may optionally be comprised of nucleic acid sequences that encode an amino acid sequence which spans the secretion peptide sequence and the therapeutic protein sequence and enables efficient adipocyte-expressed endopeptidase processing at one or more position along the stretch of the amino acid sequence.

[0043] In some embodiments of the present invention the nucleic acid sequences may encode an efficiently processable secretion peptide, said peptide may contain a stretch of 2-4 leucines in the N-region, with a downstream motif of LLLXL in the H-region, and a C-region motif of AXX, where A is the n=-3 amino acid preceding the cleavage site (where X is any amino acid, C-region is the C-terminal region of the secretion signal, and n-3 amino acid is the third position amino acid prior to the signal cleavage site). In an alternative embodiment the nucleic acid sequences may encode a peptide motif LL-(5 to 8 X)-LLLXL-(4 to 6 X)-AXX or an LL motif separated by 5 to 8 amino acids from a motif LLLXL, which is separated by 4 to 6 amino acids from a peptide motif AXX, where the final X is the n=-l or n=-2 amino acid or the last or second last amino acid prior to the cleavage site respectively.

[0044] In other embodiments the nucleic acid sequences may encode at least one efficiently processable secretion peptide, which contain 4 characteristic regions starting with an M[P / E]XXXXXX containing at least two leucines, followed by two leucine-rich regions containing at least two leucines in a row, followed by an LLL[A / L]L stretch and an AXX terminal three amino acid motif. Alternatively, the encoded amino acid sequence may contain at least 3 characteristic regions starting with an M[P / E] motif, which is later followed by at least one leucine-alanine stretch of either LLLAL, LLAL, LLALL, LALL and anGX[L / V / A] AXX terminal motif. In other embodiments the encoded secretion peptide sequence may contain at least 3 characteristic regions starting with an M[P / E] motif, which is later followed by at least one leucine-alanine stretch containing at least one dileucine and one trileucine flanked by an alanine or glycine and an GX[L / V / A]AXX terminal motif. And yet alternatively, the encoded secretion peptide sequence may contain the GX[L / V / A]AXX terminal motif derived at least 50% from an adipocyte secreted protein. The aforementioned motifs have been identified from secreted proteins that are expressed in adipocytes and appear processable and efficient in adipocytes, which are the predominant resident cell of the subcutis, as a result, the addition of aforementioned motifs to the secretion signals can greatly increase the productivity of the final gene therapy product and / or can enable the delivery of a lower dose to attain a given protein level in the general circulation, which can increase the safety profile of the treatment.

[0045] In other embodiments the nucleic acid construct delivered may encode a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is the same as the last amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is the same as the first amino acid of the therapeutic protein. Matching of the n=+l, n=+2, n=+3, and n=-l, n=-2, and n=-3 amino acids between the proteins from which the original secretion signal was derived from and the therapeutic protein intended to be coupled with said secretion signal increases processability and improves the reliability of releasing the appropriate amino acid N-terminal position.

[0046] In other embodiments of the present invention the nucleic acid constructs may encode a secretion signal and a therapeutic protein, and the said secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are the same as the last two amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is the same as the first amino acid of the therapeutic protein. In this one exemplary example, the n=-2 through n=+2 amino acids can be matched between the secreted protein and its secretion signal and the therapeutic protein and its natural secretion signal. Any amount of matching between the two in the n=-3 to n=+3 position can enhance processivity, and the matching may be exact or by charge, polarity, or steric hinderance (similarity of side chains).In other embodiments the nucleic acid sequence may encode at least a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is the same as the last amino acid of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are the same as the first two amino acids of the therapeutic protein. Alternatively, the nucleic acid construct may encode a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are the same as the last two amino acids of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are the same as the first two amino acid of the therapeutic protein. Yet alternatively, the nucleic acid construct may encode a secretion signal and a therapeutic protein, and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is of a similar charge or hydrophobicity as the last amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is of a similar charge or hydrophobicity as the first amino acid of the therapeutic protein. Alternatively, the last two amino acids of the secretion signal can be of a similar charge or hydrophobicity as the last two amino acid of the natural secretion signal of the therapeutic protein, and the first amino acid of the secreted protein from which the secretion signal is derived can be of a similar charge or hydrophobicity as the first amino acid of the therapeutic protein. Alternatively, the last amino acid of the secretion signal can be of a similar charge or hydrophobicity as the last amino acid of the natural secretion signal of the therapeutic protein, and the first two amino acids of the secreted protein from which the secretion signal is derived can be of a similar charge or hydrophobicity as the first two amino acid of the therapeutic protein. Alternatively, the last two amino acids of the secretion signal can be of a similar charge or hydrophobicity as the last two amino acids of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived can be of a similar charge or hydrophobicity as the first two amino acid of the therapeutic protein. Alternatively, the first three amino acids of the original secreted protein, which follows the secretion signal, can be of approximately the same charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following thesecretion signal. Alternatively, the first two amino acids of the original secreted protein, which follows the secretion signal, is of approximately the same charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. In another embodiment the nucleic acid encodes a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first amino acid of said protein, which follows the secretion signal is an amino acid of a similar charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. Alternatively, the first three amino acids of the adipocyte secreted protein, which follows the secretion signal are the same amino acid as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. Alternatively, the nucleic acid sequence encodes a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first two amino acids of said protein, which follows the secretion signal is the same amino acid as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal. The process of amino acid matching between the native adipocyte expressed and secreted protein and secretion signal to the native secretion signal of the therapeutic protein of interest can enhance processability and secretion of the therapeutic protein. Said matching can take place between the approximately n=-3 position to approximately n=+3 position and may be based on exact matching, matching of charge, pKa, hydrophilicity, hydrophobicity, or geometry (steric hindrance or radius of gyration of the amino acid side chain). Said matching may be efficient when only one amino acid is matched, even if the exact position in the sequence is not the same, but can be most efficient when the n=+l or n=-l amino acids are matched, and can be further enhanced when other amino acids are additionally matched.

[0047] The nucleic acid construct may contain a stretch of 24 to 240 base pairs, but preferably a stretch of 36 to 120 base pairs encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 15 or ideally the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, where the second X is the first pre-cleavage site amino acid or the first post-cleavage site amino acid. Alternatively, the nucleic acid construct can be comprised at least in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 24 to 240 base or preferably a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 15 or 12 base pairs of the secretion signal encoding nucleic acidsequence encoding an amino acid motif [A / V / S]X[A / G / S]X, where the second X is the first pre-cleavage site or first post-cleavage site amino acid. Alternatively, the motif [A / V7S]X[A / G / S]X can be followed in any scenario by 3 to 6 base pairs encoding an amino acid, which is the same the n=+l or n=+2 amino acid from either the secreted protein that the secretion signal was derived from or the therapeutic protein, or matches both proteins identically, by charge similarity, by hydrophobicity similarity, or by side chain similarity. The sequences may be semi-synthetic, natural, or fully synthetic and the motif may be [A / V / S]X[A / G / S]X, [A / V / T]X[A / G / S]X, [A / V / G]X[A / G / S]X, [A / V / S]X[A / G / T]X, [A / V7T]X[A / G / T]X, or [A / V / G]X[A / G / T]X. The aforementioned motif may end at the n=-2, n=-l, n=+l, or n=+2 position, but preferably at the n=-l or n=+l position and may optionally have one amino acid substituted by a similar amino acid. In another embodiment the nucleic acid construct may be comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 24 to 240 base pair sequence or ideally a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semisynthetic sequence derived from said protein, which contains at least a stretch encoding 5 amino acids with the motif containing at least two Methionines and at least an Alanine, Glycine, Lysine, or Arginine, followed by a downstream stretch encoding a sequence of at least 5 amino acids with at least two Lysines and at least an Alanine, Valine, or Isoleucine, followed by a sequence encoding the motif [A / V / S / T / G]X[A / G / S / T]X where the last X amino acid is the precleavage or post-cleavage amino acid.

[0048] In an alternative embodiment, the nucleic acid construct can encode at least a part of a secretion signal that ends in the following motif [A / V / S / T / G]X[A / G / S / T]Z where Z is the last pre-cleavage site amino acid or the first post cleavage site amino acid and is either the same as the last amino acid of the secretion signal from the therapeutic protein to be delivered following the secretion signal or the same as the therapeutic protein of interest’s first amino acid post its natural cleavage site. The said amino acid may be the same or similar based on charge, hydrophobicity, or side chain and may be optionally followed by another amino acid that is the same as the next amino acid in sequence when comparing the natural secretion signal and the original protein that it was derived from and the therapeutic protein of interest that it was appended to and its original secretion signal. Any signal preceding that may be natural, synthetic, or semi-synthetic, but is preferably similar to a natural secretion signal from a secreted protein naturally expressed in white subcutaneous adipocytes.In an alternative embodiment, the secretion signal sequence encoded by the nucleic acid, which is intended for subcutaneous delivery, may encode at least the first three amino acids, and another 3-7 amino acid stretch, and the last 4 amino acids of at least one of the following sequences: MPPWGAALALILAVLALLGLLGP, MLLLGAVLLLLALPGHDQ, MDPPRPALLALLALPALLLLLLAGARA, MVRMVPVLLSLLLLLGPAVP, MAHAHIQGGRRAKSRFVVCIMSGARSKLALFLCGCYVVALG, MLLLVTSLLLCELPHPAFLLIP, MKFLLLVLAALGFLTQVIPASA, MELDRAVGVLGAATLLLSFLGMAWA, MDLWQLLLTLALAGSSDA, MARLLQASCLLSLLLAGFVS, MRPFFLLCFALPGLLHA, MHWGTLCGFLWLWPYLFYVQA, MESKALLVLTLAVWLQSLTASRGGVAA, MECLYYFLGFLLLAARLPLDAA, MQLKIMPKKKRLSAGRVPLILFLC, MRGANAWAPLCLLLAAATQLSRQ, MVPGAAGWCCLVLWLPACVAA, MAPTWGPGMVSVVGPMGLLVVLLVGGCAA, MAKRSRGPGRRCLLALVLFCAWGTLA, MAPSAWAICWLLGGLLLHGGSS, MRLSPAPLKLSRTPALLALALPLAAALA, MHKEAEMLIGPQLDEKRWGWRLGDGSAAPPFLPQALSFLLLLPLASA, METPAWPRVPRPETAVARTLLLGWVFAQVAGA, MPGSPRPAPSWVLLLRLLALLRPPGLGEA, MPWPLLLLLAVSGAQT, MSPLLRRLLLAALLQLAPAQA, MRSGEPACTMDQARGLDDAAA, MELWGAYLLLCLFSLLTQVTT, MLAATVLTLALLGNAHA, MHSWERLAVLVLLGAAACAA, MKFVPCLLLVTLSCLGTLG, MGSRGQGLLLAYCLLLAFASGLVLS, MNQLSFLLFLIATTRGWS, MSGAPTAGAALMLCAATAVLLSAQG, MKGLRSLAATTLALFLVFVFLGNSSC, MKSLPILLLLCVAVCSA, MHLLAILFCALWSAVLA, MKTLLLLAVIMIFGLLQAHG, MKASSLAFSLLSAAFYLLWTPSTG, MKLVSVALMYLGSLAFLGADT, MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAG, MDYLLMIFSLLFVACQG, MNSFSTSAFGPVAFSLGLLLVLPAAFPAP, MHSSALLCCLVLLTGVRA, MLLLLLLLLLLALALA, MWWRLWWLLLLLLLLWPMVWA, MDMRVPAQLLGLLLLWLRGARC, MPLLLLLPLLWAGALA, MDAMKRGLCCVLLLCGAVFVSPS, MLLLLLLLGLRLQLSLG. Said sequences may be combined with any of the aforementioned downstream or cleavage site or cleavage region motifs and with any downstream therapeutic protein of interest. In general, one or more of any secretion signal can be used with one or more of any other secretion signal disclosed herein inwhole or in part such that a new combined signal is formed and precedes or follows the therapeutic protein or peptide of interest to enable the secretion of said therapeutic protein or peptide by adipocytes in vivo.

[0049] The aforementioned sequences may be combined in part or in whole with an adipocyte-processable cleavage site motif of [A / G / S]X[A / G / S / L]R immediately preceding the signal peptide cleavage site. Any cleavage site motif can be combined with at least 20% or preferably at least 60% of a secretion signal from a protein expressed in adipocytes and optionally followed by the first amino acid of the therapeutic peptide.

[0050] The nucleic acid sequences can encode a secretion signal, therapeutic protein or peptide of interest and optionally one or more spacer sequences between them, which may be flexible, rigid, or semi-rigid, and may contain at least one and up to 4 endopeptidase cleavage site, which may be recognized by intracellular or extracellular endopeptidases. Optionally, exopeptidase cleavage sites may be added to trim sequences in order to expose an appropriate N-terminal amino acid for therapeutic protein functionality. In line with said architecture any adipocyte processable motif, such as [A / G / S]X[A / G / S / L]R, where R is a pre-cleavage site amino acid may be coupled with a spacer sequence which has a leading amino acid that is the same or similar to the amino acid of the natural secreted protein that the secretion signal is derived from. The secretion signal can have an N-region (or N-terminal region), H-region (or generally hydrophobic region) and a C-region (or a C-terminal region that is nearest to the cleavage site). Some sequences that enhance processability of the secretion signal in adipocytes that span one or more of the aforementioned regions may include the following motif [LAV]L[X]nL[L / F / V][L / A]L[L / A][X]m preceding the last 3-5 amino acids of the secretion signal such that n is between 2 and 6 and m is between 2 and 7.

[0051] The nucleic acid sequence can encode a secretion signal that contains within it an efficiently processable motif derived from adipocyte secreted proteins, which contains [L / F / V][L / A]L[L / A][X]npreceding the last 4 amino acids of the secretion signals such that n is between 1 and 8. Said motif can be preceded by a synthetic, semi- synthetic, or natural secretion signal sequence that spans all of the N-region, and at least some of the H-region. The motif can be followed by a 2 to 8 amino acid stretch that encodes the C-region of the secretion signal in whole or in part, a cleavage site, and at least the starting sequence of the therapeutic protein or spacer sequence, which may be flexible, rigid, or semi-rigid. Flexible sequences are defined as those generally not forming a secondary structure such as a beta sheet or alpha helixor other secondary structure when appended to the therapeutic protein of interest. Rigid sequences are sequences that generally form at least one motif such as a beta sheet, barrel, or alpha helix, or other structure that is defined with respect to itself or other structures on the therapeutic protein with a low degree of flexibility. A semi-rigid structure is defined as a structure that is combined from a flexible and a rigid structure or is a rigid structure that can take on more than one natural conformation when combined with the therapeutic protein of interest.

[0052] In other embodiments the nucleic acid sequence can encode at least the leading two amino acids, followed by at least 4 consecutive amino acids from the non-leading core, and the last 4 amino acids from the C-region from one or more of the following amino acid sequences MLLLGILTLAFAGRTAG, MSQTGSHPGRGLAGRWLWGAQPCLLLPIVPLSWLVWLLLLLLASLLPSARL, MQFVSWATLLTLLVRDLA, MKRLPLLVVFSTLLNCSYT, MLLLGAVLLLLALPGHDQ, MKLVSVALMYLGSLAFLGADT, MLSQLAMLQGSLLLVVATMSVAQQ, MSGAPTAGAALMLCAATAVLLSAQG, MLKKPLSAVTWLCIFIVAFVSHPAWL, MGSRGQGLLLAYCLLLAFASGLVLS, MIIDSSRIPSFTQLHSTMTRAPLLLLCVALVLLGHVNG, MEKKCTLYFLVLLPFFMILVTA, MRGTPKTHLLAFSLLCLLSKVRT, MLAATVLTLALLGNAHA, MHSWERLAVLVLLGAAACAA, MELWGAYLLLCLFSLLTQVTT, MVPDTACVLLLTLAALGASG, MDFPCLWLGLLLPLVAA, MGASSPRSPEPVGPPAPGLPFCCGGSLLAVVVLLALPVAWG, MRPQGPAASPQRLRGLLLLLLLQLPAPSSA, MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAG, MKIAVLFCFFLLIIFQTDFG, MDYLLMIFSLLFVACQG, MWGRLLLWPLVLGFSLS, MRRGRLLEIALGFTVLLASYTSHG, MKFVPCLLLVTLSCLGTLG, MDLWQLLLTLALAGSSDA, MHSSALLCCLVLLTGVRA, MKASSLAFSLLSAAFYLLWTPSTG, MDSYLLMWGLLTFIMVPGCQA, MNSFSTSAFGPVAFSLGLLLVLPAAFPAP, MDGLPGRALGAACLLLLAAGWLGPEAWG, MLLLLGLCLGLSLCVG, MNQLSFLLFLIATTRGWS, MHWGTLCGFLWLWPYLFYVQA, MTLTLSVLICLGLSVGPRTCVQA, MRFAWTVLLLGPLQLCALVHC, MESKALLVLTLAVWLQSLTASRGGVAA, MLTPPLLLLLPLLSALVAA,MARCFSLVLLLTSIWTTRL, MNCQQLWLGFLLPMTVSG, MKSLPILLLLCVAVCSA, MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRT, MARRAGGARMFGSLLLFALLAAGV, MRGANAWAPLCLLLAAATQLSRQ, MKTLLLLAVIMIFGLLQAHG, MAWKTLPIYLLLLLSVFVIQQVSS, MLTTLLPILLLSGWAFC, MHLLAILFCALWSAVLA, MAPSAWAICWLLGGLLLHGGSS, MKGLRSLAATTLALFLVFVFLGNSSC, MGSPAHRPALLLLLPPLLLLLLLRVPPSRS, MASQLTQRGALFLLFFLTPAVTP, MLAPRGAAVLLLHLVLQRWLAAGAQA, MPGSPRPAPSWVLLLRLLALLRPPGLGEA, MRVLLAALGLLFLGALRA, MSLQEMFRFPMGLLLGSVLLVASAPATL, MRTVVLTMKASVIEMFLVLLVTGVHS, MRARPQVCEALLFALALQTGVCYG, MLLLLLLLLLLALALA, MWWRLWWLLLLLLLLWPMVWA, MDMRVPAQLLGLLLLWLRGARC, MPLLLLLPLLWAGALA, MDAMKRGLCCVLLLCGAVFVSPS, MLLLLLLLGLRLQLSLG, MGPTSGPSLLLLLLTHLPLALG, MSSFSTTTVSFLLLLAFQLLGQTRA, MLLFLLSALVLLTQPLGYLE, MARRSSFQSCQIISLFTFAVGVNICLG, MRRLLIPLALWLGAVGVGVA, MFTIKLLLFIVPLVIS, MPPWGAALALILAVLALLGLLGP, MDPPRPALLALLALPALLLLLLAGARA, MVRMVPVLLSLLLLLGPAVP, MAHAHIQGGRRAKSRFVVCIMSGARSKLALFLCGCYVVALG, MLLLVTSLLLCELPHPAFLLIP, MKFLLLVLAALGFLTQVIPASA, MELDRAVGVLGAATLLLSFLGMAWA, MARLLQASCLLSLLLAGFVS, MRPFFLLCFALPGLLHA, MECLYYFLGFLLLAARLPLDAA, MQLKIMPKKKRLSAGRVPLILFLC, MVPGAAGWCCLVLWLPACVAA, MAPTWGPGMVSVVGPMGLLVVLLVGGCAA, MAKRSRGPGRRCLLALVLFCAWGTLA, MRLSPAPLKLSRTPALLALALPLAAALA, MHKEAEMLIGPQLDEKRWGWRLGDGSAAPPFLPQALSFLLLLPLASA, METPAWPRVPRPETAVARTLLLGWVFAQVAGA, MPWPLLLLLAVSGAQT, MSPLLRRLLLAALLQLAPAQA, MRSGEPACTMDQARGLDDAAA, MRKRAPQSEMAPAGVSLRATILCLLAWAGLAAG, MTSKLAVALLAAFLISAALC, MKIILWLCVFGLFLATLFPISWQ, MPVESGLSSEDSASSESFA, METPAQLLFLLLLWLPDTTG, MKSIYFVAGLFVMLVQGSWQ, MALWMRLLPLLALLALWGPDPAAA, MKWVTFISLLFLFSSAYSRGVFRRD, MKWVTFISLLFSSAYS, MKALCLLLLPVLGLLVSS, MYSAPSACTCLCLHFLLLCFQVQVLVA, MKCLLYLAFLFIGVNC,MPGRAPLRTVPGALGAWLLGGLWAWTLCGLCSLGAVG MKIILWLCVFGLFLATLFPISWQMPVESGLSSEDSASSESFA, which are sequences derived from adipocyte secreting proteins and specifically from their secretion signals. The aforementioned sequences can be used in part or in whole and followed by one or more cleavage sites, one or more spacer sequences, and one or more therapeutic proteins or peptides separated by one or more cleavage sites or one or more spacer sequences and one or more cleavage sites.

[0053] In yet another embodiment, the nucleic acid sequence can encode secretion peptide sequences with at least 3 amino acids that follow the secretion peptide sequence from one or more proteins secreted by adipocytes including ADIPOQ, ADM, ANGPTL4, AOC3, APOB, AZGP1, BTD, C1QTNF1, CETP, CFD, CLEC3B, CSF2RA, CXCL3, DEFB132, EDN1, ENPP1, FCN2, FGFBP2, GHR, GPX3, IL10, IL20, IL6, ITLN1, LAMB3, LEP, LPL, MAPT, MMP3, NMB, NRCAM, PCOLCE2, PLA2G2A, PRADC1, PRXL2A, PTPRS, PTX3, PXDN, RBP4, SEMA3G, SPON1, SPX, SSC4D, TF, TIMP4, TSKU, VEGFB, or ZBED3 from which at a minimum 5 consecutive amino acids is taken to construct a semi-synthetic sequence or at most the entire secretion peptide sequence is taken with up to 3 amino acids following the cleavage site.

[0054] All aforementioned sequences can be appended in whole or in part to spacer sequences that are encoded by the nucleic acid sequence. Such optional spacer sequences can encode one or more flexible amino acid spacer sequence that follow the signal peptide cleavage site and are comprised of an amino acid sequence of 2-55 amino acids in length and do not form a structured alpha helix, beta strand, hairpin loop, or other structured motif when followed by the therapeutic protein or peptide sequence. Such an optional spacer sequence may include at least a 4 amino acid stretch of one or more of GGGGSLVPRGSGGGGS, GSGSGS, GSGSGSGSGSGSGSGS, GGGGSLVPRGSGGGG, GGSGGHMGSGG, GGSGGSGGSGG, GGSGG, GGSGGGGG, GSGSGSGS, GGGSEGGGSEGGGSEGGG, AAGAATAA, GGGGG, GGSSG, GSGGGTGGGSG, GT, GSGSGSGSGGSG, GSGGSGGSGGSGGS, GSGGSGSGGSGGSG, SPNSASHSGSAPQTSSAPGSQ, GGGSGGGSGGGSGGGS, GGSG, GGSGGSG, GGSGGSGGSG, GGSGGSGGSGGSGSG, QPELQKPFKYTTVTKRSRRIRPTHPA, GGGGS, SGSG, APSVAPEPDGC, AAAAA, PAAAA, GEAAEGPAAA, AAGVGGERSS, GGPSGAGAGDE, VRTHGTLESVNGPKA, DQKVRPNEENNKDADL, GVKDTD, LPVQNGCPESAMEMN, (GGGGS)n, GGGG, or GGGGGG. Alternatively, or used in conjunction with a flexible spacer, a nucleic acid canencode a semi-flexible amino acid spacer. Such a semi-flexible spacer may be comprised of an amino acid sequence of 2-55 amino acids in length and features a semi-structured motif enriched with a combination of proline, alanine, and serine. Some examples of such semiflexible spacers may include sequences with an at least a 4 amino acid stretch of one or more of GSPAG, GGASPAGG, or GGASPAAPAPAG. Alternatively, or for use in tandem with flexible or semi-flexible spacers, the nucleic acid sequence can encode a rigid amino acid spacer. Such a rigid spacer may be comprised at least in part of an amino acid sequence of 2-55 amino acids that can form a structured alpha helix, beta strand, hairpin loop, or other structured motif when followed by the therapeutic protein or peptide sequence. Some examples of such rigid amino acid spacers can include sequences that contain features such as polyproline, poly-proline-threonine, poly-alanine, and may be stabilized by glutamic acid / lysine salt bridges. In some embodiments the optional spacer sequence encoded by the nucleotide sequence delivered subcutaneously can be rigid and be comprised of the following motif (XP)n where n is between 2 and 28 inclusively and X is an Alanine, Lysine, or Glycine. In other embodiments, the rigid amino acid spacer sequence can be comprised of at least a 4 amino acid stretch of one or more of GPPPG, GPPPPPPPG, GTPTPTPTPTG, or GGAEAAAKEAAAKAGG.

[0055] In other embodiments of the present invention the optional spacer sequence can be comprised of least one endopeptidase cleavage site, which may contain one or more of the following consensus motifs PPTIFFRL, KPIEFFRL, R[S / K][R / S / K][R / K]XXX[G / E], XXX[L / F]VXXX, PFHL[L / V / K][V / I / Y][Y / H / G][S / N], DEVD[G / S]XXX, X[E / D]XDXXXX, QTGGKXEX, PQGIAGQ, DXXD, XRXKRRX, KRIKR, RGRR, RGKR, LRGGKXXX, [D / L]ETD[G / S / A]XXX, or another motif processable by an intracellular or extracellular endopeptidase. An intracellular endopeptidase may process one cleavage site and an extracellular endopeptidase or exopeptidase may process optionally one or more other cleavage sites to for example liberate an appropriate N-terminal amino acid on the therapeutic protein or peptide of interest. In some embodiments of the present invention the nucleic acid sequence can encode an amino acid sequence which is part of the optional spacer or near or at the end of the signal peptide and is processable by one or more of trypsin, chymotrypsin, caspases, elastase, thermolysin, pepsin, glutamyl endopeptidase, plasmin, MMPs, calpain, furin, renin, neprilysin or related or other mammalian endopeptidase.

[0056] As noted, the spacer sequence or other encoded amino acid sequences can contain endopeptidase cleavage sites, which may be specifically extracellular or predominantlyextracellular endopeptidases. Some examples of said endopeptidases include one or more of Neprilysin, kallikreins, cathepsins, MMPs, AD AMTS, or plasmin following secretion from the cell, or other extracellular endopeptidase. As such, the nucleic acid sequence can encode an amino acid sequence that follows the secretion peptide that specifically contains a motif processable by an extracellular endopeptidase.

[0057] The present invention generally pertains to the delivery of nucleic acid sequences that encode at least one therapeutic protein or peptide and said therapeutic protein or peptide is designed to be secreted with or without a secretion signal. When with a section signal, the secretion signal can be processable by subcutaneous white adipocytes with high efficiency. In line with this, the sequence can encode at least a part of at least one therapeutic protein or peptide. Some examples of said therapeutic proteins or peptides include glucagon-like peptide 1, dipeptidyl peptidase-4-resistant glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, exendin-4, or derivatives, or dual- or triple-agonists, at least one of the antagonistic targets of which is the GLP-1 receptor. Other examples of said therapeutic proteins or peptides includes insulin, including furin-cleavable insulin, enhanced- stability insulin, seleno-insulin, four-disulfide insulin analog, or derivatives. Other examples of said therapeutic proteins or peptides include human growth hormone, human growth hormone antagonist, human growth hormone agonist, insulin receptor agonist or antagonist, Thyroid Stimulating Hormone (TSH), Adrenocorticotropic Hormone (ACTH), Vasopressin (Antidiuretic Hormone - ADH), Oxytocin, Parathyroid Hormone (PTH), Calcitonin, Glucagon, Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), somatostatin receptor agonist or antagonist, Corticotropin-Releasing Hormone (CRH), Amylin, Prolactin, Natriuretic Peptides (ANP and BNP), pancreatic polypeptide, Insulin-like Growth Factor 1 (IGF-1), Leptin, Peptide YY, Ghrelin, Cholecystokinin (CCK), Adiponectin, peptide from the Relaxin family, Secretin, Gastrin, GIP agonist, GIP-GLP dual agonist or other peptide hormone, polypeptide hormone, dual or triple agonist, or a fusion thereof with at least one fragment of another functional protein. The therapeutic protein or peptides, which may be encoded following the secretion sequence or may be preceding the secretion signal, may be a peptide hormone or peptide fragment of a natural protein that has or has potential therapeutic activity. Some other examples of therapeutic proteins that can be encoded by the nucleic acid sequence and that may follow or precede the secretion signal directly or following one or more spacer and one or more cleavage site sequences or endopeptidase consensus sequence include without limitation at least a part if not whole of one or more of Adalimumab, Rituximab, Trastuzumab,Bevacizumab, Infliximab, Pembrolizumab, Atezolizumab, Etanercept, Cetuximab, Ranibizumab, Omalizumab, Natalizumab, Golimumab, Tocilizumab, Ustekinumab, Daratumumab, Ixekizumab, Secukinumab, Alemtuzumab, Eculizumab, Palivizumab, Abciximab, Basiliximab, Tositumomab, Panitumumab, Ofatumumab, Trastuzumab emtansine, Mogamulizumab, Obinutuzumab, Sarilumab, Brentuximab vedotin, Dinutuximab, Efgartigimod or other monoclonal, bispecific, or multispecific antibody, antibody fragment, nanobody, or fusion thereof with at least one other functional fragment of another protein. Other examples of said therapeutic proteins or fragments thereof include IL-2, alpha-L-iduronidase, iduronate-2-sulfatase, alpha-glucosidase, alpha-galactosidase, glucocerebrosidase, phenylalanine hydroxylase, Factor VIII, Factor IX, Factor XI, or a secreted protein or enzyme the function of which is lost in a human genetic disorder, or their analog, homolog, ortholog, or their derivative. Other examples of therapeutic proteins or peptides encoded by the nucleic acid sequence include generally at least one functional protein or protein fragment or subunit, and at most several therapeutic proteins up to 12 therapeutic proteins or fragments or subunits. The therapeutic protein of interest may be a fusion of at least one functional fragment with at least one other fragment and optionally one or more functional fragments or fragment and full-length proteins. Some examples of said fusion proteins without limitation include a fusion of a fragment of IL-12 and IL-23, an anti-CD30 antibody and monomethyl auristatin E, alpha-L-iduronidase enzyme and IGF-1, TNF-alpha receptor and an Fc fragment of an antibody, VEGFR1 and VEGFR2 or other fusion protein between two or more fragments of a protein at least one of which is functional. In general, sequences encoding fusion proteins can encode fusions between at least one protein or functional protein fragment and another protein or protein fragment, either of which has or has potential therapeutic activity.

[0058] The encoded secretion peptides and therapeutic proteins or spacers that follow the secretion peptides will have spanning sequences that overlap with at least one amino acid of the secretion signal and at least one amino acid of the spacer or therapeutic protein or peptide. Such spanning sequences can be comprised of one or more of the following amino acid sequences, AXAXYXXX, AXAXXXEG, AXAXXXQG, AXAX[P / K]P[T / I][I / E], AXAXR[S / K][R / S / K][R / K], AXAXXXX[L / F], AXAX[P / D][F / E][H / V][L / D], AXAXX[E / D]XD, AXAX[Q / P][T / Q]G[G / I], AXAXDXXD, AXAXXRXK, [A / V / S / T / G]X[A / G / S / T]XYXXX, [A / V / S / T / G]X[A / G / S / T]XXXEG, [A / V / S / T / G]X[A / G / S / T]XXXQG, [A / V / S / T / G]X[A / G / S / T]X[P / K]P[T / I] [EE] , [A / V / S / T / G]X[A / G / S / T]XR[S / K] [R / S / K] [R / K] , [A / V / S / T / G]X[A / G / S / T]XXXX[L / F] ,[A / V / S / T / G]X[A / G / S / T]X[P / D][F / E][H / V][L / D], [A / V / S / T / G]X[A / G / S / T]XX[E / D]XD, [A / V / S / T / G]X[A / G / S / T]X[Q / P] [T / Q]G[G / I], [A / V / S / T / G]X[A / G / S / T]XDXXD, or [A / V / S / T / G]X[A / G / S / T]XXRXK Said spanning sequences can span the secretion signal cleavage site starting at the n=-4, n=-3, n=-2, or n=-l position, where n=-l is the amino acid immediately preceding the cleavage site (for ease of notation n=-X and n-X can be used interchangeably for the purpose of this invention, when X is a positive or negative integer). In general, a spanning sequence can be a sequence of amino acids that spans 1 to 4 amino acids of the therapeutic protein and 1 to 4 amino acids of the secretion peptide and can enhance cleavage, processing, export, expose the appropriate amino acid on the N-terminal or be specifically processed by an endopeptidase or exopeptidase.

[0059] The present invention is generally intended to deliver genetic cargo to subcutaneous tissues via a subcutaneous injection, but other approaches may be utilized to deliver the genetic cargo to adipocytes including ex vivo transduction or transfection, jet injection, gold particle bombardment, or any other approach of introducing the genetic cargo to subcutaneous adipocytes. White subcutaneous adipocytes are the preferred target tissue, but other tissues of the subcutis connective tissues, or brown adipocytes, or preadipocytes may be the target or the predominant target of the nucleic acid. Similarly, the subcutaneous adipocytes may be of any animal that the therapy is intended for including for example without limitation human, canine, feline, equine, or other mammalian tissues. To enable durable therapy the nucleic acid is preferably DNA, however, the nucleic acid can be RNA, DNA, or RNA-DNA hybrid construct, with or without chemical modifications. The DNA construct is intended to be generally nonintegrating however integrating DNA constructs are possible and can be used within the scope of the present invention, similarly, RNA therapy that delivers integrated DNA via reverse transcription and integration into genomic DNA or mitochondrial DNA or delivers proteins and RNA for genetic or epigenetic modification can also be used for the purpose of providing therapy within the scope of the present invention so long as the eventual therapeutic protein or peptide is secreted and aimed for export from the adipocyte or other cell which received the nucleic acid construct. The nucleic acid construct can be comprised of one or more strands and can be delivered in a vector which is non-antigenic or minimally antigenic, which ideally does not elicit a neutralizing immune response following subcutaneous administration at levels anticipated to be clinically effective. The delivered nucleic acid construct is generally preferably delivered by a subcutaneous injection and generally in a volume of at least 0.05 to at most 100 mL, but more preferably 0.1 to 50 mL, and most preferably 0.5 to 5 mL, in someembodiments, which may be delivered over one or more injection sites. Microneedle or jet injection can be used to deliver the therapeutic nucleic acid construct such that preferably the majority of the delivery is administered subcutaneously. In general, any physical, chemical, biological, biochemical, or biophysical means can be used to deliver the nucleic acid construct. Viral vectors and non- viral vectors may be used to deliver the nucleic acid constructs, including for example viruses, lipid nanoparticles (LNPs), virus like particles (VLPs), liposomes, endosomes, exosomes, or other carriers or encapsulators, such as for example polymeric nanoparticles, liposomes, polymeric carriers, polyelectrolytic complexes, solid lipid particles, functionalized LNPs, functionalized polymers, polymer-lipid hybrid particles or other nano or microparticles. Optionally, the nucleic acid sequences can be injected directly with or without a carrier fluid or formulation with or without the aid of transfection reagents, with or without the use of electroporation, with or without the use of charged or uncharged particle bombardment, and with or without the use of a physical or magnetic force.

[0060] Some specific examples of enabling the present invention are outlined in detail in the Example section, but are not intended to be limiting to the scope of the invention. Nucleic acid sequences can be codon optimized using general codon optimization tools using artificial intelligence-based tools and specifically for a given species of interest. Auxiliary sequences may be delivered in cis or in trans with the nucleic acid sequences encoding the therapeutic protein of interest and the secretion signal and may include one or more of promoters, terminators, insulators, 3’UTRs, 5’UTRs, enhancers, or combinations thereof.

[0061] The nucleic acid sequences encoding at least the secretion signal and the therapeutic peptide or protein of interest are intended for the treatment of human or veterinary pathologies and may be generally derived from natural sequences, synthetic sequences, or semi- synthetic sequences with or without in silico optimization, with or without the use of codon optimization, with or without the use of artificial intelligence tools or systems and with or without the use of directed evolution.

[0062] It should be understood that within the scope of this invention the amino acid sequences, nucleic acid sequences, motifs, biological and chemical compositions, or methods of use may be varied by one skilled in the art, to the extent that the structures described herewithin perform the desired function and remain within the scope of the present invention. Various parts, components or characteristics may be used in combination, with or without modification bysomeone skilled in the art to achieve the desired functionality of the aforedescribed formulation.

[0063] Moreover, all individual features and methods of use described herein, and each and every combination of two or more of such features and methods of use, are included within the scope of the present invention provided that these features and methods of use in such a combination are not mutually inconsistent. It is understood that certain portions or combinations of such portions can be varied by someone trained in the art while still achieving the main goal of the invention.

[0064] Finally, it is understood that the specific ranges provided in the current invention are not restrictive and are for example purposes only, values outside of the specified ranges may be used to achieve the goal of the invention without modification to the proposed mechanistic principals.

[0065] EXAMPLES

[0066] Example 1

[0067] In the present example Exenatide and a modified GLP-1 Receptor Agonist are delivered in DNA form for subcutaneous expression, followed by secretion, and eventual systemic biodistribution. The exenatide construct contains a CBh promoter, mouse growth hormone secretion signal (MATDSRTSWLLTVSLLCLLWPQEASA), a spacer sequence (FPAMPLSSLFSNAVLRA), a furin cleavage site (RGKR), and exenatide, as well as a polyadenylation signal and regulatory sequences. Following optimization, the final amino acid sequence encoded by the therapeutic nanoplasmid is as follows:

[0068] MATDSRTSWLLTVSLLCLLWPQEASAFPAMPLSSLFSNAVLRARGKRHGEGTFTSDLS KQMEEEAVRLFIEWLKNGGPSSGAPPPS.

[0069] The GLP-1 Receptor Agonist construct contains a CBh promoter, secreted alkaline phosphatase signal peptide (MLLLLLLLGLRLQLSLG), and trypsin and dipeptidyl peptidase-4-resistant GLP-1, as well as a poly adenylation signal and regulatory sequences. Following optimization, the final amino acid sequence encoded by the therapeutic nanoplasmid is as follows:

[0070] MLLLLLLLGLRLQLSLGHGEGTFTSDVSSYLEGQAAQEFIAWLVDGR.The nanoplasmids can be formulated into a lipid nanoparticle vector and delivery formulation and injected subcutaneously at a dose of 3.5ug per injection to treat type 2 diabetes.

[0071] The Exenatide construct was transfected into primary human adipocytes at 0.5 pg and 1 pg of construct per well of a 96-well plate. Supernatant was collected 168 hours posttransfection and measured by ELISA.

[0072] Example 2

[0073] Several signal peptides confirmed as functional in primary human adipocytes demonstrated a number of motifs important for high secretion efficiency in adipocytes. Among them were a charted, polar alternating motif in the N-region (Leader Motif), which was comprised of a minimum of 3 amino acids including the leading methionine, a hydrophobic H-region rich in leucine, which unlikely other secretion peptides did not generally exceed 10 amino acids in length, and a tail motif (C-region), which was as short as one amino acid and as long as 8 amino acids, contained at least one polar amino acid or glycine, and either an internal or alternating hydrophobic stretch of up to 5 amino acids.

[0074] In this specific example the secretion signals in FIG. 2 are encoded in a nucleic acid sequence comprised of DNA, followed by a GLP-1 Receptor Agonist with or without a spacer sequence. The spacer sequence is cleaved from the GLP-1 Receptor agonist to expose the functional site by an intracellular or extracellular endopeptidase, enabling efficient processing in adipocytes and secretion of the therapeutic peptide (GLP-1 Receptor Agonist) in functional form.

[0075] Example 3

[0076] In the present example, an amino acid sequence is selected from the following set and encoded into DNA nanoplasmids, which were tested in vitro for expression and secretion of the therapeutic protein of interest. Following lead selection, a sequence containing a high Singal P score and optimal cleavage site is selected and confirmed as functional in vivo, in C57BL / 6J mice of 30-35 g weight following administration of the nanoplasmid in an LNP formulation at a total injection dose of 20 ug per animal, administered subcutaneously.

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[0091] The nanoplasmid encodes the adipsin secretion signal MHSWERLAVLVLLGAAACAA, followed by the GLP- 1 RA with the following amino acid sequence: YGEGTFTSDLSKQMEEEAVRLFIEWLMNTKRNRNNIA.

[0092] Example 4

[0093] In the present example, the secretion and processing of the N-terminal is optimized by utilizing the sequence in Example 3, but additionally inserting an HA and Furin Cleavage Site (FCS) prior to the GLP-1 RA active sequence with an A8G substitution: Adipsin+HA+FCS+GLPl(7-37) A8G. Following optimization, the final amino acid sequence encoded by the therapeutic nanoplasmid is as follows: MHSWERLAVLVLLGAAACAAYPYDVPDYARGRRHGEGTFTSDVSSYLEGQAAKEFI AWLVKGRG and the required dose to attain a similar therapeutic effect is reduced by approximately 5-6x.

[0094] Example 5In the present example, the following DNA construct is delivered to the subcutaneous tissues in order to express adalimumab for secretion and eventual systemic biodistribution. The construct includes a CBh promoter, secretion signal from C1QTNF1, and Adalimumab in addition to a number of regulatory sequences.

[0095] Example 6

[0096] In the present example, a genetic construct is constructed for subcutaneous administration for production and expression of Follistatin in adipocytes to enable eventual systemic biodistribution for the treatment of muscular dystrophy, age related atrophy, or general muscle loss or insufficiency. The secretion signal, cleavage sites, and optional flexible spacer are obtained from the sets below and assembled in a form secretion signal - cleavage site -spacer - cleavage site - Follistatin to expose the GNCW leader sequence on the secreted form of Follistatin.

[0097] Table: Secretion Signals

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] Table: Cleavage Sites

[0104]

[0105]

[0106] Table: Spacers

[0107]

[0108]

[0109]

[0110] The alignment, followed by amino acid frequency analysis determined two additional motifs. The first, within the last 4 amino acids prior to the cleavage site of [A / G / S]X[A / G / S / L]R and the second, within the H-region of [L / W]LXXL[L / F / V][L / A]L[L / A] within at least 3 amino acids of each other.

[0111] Example 7

[0112] In the present example, an Adipsin secretion signal is followed by a furin cleavage site to enable efficient production, secretion, and N-terminal processing of a Cdk5-derived peptide ARAFGIPVRCYS for the treatment of neurodegenerative diseases. The full sequence of the encoded peptide translates to: MHSWERLAVLVLLGAAACAARRARARAFGIPVRCYS andis encoded on a double stranded linear DNA for subsequent incorporation into adipocytes via electron-flux vector mediated transfection. The treatment is administered via subcutaneous electroporation and offers a high degree of transgene localization to subcutaneous adipose tissues with optimal systemic biodistribution of the secreted peptide.

[0113] Example 8

[0114] In the present example, the motif (A / V / S / T)X(A / G / S / T)X is utilized by appending the Tissue Factor (TF) secretion signal (METPAWPRVPRPETAVARTEEEGWVFAQVAGA) to the modified GLP-1 Receptor Agonist sequence HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG for high precision liberation of the Histidine at the GLP-1 Receptor Agonist N-terminal as confirmed by Signal P 6.0.

[0115] Example 9

[0116] In the present example a set of sequences is constructed using motifs outlined in the present invention and outlined in the table below. The Angiotensinogen secretion sequence: MRKRAPQSEMAPAGVSLRATILCLLAWAGLAAG is appended to the GLP1RA sequence: YGEGTFTSDYSIAMDKIAQKAFVQWLIAGGPSSGAPPPS. The codon optimized DNA sequence is derived as:

[0117] ATGAGAAAGCGGGCCCCCCAGAGCGAGATGGCCCCCGCCGGAGTGTCCCTGAGA GCTACAATCCTGTGCCTGCTGGCCTGGGCCGGCCTGGCTGCTGGCTACGGCGAAG GCACCTTCACCAGCGACTACAGCATCGCCATGGATAAGATCGCCCAGAAAGCCTT CGTGCAGTGGCTGATCGCCGGCGGACCTAGCAGCGGCGCCCCTCCTCCATCT and GC% content is confirmed as less than 68%. The sequence is inserted into a DNA minicircle under the regulation of the CBh promoter and formulated in a lipid nanoparticle formulation for therapeutic administration. The formulation is confirmed as active at a dose of 1.5-200 ug / kg body weight and can be redosed for increased therapeutic effect via subcutaneous administration.

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[0127] Example 10

[0128] In the present example, secreted proteins expressed by adipocytes and confirmed at an RNA and IHC level are obtained, and 4 regional motifs and 2 high-consensus motifs are identified as important for efficient secretion signal processing. The proteins and secretion signals are listed in the table below:

[0129]

[0130] Their alignments (using the MUSCLE alignment algorithm) are shown in FIG. 7. The alignments resulted in a high consensus motif in the H-region of LL-(5 to 8 X)-LLLXL-(4 to 6 X)-AXX, in this motif two Leucines are followed by a 5-8 amino acid stretch, and then byan LLLXL motif, with another 4-6 amino acid stretch and finally by a cleavage site C-region motif of AXX (where X is a wildcard amino acid). More detailed analysis results in the identification of 4 characteristic regions with a high percentage of M(P / E) starting amino acids, two distinct leucine-rich regions in the H-region and a characteristic C-region containing a GX(L / V / A)AXX motif.

[0131] In line with the identified patterns a DNA sequence is constructed encoding:

[0132] MPDMMRLGLLLALLLVLLLALPGALA followed by YGEGTFTSDLSKQMEEEAVRLFIEWLMNTKRNRNNIA for the treatment of Type 2 Diabetes and Obesity. The combined sequence performed well in both Singal P and in vitro screens producing a secreted protein with a free Y at the N-terminal.

[0133] The DNA encoding the said secretion signal coupled with the agonist is structured into a DNA nanoplasmid, encapsulated in an LNP formulation and dosed subcutaneously resulting in transfection of adipocytes for local expression and systemic biodistribution of the therapeutic peptide.

[0134] Example 11

[0135] To further confirm utility of the secretion signals optimized for expression in adipocytes, secretion signals were generated by multiple algorithmic and non-algorithmic approaches using rational design and non-rational design methods to include adipocyte specific and adipocyte selective motifs. Said secretion signals were encoded in nucleic acid sequences with and without codon optimization to produce nucleic acid sequences encoding secretion signals and a therapeutic protein (or reporter protein). Algorithmic approaches and non-algorithmic approaches as well as rational design approaches were informed by characterizing proteins expressed at high and medium levels in adipocytes and those proteins that are specifically destined for secretion (or cellular export). A library of signals was created to fully cover the identified set of adipocyte specific secretion signal motifs.

[0136] The designed signals were tested in vitro in adipocytes and in a non-adipocyte cell line (HEK293). Secretion signals utilizing adipocyte specific motifs were able to outperform rational design by a magnitude of up to 5x indicating utility and applicability to subcutaneously delivered or adipocyte targeting gene therapies. In contrast, in HEK cells, a cell line, which the secretion signals were not optimized or adapted for, the adipocyte-optimized secretion signals performed comparable to a biological, rationally designed signal. No statistically significant correlation was present between adipocytes and HEK cells indicating that the use of adipocyte specific motifs improves potency in adipocytes and selectivity of expression in favor of adipocytes.

[0137] A set of adipocyte optimized secretion signals were then tested in vitro using a secreted therapeutic protein. The treatment was administered subcutaneously, using double stranded circular DNA delivered via a non- viral vector via a subcutaneous injection. As shown in FIG. 11, the secretion signals optimized for adipocytes correlated in expression level between in vitro and in vivo experiments when in vivo titer was correlated to expression levels in adipocytes in vitro. Conversely, no statistically significant correlation was observed when in vivo titers were correlated to expression levels in HEK cells in vitro. This finding confirms that not only were the in vitro experiments in adipocytes predictive of in vivo results, but also the optimization of secretion signals for adipocytes via the use of adipocyte specific motifs in novel sequence design results in increase potency of the treatment following subcutaneous delivery and selectivity of expression in adipocytes.

[0138] Follow-up in vivo analysis further confirmed that secretion signals, which were specifically optimized for processability and secretion efficiency in adipocytes (e.g. Gil, G12, G13) outperformed rational design secretion signal in productivity by up to 6x. This experiment further confirms that secretion signal optimization and de novo design using adipocyte specific and adipocyte efficient motifs enhances processability in adipocytes and therefore the potency of gene therapy treatment, as well as selectivity of processability in adipocytes enabling the use of such signals in subcutaneously administered and / or adipocyte targeting gene therapies where adipocytes are the preferential cell factory for the production of secreted therapeutic proteins in vivo.

Claims

CLAIMSWe claim:

1. A nucleic acid construct for vector-mediated delivery via a subcutaneous injection to white subcutaneous adipocytes intended to express secreted therapeutic proteins for eventual systemic or predominantly systemic biodistribution, which is comprised of:• One or more sequences encoding at least one efficiently processable secretion peptide which contains an adipocyte-processable motif, and• One or more optional sequences encoding at least one amino acid spacer sequence, which optionally contains a cleavage-enhancing sequence, and • One or more sequences encoding a therapeutic peptide, protein, or fusion protein, and• One or more spanning sequences comprised of a nucleic acid sequence that encodes an amino acid sequence which spans the secretion peptide sequence and the therapeutic protein sequence and enables efficient adipocyte-expressed endopeptidase processing.

2. One or more sequences encoding at least one efficiently processable secretion peptide, which contain a stretch of 2-4 leucines in the N-region, with a downstream motif of LLLXL in the H-region, and a C-region motif of AXX, where A is the n=-3 amino acid preceding the cleavage site.

3. One or more sequences encoding at least one efficiently processable secretion peptide, which contain the motif LL-(5 to 8 X)-LLLXL-(4 to 6 X)-AXX.

4. One or more sequences encoding at least one efficiently processable secretion peptide, which contain 4 characteristic regions starting with an M(P / E)XXXXXX containing at least two leucines, followed by two leucine-rich regions containing at least two leucines in a row, followed by an LLL(A / L)L stretch and an AXX terminal three amino acid motif.

5. One or more sequences encoding at least one efficiently processable secretion peptide, which contains at least 3 characteristic regions starting with an M(P / E) motif, which is later followed by at least one leucine-alanine stretch of either LLLAL, LLAL, LLALL, LALL and an GX(L / V / A)AXX terminal motif.

6. One or more sequences encoding at least one efficiently processable secretion peptide, which contains at least 3 characteristic regions starting with an M(P / E) motif, which is later followed by at least one leucine- alanine stretch containing at least one dileucine and one trileucine flanked by an alanine or glycine and an GX(L / V / A)AXX terminal motif.

7. One or more sequences encoding at least one efficiently processable secretion peptide, which contains the GX(L / V / A)AXX terminal motif derived at least 50% from an adipocyte secreted protein.

8. A nucleic acid construct in Claim 1, which encodes a secretion signal and a therapeutic protein and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is the same as the last amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is the same as the first amino acid of the therapeutic protein.

9. A nucleic acid construct in Claim 1, which encodes a secretion signal and a therapeutic protein and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are the same as the last two amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is the same as the first amino acid of the therapeutic protein.

10. A nucleic acid construct in Claim 1, which encodes a secretion signal and a therapeutic protein and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is the same as the last amino acid of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are the same as the first two amino acid of the therapeutic protein.

11. A nucleic acid construct in Claim 1, which encodes a secretion signal and a therapeutic protein and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are the same as the last two amino acids of the natural secretion signal of the therapeutic protein and the first two amino acids of the secretedprotein from which the secretion signal is derived are the same as the first two amino acid of the therapeutic protein.

12. A nucleic acid construct in Claim 1, which encodes a secretion signal and a therapeutic protein and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is of a similar charge or hydrophobicity as the last amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is of a similar charge or hydrophobicity as the first amino acid of the therapeutic protein.

13. A nucleic acid construct in Claim 1, which encodes a secretion signal and a therapeutic protein and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are of a similar charge or hydrophobicity as the last two amino acid of the natural secretion signal of the therapeutic protein and the first amino acid of the secreted protein from which the secretion signal is derived is of a similar charge or hydrophobicity as the first amino acid of the therapeutic protein.

14. A nucleic acid construct in Claim 1, which encodes a secretion signal and a therapeutic protein and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last amino acid of the secretion signal is of a similar charge or hydrophobicity as the last amino acid of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are of a similar charge or hydrophobicity as the first two amino acid of the therapeutic protein.

15. A nucleic acid construct in Claim 1, which encodes a secretion signal and a therapeutic protein and the secretion signal is derived from a secreted protein, which is different from the therapeutic protein, and is expressed in adipocytes, and the last two amino acids of the secretion signal are of a similar charge or hydrophobicity as the last two amino acids of the natural secretion signal of the therapeutic protein and the first two amino acids of the secreted protein from which the secretion signal is derived are of a similar charge or hydrophobicity as the first two amino acid of the therapeutic protein.

16. A nucleic acid construct in Claim 1, which is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first three amino acids of said protein, which follows the secretion signal is ofapproximately the same charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal.

17. A nucleic acid construct in Claim 1, which is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first two amino acids of said protein, which follows the secretion signal is of approximately the same charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal.

18. A nucleic acid construct in Claim 1, which is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first amino acid of said protein, which follows the secretion signal is an amino acid of a similar charge or hydrophobicity as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal.

19. A nucleic acid construct in Claim 1, which is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first three amino acids of said protein, which follows the secretion signal is the same amino acid as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal.

20. A nucleic acid construct in Claim 1, which is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first two amino acids of said protein, which follows the secretion signal is the same amino acid as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal.

21. A nucleic acid construct in Claim 1, which is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

22. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif[A / V / S]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

23. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

24. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes.

25. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

26. A nucleic acid construct in Claim 1, which is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

27. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

28. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

29. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes.

30. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

31. A nucleic acid construct in Claim 1, which is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytesor a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

32. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

33. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

34. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes.

35. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / S]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed inadipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

36. A nucleic acid construct in Claim 1, which is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

37. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

38. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

39. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes.

40. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes ora semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / S]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

41. A nucleic acid construct in Claim 1, which is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

42. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

43. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

44. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which isthe first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes.

45. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / T]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

46. A nucleic acid construct in Claim 1, which is comprised in part of a 36 to 120 base pair sequence encoding a secretion signal from a secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, where the second X is the last pre-cleavage or first post-cleavage site amino acid.

47. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, where the second X is the last pre-clevage or first post-cleavage site amino acid.

48. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

49. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequenceencoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes.

50. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, with the last 12 base pairs of the secretion signal encoding nucleic acid sequence encoding an amino acid motif [A / V / G]X[A / G / T]X, and the subsequent 3 base pairs encoding an amino acid, which is the first or second post-cleavage site amino acid from the secreted protein expressed in adipocytes and is the same as the first or second post-cleavage site amino acid from the therapeutic protein or peptide.

51. A nucleic acid construct in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, which contains at least a stretch encoding 5 amino acids with the motif containing at least two Methionines and at least an Alanine, Glycine, Lysine, or Arginine, followed by a downstream stretch encoding a sequence of at least 5 amino acids with at least two Lysines and at least an Alanine, Valine, or Isoleucine, followed by a sequence encoding the motif [A / V / S / T / G]X[A / G / S / T]X where the last X amino acid is the last pre-cleavage or first post-cleavage amino acid.

52. A nucleic acid constructs in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, which contains at least the following motif [A / V / S / T / G]X[A / G / S / T]Z where Z is the first amino acid of the therapeutic protein or peptide and is the same as the first amino acid of the secreted adipocyte expressed protein, or Z is the last amino acid of the secretion signal and is the same as the last amino acid of the therapeutic protein’s natural secretion signal.

53. A nucleic acid constructs in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, which contains at least the following motif [A / V / S / T / G]X[A / G / S / T]XZ where the second X is the first amino acid of the therapeutic protein or peptide and is the same as the first amino acid of the secreted adipocyte expressed protein and Z is the second amino acid of the therapeutic protein or peptide and is of a similar charge or polarity to the second amino acid of the secreted adipocyte expressed protein.

54. A nucleic acid constructs in Claim 1, which is comprised in part of a nucleic acid sequence encoding a therapeutic protein or peptide and a 36 to 120 base pair sequence encoding a secretion signal from a different secreted protein expressed in adipocytes or a semi- synthetic sequence derived from said protein, which contains at least the following motif [A / V / S / T / G]X[A / G / S / T]XZ where the second X is the first amino acid of the therapeutic protein or peptide and is the same as the first amino acid of the secreted adipocyte expressed protein and Z is the second amino acid of the expressed adipocyte secreted protein and is of a similar charge or polarity to the second amino acid of the therapeutic peptide or protein.

55. A nucleic acid construct in Claim 1, which is comprised of a DNA sequence encoding a secretion signal from a secreted protein expressed in adipocytes and is a protein where the first amino acid of said protein, which follows the secretion signal is the same amino acid as in the therapeutic protein encoded by the nucleic acid construct, immediately following the secretion signal.

56. A sequence encoding an efficiently processable secretion peptide in Claim 1, which encodes in addition to the therapeutic protein or peptide of interest, at least the first three amino acids, and another 5 amino acid stretch, and the last 4 amino acids of at least one of the following sequences: MPPWGAALALILAVLALLGLLGP, MLLLGAVLLLLALPGHDQ, MDPPRPALLALLALPALLLLLLAGARA, MVRMVPVLLSLLLLLGPAVP, MAHAHIQGGRRAKSRFVVCIMSGARSKLALFLCGCYVVALG, MLLLVTSLLLCELPHPAFLLIP, MKFLLLVLAALGFLTQVIPASA, MELDRAVGVLGAATLLLSFLGMAWA, MDLWQLLLTLALAGSSDA, MARLLQASCLLSLLLAGFVS, MRPFFLLCFALPGLLHA,MHWGTLCGFLWLWPYLFYVQA, MESKALLVLTLAVWLQSLTASRGGVAA, MECLYYFLGFLLLAARLPLDAA, MQLKIMPKKKRLSAGRVPLILFLC, MRGANAWAPLCLLLAAATQLSRQ, MVPGAAGWCCLVLWLPACVAA, MAPTWGPGMVSVVGPMGLLVVLLVGGCAA, MAKRSRGPGRRCLLALVLFCAWGTLA, MAPSAWAICWLLGGLLLHGGSS, MRLSPAPLKLSRTPALLALALPLAAALA, MHKEAEMLIGPQLDEKRWGWRLGDGSAAPPFLPQALSFLLLLPLASA, METPAWPRVPRPETAVARTLLLGWVFAQVAGA, MPGSPRPAPSWVLLLRLLALLRPPGLGEA, MPWPLLLLLAVSGAQT, MSPLLRRLLLAALLQLAPAQA, MRSGEPACTMDQARGLDDAAA, MELWGAYLLLCLFSLLTQVTT, MLAATVLTLALLGNAHA, MHSWERLAVLVLLGAAACAA, MKFVPCLLLVTLSCLGTLG, MGSRGQGLLLAYCLLLAFASGLVLS, MNQLSFLLFLIATTRGWS, MSGAPTAGAALMLCAATAVLLSAQG, MKGLRSLAATTLALFLVFVFLGNSSC, MKSLPILLLLCVAVCSA, MHLLAILFCALWSAVLA, MKTLLLLAVIMIFGLLQAHG, MKASSLAFSLLSAAFYLLWTPSTG, MKLVSVALMYLGSLAFLGADT, MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAG, MDYLLMIFSLLFVACQG, MNSFSTSAFGPVAFSLGLLLVLPAAFPAP, MHSSALLCCLVLLTGVRA, MLLLLLLLLLLALALA, MWWRLWWLLLLLLLLWPMVWA, MDMRVPAQLLGLLLLWLRGARC, MPLLLLLPLLWAGALA, MDAMKRGLCCVLLLCGAVFVSPS, MLLLLLLLGLRLQLSLG.

57. A sequence encoding an efficiently processable secretion peptide in Claim 1, which contains an adipocyte-processable motif [A / G / S]X[A / G / S / L]R immediately preceding the signal peptide cleavage site.

58. A sequence encoding an efficiently processable secretion peptide in Claim 1, which contains an adipocyte-processable motif [A / G / S]X[A / G / S / L]R and at least 60% of a secretion signal from a protein expressed in adipocytes and is followed by the first amino acid of the therapeutic peptide or spacer sequence, which is the same as the first amino acid of the aforementioned adipocyte expressed protein.

59. A sequence encoding an efficiently processable secretion peptide in Claim 1, which contains an adipocyte-processable motif [A / G / S]X[A / G / S / L]R and at least 60% of a secretion signal from a protein expressed in adipocytes and is followed by the firstamino acid of the therapeutic peptide or spacer sequence, which has approximately the same charge, polarity, or hydrophobicity as the first amino acid of the aforementioned adipocyte expressed protein.

60. A sequence encoding an efficiently processable secretion peptide in Claim 1, which contains the following motif [ L / W | L[ X |nL[ L / F / V ] [ L / A | L[ L / A | [ X |mpreceding the last 4 amino acids of the secretion signals such that n is between 2 and 6 and m is between 2 and 7.

61. A sequence encoding an efficiently processable secretion peptide in Claim 1, which contains the following motif [L / F / V][L / A]L[L / A][X]npreceding the last 4 amino acids of the secretion signals such that n is between 1 and 8.

62. A nucleic acid sequence in Claim 1 encoding at least the leading two amino acids, followed by at least 4 consecutive amino acids from the non-leading core, and the last 4 amino acids from the C-region from one or more of the following amino acid sequences MLLLGILTLAFAGRTAG, MSQTGSHPGRGLAGRWLWGAQPCLLLPIVPLSWLVWLLLLLLASLLPSARL, MQFVSWATLLTLLVRDLA, MKRLPLLVVFSTLLNCSYT, MLLLGAVLLLLALPGHDQ, MKLVSVALMYLGSLAFLGADT, MLSQLAMLQGSLLLVVATMSVAQQ, MSGAPTAGAALMLCAATAVLLSAQG, MLKKPLSAVTWLCIFIVAFVSHPAWL, MGSRGQGLLLAYCLLLAFASGLVLS, MIIDSSRIPSFTQLHSTMTRAPLLLLCVALVLLGHVNG, MEKKCTLYFLVLLPFFMILVTA, MRGTPKTHLLAFSLLCLLSKVRT, MLAATVLTLALLGNAHA, MHSWERLAVLVLLGAAACAA, MELWGAYLLLCLFSLLTQVTT, MVPDTACVLLLTLAALGASG, MDFPCLWLGLLLPLVAA, MGASSPRSPEPVGPPAPGLPFCCGGSLLAVVVLLALPVAWG, MRPQGPAASPQRLRGLLLLLLLQLPAPSSA, MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAG, MKIAVLFCFFLLIIFQTDFG, MDYLLMIFSLLFVACQG, MWGRLLLWPLVLGFSLS, MRRGRLLEIALGFTVLLASYTSHG, MKFVPCLLLVTLSCLGTLG, MDLWQLLLTLALAGSSDA, MHSSALLCCLVLLTGVRA, MKASSLAFSLLSAAFYLLWTPSTG, MDSYLLMWGLLTFIMVPGCQA, MNSFSTSAFGPVAFSLGLLLVLPAAFPAP, MDGLPGRALGAACLLLLAAGWLGPEAWG, MLLLLGLCLGLSLCVG,MNQLSFLLFLIATTRGWS, MHWGTLCGFLWLWPYLFYVQA, MTLTLSVLICLGLSVGPRTCVQA, MRFAWTVLLLGPLQLCALVHC, MESKALLVLTLAVWLQSLTASRGGVAA, MLTPPLLLLLPLLSALVAA, MARCFSLVLLLTSIWTTRL, MNCQQLWLGFLLPMTVSG, MKSLPILLLLCVAVCSA, MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRT, MARRAGGARMFGSLLLFALLAAGV, MRGANAWAPLCLLLAAATQLSRQ, MKTLLLLAVIMIFGLLQAHG, MAWKTLPIYLLLLLSVFVIQQVSS, MLTTLLPILLLSGWAFC, MHLLAILFCALWSAVLA, MAPSAWAICWLLGGLLLHGGSS, MKGLRSLAATTLALFLVFVFLGNSSC, MGSPAHRPALLLLLPPLLLLLLLRVPPSRS, MASQLTQRGALFLLFFLTPAVTP, MLAPRGAAVLLLHLVLQRWLAAGAQA, MPGSPRPAPSWVLLLRLLALLRPPGLGEA, MRVLLAALGLLFLGALRA, MSLQEMFRFPMGLLLGSVLLVASAPATL, MRTVVLTMKASVIEMFLVLLVTGVHS, MRARPQVCEALLFALALQTGVCYG MLLLLLLLLLLALALA, MWWRLWWLLLLLLLLWPMVWA, MDMRVPAQLLGLLLLWLRGARC, MPLLLLLPLLWAGALA, MDAMKRGLCCVLLLCGAVFVSPS, MLLLLLLLGLRLQLSLG, MGPTSGPSLLLLLLTHLPLALG, MSSFSTTTVSFLLLLAFQLLGQTRA, MLLFLLSALVLLTQPLGYLE, MARRSSFQSCQIISLFTFAVGVNICLG, MRRLLIPLALWLGAVGVGVA, MFTIKLLLFIVPLVIS, MPPWGAALALILAVLALLGLLGP, MDPPRPALLALLALPALLLLLLAGARA, MVRMVPVLLSLLLLLGPAVP, MAHAHIQGGRRAKSRFVVCIMSGARSKLALFLCGCYVVALG, MLLLVTSLLLCELPHPAFLLIP, MKFLLLVLAALGFLTQVIPASA, MELDRAVGVLGAATLLLSFLGMAWA, MARLLQASCLLSLLLAGFVS, MRPFFLLCFALPGLLHA, MECLYYFLGFLLLAARLPLDAA, MQLKIMPKKKRLSAGRVPLILFLC, MVPGAAGWCCLVLWLPACVAA, MAPTWGPGMVSVVGPMGLLVVLLVGGCAA, MAKRSRGPGRRCLLALVLFCAWGTLA, MRLSPAPLKLSRTPALLALALPLAAALA, MHKEAEMLIGPQLDEKRWGWRLGDGSAAPPFLPQALSFLLLLPLASA, METPAWPRVPRPETAVARTLLLGWVFAQVAGA, MPWPLLLLLAVSGAQT, MSPLLRRLLLAALLQLAPAQA, MRSGEPACTMDQARGLDDAAA,MRKRAPQSEMAPAGVSLRATILCLLAWAGLAAG, MTSKLAVALLAAFLISAALC, MKIILWLCVFGLFLATLFPISWQ, MPVESGLSSEDSASSESFA, METPAQLLFLLLLWLPDTTG, MKSIYFVAGLFVMLVQGSWQ, MALWMRLLPLLALLALWGPDPAAA, MKWVTFISLLFLFSSAYSRGVFRRD, MKWVTFISLLFSSAYS, MKALCLLLLPVLGLLVSS, MYSAPSACTCLCLHFLLLCFQVQVLVA, MKCLLYLAFLFIGVNC, MPGRAPLRTVPGALGAWLLGGLWAWTLCGLCSLGAVG MKIILWLCVFGLFLATLFPISWQMPVESGLSSEDSASSESFA.

63. A nucleic acid sequence in Claim 1 encoding at least 5 consecutive amino acids from the secretion signals of the following human proteins ADIPOQ, ADM, ANGPTL4, AOC3, APOB, AZGP1, BTD, C1QTNF1, CETP, CFD, CLEC3B, CSF2RA, CXCL3, DEFB132, EDN1, ENPP1, FCN2, FGFBP2, GHR, GPX3, IL10, IL20, IL6, ITLN1, LAMB3, LEP, LPL, MAPT, MMP3, NMB, NRCAM, PCOLCE2, PLA2G2A, PRADC1, PRXL2A, PTPRS, PTX3, PXDN, RBP4, SEMA3G, SPON1, SPX, SSC4D, TF, TIMP4, TSKU, VEGFB, or ZBED3.

64. An optional spacer sequence in Claim 1, which encodes a flexible amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of an amino acid sequence of 2-55 amino acids in length and does not form a structured alpha helix, beta strand, hairpin loop, or other structured motif when followed by the therapeutic protein or peptide sequence.

65. An optional spacer sequence in Claim 1, which encodes a flexible amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of at least a 4 amino acid stretch of one or more of GGGGSLVPRGSGGGGS, GSGSGS, GSGSGSGSGSGSGSGS, GGGGSLVPRGSGGGG, GGSGGHMGSGG, GGSGGSGGSGG, GGSGG, GGSGGGGG, GSGSGSGS, GGGSEGGGSEGGGSEGGG, AAGAATAA, GGGGG, GGSSG, GSGGGTGGGSG, GT, GSGSGSGSGGSG, GSGGSGGSGGSGGS, GSGGSGSGGSGGSG, SPNSASHSGSAPQTSSAPGSQ, GGGSGGGSGGGSGGGS, GGSG, GGSGGSG, GGSGGSGGSG, GGSGGSGGSGGSGSG, QPELQKPFKYTTVTKRSRRIRPTHPA, GGGGS, SGSG, APSVAPEPDGC, AAAAA, PAAAA, GEAAEGPAAA, AAGVGGERSS, GGPSGAGAGDE, VRTHGTLESVNGPKA,DQKVRPNEENNKDADL, GVKDTD, LPVQNGCPESAMEMN, (GGGGS)n, GGGG, or GGGGGG.

66. An optional spacer sequence in Claim 1, which encodes a semi-flexible amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of an amino acid sequence of 2-55 amino acids in length and features a semi- structured motif enriched with a combination of proline, alanine, and serine.

67. An optional spacer sequence in Claim 1, which encodes a semi-flexible amino acids spacer sequence that follows the signal peptide cleavage site and is comprised of at least a 4 amino acid stretch of one or more of GSPAG, GGASPAGG, or GGASPAAPAPAG.

68. An optional spacer sequence in Claim 1, which encodes a rigid amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of an amino acid sequence of 2-55 amino acids in length that can form a structured alpha helix, beta strand, hairpin loop, or other structured motif when followed by the therapeutic protein or peptide sequence.

69. An optional spacer sequence in Claim 1, which encodes a rigid amino acid spacer sequence that follows the signal peptide cleavage site and features poly-proline, poly- proline-threonine, poly-alanine, and may be stabilized by glutamic acid / lysine salt bridges.

70. An optional spacer sequence in Claim 1, which encodes a rigid amino acid spacer sequence that follows the signal peptide cleavage site and features the following motif (XP)n where n is between 2 and 28 inclusively and X is an Alanine, Lysine, or Glycine.

71. An optional spacer sequence in Claim 1, which encodes a rigid amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of at least a 4 amino acid stretch of one or more of GPPPG, GPPPPPPPG, GTPTPTPTPTG, or GGAEAAAKEAAAKAGG.

72. An optional spacer sequence in Claim 1, which encodes an amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of at least one endopeptidase cleavage site.

73. An optional spacer sequence in Claim 1, which encodes an amino acid spacer sequence that follows the signal peptide cleavage site and is comprised of PPTIFFRL, KPIEFFRL, R[S / K][R / S / K][R / K]XXX[G / E], XXX[L / F]VXXX, PFHL[L / V / K][V / I / Y][Y / H / G][S / N], DEVD[G / S]XXX, X[E / D]XDXXXX,QTGGKXEX, PQGIAGQ, DXXD, XRXKRRX, KRIKR, RGRR, RGKR, LRGGKXXX, or [D / L]ETD[G / S / A]XXX.

74. An amino acid sequence in Claim 1, which is part of the optional spacer sequence or near or at the end of the signal peptide and is processable by one or more of trypsin, chymotrypsin, caspases, elastase, thermolysin, pepsin, glutamyl endopeptidase, plasmin, MMPs, calpain, furin, renin, neprilysin or related or other mammalian endopeptidase.

75. An amino acid sequence in Claim 1, which is part of the optional spacer sequence and is specifically processable by mammalian extracellular endopeptidases, following secretion to expose one or more active sites at the N-terminal of the therapeutic protein.

76. An amino acid sequence in Claim 1, which is part of the optional spacer sequence and is specifically processable by Neprilysin, kallikreins, cathepsins, MMPs, AD AMTS, or plasmin following secretion from the cell.

77. A sequence encoding a therapeutic peptide in Claim 1, which encodes glucagon-like peptide 1, dipeptidyl peptidase-4-resistant glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, exendin-4, or derivatives, or dual- or triple-agonists, at least one of the antagonistic targets of which is the GLP-1 receptor.

78. A sequence encoding a therapeutic peptide in Claim 1, which encodes insulin, including furin-cleavable insulin, enhanced-stability insulin, seleno-insulin, four-disulfide insulin analog, or derivatives.

79. A sequence encoding a therapeutic peptide in Claim 1, which encodes human growth hormone, human growth hormone antagonist, human growth hormone agonist, insulin receptor agonist or antagonist, Thyroid Stimulating Hormone (TSH), Adrenocorticotropic Hormone (ACTH), Vasopressin (Antidiuretic Hormone - ADH), Oxytocin, Parathyroid Hormone (PTH), Calcitonin, Glucagon, Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), somatostatin receptor agonist or antagonist, Corticotropin-Releasing Hormone (CRH), Amylin, Prolactin, Natriuretic Peptides (ANP and BNP), pancreatic polypeptide, Insulin-like Growth Factor 1 (IGF- 1), Leptin, Peptide YY, Ghrelin, Cholecystokinin (CCK), Adiponectin, peptide from the Relaxin family, Secretin, Gastrin, GIP agonist, GIP-GLP dual agonist or other peptide hormone, polypeptide hormone, dual or triple agonist, or a fusion thereof with at least one fragment of another functional protein.

80. A sequence encoding a therapeutic peptide in Claim 1, which encodes a peptide hormone or peptide fragment of a natural protein that has potential therapeutic activity.

81. A sequence encoding a therapeutic protein in Claim 1, which encodes at least a fraction of one or more of Adalimumab, Rituximab, Trastuzumab, Bevacizumab, Infliximab, Pembrolizumab, Atezolizumab, Etanercept, Cetuximab, Ranibizumab, Omalizumab, Natalizumab, Golimumab, Tocilizumab, Ustekinumab, Daratumumab, Ixekizumab, Secukinumab, Alemtuzumab, Eculizumab, Palivizumab, Abciximab, Basiliximab, Tositumomab, Panitumumab, Ofatumumab, Trastuzumab emtansine, Mogamulizumab, Obinutuzumab, Sarilumab, Brentuximab vedotin, Dinutuximab, Efgartigimod or other monoclonal, bispecific, or multispecific antibody, antibody fragment, nanobody, or fusion thereof with at least one other functional fragment of another protein.

82. A sequence encoding a therapeutic protein in Claim 1, which encodes at least IL-2, alpha-L-iduronidase, iduronate-2- sulfatase, alpha-glucosidase, alpha-galactosidase, glucocerebrosidase, phenylalanine hydroxylase, Factor VIII, Factor IX, Factor XI, or a secreted protein or enzyme the function of which is lost in a human genetic disorder, or their analog, homolog, ortholog, or their derivative.

83. A sequence encoding a therapeutic protein in Claim 1, which encodes a protein or a functional protein fragment or subunit with potential therapeutic activity.

84. A sequence encoding a fusion protein in Claim 1, which encodes a fusion of a fragment of IL- 12 and IL-23, an anti-CD30 antibody and monomethyl auristatin E, alpha-L- iduronidase enzyme and IGF-1, TNF-alpha receptor and an Fc fragment of an antibody, VEGFR1 and VEGFR2 or other fusion protein between two or more fragments of a protein at least one of which is functional.

85. A sequence encoding a fusion protein in Claim 1, which encodes a fusion between at least one protein or functional protein fragment and another protein or protein fragment, either of which has potential therapeutic activity.

86. A spanning sequence in Claim 1, which encodes one or more of the following amino acid sequences, AXAXYXXX, AXAXXXEG, AXAXXXQG, AXAX[P / K]P[T / I][FE], AXAXR[S / K][R / S / K][R / K], AXAXXXX[L / F], AXAX[P / D][F / E][H / V][L / D], AXAXX[E / D]XD, AXAX[Q / P][T / Q]G[G / I], AXAXDXXD, AXAXXRXK, [A / V / S / T / G]X[A / G / S / T]XYXXX, [A / V / S / T / G]X[A / G / S / T]XXXEG, [A / V / S / T / G]X[A / G / S / T]XXXQG, [A / V / S / T / G]X[A / G / S / T]X[P / K]P[T / I] [I / E] , [A / V / S / T / G]X[A / G / S / T]XR[S / K] [R / S / K] [R / K] ,[A / V / S / T / G]X[A / G / S / T]XXXX[L / F] ,[A / V / S / T / G]X[A / G / S / T]X[P / D] [F / E] [H / V] [L / D] , [A / V / S / T / G]X[A / G / S / T]XX[E / D]XD, [A / V / S / T / G]X[A / G / S / T]X[Q / P] [T / Q]G[G / I] , [A / V / S / T / G]X[A / G / S / T]XDXXD, or [A / V / S / T / G]X[A / G / S / T]XXRXK, which span the secretion signal cleavage site starting at the n-4, n-3, n-2, or n-1 position, where n- 1 is the amino acid immediately preceding the cleavage site.

87. A spanning sequence in Claim 1, that encodes an amino acid sequence, which spans 1 to 4 amino acids of the therapeutic protein and 1 to 4 amino acids of the secretion peptide.

88. White subcutaneous adipocytes in Claim 1, which are part of human, canine, feline, equine, or other mammalian tissues.

89. A nucleic acid construct in Claim 1, which is an RNA, DNA, or RNA-DNA hybrid construct, with or without chemical modifications.

90. A vector in Claim 1, which is a non-antigenic or minimally antigenic vector that does not elicit a neutralizing immune response following subcutaneous administration at levels anticipated to be clinically effective.

91. Sequences and constructs in Claim 1, which are delivered as part of a 0.1 to 50mL subcutaneous injection.

92. Sequences and constructs in Claim 1, which are delivered by physical, chemical, or biochemical methods.

93. Sequences and constructs in Claim 1, which are delivered by viral vectors.

94. Sequences and constructs in Claim 1, which are delivered by virus like particles.

95. Sequences and constructs in Claim 1, which are delivered by polymeric nanoparticles.

96. Sequences and constructs in Claim 1, which are delivered by lipid nanoparticles.

97. Sequences and constructs in Claim 1, which are delivered by liposomes.

98. Sequences and constructs in Claim 1, which are delivered by transfection.

99. Sequences and constructs in Claim 1, which are delivered by electroporation or particle mediated transfer.

100. Sequences and constructs comprising Claim 1, which are described in the Examples of the present invention.

101. Sequences in Claim 1, which have been codon optimized.

102. Sequences in Claim 1, which have been codon optimized for a given species using codon frequency usage algorithms for said species.

103. Sequences in Claim 1, which are used in tandem with other sequences promoting increased gene expression including one or more of promoters, terminators, insulators, 3’UTRs, 5’UTRs, enhancers, or combinations thereof.

104. Sequences in Claim 1, which are used as part of therapies for human or veterinary pathologies.

105. Nucleic acid sequences in Claim 1, which encode more than one natural or in silico optimized secretion signal.

106. Nucleic acid sequences in Claim 1, which encode at least a fragment of one secretion signal and at least a fragment of one other secretion signal.

107. Nucleic acid and amino acid sequences or combinations thereof in Claim 1 described in the examples.

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