Compositions and methods for ADP-ribosylation factor molecular trafficking

Abstract

Described herein are compositions comprising a homologous ADP-ribosylation factor (ARF) protein sequence useful for the trafficking of proteins, heterologous peptide sequences, covalently-linked small molecules, and covalently-linked nucleic acids. Also described herein are methods for trafficking an ARF shuttle protein outside of a cell. Also described herein are methods for trafficking an ARF shuttle protein, a drug-peptide conjugate ARF shuttle protein, or a nucleic acid-peptide conjugate ARF shuttle protein to an intracellular location of a target cell.

Description

COMPOSITIONS AND METHODS FOR ADP-RIBOSYLATION FACTOR MOLECULAR TRAFFICKINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 609,290, filed on December 12, 2023 which is incorporated by reference herein in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 54275-715_601_SL.xml, created on December 11, 2024, which is 116,296 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0003] Proteins functioning as shuttle proteins can be involved in controlling molecular transport within cells and between cells. Shuttle proteins may act as carriers, facilitating the transport of specific cargo molecules, such as proteins or nucleic acids, to precise locations within a cell. Shuttle proteins are involved in the generation, regulation, and movement of vesicular components inside of and outside of cell plasma membranes. Shuttle proteins contribute to the organization of cellular function through the regulation of controlled intracellular and extracellular movement of molecules. Engineering of shuttle proteins provides an opportunity to modulate endogenous molecular trafficking pathways within cells, between cellular compartments, and between different cells and cell types. There remains a need for improved engineered shuttle proteins to modulate innate cellular functions for potential therapeutic effects or improvements in industrial biotechnology.SUMMARY

[0004] Small GTPases of the ADP-ribosylation factor (ARF) family are involved in the formation of transport vesicles within eukaryotic cells and the trafficking of vesicles between certain cellular compartments. ARF proteins participate in the assembly and disassembly of coat proteins which are critical for the budding and sorting of vesicles during intracellular transport. ARF proteins also have roles in the regulation of membrane lipid metabolism, being involved in the recruitment and activation of enzymes controlling phospholipid synthesis. By these means, ARF proteins function to maintain the integrity and identity of cellular organelles by influencing membrane structure and dynamics. ARF1 has been identified as a Golgi complex localized protein involved in the regulation of coatomer-coated vesicles mediating Coat protein (COPI)vesicle transport from the cis end of the Golgi complex back to the rough endoplasmic reticulum (ER). A broader Ras superfamily of small GTPases sharing sequence homologous to ARF proteins include the ARF-like protein family members, ADP ribosylation factor related proteins, and secretion associated Ras related GTPases. This family of proteins plays roles in intracellular membrane trafficking, vesicle formation, lipid metabolism, cellular signaling, and stress responses, all of which contribute to the control of cellular function and the maintenance of cellular homeostasis.

[0005] A method was discovered and developed for utilizing members of the ARF protein family to create proteins capable of trafficking covalently associated molecules out of cells via non-canonical secretion and into cells in a receptor-independent fashion.

[0006] In certain aspects, disclosed herein are shuttle proteins comprising a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently -coupled heterologous peptide sequence comprising one or more functional protein domains of a therapeutic peptide. In some embodiments, the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36 or 54-55. In some embodiments, the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55, or 68-106 having one or more amino acid substitutions, deletions, or insertions. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36 or 54-55. In some embodiments, the covalently -coupled heterologous peptide sequence comprises one or more functional protein domains of a therapeutic peptide. In some embodiments, the one or more protein functional domains of the therapeutic peptide comprise an amino acid sequence at least about 90% identical to the sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the one or more protein functional domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide, with one or more amino acid substitutions, insertions, or deletions.In some embodiments, the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the covalently -coupled heterologous peptide sequence is positioned N-terminal to the homologous ARF superfamily sequence. In some embodiments, the covalently-coupled heterologous peptide sequence is positioned C-terminal to the homologous ARF superfamily sequence. In some embodiments, the homologous ARF superfamily sequence and the covalently- coupled heterologous peptide sequence are separated by a linker peptide sequence. In some embodiments, the linker peptide sequence comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the linker peptide sequence comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, a signal peptide positioned at the N-terminus of the shuttle protein. In some embodiments, the signal peptide comprises an amino acid sequence selected from SEQ ID NOs: 58-67. In some embodiments, the shuttle protein comprises a site for myristoylation. In some embodiments, the site for myristoylation is located at or near the N- terminus of the shuttle protein. In some embodiments, the site for myristoylation comprises a glycine residue. In some embodiments, the site for myristoylation comprises the peptide sequence MGSS. In some embodiments, the shuttle protein comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N-terminus of the shuttle protein. In some embodiments, the shuttle protein comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the shuttle protein. In some embodiments, the shuttle protein comprises a cleavage site. In some embodiments, the cleavage site comprises a TEV protease recognition motif. In some embodiments, the TEV protease recognition motif comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the shuttle protein comprises a half-life extending moiety. In some embodiments, the half-life extending moiety comprises an amino acid sequence at least about 95% identical to a human serum albumin (HSA) sequence. In some embodiments, the halflife extending moiety comprises an amino acid sequence identical to SEQ ID NO: 49. In some embodiments, the half-life extending moiety comprises an amino acid sequence at least about 95% identical to an Fc portion of a human immunoglobulin. In some embodiments, the half-life extending moiety comprises an amino acid sequence identical to SEQ ID NO: 48.

[0007] In certain aspects, disclosed herein are drug-peptide conjugate shuttle proteins comprising a homologous ADP-ribosylation factor (ARF) superfamily sequence, and acovalently-coupled small molecule drug. In some embodiments, the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36 or 54-55. In some embodiments, the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55, or 68-106 having one or more amino acid substitutions, deletions, or insertions. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36 or 54-55. In some embodiments, the small molecule drug is selected from the group consisting of acetylsalicylic acid, ibuprofen, acetaminophen, simvastatin, lisinopril, metformin, atorvastatin, clopidogrel, omeprazolec, ciprofloxacin, metoprolol, diazepam, cetirizine, alprazolam, levothyroxine, warfarin, ranitidine, hydrochlorothiazide, fluoxetine, ranitidine, morphine, allopurinol, furosemide, hydralazine, methotrexate, losartan, montelukast, cephalexin, digoxin, phenytoin, carvedilol, isosorbide mononitrate, tamsulosin, sitagliptin, cyclophosphamide, 5 -fluorouracil, doxorubicin, vincristine, paclitaxel, docetaxel, cisplatin, carboplatin, ifosfamide, etoposide, imatinib, tamoxifen, flutamide, bortezomib, lenalidomide, erlotinib, sorafenib, irinotecan, topotecan, gemcitabine, dasatinib, and everolimus. In some embodiments, the small molecule drug is positioned at or near the N-terminus of the homologous ARF superfamily sequence. In some embodiments, the small molecule drug is positioned at or near the C-terminus of the homologous ARF superfamily sequence.

[0008] In certain aspects, disclosed herein are nucleic acid-peptide conjugate shuttle proteins comprising a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently-coupled nucleic acid sequence. In some embodiments, the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36 or 54-55. In some embodiments, the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55, or 68-106 having one or more amino acid substitutions, deletions, or insertions. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36 or 54-55. In some embodiments, the covalently-coupled nucleic acid sequence comprises a therapeutic DNA sequence. In some embodiments, the covalently -coupled nucleic acid sequence comprises a therapeutic RNA sequence. In some embodiments, the therapeutic RNA sequence comprises a small interfering RNA sequence. In some embodiments, the covalently-coupled nucleic acid sequence comprises a therapeutic peptide nucleic acid (PNA) sequence. In some embodiments, the covalently-couplednucleic acid sequence comprises an antisense oligonucleotide sequence. In some embodiments, the covalently-coupled nucleic acid sequence is positioned at or near the N-terminus of the homologous ARF superfamily sequence. In some embodiments, the covalently-coupled nucleic acid sequence is positioned at or near the C-terminus of the homologous ARF superfamily sequence.

[0009] In certain aspects, disclosed herein are methods of trafficking a shuttle protein outside of a cell, the methods comprising expressing a recombinant nucleic acid coding for a shuttle protein described herein in vitro in cultured cells from a cell line, wherein the shuttle protein is efficiently transported into supernatant surrounding the cultured cells.

[0010] In certain aspects, disclosed herein are methods of trafficking a shuttle protein into a target cell, the methods comprising contacting a shuttle protein described herein to the surface of the target cell, wherein the shuttle protein is efficiently internalized into an intracellular location within the target cell.

[0011] In certain aspects, disclosed herein are methods of trafficking a drug-peptide conjugate shuttle protein into a target cell, the methods comprising contacting a drug-peptide conjugate shuttle protein described herein to the surface of the target cell, wherein the drug- peptide conjugate shuttle protein is efficiently internalized into an intracellular location within the target cell.

[0012] In certain aspects, disclosed herein are methods of trafficking a nucleic acid-peptide conjugate shuttle protein into a target cell, the methods comprising contacting a nucleic acid- peptide conjugate shuttle protein described herein to the surface of the target cell, wherein the nucleic acid-peptide conjugate shuttle protein is efficiently internalized into an intracellular location within the target cell.

[0013] In certain aspects, disclosed herein are shuttle proteins comprising: a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently -coupled heterologous peptide sequence comprising one or more functional protein domains of a therapeutic peptide. In some embodiments, the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36, 54-55, or 68-106. In some embodiments, the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55, or 68-106 having one or more amino acid substitutions, deletions, or insertions. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36, 54- 55, or 68-106. In some embodiments, the covalently-coupled heterologous peptide sequence comprises one or more functional protein domains of a therapeutic peptide. In someembodiments, the covalently -coupled heterologous peptide sequence comprises one or more functional protein domains of a therapeutic peptide. In some embodiments, the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence at least about 90% identical to the sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide, with one or more amino acid substitutions, insertions, or deletions. In some embodiments, the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the covalently -coupled heterologous peptide sequence is positioned N-terminal to the homologous ARF superfamily sequence. In some embodiments, the covalently-coupled heterologous peptide sequence is positioned C-terminal to the homologous ARF superfamily sequence. In some embodiments, the homologous ARF superfamily sequence and the covalently- coupled heterologous peptide sequence are separated by a linker peptide sequence. In some embodiments, the shuttle protein further comprises a signal peptide positioned at the N-terminus. In some embodiments, the shuttle protein is lacking signal peptide positioned at the N-terminus. In some embodiments, the shuttle protein further comprises a site for myristoylation. In some embodiments, the site for myristoylation comprises the peptide sequence MGSS. In some embodiments, the shuttle protein is lacking a site for myristoylation. In some embodiments, the shuttle protein further comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N-terminus of the shuttle protein. In some embodiments, the shuttle protein further comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the shuttle protein.In some embodiments, the shuttle protein further comprises a cleavage site. In some embodiments, the shuttle protein further comprises a half-life extending moiety. In some embodiments, the half-life extending moiety comprises an amino acid sequence at least about 95% identical to a human serum albumin (HSA) sequence. In some embodiments, the half-life extending moiety comprises an amino acid sequence of SEQ ID NO: 49. In some embodiments, the half-life extending moiety comprises an amino acid sequence of SEQ ID NO: 50. In some embodiments, the half-life extending moiety comprises an amino acid sequence at least about 95% identical to an Fc portion of a human immunoglobulin. In some embodiments, the half-life extending moiety comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 48. In some embodiments, the half-life extending moiety comprises an amino acid sequence of SEQ ID NO: 48. In some embodiments, the half-life extending moiety comprises an amino acid sequence of SEQ ID NO: 48 with L234A / L235A substitution. In some embodiments, the half-life extending moiety comprises an amino acid sequence of SEQ ID NO: 48 with L234A / L235A / P329G substitution. In some embodiments, the homologous ARF superfamily sequence comprises T31N substitution. In some embodiments, the homologous ARF superfamily sequence comprises C159S substitution. In some embodiments, the homologous ARF superfamily sequence comprises N60G substitution. In some embodiments, the homologous ARF superfamily sequence comprises N60G / C159S substitution. In some embodiments, the homologous ARF superfamily sequence comprises T48I substitution. In some embodiments, the homologous ARF superfamily sequence comprises F51L substitution. In some embodiments, the homologous ARF superfamily sequence comprises Q71L substitution. In some embodiments, the homologous ARF superfamily sequence comprises R99H substitution.

[0014] In certain aspects, disclosed herein are drug-peptide conjugate shuttle proteins comprising: a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently-coupled small molecule drug. In some embodiments, the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36, 54-55, or 68-106. In some embodiments, the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55, or 68-106 having one or more amino acid substitutions, deletions, or insertions. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36, 54-55, or 68-106. In some embodiments, the small molecule drug is selected from the group consisting of acetylsalicylic acid, ibuprofen, acetaminophen, simvastatin, lisinopril, metformin, atorvastatin, clopidogrel, omeprazolec, ciprofloxacin, metoprolol, diazepam, cetirizine, alprazolam, levothyroxine, warfarin, ranitidine,hydrochlorothiazide, fluoxetine, ranitidine, morphine, allopurinol, furosemide, hydralazine, methotrexate, losartan, montelukast, cephalexin, digoxin, phenytoin, carvedilol, isosorbide mononitrate, tamsulosin, sitagliptin, cyclophosphamide, 5 -fluorouracil, doxorubicin, vincristine, paclitaxel, docetaxel, cisplatin, carboplatin, ifosfamide, etoposide, imatinib, tamoxifen, flutamide, bortezomib, lenalidomide, erlotinib, sorafenib, irinotecan, topotecan, gemcitabine, dasatinib, and everolimus. In some embodiments, the small molecule drug is positioned at or near the N-terminus of the homologous ARF superfamily sequence. In some embodiments, the small molecule drug is positioned at or near the C-terminus of the homologous ARF superfamily sequence.

[0015] In certain aspects, disclosed herein are methods of trafficking a shuttle protein outside of a cell, the method comprising expressing a recombinant nucleic acid coding for a shuttle protein described herein in vitro in cultured cells from a cell line, wherein the shuttle protein is efficiently transported into supernatant surrounding the cultured cells.

[0016] In certain aspects, disclosed herein are methods of trafficking a shuttle protein into a target cell, the method comprising contacting a shuttle protein described herein to the surface of the target cell, wherein the shuttle protein is efficiently internalized into an intracellular location within the target cell. In some embodiments, the target cell is an adipocyte. In some embodiments, the target cell is a preadipocyte.

[0017] In certain aspects, disclosed herein are methods of trafficking a drug-peptide conjugate shuttle protein into a target cell, the method comprising contacting a drug-peptide conjugate shuttle protein described to the surface of the target cell, wherein the drug-peptide conjugate shuttle protein is efficiently internalized into an intracellular location within the target cell. In some embodiments, the target cell is an adipocyte. In some embodiments, the target cell is a preadipocyte.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the features described herein are utilized, and the accompanying drawings of which:

[0019] FIG. 1 shows alignment of human ARF1, ARF3, and ARF5 protein sequences indicating homologous and divergent amino acids residues at positions within the proteins. Human ARF1 protein sequence corresponds to P84077 (SEQ ID NO: 1). Human ARF3 protein sequence corresponds to P61204 (SEQ ID NO: 12). Human ARF5 protein sequence correspondsto P84085 (SEQ ID NO: 14).

[0020] FIG. 2A-FIG. 2B show results of engineered ARF1 protein production in Expi239F™ cells (ThermoFisher). FIG. 2A shows a Western blot of MGSS HIS TEV ARFl wt protein produced three days following transfection with an ARF1 expression construct detected using an anti-ARFl antibody. Protein samples from supernatant and cell pellet were assayed. FIG. 2B shows a chart measuring the amount of engineered ARF1 protein produced from the respective lanes shown in the Western blot of FIG. 2A.

[0021] FIG. 3A-FIG. 3B show results of His-tagged engineered ARF1 protein production. FIG. 3A shows a Western blot of using anti -His antibody to assess production of His-tagged ARF1 polypeptides from the supernatants of CHO cells at Day 5 post-transfection or in Expi293F cells at Day 4 post-transfection to assess if His-tagged engineered ARF1 polypeptides are secreted. FIG. 3B shows a Western blot using anti -His antibody to assess production of His- tagged ARF1 polypeptides from the cell lysates of CHO cells at Day 5 post-transfection or in Expi293F cells at Day 4 post-transfection to assess if His-tagged engineered ARF1 polypeptides are produced and maintained intracellularly.

[0022] FIG. 4A-FIG. 4B show results of engineered ARFl-Fc fusion protein and Flag- tagged ARF1 polypeptide production in Expi293F cells in supernatant and in cell lysates. FIG. 4A shows an image of a Western blot using an anti-His antibody to assay His-tagged engineered ARF1 polypeptides, non-tagged engineered ARF1 polypeptides, and FL AG-tagged engineered ARF1 polypeptides found in the supernatant (sup) or cell pellet (pellet) from Day 3 posttransfection. FIG. 4B shows an image of a Western blot using an anti-ARFl antibody for detection of His-tagged engineered ARF1 polypeptides, ARFl-Fc fusions, and FLAG-tagged engineered ARF1 polypeptides.

[0023] FIG. 5 shows results of Western blot (left) of completed medium extracted protein samples and Western blot (right) of cell pellet extracted protein samples using an anti-ARFl antibody for detection of engineered ARF1 polypeptides with HSA fusions and engineered ARF1 polypeptides with amino acid substitutions testing production and secretion in Expi293F cells in Day 4 post-transfection.

[0024] FIG. 6 shows results of Western blot (left) and charting of results (right) for engineered ARF1 polypeptides testing intracellular expression and secretion in Expi293F cells on Day 3 post-transfection.

[0025] FIG. 7 shows a graph of Western blot results from various engineered ARF1 protein constructs quantitating protein expression levels in 293T cells within the supernatant, the pellet, and a calculated ratio of supernatant / pellet.

[0026] FIG. 8 shows results of engineered ARF1 protein production via Western blot on anon-reducing SDS-PAGE gel. Gel lanes and SEQ ID NOs are indicated in the figure.

[0027] FIG. 9 shows results of engineered ARF1 protein production via Western blot on reducing and non-reducing SDS-PAGE gel. Gel lanes and SEQ ID NOs are indicated in the figure.

[0028] FIG. 10A-FIG. 10C show results from testing ARF1-T3 IN constructs using SDS- PAGE, analytical SEC-HPLC, and adipose cell droplet reduction quantitation. FIG. 10A shows Western blot results on SDS-PAGE. FIG. 10B shows a graph of analytical SEC-HPLC results. FIG. 10B shows results from adipose cell droplet reduction quantitation.

[0029] FIG. 11A-FIG.11F show uptake of engineered ARF1 proteins and ARF1 fusion proteins in 3T3-L1 mouse preadipocytes. FIG. HA shows a graph of protein uptake in preadipocytes for various engineered ARF1 proteins at three test doses. FIG. 11B shows representative images of immunofluorescence protein detection and localization for various engineered ARF1 proteins at three test doses. FIG. 11C shows a graph of protein uptake in preadipocytes for various engineered ARF1 proteins and ARF1 fusion proteins at two test doses. FIG. HD shows representative images of immunofluorescence protein detection and localization for various engineered ARF1 proteins and ARF1 fusion proteins at two test doses. FIG. HE shows a graph of protein uptake in preadipocytes for various engineered ARF1 proteins and ARF1 fusion proteins at two test doses. FIG. HF shows representative images of immunofluorescence protein detection and localization for various engineered ARF1 proteins and ARF1 fusion proteins at two test doses.

[0030] FIG. 12 shows results from Western blot via non-reducing SDS-PAGE to analyze dimerization and glycosylation of several engineered ARF1 constructs to improve homogeneity of production. Proteins of SEQ ID NOs 91-96 were tested.

[0031] FIG. 13 shows results from Western blot via non-reducing and reducing SDS-PAGE to analyze protein aggregation to improve homogeneity of production. Proteins of SEQ ID NOs 77, 78, and 87 were tested.

[0032] FIG. 14A-FIG. 14C shows the results of analyzing engineered ARF1 protein biochemistry and in vitro activity on Days 3, 4, and 6 produced from HEK293 cells. FIG. 14A shows non-reducing and reducing SDS-PAGE assessed by Western blot. FIG. 14B shows a graph analytical SEC-HPLC results. In vitro activity in adipose lipid drop reduction was assessed in the chart in FIG. 14C.DETAILED DESCRIPTION

[0033] ADP-ribosylation factor 1 (ARF1) is a member of the human ARF gene family. The ARF family members encode small guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity. ARF1 protein is produced from the ARF1 gene locus (official full name: ADP ribosylation factor 1; also known an PVNH8). The ARF1 protein was known to be localized to the Golgi apparatus and has been determined to have a central role in intra-Golgi transport. ARF1 protein is herein identified as a molecule that can be secreted from stem cells. Blood profiling data demonstrates that ARF proteins including ARF1, ARF3, ARF4, and ARF5 have been detected circulating in the blood.

[0034] In mammals, there are typically at least 5 ARF proteins and 11 ARF-like proteins, which together constitute a family of the RAS superfamily. The RAS superfamily are referred to as the ADP -ribosylation factor (ARF) superfamily of proteins. Class 1 ARF proteins include ARF1, and ARF3. Class 2 ARF proteins include ARF4 (formerly known as ARF2) and ARF5. Class 3 ARF proteins include ARF6. ARF-like proteins include ARL2, ARL3, ARL5, ARL6, ARL8, ARP, SARI A, and SAR1B, among others. ARF and ARF-like proteins have demonstrated activities in early stages of COP1 vesicle-mediated secretory pathways with roles functioning in the cytoplasm or in concert with the Golgi apparatus or other organelle membranes. ARF and ARF-like proteins had not previously been reported to be exported from the cells in which they were produced. Described herein are shuttle proteins comprising homologous ADP-ribosylation factor (ARF) superfamily sequences that are trafficked extracellularly. These shuttle proteins are useful for aiding in manufacturing of ARF and ARF- like protein sequences allowing for purification from supernatant of in vitro cultured production cells. These shuttle proteins are useful for trafficking of a homologous ARF superfamily sequence outside of cell. These shuttle proteins are useful for trafficking of a heterologous peptide sequence covalently-coupled to the homologous ARF superfamily sequence outside of cell. These shuttle proteins are useful for trafficking of a small molecule drug covalently-coupled to the homologous ARF superfamily sequence outside of cell. These shuttle proteins are useful for trafficking of a nucleic acid covalently-coupled to the homologous ARF superfamily sequence outside of cell. In some aspects described herein are ARF superfamily shuttle proteins comprising a homologous ADP-ribosylation factor (ARF) superfamily sequence and a covalently-coupled heterologous peptide sequence. In some embodiments, the covalently- coupled heterologous peptide sequence comprises one or more functional protein domains of a therapeutic peptide. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARF1. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARF3. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARF4. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at leastabout 90% identical to human ARF5. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARF6. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARL2. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARL3. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARIA. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARIA. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARL8. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARP. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human SARI A. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human SAR1B. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF1, wherein the ARF superfamily sequence comprises a sequence of human ARF1 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF3, wherein the ARF superfamily sequence comprises a sequence of human ARF3 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF4, wherein the ARF superfamily sequence comprises a sequence of human ARF4 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF5, wherein the ARF superfamily sequence comprises a sequence of human ARF5 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF6, wherein the ARF superfamily sequence comprises a sequence of human ARF6 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARL2, wherein the ARF superfamily sequence comprises a sequence of human ARL2 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARIA, wherein the ARF superfamily sequence comprises a sequence of human ARIA having one or more amino acid substitutions, deletions, or additions.In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARL5, wherein the ARF superfamily sequence comprises a sequence of human ARIA having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARIA, wherein the ARF superfamily sequence comprises a sequence of human ARIA having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARL8, wherein the ARF superfamily sequence comprises a sequence of human ARL8 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARP, wherein the ARF superfamily sequence comprises a sequence of human ARP having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human SARI A, wherein the ARF superfamily sequence comprises a sequence of human SARI A having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human SAR1B, wherein the ARF superfamily sequence comprises a sequence of human SAR1B having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF1 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF3 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF4 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF5 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF6 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARL2 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARL3 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARIA protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARIA protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARL8 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARP protein sequence. In some embodiments, thehomologous ARF superfamily sequence comprises a protein sequence identical to human SARI A protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human SAR1B protein sequence. In some embodiments, the covalently -coupled heterologous peptide sequence comprises a peptide tag. In some embodiments, the covalently-coupled heterologous peptide sequence comprises a linker sequence. In some embodiments, the covalently-coupled heterologous peptide sequence comprises a site for cleavage. In some embodiments, the site for cleavage comprises a TEV protease recognition domain. In some embodiments, the covalently-coupled heterologous peptide sequence comprises a Fc portion of an IgG molecule. In some embodiments, the covalently- coupled heterologous peptide sequence comprises a serum albumin domain. In some embodiments, the serum albumin domain is a human serum albumin (HSA) domain. In some embodiments, the serum albumin domain is a variant of a human serum albumin domain. In some embodiments, the HSA domain comprises a sequence at least about 99% identical to SEQ ID NO: 49 having one or more amino acid substitutions, additions, or deletions. In some embodiments, the HSA domain comprises a sequence at least about 99% identical to SEQ ID NO: 50 having one or more amino acid substitutions, additions, or deletions. In some embodiments, the HSA domain comprises a sequence identical to SEQ ID NO: 49. In some embodiments, the HSA domain comprises a sequence identical to SEQ ID NO: 50. In some embodiments, the one or more functional protein domains of the therapeutic peptide alters serum half-life of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide increases serum half-life of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide alters protein trafficking dynamics of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide is designed to allow myristoylation of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide is designed to prevent myristoylation of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide correspond to one or more functional protein domains of the therapeutic peptide. In some embodiments, the one or more functional protein domains of the therapeutic peptide correspond one protein domain of the therapeutic peptide. In some embodiments, the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence at least about 90% identical to the sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide,lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide, with one or more amino acid substitutions, insertions, or deletions. In some embodiments, the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the covalently- coupled heterologous peptide sequence is positioned N-terminal to the homologous ARF superfamily sequence. In some embodiments, the covalently-coupled heterologous peptide sequence is positioned C-terminal to the homologous ARF superfamily sequence. In some embodiments, the shuttle protein is an ARF1 shuttle protein. In some embodiments, the shuttle protein is an ARF3 shuttle protein. In some embodiments, the shuttle protein is an ARF4 shuttle protein. In some embodiments, the shuttle protein is an ARF5 shuttle protein. In some embodiments, the shuttle protein is an ARF6 shuttle protein. In some embodiments, the shuttle protein is an ARL2 shuttle protein. In some embodiments, the shuttle protein is an ARL3 shuttle protein. In some embodiments, the shuttle protein is an ARIA shuttle protein. In some embodiments, the shuttle protein is an ARL6 shuttle protein. In some embodiments, the shuttle protein is an ARL8 shuttle protein. In some embodiments, the shuttle protein is an ARP shuttle protein. In some embodiments, the shuttle protein is an SARI A shuttle protein. In some embodiments, the shuttle protein is an SAR1B shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide comprise a recognized protein domain motif and convey a functional property to the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide do not comprise a recognized protein domain motif and convey a functional property to the shuttle protein. In some embodiments, the functional property alters intracellular trafficking of the shuttle protein. In some embodiments, the functional property alters extracellular trafficking of the shuttle protein. In some embodiments, the functional property alters secretion of the shuttle protein. In some embodiments, the functional property increases a percentage of shuttle protein that is secretedcompared to a total amount synthesized in a cell.

[0035] In some aspects described herein are drug-peptide conjugate shuttle proteins comprising a homologous ADP-ribosylation factor (ARF) superfamily sequence and a covalently-coupled small molecule drug. In some embodiments, the drug-peptide conjugate shuttle protein further comprises a covalently-coupled heterologous peptide sequence comprising one or more functional protein domains of a therapeutic peptide. In some embodiments, the covalently-coupled heterologous peptide sequence comprises one or more functional protein domains of a therapeutic peptide. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARF1. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARF3. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARFA In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARF5. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARF6. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARL2. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARL3. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARIA. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARIA. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARL8. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human ARP. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human SARI A. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 90% identical to human SAR1B. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF1, wherein the ARF superfamily sequence comprises a sequence of human ARF1 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF3, wherein the ARF superfamily sequence comprises a sequence of human ARF3 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF4, wherein the ARF superfamily sequence comprises asequence of human ARF 4 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF5, wherein the ARF superfamily sequence comprises a sequence of human ARF5 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARF6, wherein the ARF superfamily sequence comprises a sequence of human ARF6 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARL2, wherein the ARF superfamily sequence comprises a sequence of human ARL2 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARL3, wherein the ARF superfamily sequence comprises a sequence of human ARL3 having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARIA, wherein the ARF superfamily sequence comprises a sequence of human ARIA having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARIA, wherein the ARF superfamily sequence comprises a sequence of human ARIA having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARIA, wherein the ARF superfamily sequence comprises a sequence of human ARIA having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human ARP, wherein the ARF superfamily sequence comprises a sequence of human ARP having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human SARI A, wherein the ARF superfamily sequence comprises a sequence of human SARI A having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence at least about 98% identical to human SAR1B, wherein the ARF superfamily sequence comprises a sequence of human SAR1B having one or more amino acid substitutions, deletions, or additions. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF1 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF3 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprisesa protein sequence identical to human ARF4 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF5 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARF6 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARL2 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARL3 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARIA protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARL6 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARL8 protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human ARP protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human SARI A protein sequence. In some embodiments, the homologous ARF superfamily sequence comprises a protein sequence identical to human SAR1B protein sequence. In some embodiments, the covalently -coupled heterologous peptide sequence comprises a peptide tag. In some embodiments, the covalently-coupled heterologous peptide sequence comprises a linker sequence. In some embodiments, the covalently-coupled heterologous peptide sequence comprises a site for cleavage. In some embodiments, the site for cleavage comprises a TEV protease recognition domain. In some embodiments, the covalently-coupled heterologous peptide sequence comprises a Fc portion of an IgG molecule. In some embodiments, the covalently- coupled heterologous peptide sequence comprises a serum albumin domain. In some embodiments, the serum albumin domain is a human serum albumin (HSA) domain. In some embodiments, the serum albumin domain is a variant of a human serum albumin domain. In some embodiments, the HSA domain comprises a sequence at least about 99% identical to SEQ ID NO: 49 having one or more amino acid substitutions, additions, or deletions. In some embodiments, the HSA domain comprises a sequence at least about 99% identical to SEQ ID NO: 50 having one or more amino acid substitutions, additions, or deletions. In some embodiments, the HSA domain comprises a sequence identical to SEQ ID NO: 49. In some embodiments, the HSA domain comprises a sequence identical to SEQ ID NO: 50. In some embodiments, the one or more functional protein domains of the therapeutic peptide alters serum half-life of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide increases serum half-life of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide alters protein traffickingdynamics of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide is designed to allow myristoylation of the shuttle protein. In some embodiments, the one or more functional protein domains of the therapeutic peptide is designed to prevent myristoylation of the shuttle protein. In some embodiments, the covalently- coupled small molecule drug is selected from the group consisting of acetylsalicylic acid, ibuprofen, acetaminophen, simvastatin, lisinopril, metformin, atorvastatin, clopidogrel, omeprazolec, ciprofloxacin, metoprolol, diazepam, cetirizine, alprazolam, levothyroxine, warfarin, ranitidine, hydrochlorothiazide, fluoxetine, ranitidine, morphine, allopurinol, furosemide, hydralazine, methotrexate, losartan, montelukast, cephalexin, digoxin, phenytoin, carvedilol, isosorbide mononitrate, tamsulosin, sitagliptin, cyclophosphamide, 5-fluorouracil, doxorubicin, vincristine, paclitaxel, docetaxel, cisplatin, carboplatin, ifosfamide, etoposide, imatinib, tamoxifen, flutamide, bortezomib, lenalidomide, erlotinib, sorafenib, irinotecan, topotecan, gemcitabine, dasatinib, and everolimus. In some embodiments, the small molecule drug is positioned at or near the N-terminus of the homologous ARF superfamily sequence. In some embodiments, the small molecule drug is positioned at or near the C-terminus of the homologous ARF superfamily sequence.

[0036] Described herein are shuttle proteins comprising homologous ADP-ribosylation factor (ARF) superfamily sequences that are trafficked intracellularly. These shuttle proteins are useful for trafficking of a homologous ARF superfamily sequence to one or more intracellular locations following contacting with the extracellular surface and cellular uptake of the homologous ARF superfamily sequence. These shuttle proteins are useful for trafficking of a heterologous peptide sequence covalently-coupled to the homologous ARF superfamily sequence to one or more intracellular locations following contacting with the extracellular surface and cellular uptake of the homologous ARF superfamily sequence. These shuttle proteins are useful for trafficking of a small molecule drug covalently-coupled to the homologous ARF superfamily sequence to one or more intracellular locations following contacting with the extracellular surface and cellular uptake of the homologous ARF superfamily sequence. These shuttle proteins are useful for trafficking of a nucleic acid sequence covalently-coupled to the homologous ARF superfamily sequence to one or more intracellular locations following contacting with the extracellular surface and cellular uptake of the homologous ARF superfamily sequence.

[0037] In an aspect, described herein is a composition comprising a shuttle protein comprising a homologous ADP-ribosylation factor (ARF) superfamily sequence and a covalently-coupled heterologous peptide sequence comprising one or more functional protein domains of a therapeutic peptide. In some embodiments, the shuttle protein comprises a half-life extending moiety. In some embodiments, the shuttle protein comprises a signal peptide sequence.In some embodiments, the shuttle protein comprises a site for myristoylation. In some embodiments, the shuttle protein comprises a peptide tag. In some embodiments, the shuttle protein comprises a therapeutic heterologous peptide sequence. In some embodiments, the halflife extending moiety comprises a serum albumin sequence or variant thereof. In some embodiments, the half-life extending moiety comprises a human serum albumin protein sequence. In some embodiments, the half-life extending moiety comprises an Fc region of an IgG molecule. In some embodiments, the half-life extending moiety comprises an Fc region of a human IgGl molecule or variant thereof. In some embodiments, the heterologous protein sequence comprises a fluorescent protein sequence. In some embodiments, the fluorescent protein sequence comprises a green fluorescent protein (GFP) or an enhanced green fluorescent protein (EGFP) sequence. In some embodiments, the shuttle protein comprises a cleavage site. In some embodiments, the cleavage site comprises a protease recognition motif. In some embodiments, the shuttle protein comprises a cleavage recognition site for a TEV protease. In some embodiments, the shuttle protein comprises a peptide tag. In some embodiments, the peptide tag comprises an amino acid sequence selected from SEQ ID NOs: 37-38 or 40-47. In some embodiments, the shuttle protein possesses an increase in at least one of stability, biological function, or in vivo half-life of the shuttle protein compared to a wild-type ARF or ARF-like protein sequence.

[0038] In some aspect, the ARF shuttle protein comprises a covalently-coupled protein trafficking domain sequence inserted within or next to the homologous ARF superfamily protein sequence that increases at least one of stability, biological function, or in vivo half-life of the ARF shuttle protein. In some embodiments, a heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF superfamily protein sequence increases stability of the shuttle protein. In some embodiments, the heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF superfamily protein sequence increases biological function of the shuttle protein. In some embodiments, the heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF superfamily protein sequence increases in vivo half-life of the shuttle protein. In some embodiments, a heterologous peptide amino acid sequence inserted within or next to the homologous ARF superfamily protein sequence improves in vitro expression of the shuttle protein. In some embodiments, a heterologous peptide amino acid sequence inserted within or next to the homologous ARF superfamily protein sequence improves in vitro purification of the shuttle protein. In some embodiments, a heterologous peptide amino acid sequence inserted within or next to the homologous ARF superfamily protein sequence improves in vitro secretion of the shuttle protein. In some embodiments, the shuttle protein comprises one or more chemical modifications. In some embodiments, the one or morechemical modifications comprise one or more site-specific covalent attachments of a fatty acid to amino acid residues of the shuttle protein. In some embodiments, a form of the shuttle protein comprising one or more fatty acids maintains a longer serum half-life. In some embodiments, the longer serum half-life allows for a subject to be administered a lower effective dose of a composition comprising the shuttle protein. In some embodiments, the lower effective dose is sufficient for treatment of a subject.

[0039] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided are practiced without these details. Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0040] As used herein a composition that is “consisting essentially” of the recited components is a composition that only has the recited elements as active ingredients, but can comprise other non-active components that do not appreciably modify the function or activity of the recited components. Any list disclosed herein that is recited as “comprising” can be recited as “consisting essentially,” to exclude non-recited polypeptide or protein components.

[0041] As used herein the term “about” refers to an amount that is near the stated amount by 10% or less.

[0042] As used herein the terms “individual” “subject,” and “patient” are interchangeable. The subject can be mammal such as a horse, cow, pig, chicken, goat, rabbit, mouse, rat, dog, or cat. In certain embodiments, the subject is a human person.

[0043] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. Polypeptides, including the provided polypeptide chains and other peptides, e.g., linkers and binding peptides, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-translational modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as throughmutations of hosts that produce the proteins, errors due to PCR amplification, or errors in protein translation.

[0044] In some embodiments, a recombinant protein is a protein expressed in a system other than a human, e.g., the protein is expressed from bacteria, yeast, or mammalian cells in culture. In some cases, the protein is expressed from Chinese Hamster Ovary cells (CHO cells). In some cases, the protein is expressed from mouse myeloma cells, e.g., (NSO) cells. In some cases, the protein is expressed from E. coli.

[0045] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0046] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acidsequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0047] Heterologous polypeptide amino acid sequences contain amino acid residue sequence not present in a polypeptide sequence originating from a different source (e.g., a different gene or a different species) in which the heterologous polypeptide amino acid sequence is now part of as a single polypeptide chain. A heterologous polypeptide amino acid sequence may comprise a short peptide tag. A heterologous polypeptide amino acid sequence may consist essentially of a short peptide tag. A heterologous polypeptide amino acid sequence may comprise part of a fusion protein or an engineered modified protein described herein that may comprise, consist, or consist essentially of a fragment of an immunoglobulin molecule, an albumin molecule, an Annexin A10 peptide molecule, a transferrin molecule, an XTEN sequence, a proline-alanine-serine polymer, a homo-amino acid polymer, a glycine-rich sequence, a gelatin-like polymer, an elastin-like peptide, a carboxy-terminal peptide, or combinations thereof.

[0048] As described herein a “ homologous ADP-ribosylation factor 1 (ARF1) protein sequence” is a polypeptide sequence sharing a certain extent of sequence identity with various ARF1 proteins derived from a vertebrate species. The extent of shared sequence identity must be sufficient to use a protein alignment software program to align at least a portion of the homologous ARF1 sequence to a known ARF1 protein derived from a vertebrate species (e.g., at least 50% identity of aligned portions of a polypeptide sequence over a minimum length of 40 amino acids). Exemplary ARF1 proteins derived from various vertebrate species to be used in an assessment of polypeptide alignment with a homologous ARF1 protein sequence can be identified through the following NCBI Accession numbers: NP_001649.1, JAA08426.1, OCT75402.1, BAE31170.1, 8SDW_A, DAA31858.1, EHH50487.1, CAI9572194.1, NP_958888.1, or XP_039540413.1.

[0049] As described herein a “homologous ADP-ribosylation factor (ARF) superfamily sequence” is a polypeptide sequence sharing a certain extent of sequence identity with various ARF or ARF-like proteins derived from a vertebrate species. The extent of shared sequence identity must be sufficient to use a protein alignment software program to align at least a portion of the homologous ARF superfamily sequence to a known ARF or ARF-like protein derived from a vertebrate species (e.g., at least 50% identity of aligned portions of a polypeptide sequence over a minimum length of 40 amino acids). Exemplary ARF and ARF-like proteins derived from various vertebrate species to be used in an assessment of polypeptide alignment with a homologous ARF superfamily protein sequence can be identified through the following NCBI Accession numbers: NP_001649.1, JAA08426.1, OCT75402.1, BAE31170.1, 8SDW_A, DAA31858.1, EHH50487.1, CAI9572194.1, NP_958888.1, XP_039540413.1, or by SEQ IDNOs: 1-36, 54-55, or 68-106.

[0050] In some embodiments, reference to a conjugate, polypeptide conjugate, or protein conjugate refers to a synthetically and / or recombinantly produced molecule comprising a chemical entity covalently bound to one or more amino acids of an amino acid sequence. In some cases, the conjugation is selective such that the chemical entity is connected to a specific amino acid of the amino acid sequence. In some embodiments, the amino acid sequence comprises a polypeptide described herein. For example, the polypeptide described herein is a polypeptide comprising a homologous ARF1 protein sequence. In another example, the polypeptide described herein is a polypeptide comprising a homologous ARF superfamily protein sequence.

[0051] In some embodiments, a polypeptide described herein is a proteoform of a protein comprising a homologous ARF1 protein sequence or a homologous ARF superfamily protein sequence. In some aspects, as used herein a proteoform describes a molecular form of a protein product arising from a gene encoding a protein, such as a protein including, for example, ARF1. In some cases, a proteoform includes proteins that arise from the same gene as a result of genetic variation, alternatively spliced RNA transcripts, post-translational modifications, or polypeptide cleavage event.Engineered ARF Superfamily Polypeptides

[0052] In some aspects, shuttle proteins described herein are engineered polypeptides useful for treating a disorder in a subject.

[0053] In an aspect, shuttle proteins described herein comprising a homologous ARF superfamily protein sequence are formulated in a pharmaceutical composition. In certain embodiments, polypeptides described herein comprising a homologous ARF superfamily protein sequence are included in a composition comprising a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, polypeptides described herein comprising a homologous ARF superfamily protein sequence are produced by in vitro techniques and included in a composition to be administered to a subject in need thereof.

[0054] In some aspects, shuttle proteins described herein comprises one or more modifications. In some embodiments, modifications to a homologous ARF superfamily polypeptide described herein comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids from the N-terminal region of the homologous ARF superfamily protein sequence. In some embodiments, modifications to a homologous ARF superfamily polypeptidedescribed herein comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids from the C-terminal region of the homologous ARF superfamily protein sequence. In some embodiments, the deletion is a deletion of a known inhibitory domain or domains of a homologous ARF superfamily protein sequence. In some embodiments, the deletion is a deletion of one or more amino acids known to be required for a GTP-GDP exchange reaction. In some embodiments, the deletion is a deletion of one or more amino acids known to be required for dissociation of GDP from the GDP -bound form of the small GTPase. In some embodiments, the deletion is a deletion of one or more amino acids known to be required for association of GTP to a guanine-nucleotide free form of the small GTPase. In some embodiments, the deletion is a deletion of one or more amino acids known to be required for the catalytic hydrolysis of GTP.

[0055] Additional modifications to the ARF shuttle proteins described herein comprise insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180 or more amino acids into the N-terminal region of the homologous ARF superfamily protein sequence. Additional modifications to the ARF shuttle proteins described herein comprise insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids into the C-terminal region of the homologous ARF superfamily protein sequence. Additional modifications to the ARF shuttle proteins described herein comprise insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids into a region internal to the N-terminus and C-terminus of the homologous ARF superfamily protein sequence.

[0056] Additional modifications to the ARF shuttle proteins described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in the N-terminal region of the homologous ARF superfamily protein sequence. Additional modifications to the ARF shuttle proteins described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in the C- terminal region of the homologous ARF superfamily protein sequence. Additional modificationsto the ARF shuttle proteins described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in a region internal to the N-terminus and C-terminus of the homologous ARF superfamily protein sequence. In some embodiments, the amino acid substitution or substitutions are conservative substitutions. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Exemplary conservative amino acid substitutions are listed in Table 3. In some embodiments, the amino acid substitution or substitutions are not conservative substitutions.

[0057] In some embodiments, the ARF shuttle proteins or a component of the ARF shuttle protein comprising the homologous ARF superfamily protein sequence can comprise cleavage products of a pro-protein. Cleavage of a pro-protein can result in activation or higher activity of said pro-protein. In certain aspects, the polypeptides comprise only the active domain of a homologous ARF or ARF -like pro-protein (e.g., a minimal portion sufficient to create a biological effect). In certain aspects, the polypeptides comprise only the active domain of a homologous ARF1 pro-protein (e.g., a minimal portion sufficient to create a biological effect). In some embodiments, a component of the modified protein comprising the homologous ARF1 protein sequence is a product or a portion corresponding to a larger modified protein (e.g., a cleavage product). In some embodiments, a covalently-coupled protein trafficking domain sequence is cleaved off of ARF shuttle protein.

[0058] In certain aspects, a composition consists essentially of an ARF shuttle protein described herein or of a component of the ARF shuttle protein comprising the homologous ARF superfamily protein sequence, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0059] In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 1, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 2, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 3, and a pharmaceutically acceptable excipient, carrier, ordiluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 4, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 5, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 6, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 7, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 8, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 9, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 10, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 11, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% aminoacid sequence identity to SEQ ID NO: 12, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 13, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 14, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 15, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 16, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 17, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 18, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 19, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 20, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20. In certain aspects, described herein is a compositioncomprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 21, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 21. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 22, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 23, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 23. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 24, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 25, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 25. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 26, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 26. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 27, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 27. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 28, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 28. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 29, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO: 29. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 30, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 31, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 32, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 32. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 33, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 33. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 34, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 35, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 35. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 36, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 36. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 54, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 54. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 55, and a pharmaceutically acceptable excipient, carrier,or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 55.

[0060] In some aspects described herein, an ARF shuttle protein comprises a homologous ARF superfamily sequence, and a covalently-coupled heterologous peptide sequence comprising one or more functional protein domains of a therapeutic peptide. In some embodiments, the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36, 54-55, or 68-106. In some embodiments, the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55, or 68-106 having one or more amino acid substitutions, deletions, or insertions. In some embodiments, the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36, 54-55, or 68-106.

[0061] In some embodiments, the covalently-coupled heterologous peptide sequence comprises one or more protein domains of a therapeutic peptide. In some embodiments, the one or more protein domains of a therapeutic peptide comprise at least one functional protein domain. In some embodiments, the one or more protein domains of a therapeutic peptide comprise at least two functional protein domains. In some embodiments, the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid-peptide conjugate shuttle protein comprise one protein domain of a therapeutic peptide. In some embodiments, a therapeutic peptide has a length of between about 2-50 amino acids. In some embodiments, the one or more protein domains are contained within a portion of the therapeutic peptide. In some embodiments, the one or more protein domains are contained within the entirety of the therapeutic peptide. In some embodiments, the therapeutic peptide is a heterologous peptide sequence distinct from an ARF superfamily derived protein sequence. In some embodiments, the therapeutic peptide serves as a linker sequence to properly space peptide or proteins within a single polypeptide chain. In some embodiments, the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid-peptide conjugate shuttle protein comprise a therapeutic peptide and a linker sequence. In some embodiments, the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid-peptide conjugate shuttle protein comprise a polypeptide sequence derived from a homologous ARF superfamily sequence and a covalently-coupled heterologous peptide sequence arranged in a single polypeptide chain. In some embodiments, the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid-peptide conjugate shuttle protein comprise a polypeptide sequence derived from a homologous ARF superfamily sequence and a covalently-coupled heterologous peptide sequence arranged in a single polypeptide chain as a fusion protein. In some embodiments, single polypeptide chains of the shuttle protein, drug-peptide conjugate shuttleprotein, or nucleic acid-peptide conjugate shuttle protein dimerize. In some embodiments, single polypeptide chains of the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid- peptide conjugate shuttle protein are predominantly in monomeric form. In some embodiments, single polypeptide chains of the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid-peptide conjugate shuttle protein are glycosylated. In some embodiments, single polypeptide chains of the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid-peptide conjugate shuttle protein are not significantly glycosylated. In some embodiments, the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid-peptide conjugate shuttle protein comprise one or more amino acid substitutions to reduce protein aggregation. In some embodiments, the one or more protein domains confer a functional property onto the shuttle protein, drug-peptide conjugate shuttle protein, or nucleic acid-peptide conjugate shuttle protein. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in glucose transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in hormone signaling in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in neurotransmitter uptake in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in ion transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in drug efflux in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in hormone transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in metal ion transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in waste product removal in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in RNA transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in vitamin transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in lipid transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in enzyme transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in metabolite transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in signaling transduction in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in nucleotide transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in intracellular vesicle transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in second messenger transport in a host cell. In some embodiments, the one or more protein domains of thetherapeutic peptide are involved in glycoprotein transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in cellular organelle transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in mitochondrial protein transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in morphogen transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in RNA interference in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in gas molecule in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in biofluorescence molecule transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in stress granule component transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in mitotic checkpoint protein transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in cholesterol efflux in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in metabolic sensor transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in immune system modulator transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in DNA repair protein transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in telomere repair protein transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in exosome cargo transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in oxidative phosphorylation component transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in Golgi apparatus enzyme transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in nuclear pore complex component transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in adhesion molecule transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in intracellular redox sensor transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in cellular NAD+ transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in plasma membrane lipid raft component transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in molecular chaperone transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in cellularheat shock response regulator transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in lysosomal enzyme transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in histone acetyltransferase transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in Protein kinase C (PKC) isoform transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide are involved in G-protein coupled receptor (GPRC) transport in a host cell. In some embodiments, the one or more protein domains of the therapeutic peptide comprise an amino acid sequence at least about 90% identical to the sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the one or more protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide, with one or more amino acid substitutions, insertions, or deletions. In some embodiments, the one or more protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the covalently -coupled heterologous peptide sequence is positioned N-terminal to the homologous ARF superfamily sequence. In some embodiments, the covalently-coupled heterologous peptide sequence is positioned C-terminal to the homologous ARF superfamily sequence. In some embodiments, the homologous ARF superfamily sequence and the covalently- coupled heterologous peptide sequence are separated by a linker peptide sequence. In some embodiments, the linker peptide sequence comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the linker peptide sequence comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, a signal peptide positioned at the N-terminus of the shuttle protein. In some embodiments, the signal peptide comprises an amino acid sequenceselected from SEQ ID NOs: 58-67. In some embodiments, the shuttle protein comprises a site for myristoylation. In some embodiments, the site for myristoylation is located at or near the N- terminus of the shuttle protein. In some embodiments, the site for myristoylation comprises a glycine residue. In some embodiments, the site for myristoylation comprises the peptide sequence MGSS. In some embodiments, the site for myristoylation is specifically blocked or removed. In some embodiments, a site typically myristoylated in wild-type ARF1 protein is configured in the engineered ARF1 protein to prevent addition of a myristoyl group to a glycine residue. Position G2 from SEQ ID NO: 1 is typically myristoylated in endogenously produced wild-type ARF1 protein. In some embodiments, the sequence corresponding to G2 in a homologous ARF protein sequence is mutated to a natural amino acid residue other than glycine. In some embodiments, the sequence corresponding to G2 in the homologous ARF protein sequence is positioned within the engineered ARF1 protein or engineered ARF superfamily protein at a location in a single polypeptide chain other than the second amino acid following an initiation methionine, thereby preventing myristoylation. In some embodiments, a site typically myristoylated in wild-type ARF family member protein is configured in the engineered ARF protein to prevent addition of a myristoyl group to a glycine residue. Engineered ARF1 protein designed to prevent myristoylation of an N-terminal glycine residue has modified protein trafficking dynamics compared to endogenously produced ARF1 protein. In some embodiments, the shuttle protein comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N-terminus of the shuttle protein. In some embodiments, the shuttle protein comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the shuttle protein. In some embodiments, the shuttle protein comprises a cleavage site. In some embodiments, the cleavage site comprises a TEV protease recognition motif. In some embodiments, the TEV protease recognition motif comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the shuttle protein comprises a half-life extending moiety. In some embodiments, the half-life extending moiety comprises an amino acid sequence at least about 95% identical to a human serum albumin (HSA) sequence. In some embodiments, the half-life extending moiety comprises an amino acid sequence identical to SEQ ID NO: 49. In some embodiments, the halflife extending moiety comprises an amino acid sequence at least about 95% identical to an Fc portion of a human immunoglobulin. In some embodiments, the half-life extending moiety comprises an amino acid sequence identical to SEQ ID NO: 48.

[0062] In some embodiments, the covalently-coupled heterologous peptide sequence may allow for plasma membrane insertion of the shuttle protein in a target cell. In some embodiments, the covalently-coupled heterologous peptide sequence may allow for plasma membrane insertionof the shuttle protein in a cell expressing the shuttle protein. In some embodiments, plasma membrane insertion allows for presentation on the cell surface of one or more protein domains of an engineered ARF shuttle fusion protein of a specific factor involved in intercellular signaling. In some embodiments, the specific factor involved in intercellular signaling is a ligand. In some embodiments, the specific factor involved in intercellular signaling is a receptor. In some embodiments, the specific factor involved in intercellular signaling is an antigen-presentation component.

[0063] Table 1 lists amino acid sequences for exemplary peptide and polypeptide components of engineered ARF shuttle proteins. In some embodiments, the engineered ARF shuttle protein comprises a six histidine peptide tag. In some embodiments, the engineered ARF shuttle protein comprises a myristoylation sequence-Six histidine short peptide. In some embodiments, the engineered ARF shuttle protein comprises a TEV protease recognition motif. In some embodiments, the engineered ARF shuttle protein comprises a myristoylation sequence- Six histidine short peptide + TEV protease recognition motif. In some embodiments, the engineered ARF shuttle protein comprises a peptide tag. In some embodiments, the engineered ARF shuttle protein comprises a human IgGl Fc mutant. In some embodiments, the engineered ARF shuttle protein comprises an HSA protein sequence. In some embodiments, the engineered ARF shuttle protein comprises an HSA protein sequence of SEQ ID NO: 49. In some embodiments, the engineered ARF shuttle protein comprises a green fluorescent protein sequence of SEQ ID NO: 56. In some embodiments, the engineered ARF shuttle protein comprises a green fluorescent protein sequence of SEQ ID NO: 57.

[0064] In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence setforth in SEQ ID NO: 11. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the engineered ARF shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the engineered ARF shuttle protein comprises the amino acidsequence set forth in SEQ ID NO: 55.Table 1: Sequences for components of engineered ART shuttle proteins

[0065] In some embodiments, the engineered ARF shuttle protein comprises one or more post-translational modifications. In some embodiments, the one or more post-translational modifications comprise site-specific glycosylation of species amino acid residues of the modified protein.

[0066] In some embodiments, the engineered ARF shuttle protein comprises one or more chemical modifications to the homologous ARF superfamily protein sequence. In some embodiments, the one or more chemical modifications comprises a site-specific attachment of one or more fatty acid residues. In some embodiments, the one or more fatty acid residues are attached via covalent attachment to one or more cysteine residues of the engineered ARF1 polypeptide.Fusion Proteins

[0067] In some aspects, a engineered ARF shuttle protein described herein comprises a signal peptide located at the N-terminus of a polypeptide chain. In some embodiments, the signal peptide comprises an amino acid sequence listed in Table 2A. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the signalpeptide comprises the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the signal peptide is cleaved from the engineered ARF shuttle protein upon or immediately after translocation of the engineered polypeptide to the plasma membrane. In some embodiments, cleavage of the signal peptide releases the engineered ARF shuttle protein from a cell in which it was produced. In some embodiments, secreted engineered ARF shuttle proteins are produced in vitro and purified for use in a therapeutic composition. Table 2A lists exemplary signal peptide sequences for use in expression and production of engineered ARF shuttle proteins described herein. In some embodiments, the signal peptide is used to express an engineered ARF1 protein described herein. In some embodiments, the signal peptide is used to express an engineered ARF Superfamily protein described herein. In some embodiments, the signal peptide is used to express an engineered ARF1 fusion protein described herein. In some embodiments, the signal peptide is used to express an engineered ARF fusion protein described herein.

[0068] In some aspects, mutation of specific residues within an ARF homologous sequence improves protein homogeneity during production. In some embodiments, mutation of one or more specific residues within an ARF1 sequence improves protein homogeneity during production. In some embodiments, an engineered ARF1 protein comprising C159S substantially prevents dimerization. In some embodiments, an engineered ARF1 protein comprising C159S prevents dimerization. In some embodiments, an engineered ARF1 protein comprising N60G significantly reduces glycosylation. In some embodiments, an engineered ARF1 protein existing predominantly in monomeric form following production is desired as an improvement in protein homogeneity. In some embodiments, an engineered ARF1 protein existing predominantly in nonglycosylated form following production is desired as an improvement in protein homogeneity. In some embodiments, monomeric engineered ARF1 proteins have improved stability. In some embodiments, non-glycosylated engineered ARF1 proteins have improved stability. In some embodiments, monomeric and non-glycosylated engineered ARF1 proteins have improved stability. In some embodiments of engineered ARF1 proteins described herein, unwanted protein aggregation is observed. In some embodiments, aggregation mainly caused by T31N significantly in C-term Fc ARF1 proteins is observed. In some embodiments, ARF1 fusion protein comprising C159S substitution in the ARF1 homology portion has reduced protein aggregation.

[0069] In some aspects, engineered ARF1 proteins described herein are transported inside cells when contacted extracellularly. In some embodiments, engineered ARF1 proteins described herein are efficiently transported inside cells when contacted extracellularly. In some embodiments, engineered ARF1 proteins described herein are transported intracellularly in a dose-dependent manner. In some embodiments, an engineered ARF1 protein comprising asubstitution in the ARF1 homology region is efficiently transported inside cells when contacted extracellularly. In some embodiments, the engineered ARF1 protein is efficiently transported inside preadipocytes. In some embodiments, the engineered ARF1 protein is efficiently transported inside adipocytes. In some embodiments, an engineered ARF1 protein comprising T3 IN is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 protein comprising T48I is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 protein comprising F51L is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 protein comprising Q71L is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 protein comprising R99H is efficiently transported inside a target cell. In some embodiments, the target cell is a preadipocyte. In some embodiments, the target cell is an adipocyte. In some embodiments, the target cells comprise preadipocytes and adipocytes. In some embodiments, an engineered ARF1 protein comprising ARF1-T31N with N-terminal His-TEV is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 protein comprising ARF1 -wild-type with N-terminal His- TEV is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 fusion protein comprising T31N with C-terminal Fc sequence is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 fusion protein comprising T31N / C159S with C-terminal Fc sequence is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 fusion protein comprising T31N / C159S with N-terminal Fc sequence is efficiently transported inside a target cell. In some embodiments, an engineered ARF1 protein comprising N60G substitution has reduced glycosylation. In some embodiments, an engineered ARF1 protein comprising C159S substitution has reduced dimerization. In some embodiments, an engineered ARF1 protein comprising T31N / C159S substitutions has reduced dimerization and reduced glycosylation. In some embodiments, the engineered ARF1 protein is selected in a format that improves homogeneity during production and isolation from cells. In some embodiments, an engineered ARF1 protein comprising C159S substitution reduces protein aggregation. In some embodiments, an engineered ARF1 protein comprising C159S substitution reduces aggregation of ARF1 proteins comprising T31N. In some embodiments, an engineered ARF1 protein comprising C159S substitution reduces aggregation of ARF1 proteins comprising T31N and C-terminal Fc. In some embodiments, the engineered ARF1 protein maintains lipid reduction activity in adipocytes 3 days after application to target cells. In some embodiments, the engineered ARF1 protein maintains lipid reduction activity in adipocytes 3 days after application to target cells. In some embodiments, the engineered ARF1 protein does not possess lipid reduction activity in adipocytes 6 days after application to target cells. In some instances, efficient transport of a shuttle protein, an ARF1 fusion protein, a drug-peptide conjugate shuttleprotein, or a nucleic acid-peptide conjugate shuttle protein into supernatant surrounding cultured cells is measured by a ratio of detected protein levels in supernatant versus pellet from cells expressing a construct that encodes the shuttle protein, the ARF1 fusion protein, the drug-peptide conjugate shuttle protein, or the nucleic acid-peptide conjugate shuttle protein. In some instances, detected protein levels are measured in mg / L. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 1.0. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 2.0. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 2.3. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 3.2. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 5.4. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 9.5. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 10.0. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 15.0. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 20.0. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 25.0. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 30.0. In some embodiments, efficient transport is a supernatant to pellet ratio of at least about 33.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 1.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 2.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 3.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 4.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 5.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 9.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 10.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 15.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 20.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 25.0. In some embodiments, efficient transport is a supernatant to pellet ratio of greater than 30.0.Table 2A: Exemplary Signal Peptide Sequences for Engineered ARE Shuttle Proteins

[0070] In some aspects the engineered ARF shuttle protein, engineered ARF1 shuttle protein, ARF1 fusion protein, or ARF fusion protein described herein does not comprise a signal peptide located at the N-terminus of a polypeptide chain. In some embodiments, the engineered ARF1 protein without a signal peptide is secreted from cells. In some embodiments, the engineered ARF1 protein without a signal peptide is efficiently secreted from cells. In some embodiments, the engineered ARF1 protein without a signal peptide is secreted from cells more efficiently than engineered ARF1 protein that remains localized intracellularly. In some embodiments, without a signal peptide (SP), engineered ARF1 protein can be secreted with N-term His and C-term His, GFP, or Fc tag. In some embodiments, without a signal peptide (SP), engineered ARF1 protein cannot be efficiently secreted with N-flag and C-terminal HSA domain (C-HSA). In some embodiments, N-flag or C-HSA hinders membrane trafficking functions of engineered ARF1 protein.

[0071] Table 2B lists constructs for engineered ARF1 shuttle protein and ARF1 fusion protein.Table 2B: Sequences for components of engineered polypeptides and full length EngineeredARF polypeptides

[0072] In some aspects, the engineered ARF1 shuttle protein comprises a heterologous peptide sequence. In some embodiments, the heterologous peptide sequence comprises a peptide tag. In some embodiments, the heterologous peptide sequence comprises a TEV protease recognition sequence. In some embodiments, the heterologous peptide sequence comprises a serum albumin domain. In some embodiments, the heterologous peptide sequence comprises an Fc protein of an IgG molecule. In some embodiments, the ARF1 shuttle protein comprises a sequence at least about 90% identical to a sequence listed in Table 2B. In some embodiments, the ARF1 shuttle protein comprises a protein sequence at least about 90% identical to any one of SEQ ID NOs: 68-106. In some embodiments, the ARF1 shuttle protein comprises a protein sequence at least about 95% identical to any one of SEQ ID NOs: 68-106. In some embodiments, the ARF1 shuttle protein comprises a protein sequence at least about 99% identical to any one of SEQ ID NOs: 68-106. In some embodiments, the ARF1 shuttle protein comprises a protein sequence at least about 99% identical to any one of SEQ ID NOs: 68-106 with one or more amino acid substitutions, deletions, or insertions. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to any one of SEQ ID NOs: 68-106. In some embodiments, the ARF1 shuttle protein consists essentially of a protein sequence identical to any one of SEQ ID NOs: 68-106. In some embodiments, the ARF1 shuttle protein is formulated with a pharmaceutically acceptable excipient or diluent. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 68. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 69. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 70. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 71. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 72. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 73. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 74. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 75. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 76. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 77. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO:78. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 79. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 80. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 81. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 82. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 83. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 84. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 85. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 86. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 87. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 88. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 89. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 90. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 91. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 92. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 93. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 94. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 95. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 96. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 97. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 98. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 99. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 100. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 101. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 102. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 103. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 104. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 105. In some embodiments, the ARF1 shuttle protein comprises a protein sequence identical to SEQ ID NO: 106.

[0073] In certain aspects, a composition comprising an engineered ARF shuttle protein comprising a homologous ARF superfamily protein sequence and a heterologous polypeptide amino acid sequence inserted within or adjacent to the homologous ARF superfamily proteinsequence comprises one of more conservative amino acid substitutions. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., arginine, lysine and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, and tryptophan), nonpolar side chains (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline, and valine), beta-branched side chains (e.g., isoleucine, threonine, and valine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine). Exemplary conservative amino acid substitutions are listed in Table 3.Table 3: Exemplary Amino Acid Modifications

[0074] In some embodiments, the amino acid substitution or substitutions are not conservative substitutions. FIG. 1 shows an alignment of human ARF1, ARF3, and ARF5 protein sequences. Human ARF1 protein sequence corresponds to P84077 (SEQ ID NO: 1). Human ARF3 protein sequence corresponds to P61204 (SEQ ID NO: 12). Human ARF5 protein sequence corresponds to P84085 (SEQ ID NO: 14). In some embodiments, non-conserved amino acid positions are mutated either by insertion, deletion, or substitution in the engineered ARF shuttle protein. In some embodiments, the substitution is a substitution representing a conservative amino acid change. In some embodiments, the substitution is not a substitution representing a conservative amino acid change. In some embodiments, non-conserved amino acid positions between ARF1 and ARF3 in FIG. 1 represent positions that when mutated can modulate function of the engineered ARF shuttle protein. In some embodiments, non-conserved amino acid positions between ARF1 and ARF5 in FIG. 1 represent positions that when mutated can modulate function of the engineered ARF shuttle protein. In some embodiments, nonconserved amino acid positions between ARF3 and ARF5 in FIG. 1 represent positions that when mutated can modulate function of the engineered ARF shuttle protein. In someembodiments, non-conserved amino acid positions between ARF1 and both ARF3 and ARF5 in FIG. 1 represent positions that when mutated can modulate function of the engineered ARF shuttle protein.

[0075] In some aspects, described herein are drug-peptide conjugate ARF shuttle proteins comprising a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently-coupled small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of acetylsalicylic acid, ibuprofen, acetaminophen, simvastatin, lisinopril, metformin, atorvastatin, clopidogrel, omeprazolec, ciprofloxacin, metoprolol, diazepam, cetirizine, alprazolam, levothyroxine, warfarin, ranitidine, hydrochlorothiazide, fluoxetine, ranitidine, morphine, allopurinol, furosemide, hydralazine, methotrexate, losartan, montelukast, cephalexin, digoxin, phenytoin, carvedilol, isosorbide mononitrate, tamsulosin, sitagliptin, cyclophosphamide, 5-fluorouracil, doxorubicin, vincristine, paclitaxel, docetaxel, cisplatin, carboplatin, ifosfamide, etoposide, imatinib, tamoxifen, flutamide, bortezomib, lenalidomide, erlotinib, sorafenib, irinotecan, topotecan, gemcitabine, dasatinib, and everolimus.

[0076] In some aspects, described herein are nucleic acid-peptide conjugate ARF shuttle proteins comprising a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently-coupled nucleic acid sequence. In some embodiments, the nucleic acid sequence comprises a therapeutic DNA sequence. In some embodiments, the nucleic acid sequence comprises a therapeutic RNA sequence. In some embodiments, the nucleic acid sequence comprises a therapeutic PNA sequence. In some embodiments, the nucleic acid sequence comprises an antisense oligonucleotide sequence. In some embodiments, the nucleic acid sequence comprises a small interfering RNA sequence.Methods of Production and Use of ARF Shuttle ProteinsProduction of polypeptides

[0077] Once a polypeptide (e.g., a modified protein, a homologous ARF superfamily protein sequence, an ARF1 protein sequence, or an engineered ARF shuttle protein) is selected it can be purified or synthesized in any suitable manner. A nucleic acid encoding the polypeptide can be cloned into a suitable vector and expressed in a suitable cellular system. In certain aspects, the cellular system is a prokaryotic cell system. In certain aspects, the cellular system is a eukaryotic cell system. In certain aspects, the cellular system is a mammalian cell system. The supernatants from such an expression system can be subjected to one or more purification steps involving centrifugation, ultracentrifugation, filtration, diafiltration, tangential -flow filtration, dialysis, chromatography (e.g., cation exchange, ion exchange, hydrophobic interaction, reverse phase,affinity, or size exclusion). The polypeptides can be purified to an extent suitable for human administration. Additionally, polypeptides can be synthesized for inclusion in a formulation to be administered to a human subject. In certain aspects, the polypeptides can be produced by a suitable peptide synthesis method, such as solid-phase synthesis. In some aspects, the ARF shuttle proteins described herein are trafficked extracellularly by a non-canonical secretion mechanism. In some embodiments, use of a signal peptide at the N-terminus of the ARF shuttle protein decreases or abolishes extracellular secretion of the ARF shuttle protein. In some embodiments, efficient secretion of the ARF shuttle protein allow purification from cell supernatants with improved yields from bioreactor based processes. In some embodiments, efficient secretion of the ARF shuttle protein maintains the ARF shuttle protein in cytoplasmic rather than ER / Golgi conditions during maturation which may improve folding or expression. In some embodiments, this maintenance in the cytoplasm may subject the ARF shuttle protein to indications with one or more different chaperone proteins as compared to wild-type ARF or ARF-like proteins.Intracellular Trafficking of ARF Shuttle Proteins

[0078] ARF shuttle proteins described here (e.g., ARF shuttle proteins comprising a homologous ARF superfamily sequence, and a covalently -coupled heterologous peptide sequence; drug-peptide conjugate ARF shuttle proteins; or nucleic acid-peptide conjugate ARF shuttle protein) are capable of cell internalization by one of several mechanisms. In some embodiments, ARF shuttle proteins traffic through vesicle, microvesicle, or exosome mediated uptake by an ability to associate with the lipid membranes and protein complexes coating the vesicle, microvesicle, or exosome. In some embodiments, ARF shuttle proteins are capable of extracellular membrane surface remodeling in such a way that actively drives internalization of an ARF shuttle protein. In some embodiments, ARF shuttle proteins are capable of interacting with one or more cell surface protein structures that promote transmembrane transport. In instances in which ARF shuttle proteins are internalized following contacting to the extracellular surface of a target cell, a therapeutic molecule covalently attached to the ARF shuttle protein is delivered to one or more intracellular locations within the target cell. In some embodiments, the therapeutic molecule covalently attached to the ARF shuttle protein is a therapeutic peptide sequence. In some embodiments, the therapeutic peptide sequence is selected from exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. In some embodiments, the therapeuticmolecule covalently attached to the ARF shuttle protein is a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of acetylsalicylic acid, ibuprofen, acetaminophen, simvastatin, lisinopril, metformin, atorvastatin, clopidogrel, omeprazolec, ciprofloxacin, metoprolol, diazepam, cetirizine, alprazolam, levothyroxine, warfarin, ranitidine, hydrochlorothiazide, fluoxetine, ranitidine, morphine, allopurinol, furosemide, hydralazine, methotrexate, losartan, montelukast, cephalexin, digoxin, phenytoin, carvedilol, isosorbide mononitrate, tamsulosin, sitagliptin, cyclophosphamide, 5-fluorouracil, doxorubicin, vincristine, paclitaxel, docetaxel, cisplatin, carboplatin, ifosfamide, etoposide, imatinib, tamoxifen, flutamide, bortezomib, lenalidomide, erlotinib, sorafenib, irinotecan, topotecan, gemcitabine, dasatinib, and everolimus. In some embodiments, the therapeutic molecule covalently attached to the ARF shuttle protein is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is a therapeutic DNA sequence. In some embodiments, the therapeutic nucleic acid is a therapeutic RNA sequence. In some embodiments, the therapeutic nucleic acid is a therapeutic PNA sequence. In some embodiments, the therapeutic nucleic acid comprises an antisense oligonucleotide sequence. In some embodiments, the therapeutic nucleic acid comprises a small interfering RNA sequence. In some embodiments, a mechanism of internalization of an ARF shuttle protein are more prevalent or active in a particular cell type and could be used for specificity of delivery (targeting) to a cell type of interest. In some embodiments, the ARF shuttle protein is trafficked intracellularly. In some embodiments, the ARF shuttle protein is trafficked intracellularly and is not significantly retained within endosomes. In some embodiments, the ARF shuttle protein is trafficked intracellularly and is not significantly targeted for degradation. In some embodiments, the ARF shuttle protein is trafficked intracellularly by endocytosis. In some embodiments, the ARF shuttle protein is able to escape endosomal retention. In some embodiments, the ARF shuttle protein is able to avoid significant cellular efflux of a covalently-coupled small molecule drug. In some embodiments, the ARF shuttle protein is able to avoid significant cellular efflux of a covalently-coupled small molecule drug after the ARF shuttle protein has contacted a target cell and become internalized.

[0079] In some aspects described herein are methods of trafficking a shuttle protein outside of a cell, the methods comprising expressing a recombinant nucleic acid coding for the shuttle protein described herein in vitro in cultured cells from a cell line, wherein the shuttle protein is efficiently transported into supernatant surrounding the cultured cells. In some embodiments, efficient transport of the shuttle protein into supernatant aids in isolation or purification of the shuttle protein.

[0080] In some aspects described herein are methods of trafficking a shuttle protein into a target cell, the methods comprising contacting a shuttle protein described herein to the surface ofthe target cell, wherein the shuttle protein is efficiently internalized into an intracellular location within the target cell. In some embodiments, the target cell is a preadipocyte. In some embodiments, the target cell is an preadipocyte. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is a muscle precursor cell. In some embodiments, the target cell is a myoblast.

[0081] In some aspects described herein are methods of trafficking a drug-peptide conjugate shuttle protein into a target cell, the methods comprising contacting a drug-peptide conjugate shuttle protein described herein to the surface of the target cell, wherein the drug-peptide conjugate shuttle protein is efficiently internalized into an intracellular location within the target cell. In some embodiments, the target cell is a preadipocyte. In some embodiments, the target cell is an preadipocyte. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is a muscle precursor cell. In some embodiments, the target cell is a myoblast.

[0082] In some aspects described herein are methods of trafficking a nucleic acid-peptide conjugate shuttle protein into a target cell, the methods comprising contacting a nucleic acid- peptide conjugate shuttle protein described herein to the surface of the target cell, wherein the nucleic acid-peptide conjugate shuttle protein is efficiently internalized into an intracellular location within the target cell. In some embodiments, the target cell is a preadipocyte. In some embodiments, the target cell is an preadipocyte. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is a muscle precursor cell. In some embodiments, the target cell is a myoblast.Pharmaceutically acceptable excipients, carriers, and diluents

[0083] The polypeptide (e.g., a modified protein, a homologous ARF superfamily protein sequence, an ARF shuttle protein sequence, or an engineered ARF1 polypeptide) described herein can be administered in a pharmaceutical composition that comprises one or more pharmaceutically acceptable excipients, carriers, or diluents. The exact components can differ based upon the preferred route of administration. The excipients used in a pharmaceutical composition can provide additional function to the polypeptide by making the polypeptide suitable for a particular route of administration (e.g., intravenous, topical, subcutaneous, or intramuscular), increasing polypeptide stability, increasing penetration of a desired tissue (e.g., skeletal muscle, vasculature, adipose tissue, or bone), increasing residence time at particular site, increasing solubility, enhancing the efficacy of the polypeptide, and / or reducing inflammatory reactions coincident with administration.

[0084] In certain aspects, the polypeptides (e.g., a modified protein, a homologous ARF superfamily protein sequence, an ARF shuttle protein sequence, or an engineered ARF1polypeptide) described herein are included in a pharmaceutical composition with a solubilizing emulsifying, or dispersing agent. In certain aspects, the solubilizing agent can allow high- concentration solutions of ARF shuttle protein that exceed at least about 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, or 20 mg / mL. Carbomers in an aqueous pharmaceutical composition serve as emulsifying agents and viscosity modifying agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a carbomer. In certain aspects, the carbomer comprises or consists of carbomer 910, carbomer 934, carbomer 934P, carbomer 940, carbomer 941, carbomer 1342, or combinations thereof. Cyclodextrins in an aqueous pharmaceutical composition serve as solubilizing and stabilizing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a cyclodextrin. In certain aspects, the cyclodextrin comprises or consists of alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, or combinations thereof. Lecithin in a pharmaceutical composition may serve as a solubilizing agent. In certain aspects, the solubilizing agent comprises or consists of lecithin. Poloxamers in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, and dispersing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a poloxamer. In certain aspects, the poloxamer comprises or consists of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or combinations thereof. Polyoxyethylene sorbitan fatty acid esters in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, surfactants, and dispersing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a polyoxyethylene sorbitan fatty acid ester. In certain aspects, the polyoxyethylene sorbitan fatty acid ester comprises or consists of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, or combinations thereof. Polyoxyethylene stearates in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, surfactants, and dispersing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a polyoxyethylene stearate. In certain aspects, the polyoxyethylene stearate comprises or consists of polyoxyl 2 stearate, polyoxyl 4 stearate, polyoxyl 6 stearate, polyoxyl 8 stearate, polyoxyl 12 stearate, polyoxyl 20 stearate, polyoxyl 30 stearate, polyoxyl 40 stearate, polyoxyl 50 stearate, polyoxyl 100 stearate, polyoxyl 150 stearate, polyoxyl 4 distearate, polyoxyl 8 distearate, polyoxyl 12 distearate, polyoxyl 32 distearate, polyoxyl 150 distearate, or combinations thereof. Sorbitan esters in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, and non-ionic surfactants, and dispersing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a sorbitan ester. In certain aspects, the sorbitan ester comprises or consists of sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan trioleate, sorbitan sesquioleate, or combinations thereof. Incertain aspects, solubility can be achieved with a protein carrier. In certain embodiments the protein carrier comprises recombinant human albumin.

[0085] In certain aspects, the polypeptide (e.g., a modified protein, a homologous ARF superfamily protein sequence, an ARF shuttle protein sequence, or an engineered ARF1 polypeptide) of the current disclosure are formulated to increase stability. Polypeptides in aqueous formulations may require stabilization to prevent degradation. In certain aspects, the stabilizer comprises pH buffers, salts, amino acids, polyols / disaccharides / polysaccharides, liposomes, surfactants, antioxidants, reducing agents, or chelating agents. In certain aspects, the stabilizer comprises or consists of a polyol / non-reducing sugar. In certain embodiments, the nonreducing sugar comprises or consists of sucrose, mannitol, trehalose, raffinose, stachyose, xylitol, starch, verbascose, or combinations thereof. Polypeptides can be encapsulated in liposomes to increase stability. In certain aspects, the stabilizer comprises or consists of liposomes. In certain aspects, the liposomes comprise or consists of ipalmitoylphosphatidylcholine (DPPC) liposomes, phosphatidylcholine:cholesterol (PC:Chol) (70:30) liposomes, or dipalmitoylphosphatidylcholine: dipalmitoylphosphatidylserine (DPPC:DPPS) liposomes (70:30). Non-ionic surfactants can increase the stability of a polypeptide. In certain aspects, the stabilizer comprises or consists of a non-ionic surfactant. In certain aspects, the non-ionic surfactant comprises or consists of polysorbates (e.g., poly sorbate 80, poly sorbate 20), alkylsaccharides alkyl ethers and alkyl glyceryl ethers, polyoxyethelene (4) lauryl ether; polyoxyethylene cetyl ethers, polyoxyethylene stearyl ethers, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, or combinations thereof. In certain aspects, the polypeptide is formulated with a protein surfactant, such as recombinant human serum albumin as a stabilizer. Antioxidants or reducing agents can increase the stability of a polypeptide. In certain aspects, the stabilizer comprises or consists of an antioxidant or reducing agent. In certain aspects, the reducing agent comprises or consists of dithiothreitol, ethylenediaminetetraacetic acid, 2- Mercaptoethanol, Tris(2-carboxyethyl)phosphine hydrochloride, Tris(hydroxypropyl)phosphine, or combinations thereof. In certain aspects, the antioxidant comprises or consists of methionine, ascorbic acid, citric acid, alpha tocopherol, sodium bisulfite, ascorbyl palmitate, erythorbic acid, or combinations thereof. Chelating agents can stabilize polypeptides by reducing the activity of proteases. In certain aspects, the stabilizer comprises or consists of a chelating agent. In certain aspects, the chelating agent comprises or consists of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), metal complexes (e.g. Zn-protein complexes), or combinations thereof. Buffer agents can stabilize polypeptides by reducing the acid hydrolysis of polypeptides. In certain aspects, the stabilizer comprises or consists of a buffer agent. In certain aspects, the buffer agent comprises or consists of sucroseocta-sulfate, ammonium carbonate, ammonium phosphate, boric acid, sodium citrate, potassium citrate, lactic acid, 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N- morpholino)ethanesulfonic acid (MES), hydroxymethylaminomethane (Tris), calcium carbonate, calcium phosphate or combinations thereof.EXAMPLES

[0086] The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way. Example 1 — Construction and testing of engineered polypeptides for secretion and intracellular trafficking

[0087] ARE superfamily (ARF, ARFL, ARL, ARP, and SAR) protein sequences are analyzed for structural features relating to biological function of ARF superfamily members and potential modifications that could modulate stability, biological function, in vivo half-life, intracellular transport, or extracellular transport (secretion) of an engineered protein. Features are also analyzed with regard to modifications that could improve in vitro expression or in vitro purification of the engineered protein.

[0088] Insertions, deletions, or amino acid substitutions of various peptide sequences are made at positions of interest in homologous ARF1 protein sequences. SEQ ID NOs: 1-10 list various vertebrate ARF1 protein sequences that are selected for engineering. Insertions, deletions, or amino acid substitutions of various peptide sequences are made at positions of interest in ARF superfamily protein sequences. SEQ ID NOs: 12-34 list various vertebrate ARF1 protein sequences that are selected for engineering. Versions of amino acid substitutions in the protein sequence of SEQ ID NO: 1 were made. As an example, SEQ ID NO: 11 has the initiation Methionine deleted from the protein sequence of SEQ ID NO: 1. As another example, SEQ ID NO: 68 has an N-terminal 6X-His tag followed by a TEV protease cleavage recognition sequence. The presence of the 6X-His tag and TEV sequence prevents myristoylation of a glycine residue corresponding to G2 in wild-type ARF1 sequence in the engineered ARF1 protein of SEQ ID NO: 68. Similarly, engineered ARF1 proteins or engineered ARF superfamily proteins comprising proteins sequence from any one of SEQ ID NOs: 1-34 in which the G2 residue corresponding to SEQ ID NO: 1 is not positioned following the initiating methionine results in an engineered protein lacking myristoylation at the G2 residue corresponding to SEQ ID NO: 1. Engineered ARF1 proteins or engineered ARF superfamily proteins lacking N- terminal myristoylation have protein trafficking characteristics by changing association of the engineered proteins with other membrane-associated proteins or by removing the myristoyl group which would otherwise serve as a membrane anchor. An another example, SEQ ID NO: 35has a T31N amino acid substitution. Engineered ARF1 proteins or engineered ARF superfamily proteins 1 comprising T3 IN amino acid substitution have a lower affinity for GTP and are preferentially GDP -bound thereby significantly reducing GTPase activity. As another example, SEQ ID NO: 36 lists in the protein sequence eleven positions relative to SEQ ID NO: 1 marked by an X which can be substituted with any amino acid or retain the wild-type amino acid relative to SEQ ID NO: 1. These engineered versions of ARF1 protein are tested either individually, or incorporated into a further engineered protein construct having additional amino acid sequences in a single polypeptide chain and then tested.

[0089] Various inserts, N-terminal additions, or C-terminal additions to wild-type and engineered ARF1 polypeptide sequence are made. Examples include inserting a peptide tag within an engineered ARF1 polypeptide sequence or positioning a peptide tag N-terminal or C- terminal to the engineered ARF1 polypeptide sequence. Examples include incorporation of a six histidine tag (SEQ ID NO: 37), a Strep-tag (SEQ ID NO: 41), a serine with a Strep tag (SEQ ID NO: 42), a hemagglutinin (HA) tag (SEQ ID NO: 43), a Myc tag (SEQ ID NO: 44), a synthetic NE-tag (SEQ ID NO: 45), a FLAG octapeptide tag (SEQ ID NO: 46), and three consecutive copies of a FLAG octapeptide tag (SEQ ID NO: 47). N-terminal addition of a peptide tag to engineered ARF1 proteins or engineered ARF superfamily proteins prevents N-terminal addition of a myrstoyl group to the engineered proteins.

[0090] Another example is an insertion, N-terminal addition, or C-terminal addition of one or more heterologous peptide sequences. Examples include inserting a human serum albumin (HSA) protein sequence or variant thereof N-terminal or C-terminal to the engineered ARF1 polypeptide sequence. An example HSA protein sequence used is listed in SEQ ID NO: 49. An another example, HSA protein sequence used is listed in SEQ ID NO: 50. Further examples include inserting a human serum albumin (HSA) protein sequence or variant thereof N-terminal or C-terminal to an engineered polypeptide sequence from the ARF superfamily of GTPases. An example HSA protein sequence used is listed in SEQ ID NO: 49. Further examples include inserting a serine residue upstream of a HSA protein sequence or variant thereof inserted N- terminal or C-terminal to the engineered ARF1 polypeptide sequence. As another example, HSA protein sequence used is listed in SEQ ID NO: 50. Examples include inserting a green fluorescent protein (GFP) sequence or variant thereof (e.g., eGFP) N-terminal or C-terminal to the engineered ARF1 polypeptide sequence. Another example is an insertion, N-terminal addition, or C-terminal addition of an immunoglobulin protein sequence to the engineered ARF1 polypeptide sequence. An example human IgGl Fc protein sequence used is listed in SEQ ID NO: 48. As an example, a human IgGl Fc wild-type or mutant sequence can be positioned upstream of, downstream of, or within the engineered ARF1 polypeptide sequence. Another example is aninsertion, N-terminal addition, or C-terminal addition of an immunoglobulin protein sequence to an engineered polypeptide sequence from the ARF superfamily of GTPases. An example human IgGl Fc protein sequence used is listed in SEQ ID NO: 48.

[0091] Another example of a modification to an engineered ARF1 polypeptide sequence is the N-terminal addition of a peptide sequence recognized for a specific type of post-translational modification. One example used here is that addition of a myristoylation recognition sequence positioned N-terminal to the engineered ARF1 polypeptide sequence. The peptide sequence MGSS was inserted at the N-terminal position of various engineered ARF1 constructs. In some instances, the myristoylation recognition sequence was positioned next to a peptide tag sequence, or next to a protease recognition sequence, or next to both a peptide tag sequence and a protease recognition sequence. An example protease recognition sequence used was the TEV protease recognition motif as shown in SEQ ID NO: 39. An example myristoylation recognition sequence and peptide tag sequence used was SEQ ID NO: 38. An example myristoylation recognition sequence, peptide tag sequence, and protease recognition sequence used was SEQ ID NO: 40. Myristoylation can occur on penultimate N-terminal glycine residues and is known to require the prior removal of the initial methionine residue. Following recognition of a myristoylation sequence, a lipid modification to the protein occurs involving the addition of a 14- carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of selected proteins. This post-translational modification promotes binding of the protein to cell membranes for a variety of biological functions. In some instances, engineered ARF1 proteins or engineered ARF superfamily proteins are designed to yield a protein comprising myristoylation of an N- terminal glycine residue. . In some instances, engineered ARF1 proteins or engineered ARF superfamily proteins are designed to yield a protein that does not comprise myristoylation of an N-terminal glycine residue.

[0092] In some examples, linker sequences separate the inserted heterologous protein sequence or sequences from the homologous ARF1 protein sequence within the engineered polypeptide. Exemplary linker sequences used in engineered polypeptide constructs include a short flexible linker sequence (SEQ ID NO: 51) and a long flexible linker sequence (SEQ ID NO: 52).

[0093] In some examples, the effect of including a signal peptide sequence at the N-terminus of an engineered ARF1 polypeptide on protein production, protein secretion, protein accumulation, or a combination thereof was examined. A signal peptide sequence was selected from exemplary sequences listed as SEQ ID NOs: 58-67. Engineered ARF1 constructs were generated with a selected signal peptide sequence N-terminal to the engineered or wild-type ARF1 protein sequence. Mammalian cells were transfected with engineered ARF1 polypeptideexpression constructs and samples were taken from supernatant and cell pellet do assay protein production levels.

[0094] Engineered ARF1 and ARF superfamily constructs are transfected into various cell lines and tested for protein localization and protein production. Three transfection methods were utilized to test the constructs and are described below:

[0095] Method 1: Expi293F™ cells, TransIT-PRO transfection:

[0096] Materials:

[0097] Expi293F™ cells (Gibco Cat A14527)

[0098] Expi293 Expression Medium (Gibco Cat A1435101)

[0099] Opti-MEM I Reduced Serum Medium (Gibco Cat 31985070)

[0100] TransIT-PRO (MirusBio Cat MIR 5750)

[0101] Method from MirusBio for Expi293F™ (25mL volume example):

[0102] Day -1 : Split cells to 2 x 106cell / ml density

[0103] Day 0:

[0104] Check cell viability > 95%.

[0105] Dilute cells to 2.5 x 106cell / ml density and 25mL volume. Incubate in incubator shaker.

[0106] Warm TransIT-PRO to room temperature and vortex gently.

[0107] Add 25 pg plasmid DNA to room temperature 2.5mL Opti-MEM. Mix.

[0108] Add 25pL TransIT-PRO. Mix.

[0109] Incubate at room temperature for 15 min.

[0110] Add mixture to cells.

[0111] Incubate in incubator shaker and harvest.

[0112] Method 2: Expi293F™ cells, ExpiFectamine 293 transfection:

[0113] Materials:

[0114] Expi293F™ cells (Gibco Cat A14527)

[0115] Expi293 Expression Medium (Gibco Cat A1435101)

[0116] Opti-MEM I Reduced Serum Medium (Gibco Cat 31985070)

[0117] ExpiFectamine 293 Transfection Kit (Gibco Cat A14524)

[0118] Method from Gibco user guide (25mL volume example):

[0119] Day -1 : Split cells to 2.5 x 106cell / ml density

[0120] Day 0:

[0121] Check cell viability > 95%.

[0122] Dilute cells to 3 x 106cell / ml density and 25mL volume. Incubate in incubator shaker.

[0123] Mix ExpiFectamine by inverting 5 times.

[0124] Add 25pg plasmid DNA to 1 ,5mL room temperature Opti-MEM. Mix.

[0125] Add 80pL ExpiFectamine to 1.4mL room temperature Opti-MEM. Mix. Incubate at room temperature for 5 min.

[0126] Combine DNA and ExpiFectamine mixtures. Mix. Incubate at room temperature for 10-20 min.

[0127] Add mixture to cells. Incubate in incubator shaker.

[0128] Day 1 : Feed 18 to 22 hours post-transfection

[0129] Combine 150pL Enhancer 1 and 1.5mL Enhancer 2. (No need to pre-warm.)

[0130] Add mixture to cells.

[0131] Incubate in incubator shaker and harvest.

[0132] Method 3: ExpiCHO-S cells, ExpiFectamine CHO transfection:

[0133] Materials:

[0134] ExpiCHO-S cells (Gibco Cat A29127)

[0135] ExpiCHO Expression Medium (Gibco Cat A2910001)

[0136] OptiPRO SFM Medium (Gibco Cat 12309019)

[0137] ExpiFectamine CHO Transfection Kit (Gibco Cat A29129)

[0138] Method from Gibco user guide (25mL volume example):

[0139] Day -1 : Split cells to 3 x 106cell / ml density

[0140] Day 0:

[0141] Check cell viability > 95%.

[0142] Dilute cells to 6 x 106cell / ml density and 25mL volume. Incubate in incubator shaker.

[0143] Mix ExpiFectamine by inverting 5 times.

[0144] Add 25 pg plasmid DNA to ImL cold (take from 4°C and use immediately on bench) OptiPRO. Mix.

[0145] Add 80pL ExpiFectamine to 920pL cold OptiPRO. Mix.

[0146] Combine DNA and ExpiFectamine mixtures immediately or up to 5 min. Mix.Incubate at room temperature for 1-5 min.

[0147] Add mixture to cells. Incubate in incubator shaker.

[0148] Day 1 : Feed 18 to 22 hours post-transfection

[0149] Combine 150pL Enhancer and 6mL Feed. (No need to pre-warm.)

[0150] Add mixture to cells.

[0151] Incubate in incubator shaker and harvest.

[0152] The expression construct for the engineered ARF1 polypeptide namedMGSS HIS TEV ARFl wt (corresponding to SEQ ID NO: 53) was transfected into Expi239F cells using Method 1 or Method 2. Supernatant samples were purified to assay Engineered ARF1 polypeptide secreted from the cells. Cell pellet samples were purified to assay Engineered ARF1 polypeptide produced and maintained within the cells. Purified protein samples were assayed by Western blot using standard techniques with an anti-ARFl antibody used for detection. FIG. 2A shows an image of a Western blot indicating that MGSS HIS TEV ARFl wt was abundantly produced and located with the supernatant and within the cell pellet when cells were harvested 3 days post-transfection. FIG. 2B shows a chart of protein production levels ofMGSS HIS TEV ARFl wt following transfection and protein purification. As shown in the chart, supernatant samples demonstrated robust and greater protein production than cell pellet samples. This was a surprising result given known roles for ARF1 as proteins localized to the Golgi apparatus. In lane 1, protein production was ~74pg protein per mL supernatant from ExpiFectamine. In lane 2, protein production was ~70pg protein per mL supernatant from ExpiFectamine. In lane 3, protein production was ~63pg protein per mL supernatant from TransIT-PRO. ~63pg protein per mL supernatant from TransIT-PRO. In lane 4, protein production was ~55pg protein per mL culture pellet from TransIT-PRO. Lane 5 was a control group with no transfection reagent added.

[0153] Expression of various His-tagged Engineered ARF1 polypeptide constructs was tested and compared in Expi239F cells and CHO cells. As shown via Western blot using an anti-HIS antibody in FIG. 3A, constructs tested with a signal sequence did not produce engineered ARF1 polypeptide in detectable levels in the cell supernatant either at Day 5 (in CHO cells) or at Day 4 (in Expi239F cells) post transfection. Constructs lacking an N-terminal signal peptide sequence did show robust production in the supernatant at Day 5 (in CHO cells) or at Day 4 (in Expi239F cells). As shown via Western blot using an anti-HIS antibody in FIG. 3B, constructs tested with a signal sequence did not produce engineered ARF1 polypeptide in detectable levels in the cell pellet either at Day 5 (in CHO cells) or at Day 4 (in Expi239F cells) post transfection. Constructs lacking an N-terminal signal peptide sequence did show robust production in the cell pellet at Day 5 (in CHO cells) or at Day 4 (in Expi239F cells). In comparing expression per cell type, the engineered ARF1 polypeptides showed high expression levels in Expi293F cells compared to CHO cells. Constructs tested in FIG. 3A-FIG. 3B are listed in Table 4.Table 4: Listing of engineered ARF1 polypeptides tested in Expi239F cells and CHO cells

[0154] Engineered ARF 1 polypeptides with IgG Fc fusions or with FLAG tags were tested for protein production with and without an N-terminal signal peptide sequence. Expi293F cells were used to assay His-tagged engineered ARF1 polypeptides found in the supernatant or cell pellet from Day 3 post-transfection via Western blot using an anti-His antibody as shown in FIG. 4A. Expi293F cells were used to assay His-tagged engineered ARF1 polypeptides, non-tagged engineered ARF1 polypeptides, and FLAG-tagged engineered ARF1 polypeptides found in the supernatant (sup) or cell pellet (pellet) from Day 3 post-transfection via Western blot using an anti-ARFl antibody as shown in FIG. 4B. Engineered ARF1 polypeptides listed in Table 5 were tested.Table 5: Listing of engineered ARF1 polypeptides tested in Expi239F cells

[0155] As shown in FIG. 4A in an image of a Western blot using anti-His antibody for detection, His-tagged engineered ARF1 polypeptides list in Table 5 were detected with the anti- His antibody. ARFl-Fc fusions and FLAG-tagged ARF1 polypeptides were not detected with the anti-His antibody. As shown in FIG. 4B in an image of a Western blot using anti-ARFl antibody for detection, His-tagged engineered ARF1 polypeptides, ARFl-Fc fusions, and FLAG-tagged engineered ARF1 polypeptides were detected. The results indicated the ARFl-Fc fusions with and without a signal peptide showed expression in both supernatant and in cell pellet indicatingthat ARFl-Fc fusion proteins were both secreted and found intracellularly.

[0156] Engineered ARF1 polypeptides with HSA fusions were tested for production and secretion in Expi293F cells in Day 4 post-transfection. Engineered ARF1 polypeptides with single amino acid substitutions were tested for production and secretion in Expi293F cells.Results of Western blot using anti-ARFl antibody are shown in FIG. 5 (expression in completed medium on left and expression in cell pellet on right). Engineered ARF1 polypeptides tested are listed in Table 6.Table 6: Listing of engineered HSA-ARF1 polypeptides and ARF1 single amino acid substitutions tested in Expi239F cells

[0157] As shown in FIG. 5, HSA0-HSA3 were all expressed and found in the cell pellet, whereas HSAO was also robustly expressed in the supernatant. Single substitution mutants were all expressed and found in the cell pellet. The ARF1-T3 IN construct, a mutant form of ARF1 that has low affinity for GTP and exists primarily in a GDP -bound state, was not detected to be strongly secreted. FLAG-tagged HSA-ARF1 (HSAO) was more strongly secreted than the ARF1- T3 IN construct.

[0158] As shown in FIG. 6, various engineered ARF1 polypeptide constructs were tested for intracellular expression and secreted expression in Expi293F cells on Day 3. FIG. 6 showsresults on Western blot using an anti-ARFl antibody for detection on Day 3 post-transfection (left) and a chart of protein levels from the Western blot (right). Engineered ARF1 polypeptides tested are listed in Table 7. Qualitative levels of expression are indicated by WB note.Table 7: Listing of engineered HSA-ARF1 polypeptides tested for secretion in Expi239F cells on Day 3

[0159] The results indicated that His TEV ARF 1_T3 IN, SP flag TEV ARF 1_T3 IN,ARFI GFP His, and ARFl_GFP_His_T31N were all strongly expressed in the supernatant at Day 3. A4_His_TEV_ARFl was detected in the supernatant but less strongly expressed.

[0160] The ExpiCHO™ Expression System Kit (ThermoFisher) is used according to the manufacturer’s instructions for expression and secretion of modified proteins. In the case of a His-tagged engineered polypeptides, protein purification proceeds by the IMAC method to rapidly purify and enrich a polyhistidine affinity -tagged protein. Various other protein purification methods are used for engineered polypeptides that do not comprises a polyhistidine tag. Next, size exclusion chromatography (SEC), also known as gel filtration, is used as a chemically inert mode of chromatography employed for biomolecule purification to furtherpurify the engineered polypeptide. Next, the purified engineered polypeptide is further concentrated using standard protein concentration techniques.

[0161] Following production, purification, and concentration of the engineered ARF1 polypeptides, testing begins through various in vitro and in vivo assays.

[0162] In FIG. 7, a graph of Western blot results from various engineered ARF1 protein constructs quantitating protein expression levels in 293T cells within the supernatant, the pellet, and a calculated ratio of supernatant / pellet. Graph order for expression level of each construct is supernatant mg / L, pellet mg / L, and a calculated supernatant / pellet ratio. SP-ARFl-no tag (SEQ ID NO: 97) had a supernatant level at 4.5 mg / L, pellet 0.0 mg / L, and supernatant / pellet ratio of 0.0. ARFl-no tag (SEQ ID NO: 98) which does not comprise a signal sequence had a supernatant level at 9.5 mg / L, pellet 19.6 mg / L, and supernatant / pellet ratio of 0.5 indicating higher intracellular protein production than secreted protein production. SP-His-TEV-ARFl (SEQ ID NO: 99) comprising a signal sequence and a 6X-His tag near the N-terminus had a supernatant level at 57.2 mg / L, pellet 6.0 mg / L, and supernatant / pellet ratio of 9.5 indicating significantly increase secreted protein production and a high supernatant / pellet ratio. This version lacks N- terminal myristoylation. Removing the signal peptide sequence in His-TEV-ARFl (SEQ ID NO: 68) lacking a signal sequence and comprising a 6X-His tag near the N-terminus has a supernatant level at 27.3 mg / L, pellet 46.1 mg / L, and supernatant / pellet ratio of 0.6 indicating significantly increase secreted protein production and a high supernatant / pellet ratio similar to that of ARFl-noTag. SP-Flag-TEV-ARFl (SEQ ID NO: 91) comprising a signal sequence and a Flag tag near the N-terminus had a supernatant level at 34.1 mg / L, pellet 1.0 mg / L, and supernatant / pellet ratio of 33.0 indicating significantly increase secreted protein production, low intracellular production, and a high supernatant / pellet ratio. Flag-TEV-ARFl (SEQ ID NO: 100) lacking a signal sequence and comprising a Flag tag near the N-terminus had a supernatant level at 6.9 mg / L, pellet 25.6 mg / L, and supernatant / pellet ratio of 0.3 indicating secreted protein production, intracellular production, and a low supernatant / pellet ratio demonstrating that higher percentage of protein were produced intracellularly. SP-ARFl-GFP-His (SEQ ID NO: 101) comprising an ARF1 wild-type-GFP fusion protein with a signal peptide and a C-terminal 6X His tag had a supernatant level at 39.1 mg / L, pellet 3.9 mg / L, and supernatant / pellet ratio of 10.1 indicating significantly increase secreted protein production, low intracellular production, and a high supernatant / pellet ratio. ARFl-GFP-His (SEQ ID NO: 102) comprising an ARF1 wild-type- GFP fusion protein with a C-terminal 6X His tag had a supernatant level at 48.0 mg / L, pellet 15.1 mg / L, and supernatant / pellet ratio of 3.2 indicating that removal of the signal peptide resulted in increased intracellular production and a lower supernatant / pellet ratio compared to SEQ ID NO: 101. SP-ARFl-Fc (SEQ ID NO: 103) comprising an ARF1 wild-type-Fc fusion proteincomprising a signal peptide had a supernatant level at 106.5 mg / L, pellet 54.7 mg / L, and supematant / pellet ratio of 2.0 indicating robust intracellular and secreted protein production. Removal of the signal peptide in ARFl-Fc (SEQ ID NO: 104) comprising an ARF1 wild-type-Fc fusion protein lacking a signal peptide produced similar results with a supernatant level at 89.1 mg / L, pellet 39.6 mg / L, and supernatant / pellet ratio of 2.3 indicating that the presence of the signal peptide in the Fc fusion protein did not substantially influence extracellular protein production. SP-HSA-ARF1 (SEQ ID NO: 105) comprising an ARF1 wild-type-HSA fusion protein comprising a signal peptide had a supernatant level at 126.2 mg / L, pellet 23.3 mg / L, and supematant / pellet ratio of 5.4 indicating robust intracellular and secreted protein production and a high ratio of secreted protein to intracellular protein production. An HSA-ARF1 construct lacking the signal peptide (SEQ ID NO: 106) did not express detectable protein in this assay. Construct formats with high supematant / pellet ratio are used to engineered ARF1 proteins that are transported extracellularly when expressed in vitro. High supematant / pellet ratios can aid in protein purification. Throughout FIG. 7, ARF1 protein expression demonstrated non-canonical secretion and protein trafficking. Constructs with and without signal peptide as well as various tags or no tag were transfected in HEK293 cells for 3 days. The proteins from supernatant and pellet were quantified by Western blot using anti ARF1 antibody; the ratio of supernatant and pellet were calculated based on signal intensity. Without SP, ARF1 protein can be secreted with N-term His and C-term His, GFP, Fc tag. Without SP, ARF1 cannot be efficiently secreted with N-flag and C-HSA, indicating these tags may hinder its membrane trafficking functions.

[0163] Western blot analysis on non-reducing SDS-PAGE analysis was used to assess production of engineered ARF1 constructs and subunit association in FIG. 8. Images of a nonreduced SDS-PAGE gel, loaded with 5-8 ug of sample protein, were examined. Results showed that C159S and N60G mutations improve protein homogeneity by removing ectopic dimerization and glycosylation, respectively. Mutations of C159S and N60G showed similar effects on His, Flag and Fc tagged proteins to improve protein homogeneity or stability. Lane 8, SP-His-TEV- ARF1-C159S (SEQ ID NO: 75), showed a high amount of glycosylation. Lane 9, SP-His-TEV- N60G-C159S (SEQ ID NO: 76), showed a reduced amount of glycosylation and no evidence of dimerization, indicating a predominantly non-glycosylated, monomeric form.

[0164] Western blot analysis on non-reducing and reducing SDS-PAGE analysis was used to assess production of engineered ARF1 constructs and subunit association in FIG. 9. Western-blot and SDS-PAGE showed that cysteine mutation (C159S) reduced the aggregation mainly caused by T31N significantly in C-term Fc ARF1 proteins. Lower cell culture temperature from 37°C to 32°C did not improve the aggregation. Proteins from SEQ ID NOs: 77-79 were assessed in this experiment.

[0165] Next, production features and effects on adipose lipid droplet intensity of ARF1- T31N constructs were assessed in FIG. 10A-FIG. 10C. In FIG. 10A, Western blot following SDS-PAGE on reducing and non-reducing gels shows that His-TEV-T31N protein was expressed in HEK293 and harvested on day 3 and 4 and 6 of post-transfection. Protein was purified by IMAC and followed by SEC with over 99% purity and a small presentation of homodimer. In FIG. 10B, analytical SEC-HPLC showed purified His-TEV-T31N protein was in monomer (>99%) and small percentage (<0.1%) in dimer using day 3 protein as an example. In FIG. 10C, adipose cell droplet reduction quantitation was analyzed. His-TEV-T31N proteins purified from supernatants harvested on day 3, 4 and 6 were tested in mature 3T3L1 adipocytes in three doses in triplicates. Dose 1, 2 and 3 corresponds to 0.5 pmol, Ipmol and 2 pmol protein. Forskolin has been used as a positive control for lipid reduction. Proteins from day 4 and 5 showed lipid droplet reduction activity in a dose-dependent manner while no activity were shown in day 6 protein. Data from 3 independent experiments. One-way ANOVA followed by Fisher’s LSD test. *, p<0.05, NS: not significant.

[0166] Next, cellular update of engineered ARF1 constructs was analyzed in FIG. 11A- FIG.11B. In FIG. 11A, 3T3-L1 mouse preadipocytes cell uptake quantitation was examined. 3T3L1 cells were plated at 10k cells / well treated with proteins in three does l. luM, 2.2uM and 4.4uM in duplicates (~16 hours) stained with anti-ARFl antibody followed by secondary antibody conjugated to Alexa Fluor 488. DAPI was used to stain nuclei. Data is normalized by nuclear count. Imaged under 40x WI magnification. Several engineered ARF1 constructs (e.g., T31N, Q71L, T48I, and F51L) showed dose-dependent increases in cellular update. The ARF1- R99H shows elevated cellular uptake in all doses tested. The ARF1-Y35H construct did not show increased cellular uptake compared to vehicle. In FIG. 11B, various engineered ARF1 constructs were examined for cellular uptake by immunofluorescence. FIG. 11B, shows representative images demonstrating that ARF1-T31N, -T48I, -F51L, Q71L, and -R99H sequence mutants have cellular uptake activity in the 3T3-L1 preadipocytes grown in vitro. In FIG. 11C, 3T3-L1 mouse preadipocytes were assayed for cell uptake quantitation. 3T3-L1 cells were plated at 10k cells / well treated with proteins in two does 2.2uM and 4.4uM in triplicate (~16 hours) stained with anti-ARFl antibody followed by secondary antibody conjugated to Alexa Fluor 488. DAPI was used to stain nuclei. Data is normalized by nuclear count. Imaged under 40x WI magnification. One-Way ANOVA followed by Bonferroni’s post-test ****, p<0.0001; ***, p<0.001. Engineered ARF1 proteins from SEQ ID NOs 70, 78, and 77 shows significantly increased cell uptake at 4.4pM compared to vehicle. In FIG. 11D, representative immunofluorescence images are shown demonstrating that ARF1-T3 IN and WT with N-term His-TEV, as well as T3 IN and T31N / C159S with C-term Fc sequence have cellular uptakeactivity in the 3T3-L1 preadipocytes grown in vitro. In FIG. HE, 3T3-L1 mouse preadipocytes cell uptake quantitation. 3T3-L1 cells were plated at 10k cells / well treated with proteins in two does 2.2uM and 4.4uM in triplicate (~16 hours) stained with anti-ARFl antibody followed by secondary antibody conjugated to Alexa Fluor 488. DAPI was used to stain nuclei. Data is normalized by nuclear count. Imaged under 40x WI magnification. One-Way ANOVA followed by Bonferroni’s post-test ****, p<0.0001; ***, p<0.001. Engineered ARF1 protein of SEQ ID NO: 70 and ARF1 fusion proteins of SEQ ID NOs: 86, 88, and 89 showed significantly increases cellular uptake. In FIG. HF, representative immunofluorescence images demonstrating that ARF1-T3 IN with N-term His-TEV, as well as T3 IN and T31N / C159S with C-term and N-term Fc sequence have cellular uptake activity in the 3T3-L1 preadipocytes grown in vitro were shown.

[0167] Next, in FIG. 12, protein engineering was assessed to improve homogeneity. Images of a non-reduced SDS-PAGE gel, loaded with 5-8 ug of sample protein, was examined. Removal of dimer and glycosylation by C159S and N60G mutation on flag tagged ARF1 was observed, indicating that both flag-tagged and HIS-tagged versions of these engineered ARF1 proteins can improve homogeneity of production.

[0168] Next, in FIG. 13, aggregation over various ARFl-Fc fusion protein constructs was analyzed. Non-reducing and reducing SDS-PAGE gels were analyzed by Western blot. Cysteine mutation (C159S) reduced the aggregation (T31N) significantly in C-terminal Fc in compared Fc fusion proteins produced from SEQ ID NO: 77 (T31N / C159S) to SEQ ID NO: 78 (T31N).

[0169] Next, in FIG. 14A-FIG. 14C, biochemistry and in vitro activity of His-TEV-T31N was analyzed. In FIG. 14A, His-TEV-T31N protein expressed in HEK293: harvest day 3, 4 and 6 was analyzed by Western blot via non-reducing SDS-PAGE and protein harvested on day 3 was analyzed by Western blots via reducing SDS-PAGE. Dimeric forms were present but were progressively reduced on Days 4 and 6. In FIG. 14B, analytical SEC-HPLC showed purified His-TEV-T3 IN protein was in monomer (>99%) and small percentage (<0.1%) in dimer using day 3 protein as an example. In FIG. 14C, adipose cell droplet reduction quantitation was assessed. His-TEV-T31N proteins purified from supernatants harvested on day 3, 4 and 6 were tested in mature 3T3L1 adipocytes in three doses in triplicates. Dose 1, 2 and 3 corresponds to 0.5 pmol, Ipmol and 2 pmol protein. Forskolin has been used as a positive control for lipid reduction. Day 3 and Day 4 protein showed lipid droplet reduction activity in a dose-dependent manner. Day 6 protein did not show the lipid droplet reduction activity. His-TEV-T31N protein expressed in HEK293: harvest day 3 and 4 showed no different in biochemistry assays but no in vitro activity to reduce lipid droplets in day 6 protein.Example 2 — Engineered ARF Superfamily Shuttle Proteins

[0170] For this example, a protein sequence from an ARF superfamily protein (e.g., SEQ ID NOs 1-36, and 54-55) is engineered to serve as a shuttle protein to aid in the intracellular or extracellular transport of a chemical or biological molecule.

[0171] A molecule is conjugated by a covalent linkage to an ARF superfamily protein to produce an engineered composite molecule that can serve as a shuttle protein. In several examples, the engineered composite molecule is produced by fusing an ARF superfamily protein (e.g., SEQ ID NOs 1-36, and 54-55) to a heterologous peptide sequence having a therapeutic effect to create an engineered ARF fusion protein in a single amino acid chain. In this example, heterologous peptide sequences having a therapeutic effect are exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide. The engineered composite molecule has a DNA coding sequence representing the engineered composite molecule cloning into an expression vector. The expression vector is used to produce recombinant engineered ARF fusion protein for in vitro and in vivo testing of therapeutic efficacy.

[0172] In another example, a small molecule drug is covalently coupled to an ARF superfamily protein (e.g., SEQ ID NOs 1-36, and 54-55) to create a drug-ARF peptide conjugate. In this example, the small molecule drug is acetylsalicylic acid, ibuprofen, acetaminophen, simvastatin, lisinopril, metformin, atorvastatin, clopidogrel, omeprazolec, ciprofloxacin, metoprolol, diazepam, cetirizine, alprazolam, levothyroxine, warfarin, ranitidine, hydrochlorothiazide, fluoxetine, ranitidine, morphine, allopurinol, furosemide, hydralazine, methotrexate, losartan, montelukast, cephalexin, digoxin, phenytoin, carvedilol, isosorbide mononitrate, tamsulosin, sitagliptin, cyclophosphamide, 5-fluorouracil, doxorubicin, vincristine, paclitaxel, docetaxel, cisplatin, carboplatin, ifosfamide, etoposide, imatinib, tamoxifen, flutamide, bortezomib, lenalidomide, erlotinib, sorafenib, irinotecan, topotecan, gemcitabine, dasatinib, or everolimus.

[0173] The drug-ARF peptide conjugate is produced and purified for in vitro and in vivo testing of therapeutic efficacy.

[0174] In another example, a nucleic acid or an oligonucleotide is covalently coupled to an ARF superfamily protein (e.g., SEQ ID NOs 1-36, and 54-55) to create a nucleic acid-ARF peptide conjugate. The nucleic acid or oligonucleotide is selected from a list of therapeutic nucleic acids including antisense oligonucleotides and small interfering RNAs. The nucleic acid-peptide conjugate is purified for in vitro and in vivo testing of therapeutic efficacy.

[0175] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein are employed in practicing the invention.

[0176] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.ASPECTSAspect 1. A shuttle protein comprising: a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently-coupled heterologous peptide sequence comprising one or more functional protein domains of a therapeutic peptide.Aspect 2. The shuttle protein of aspect 1, wherein the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36, 54-55 or 68-106.Aspect 3. The shuttle protein of aspect 1 or 2, wherein the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1.Aspect 4. The shuttle protein of any one of aspects 1-3, wherein the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55 or 68-106 having one or more amino acid substitutions, deletions, or insertions.Aspect 5. The shuttle protein of any one of aspects 1-4, wherein the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36, 54- 55 or 68-106.Aspect 6. The shuttle protein of any one of aspects 1-5, wherein the covalently-coupled heterologous peptide sequence comprises one or more protein domains of a therapeutic peptide. Aspect 7. The shuttle protein of aspect 6, wherein the one or more protein domains of the therapeutic peptide comprise an amino acid sequence at least about 90% identical to the sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide,angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide.Aspect 8. The shuttle protein of aspect 6 or 7, wherein the one or more protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide, with one or more amino acid substitutions, insertions, or deletions. Aspect 9. The shuttle protein any one of aspects 6-8, wherein the one or more protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide.Aspect 10. The shuttle protein of any one of aspects 1-9, wherein the covalently-coupled heterologous peptide sequence is positioned N-terminal to the homologous ARF superfamily sequence.Aspect 11. The shuttle protein of any one of aspects 1-9, wherein the covalently-coupled heterologous peptide sequence is positioned C-terminal to the homologous ARF superfamily sequence.Aspect 12. The shuttle protein of any one of aspects 1-11, wherein the homologous ARF superfamily sequence and the covalently-coupled heterologous peptide sequence are separated by a linker peptide sequence.Aspect 13. The shuttle protein of aspect 12, wherein the linker peptide sequence comprises the amino acid sequence of SEQ ID NO: 51.Aspect 14. The shuttle protein of aspect 12, wherein the linker peptide sequence comprises the amino acid sequence of SEQ ID NO: 52.Aspect 15. The shuttle protein of any one of aspects 1-14, further comprising a signal peptide positioned at the N-terminus.Aspect 16. The shuttle protein of aspect 15, wherein the signal peptide comprises an amino acid sequence selected from SEQ ID NOS: 58-67.Aspect 17. The shuttle protein of any one of aspects 1-16, further comprising a site for myristoylation.Aspect 18. The shuttle protein of aspect 17, wherein the site for myristoylation is located at or near the N-terminus of the shuttle protein.Aspect 19. The shuttle protein of aspect 17 or 18, wherein the site for myristoylation comprises a glycine residue.Aspect 20. The shuttle protein of any one of aspects 17-19, wherein the site for myristoylation comprises the peptide sequence MGSS.Aspect 21. The shuttle protein of any one of aspects 1-20, further comprising a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N-terminus of the shuttle protein.Aspect 22. The shuttle protein of any one of aspects 1-20, further comprising a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the shuttle protein.Aspect 23. The shuttle protein of any one of aspects 1-22, further comprising a cleavage site.Aspect 24. The shuttle protein of aspect 23, wherein the cleavage site comprises a TEV protease recognition motif.Aspect 25. The shuttle protein of aspect 23 or 24, wherein the TEV protease recognition motif comprises the amino acid sequence of SEQ ID NO: 39.Aspect 26. The shuttle protein of any one of aspects 1-25, further comprising a half-life extending moiety.Aspect 27. The shuttle protein of aspect 26, wherein the half-life extending moiety comprises an amino acid sequence at least about 95% identical to a human serum albumin (HSA) sequence. Aspect 28. the shuttle protein of aspect 26 or 27, wherein the half-life extending moiety comprises an amino acid sequence identical to SEQ ID NO: 49.Aspect 29. The shuttle protein of aspect 26, wherein the half-life extending moiety comprises an amino acid sequence at least about 95% identical to an Fc portion of a human immunoglobulin. Aspect 30. The shuttle protein of aspect 26 or 29, wherein the half-life extending moiety comprises an amino acid sequence identical to SEQ ID NO: 48.Aspect 31. A drug-peptide conjugate shuttle protein comprising: a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently -coupled small molecule drug.Aspect 32. The drug-peptide conjugate shuttle protein of aspect 31, wherein the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36, 54-55 or 68-106.Aspect 33. The drug-peptide conjugate shuttle protein of aspect 31 or 32, wherein the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1.Aspect 34. The drug-peptide conjugate shuttle protein of any one of aspects 31-33, wherein thehomologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55 or 68-106 having one or more amino acid substitutions, deletions, or insertions.Aspect 35. The drug-peptide conjugate shuttle protein of any one of aspects 31-34, wherein the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36, 54-55 or 68-106.Aspect 36. The drug-peptide conjugate shuttle protein of any one of aspects 31-35, wherein the small molecule drug is selected from the group consisting of acetylsalicylic acid, ibuprofen, acetaminophen, simvastatin, lisinopril, metformin, atorvastatin, clopidogrel, omeprazolec, ciprofloxacin, metoprolol, diazepam, cetirizine, alprazolam, levothyroxine, warfarin, ranitidine, hydrochlorothiazide, fluoxetine, ranitidine, morphine, allopurinol, furosemide, hydralazine, methotrexate, losartan, montelukast, cephalexin, digoxin, phenytoin, carvedilol, isosorbide mononitrate, tamsulosin, sitagliptin, cyclophosphamide, 5 -fluorouracil, doxorubicin, vincristine, paclitaxel, docetaxel, cisplatin, carboplatin, ifosfamide, etoposide, imatinib, tamoxifen, flutamide, bortezomib, lenalidomide, erlotinib, sorafenib, irinotecan, topotecan, gemcitabine, dasatinib, and everolimus.Aspect 37. The drug-peptide conjugate shuttle protein of any one of aspects 31-36, wherein the small molecule drug is positioned at or near the N-terminus of the homologous ARF superfamily sequence.Aspect 38. The drug-peptide conjugate shuttle protein of any one of aspects 31-36, wherein the small molecule drug is positioned at or near the C-terminus of the homologous ARF superfamily sequence.Aspect 39. A nucleic acid-peptide conjugate shuttle protein comprising: a homologous ADP- ribosylation factor (ARF) superfamily sequence, and a covalently-coupled nucleic acid sequence. Aspect 40. The nucleic acid-peptide conjugate shuttle protein of aspect 39, wherein the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36, 54-55 or 68-106.Aspect 41. The nucleic acid-peptide conjugate shuttle protein of aspect 39 or 40, wherein the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1.Aspect 42. The nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-41, wherein the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55 or 68-106 having one or more amino acid substitutions, deletions, or insertions.Aspect 43. The nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-42, wherein the homologous ARF superfamily sequence comprises an amino acid sequence selectedfrom SEQ ID NOs: 1-36, 54-55 or 68-106.Aspect 44. The nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-43, wherein the covalently-coupled nucleic acid sequence comprises a therapeutic DNA sequence. Aspect 45. The nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-43, wherein the covalently-coupled nucleic acid sequence comprises a therapeutic RNA sequence. Aspect 46. The nucleic acid-peptide conjugate shuttle protein of aspect 45, wherein the therapeutic RNA sequence comprises a small interfering RNA sequence.Aspect 47. The nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-43, wherein the covalently-coupled nucleic acid sequence comprises a therapeutic peptide nucleic acid (PNA) sequence.Aspect 48. The nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-47, wherein the covalently-coupled nucleic acid sequence comprises an antisense oligonucleotide sequence.Aspect 49. The nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-48, wherein the covalently-coupled nucleic acid sequence is positioned at or near the N-terminus of the homologous ARF superfamily sequence.Aspect 50. The nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-48, wherein the covalently-coupled nucleic acid sequence is positioned at or near the C-terminus of the homologous ARF superfamily sequence.Aspect 51. A method of trafficking a shuttle protein outside of a cell, the method comprising expressing a recombinant nucleic acid coding for the shuttle protein of any one of aspects 1-30 in vitro in cultured cells from a cell line, wherein the shuttle protein is efficiently transported into supernatant surrounding the cultured cells.52. A method of trafficking a shuttle protein into a target cell, the method comprising contacting the shuttle protein of any one of aspects 1-30 to the surface of the target cell, wherein the shuttle protein is efficiently internalized into an intracellular location within the target cell.53. A method of trafficking a drug-peptide conjugate shuttle protein into a target cell, the method comprising contacting the drug-peptide conjugate shuttle protein of any one of aspects 31-38 to the surface of the target cell, wherein the drug-peptide conjugate shuttle protein is efficiently internalized into an intracellular location within the target cell.54. A method of trafficking a nucleic acid-peptide conjugate shuttle protein into a target cell, the method comprising contacting the nucleic acid-peptide conjugate shuttle protein of any one of aspects 39-50 to the surface of the target cell, wherein the nucleic acid-peptide conjugate shuttle protein is efficiently internalized into an intracellular location within the target cell.55. The method of aspect 53 or 54, wherein the target cell is an adipocyte.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A shuttle protein comprising: a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently-coupled heterologous peptide sequence comprising one or more functional protein domains of a therapeutic peptide.

2. The shuttle protein of claim 1, wherein the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36, 54-55, or 68-106.

3. The shuttle protein of claim 1, wherein the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1.

4. The shuttle protein of claim 1, wherein the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54-55, or 68-106 having one or more amino acid substitutions, deletions, or insertions.

5. The shuttle protein of claim 1, wherein the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36, 54-55, or 68-106.

6. The shuttle protein of claim 1, wherein the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence at least about 90% identical to the sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide.

7. The shuttle protein of claim 1, wherein the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide, with one or more amino acid substitutions, insertions, or deletions.

8. The shuttle protein any claim 1, wherein the one or more functional protein domains of the therapeutic peptide comprise an amino acid sequence of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, tirzepatide, teduglutide, linaclotide, pramlintide, abarelix, degarelix, carfilzomib, mifamurtide, aviptadil, atosiban, carbetocin, taltirelin, bremelanotide, teriparatide, abaloparatide, plecanatide, nesirtide, angiotensin II, icatibant, enfuvirtide, tesamorelin, ziconotide, romiplostim, peginesatide, lucinactant, etelcalcetide, afamelanotide, pasireotide, or setmelantide.

9. The shuttle protein of any one of claims 1-8, wherein the covalently-coupled heterologous peptide sequence is positioned N-terminal to the homologous ARF superfamily sequence.

10. The shuttle protein of any one of claims 1-8, wherein the covalently-coupled heterologous peptide sequence is positioned C-terminal to the homologous ARF superfamily sequence.

11. The shuttle protein of any one of claims 1-10, wherein the homologous ARF superfamily sequence and the covalently -coupled heterologous peptide sequence are separated by a linker peptide sequence.

12. The shuttle protein of any one of claims 1-11, further comprising a signal peptide positioned at the N-terminus.

13. The shuttle protein of any one of claims 1-11, wherein the shuttle protein is lacking signal peptide positioned at the N-terminus.

14. The shuttle protein of any one of claims 1-13, further comprising a site for myristoylation.

15. The shuttle protein of any claim 14, wherein the site for myristoylation comprises the peptide sequence MGSS.

16. The shuttle protein of any one of claims 1-13, wherein the shuttle protein is lacking a site for myristoylation.

17. The shuttle protein of any one of claims 1-16, further comprising a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N-terminus of the shuttle protein.

18. The shuttle protein of any one of claims 1-17, further comprising a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the shuttle protein.

19. The shuttle protein of any one of claims 1-18, further comprising a cleavage site.

20. The shuttle protein of any one of claims 1-19, further comprising a half-life extending moiety.

21. The shuttle protein of claim 20, wherein the half-life extending moiety comprises an amino acid sequence at least about 95% identical to a human serum albumin (HSA) sequence.

22. The shuttle protein of claim 20, wherein the half-life extending moiety comprises an amino acid sequence at least about 95% identical to an Fc portion of a human immunoglobulin.

23. A drug-peptide conjugate shuttle protein comprising: a homologous ADP-ribosylation factor (ARF) superfamily sequence, and a covalently -coupled small molecule drug.

24. The drug-peptide conjugate shuttle protein of claim 23, wherein the homologous ARF superfamily sequence is at least about 90% identical to an ARF superfamily sequence selected from SEQ ID NOs: 1-36, 54-55, or 68-106.

25. The drug-peptide conjugate shuttle protein of claim 23, wherein the homologous ARF superfamily sequence is at least about 95% identical to SEQ ID NO: 1.

26. The drug-peptide conjugate shuttle protein of claim 23, wherein the homologous ARF superfamily sequence comprises an amino acid sequence of any one of SEQ ID NOs: 1-36, 54- 55, or 68-106 having one or more amino acid substitutions, deletions, or insertions.

27. The drug-peptide conjugate shuttle protein claim 23, wherein the homologous ARF superfamily sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-36, 54- 55, or 68-106.

28. The drug-peptide conjugate shuttle protein of any one of claims 23-27, wherein the small molecule drug is selected from the group consisting of acetylsalicylic acid, ibuprofen, acetaminophen, simvastatin, lisinopril, metformin, atorvastatin, clopidogrel, omeprazolec, ciprofloxacin, metoprolol, diazepam, cetirizine, alprazolam, levothyroxine, warfarin, ranitidine, hydrochlorothiazide, fluoxetine, ranitidine, morphine, allopurinol, furosemide, hydralazine, methotrexate, losartan, montelukast, cephalexin, digoxin, phenytoin, carvedilol, isosorbide mononitrate, tamsulosin, sitagliptin, cyclophosphamide, 5 -fluorouracil, doxorubicin, vincristine, paclitaxel, docetaxel, cisplatin, carboplatin, ifosfamide, etoposide, imatinib, tamoxifen,flutamide, bortezomib, lenalidomide, erlotinib, sorafenib, irinotecan, topotecan, gemcitabine, dasatinib, and everolimus.

29. The drug-peptide conjugate shuttle protein of any one of claims 23-28, wherein the small molecule drug is positioned at or near the N-terminus of the homologous ARF superfamily sequence.

30. The drug-peptide conjugate shuttle protein of any one of claims 23-28, wherein the small molecule drug is positioned at or near the C-terminus of the homologous ARF superfamily sequence.

31. A method of trafficking a shuttle protein outside of a cell, the method comprising expressing a recombinant nucleic acid coding for the shuttle protein of any one of claims 1-22 in vitro in cultured cells from a cell line, wherein the shuttle protein is efficiently transported into supernatant surrounding the cultured cells.

32. A method of trafficking a shuttle protein into a target cell, the method comprising contacting the shuttle protein of any one of claims 1-22 to the surface of the target cell, wherein the shuttle protein is efficiently internalized into an intracellular location within the target cell.

33. A method of trafficking a drug-peptide conjugate shuttle protein into a target cell, the method comprising contacting the drug-peptide conjugate shuttle protein of any one of claims 23-30 to the surface of the target cell, wherein the drug-peptide conjugate shuttle protein is efficiently internalized into an intracellular location within the target cell.

34. The method of claim 32 or 33, wherein the target cell is an adipocyte.

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