Hydrophobic modifications to proteins

Abstract

The disclosure provides, in various embodiments, polynucleotides encoding a fusion protein, wherein the polynucleotides comprise: (a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and (b) at least one of the following: (i) a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase, wherein the polypeptide is N-terminal to the non-Hedgehog therapeutic protein, (ii) a sequence encoding a Hedgehog auto-processing domain having sterol modification activity, wherein the domain is C-terminal to the non-Hedgehog therapeutic protein, and (iii) a sequence encoding a polypeptide sufficient for palmitoylation by a membrane-bound O-acyltransferase enzyme. The disclosure also provides, in various embodiments, vectors, cells, and kits comprising the polynucleotides, and methods of using the polynucleotides, vectors, cells, and / or kits.

Description

0399.2076001HYDROPHOBIC MODIFICATIONS TO PROTEINSRELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 698,319, filed on September 24, 2024. The entire teachings of the above application are incorporated herein by reference.INCORPORATION BY REFERENCE OF MATERIAL IN XML

[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith: File name: 0399-2076001_SL.xml; created September 24, 2025, 50,369 Bytes in size.GOVERNMENT SUPPORT

[0003] This invention was made with government support under HD108777 and GM151487 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Engineers seeking to build therapeutic molecules, including small molecules and proteins, have found that a molecule is more stable in circulation if it interacts with albumin, a major protein in blood. There are many ways to force a molecule to interact with albumin, but a recent example is to covalently modify a protein with a hydrophobic group, which then binds hydrophobic pockets present in albumin. This strategy was used to convert GLP-1, which has a serum half-life of about 5 minutes, into Semaglutide (z.e., OZEMPIC®), which has a serum halflife of approximately 7 days.

[0005] In the case of GLP-1, chemists generated Semaglutide by synthesizing the total protein sequence, and inserted a lipid-modified amino acid in a region of the protein that would not affect the receptor interaction. This strategy is very low throughput and of limited utility for proteins longer than a few dozen amino acids, because total synthesis of long proteins is prohibitively difficult.SUMMARY

[0006] There is a critical need for compositions and methods useful for hydrophobic modification to therapeutic molecules. The disclosure provides such compositions and methods.- 1 -4223116.V10399.2076001

[0007] Provided herein, among other things, are polynucleotides encoding fusion proteins. In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) at least one of the following: i. a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase, wherein the polypeptide is N-terminal to the non-Hedgehog therapeutic protein, ii. a sequence encoding a Hedgehog auto-processing domain having sterol modification activity, wherein the domain is C-terminal to the nonHedgehog therapeutic protein, and iii. a sequence encoding a polypeptide sufficient for palmitoylation by a membrane-bound O-acyltransferase enzyme.

[0008] In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase, wherein the polypeptide is N-terminal to the non-Hedgehog therapeutic protein.

[0009] In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a Hedgehog auto-processing domain having lipophilic modification activity, wherein the domain is C-terminal to the non-Hedgehog therapeutic protein.

[0010] In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a Hedgehog auto-processing domain having sterol modification activity, wherein the domain is C-terminal to the non-Hedgehog therapeutic protein.

[0011] In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a polypeptide sufficient for palmitoylation by a membranebound O-acyltransferase enzyme.

[0012] Also provided herein, among other things, are vectors comprising one or more of the polynucleotides disclosed herein.- 2 -4223116.V10399.2076001

[0013] Also provided herein, among other things, are cells comprising one or more of the polynucleotides or vectors disclosed herein.

[0014] Also provided herein, among other things, are kits comprising a container and, optionally, an instruction for use, wherein the container comprises any one or more of the polynucleotides, vectors, or cells disclosed herein, or any combination of the foregoing.

[0015] Also provided herein, among other things, are methods modifying a non-Hedgehog therapeutic protein in a cell.

[0016] In some embodiments, a method comprises expressing a polynucleotide or vector in a cell expressing a Hedgehog acyltransferase, wherein the polynucleotide or vector comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a polypeptide sufficient for palmitoylation by the Hedgehog acyltransferase.

[0017] In some embodiments, a method comprises expressing a polynucleotide or vector in a cell, wherein the polynucleotide or vector comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a Hedgehog auto-processing domain having sterol modification activity.

[0018] In some embodiments, a method comprises expressing a polynucleotide or vector in a cell expressing a membrane-bound O-acyltransferase enzyme, wherein the polynucleotide or vector comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a polypeptide sufficient for palmitoylation by the membrane-bound O-acyltransferase enzyme.

[0019] Also provided herein, among other things, are methods of modifying a purified nonHedgehog therapeutic protein.

[0020] In some embodiments, a method comprises: a) expressing a polynucleotide in a host cell, wherein the polynucleotide comprises: i. a protein coding sequence encoding a non-Hedgehog therapeutic protein, and ii. a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase, b) purifying a non-Hedgehog therapeutic protein, and- 3 -4223116.V10399.2076001 c) contacting the purified non-Hedgehog therapeutic protein with the Hedgehog acyltransferase and palmityl-CoA.

[0021] In some embodiments, a method comprises: a) expressing a polynucleotide in a host cell, wherein the polynucleotide comprises: i. a protein coding sequence encoding a non-Hedgehog therapeutic protein, and ii. a sequence encoding a polypeptide sufficient for palmitoylation by a membrane-bound O-acyltransferase enzyme, b) purifying a non-Hedgehog therapeutic protein, and c) contacting the purified non-Hedgehog therapeutic protein with the membranebound O-acyltransferase enzyme and palmityl-CoA.

[0022] Also provided herein, among other things, are proteins produced by one of more of the methods described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0024] FIGs. 1A-1C. Illustrations of non-limiting examples of protein fusions. A post- translational modification could either occur in a cell (e.g., a culture cell) or in vitro, for example, on proteins biochemically purified from host cells (e.g., bacteria).

[0025] FIGs. 2A-2B. Histograms of instantaneous diffusion rates.

[0026] FIGs. 3A-3B. Survival curves for each fusion protein in the slowest (extracellular matrix-associated) subpopulation.

[0027] FIG. 4. Signaling gradients formed by each fusion protein.

[0028] FIG. 5. Signaling potency of each fusion protein.DETAILED DESCRIPTION

[0029] A description of example embodiments follows.Polynucleotides

[0030] Provided herein, among other things, is a polynucleotide encoding a fusion protein, wherein the polynucleotide comprises:- 4 -4223116.V10399.2076001 a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) at least one of the following: i. a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase, wherein the polypeptide is N-terminal to the non-Hedgehog therapeutic protein, ii. a sequence encoding a Hedgehog auto-processing domain having sterol modification activity, wherein the domain is C-terminal to the nonHedgehog therapeutic protein, and iii. a sequence encoding a polypeptide sufficient for palmitoylation by a membrane-bound O-acyltransferase enzyme.

[0031] Non-limiting examples of non-Hedgehog proteins (e.g., therapeutic proteins) include peptide hormones, developmental morphogens, or peptide-based agonists (e.g., for a signaling receptor).

[0032] In some embodiments, a non-Hedgehog therapeutic protein is an antibody (e.g., a therapeutic antibody). In some embodiments, an antibody (e.g., a therapeutic antibody) is a nanobody.

[0033] In some embodiments, a non-Hedgehog therapeutic protein is a cytokine (e.g., an anti-inflammatory cytokine or a pro-inflammatory cytokine). In some embodiments, a cytokine is a chemokine. In some embodiments, a cytokine is an interleukin (e.g., IL4, IL10, or IL13). In some embodiments, an interleukin is IL4. In some embodiments, an interleukin is IL10. In some embodiments, an interleukin is IL13.

[0034] In some embodiments, a fusion protein comprises a protein tag. Non-limiting examples of protein tags include HALOTAG® protein fusion tag (HALO), SNAP-TAG® protein tag that reacts specifically and rapidly with benzylguanine (BG) derivatives (SNAP), CLIP, NANOLUC® luciferase (nLUC), ALFA, epitope tags (e.g., a V5 tag), and protein purification tags (e.g., a His tag). In some embodiments, a fusion protein comprises an epitope tag, a self-labeling protein tag, or both. In some embodiments, a fusion protein comprises a minimal tag (e.g., a 6xHis tag). In some embodiments, a fusion protein comprises a protein purification tag.

[0035] In some embodiments, a fusion protein encoded by a polynucleotide disclosed herein comprises a signal sequence. In some embodiments, a signal sequence is a Sonic Hedgehog signal sequence (for example, based on protein NP 033196.1). In some embodiments, a signal sequence comprises MLLLLARCFLVILASSLLVCPGLA (SEQ ID NO:21).- 5 -4223116.V10399.2076001N-terminal Palmitoylation

[0036] In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase.

[0037] In some embodiments, a polypeptide is N-terminal to a non-Hedgehog therapeutic protein. In some embodiments, a polypeptide is at the N-terminus of a fusion protein.

[0038] In some embodiments, a Hedgehog acyltransferase is a mammalian Hedgehog acyltransferase.

[0039] In some embodiments, a Hedgehog acyltransferase is a mouse Hedgehog acyltransferase (e.g., NCBI ID NP 659130.2 (SEQ ID NO:1), translated from NCBI Gene ID 226861).

[0040] MLPGWELTLCLLVSLGFHFRSFYEVYKVSREHEEELDQEFELEMDTLFGGLKKDPTDFEWNFWMEWGKRRLVWLFIGHMAVSQLATLLTKKHRPWIVMVYGMWACWC VLGAPGVVMVLLHSTIAFCVAQFRSVLLSWLCSLLLLSTLRLQSVEEVKRRWYKTENEY YLLQFTLTVRCLYYTSFSLELCRQPPSAQPTPSAQGASHSYPWLLTYVFYYPVFHNGPIL NFPEFFRQMQQPELNSLQHSLCIVAKGLGRLLCWWWLAELMVHLMYMHALYSSAPLL ESVSCWTLGGLALAQVLFFYVKYLVLFGVPALLMRLDGLTPPPLPRCVSTMFSFTGMW RYFDVGLHNFLIRYVYIPLGGSQHGLLGTLLSTATTFAFVSYWHGSYEDLWCWAALNW LGVTVESGVRRLLETPCVRETLARHLSPQAHHRLHALLAACSTSMLILFNLVFLGGIQVG KTYWNRIFLQGWPWVTLSVLGFLYCYSHVDIAWAQTYTVL (SEQ ID NO:1)

[0041] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 1 (e.g., NCBI ID NP 001116306.1 (SEQ ID NO:2)).

[0042] MLPRWELALYLLASLGFHFYSFYEVYKVSREHEEELDQEFELETDTLFGGLKKDATDFEWSFWMEWGKQWLVWLLLGHMVVSQMATLLARKHRPWILMLYGMWACW CVLGTPGVAMVLLHTTISFCVAQFRSQLLTWLCSLLLLSTLRLQGVEEVKRRWYKTENE YYLLQFTLTVRCLYYTSFSLELCWQQLPAASTSYSFPWMLAYVFYYPVLHNGPILSFSEF IKQMQQQEHDSLKASLCVLALGLGRLLCWWWLAELMAHLMYMHAIYSSIPLLETVSC WTLGGLALAQVLFFYVKYLVLFGVPALLMRLDGLTPPALPRCVSTMFSFTGMWRYFDV GLHNFLIRYVYIPVGGSQHGLLGTLFSTAMTFAFVSYWHGGYDYLWCWAALNWLGVT VENGVRRLVETPCIQDSLARYFSPQARRRFHAALASCSTSMLILSNLVFLGGNEVGKTY WNRIFIQGWPWVTLSVLGFLYCYSHVGIAWAQTYATD (SEQ ID NO:2)- 6 -4223116.V10399.2076001

[0043] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 2 e.g., NCBI ID NP_001164035.1 (SEQ ID NO:3)).

[0044] MLPRWELALYLLASLGFHFYSFYEVYKVSREHEEELDQEFELETDTLFGGLKKDATDFEWSFWMEWGKQWLVWLLLGHMVVSQMATLLARKMQQQEHDSLKASLCVL ALGLGRLLCWWWLAELMAHLMYMHAIYSSIPLLETVSCWTLGGLALAQVLFFYVKYL VLFGVPALLMRLDGLTPPALPRCVSTMFSFTGMWRYFDVGLHNFLIRYVYIPVGGSQHG LLGTLFSTAMTFAFVSYWHGGYDYLWCWAALNWLGVTVENGVRRLVETPCIQDSLAR YFSPQARRRFHAALASCSTSMLILSNLVFLGGNEVGKTYWNRIFIQGWPWVTLSVLGFL YCYSHVGIAWAQTYATD (SEQ ID NO:3)

[0045] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 3 (e.g., NCBI ID NP_001164059.1 (SEQ ID NO:4)).

[0046] MLPRWELALYLLASLGFHFYSFYEVYKVSREHEEELDQEFELETDTLFGGLKKDATDFEWSFWMEWGKQWLVWLLLGHMVVSQMATLLARKRRWYKTENEYYLLQFT LTVRCLYYTSFSLELCWQQLPAASTSYSFPWMLAYVFYYPVLHNGPILSFSEFIKQMQQ QEHDSLKASLCVLALGLGRLLCWWWLAELMAHLMYMHAIYSSIPLLETVSCWTLGGL ALAQVLFFYVKYLVLFGVPALLMRLDGLTPPALPRCVSTMFSFTGMWRYFDVGLHNFL IRYVYIPVGGSQHGLLGTLFSTAMTFAFVSYWHGGYDYLWCWAALNWLGVTVENGVR RLVETPCIQDSLARYFSPQARRRFHAALASCSTSMLILSNLVFLGGNEVGKTYWNRIFIQ GWPWVTLSVLGFL YCYSHVGIAWAQTYATD (SEQ ID NO:4)

[0047] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 4 (e.g., NCBI ID NP_001164058.1 (SEQ ID NO:5)).

[0048] MSLGLGSAERGVLGTRGARERCRRRRPGQPGEHEEELDQEFELETDTLFGGLKKDATDFEWSFWMEWGKQWLVWLLLGHMVVSQMATLLARKHRPWILMLYGMWAC WCVLGTPGVAMVLLHTTISFCVAQFRSQLLTWLCSLLLLSTLRLQGVEEVKRRWYKTE NEYYLLQFTLTVRCLYYTSFSLELCWQQLPAASTSYSFPWMLAYVFYYPVLHNGPILSFS EFIKQMQQQEHDSLKASLCVLALGLGRLLCWWWLAELMAHLMYMHAIYSSIPLLETVS CWTLGGLALAQVLFFYVKYLVLFGVPALLMRLDGLTPPALPRCVSTMFSFTGMWRYFD VGLHNFLIRYVYIPVGGSQHGLLGTLFSTAMTFAFVSYWHGGYDYLWCWAALNWLGV TVENGVRRLVETPCIQDSLARYFSPQARRRFHAALASCSTSMLILSNLVFLGGNEVGKTY WNRIFIQGWPWVTLSVLGFL YCYSHVGIAWAQTYATD (SEQ ID NO: 5)

[0049] In some embodiments, a Hedgehog acyltransferase is an insect Hedgehog acyltransferase, rasp (e.g., NP_523898.1 (SEQ ID NO:6)).- 7 -4223116.V10399.2076001

[0050] MSRLPDRSLLTRCEIFVYFGVYIAYIVVGLYKIYGLRDHIVKEAKFQFPEGWSLYPFSQRRRDDSNDELENFGDFIVSFWPFYLLHVAVQGFIRWKRPRLQCLGFIGVCALAL SVNLDWSSMVLLVTLIASYYIVSLLSLKFLVWLLSAGWILCINVMQKNVWWTDRVGYT EYVLVIVTMSWSVLRGCSYSLSKIGAKQEDLTRYSLVQYLGYAMYFPCLTYGPIISYQRF AARREDEVQNWLGFVGGVLRSAIWWLVMQCALHYFYIHYMSRDVRMVEMMDSVFW QHSAGYFMGQFFFLYYVVTYGLGIAFAVQDGIPAPNRPRCIGRIHFYSDMWKYFDEGLY EFLFQNIYAELCGKRSSAAAKFGATALTFAFVFVWHGCYTYVLIWSILNFLCLAAEKVF KTFTAMPEYQRWTQRHLGAVGAQRLYAMLATQLFIPAAFSNVYFIGGQEIGDFLMRGA YLSGVGNYVALCFCSYCFFQCSELLLTKSDGRSKTKTF (SEQ ID NO:6)

[0051] In some embodiments, a Hedgehog acyltransferase is selected from SEQ ID NOs: l-6.In some embodiments, a Hedgehog acyltransferase is selected from SEQ ID NOs:l-5. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:1. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:2. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:3. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:4. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:5. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:6.

[0052] In some embodiments, a polypeptide comprises an N-terminal sequence of a preproprotein or proprotein (e.g., comprising CGPGRG at the N-terminus) of a vertebrateHedgehog protein (e.g., a vertebrate sonic, desert, or Indian Hedgehog protein).

[0053] In some embodiments, a polypeptide comprises an N-terminal sequence of a vertebrate Sonic Hedgehog preproprotein. In some embodiments, a polypeptide comprises an N- terminal sequence of a vertebrate Sonic Hedgehog proprotein.

[0054] In some embodiments, a polypeptide comprises an N-terminal sequence of a human sonic hedgehog protein isoform 1 preproprotein (e.g., NP 000184.1 (SEQ ID NO:7)).

[0055] MLLLARCLLLVLVSSLLVCSGLACGPGRGFGKRRHPKKLTPLAYKQFIPNVAEKTLGASGRYEGKISRNSERFKELTPNYNPDIIFKDEENTGADRLMTQRCKDKLNALAIS VMNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRSKYGMLARLAVEA GFDWVYYESKAHIHCSVKAENSVAAKSGGCFPGSATVHLEQGGTKLVKDLSPGDRVLA ADDQGRLLYSDFLTFLDRDDGAKKVFYVIETREPRERLLLTAAHLLFVAPHNDSATGEP EASSGSGPPSGGALGPRALFASRVRPGQRVYVVAERDGDRRLLPAAVHSVTLSEEAAGA YAPLTAQGTILINRVLASCYAVIEEHSWAHRAFAPFRLAHALLAALAPARTDRGGDSGG GDRGGGGGRVALTAPGAADAPGAGATAGIHWYSQLLYQIGTWLLDSEALHPLGMAVK SS (SEQ ID NO:7)- 8 -4223116.V10399.2076001

[0056] In some embodiments, a polypeptide comprises an N-terminal sequence of a human Indian hedgehog protein isoform 1 preproprotein (e.g., NP 002172.2 (SEQ ID NO:8)).

[0057] MSPARLRPRLHFCLVLLLLLVVPAAWGCGPGRVVGSRRRPPRKLVPLAYKQF SPNVPEKTLGASGRYEGKIARSSERFKELTPNYNPDIIFKDEENTGADRLMTQRCKDRLN SLAISVMNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRNKYGLLARLA VEAGFDWVYYESKAHVHCSVKSEHSAAAKTGGCFPAGAQVRLESGARVALSAVRPGD RVLAMGEDGSPTFSDVLIFLDREPHRLRAFQVIETQDPPRRLALTPAHLLFTADNHTEPA ARFRATFASHVQPGQYVLVAGVPGLQPARVAAVSTHVALGAYAPLTKHGTLVVEDVV ASCFAAVADHHLAQLAFWPLRLFHSLAWGSWTPGEGVHWYPQLLYRLGRLLLEEGSFHPLGMSGAGS (SEQ ID NO: 8)

[0058] In some embodiments, a polypeptide comprises an N-terminal sequence of a human desert hedgehog protein isoform 1 preproprotein (e.g., NP 066382.1 (SEQ ID NO:9)).

[0059] MALLTNLLPLCCLALLALPAQSCGPGRGPVGRRRYARKQLVPLLYKQFVPG VPERTLGASGPAEGRVARGSERFRDLVPNYNPDIIFKDEENSGADRLMTERCKERVNALAIAVMNMWPGVRLRVTEGWDEDGHHAQDSLHYEGRALDITTSDRDRNKYGLLARLA VEAGFDWVYYESRNHVHVSVKADNSLAVRAGGCFPGNATVRLWSGERKGLRELHRGDWVLAADASGRVVPTPVLLFLDRDLQRRASFVAVETEWPPRKLLLTPWHLVFAARGPA PAPGDFAPVFARRLRAGDSVLAPGGDALRPARVARVAREEAVGVFAPLTAHGTLLVND VLASCYAVLESHQWAHRAFAPLRLLHALGALLPGGAVQPTGMHWYSRLLYRLAEELL G (SEQ ID NO: 9)

[0060] In some embodiments, an N-terminal sequence (e.g., an N-terminal sequence of a vertebrate Sonic Hedgehog proprotein) is at least 6 amino acids in length, for example, at least:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, an N-terminal sequence is at least 8 amino acids in length. In some embodiments, an N-terminal sequence is at least 13 amino acids in length.

[0061] In some embodiments, an N-terminal sequence is about: 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 amino acids in length, for example, about: 6-20, 6-19, 6-18, 6-17, 6-16,6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13,7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-- 9 -4223116.V10399.207600116, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 amino acids in length.

[0062] In some embodiments, an N-terminal sequence is about 6 amino acids in length. In some embodiments, an N-terminal sequence is about 7 amino acids in length. In some embodiments, an N-terminal sequence is about 8 amino acids in length. In some embodiments, an N-terminal sequence is about 9 amino acids in length. In some embodiments, an N-terminal sequence is about 10 amino acids in length. In some embodiments, an N-terminal sequence is about 11 amino acids in length. In some embodiments, an N-terminal sequence is about 12 amino acids in length. In some embodiments, an N-terminal sequence is about 13 amino acids in length. In some embodiments, an N-terminal sequence is about 14 amino acids in length. In some embodiments, an N-terminal sequence is about 15 amino acids in length. In some embodiments, an N-terminal sequence is about 16 amino acids in length. In some embodiments, an N-terminal sequence is about 17 amino acids in length. In some embodiments, an N-terminal sequence is about 18 amino acids in length. In some embodiments, an N-terminal sequence is about 19 amino acids in length. In some embodiments, an N-terminal sequence is about 20 amino acids in length.

[0063] In some embodiments, an N-terminal sequence comprises at least 1 polar residue, for example, at least: 2, 3, 4, or 5 polar residues. In some embodiments, an N-terminal sequence comprises about: 1, 2, 3, 4, or 5 polar residues. In some embodiments, an N-terminal sequence comprises about: 1-5, 1-4, 1-5, 1-6, 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 polar residues.

[0064] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog protein proprotein of X1X2X3X4RG, wherein: Xi is C or S; X2 is G, A, S, Q, or P; X3 is P or A; and X4 is G or A.

[0065] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog proprotein of CGPGRG (SEQ ID NO: 11).

[0066] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog proprotein of CGPGRGFG (SEQ ID NO: 12).Auto-Processing Domain-Mediated Modification

[0067] In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a Hedgehog auto-processing domain having lipophilic modification activity, wherein the domain is C-terminal to the non-Hedgehog therapeutic protein.- 10 -4223116.V10399.2076001

[0068] In some embodiments, a lipophilic modification is a cholesterol modification.

[0069] In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a Hedgehog auto-processing domain having sterol modification activity, wherein the domain is C-terminal to the non-Hedgehog therapeutic protein.

[0070] In some embodiments, a sterol modification is a cholesterol modification.

[0071] In some embodiments, a polypeptide is C-terminal to a non-Hedgehog therapeutic protein. In some embodiments, a polypeptide is at the C-terminus of a fusion protein.

[0072] In some embodiments, a Hedgehog auto-processing domain is a mammalian SHH- derived Hint domain. In some embodiments, a Hedgehog auto-processing domain is a primate (e.g., human) SHH-derived Hint domain. In some embodiments, a Hedgehog auto-processing domain is a rodent e.g., mouse) SHH-derived Hint domain.

[0073] In some embodiments, a Hedgehog auto-processing domain comprises any one of SEQ ID NOs: 13-16. In some embodiments, a Hedgehog auto-processing domain comprises SEQ ID NO: 13. In some embodiments, a Hedgehog auto-processing domain comprises SEQ ID NO: 14. In some embodiments, a Hedgehog auto-processing domain comprises SEQ ID NO: 15. In some embodiments, a Hedgehog auto-processing domain comprises SEQ ID NO: 16.

[0074] GGCFPGSATVHLEQGGTKLVKDLRPGDRVLAADDQGRLLYSDFLTFLDRDE GAKKVFYVIETLEPRERLLLTAAHLLFVAPHNDSGPTPGPSALFASRVRPGQRVYVVAE RGGDRRLLPAAVHSVTLREEEAGAYAPLTAHGTILINRVLASCYAVIEEHSWAHRAFAP FRLAHALLAALAPARTDGGGGGSIPAAQSATEARGAEPTAGIHWYSQLLYHIGTWLLDS ETMHPLGMAVKSS (SEQ ID NO: 13)

[0075] Human SHH-derived Hint domain (NP 000184 .1)

[0076] GGCFPGSATVHLEQGGTKLVKDLSPGDRVLAADDQGRLLYSDFLTFLDRDD GAKKVFYVIETREPRERLLLTAAHLLFVAPHNDSATGEPEASSGSGPPSGGALGPRALFA SRVRPGQRVYVVAERDGDRRLLPAAVHSVTLSEEAAGAYAPLTAQGTILINRVLASCYA VIEEHSWAHRAFAPFRLAHALLAALAPARTDRGGDSGGGDRGGGGGRVALTAPGAAD APGAGATAGIHWYSQLLYQIGTWLLDSEALHPLGMAVKSS (SEQ ID NO: 14)

[0077] Mouse IHH-derived Hint domain (NP 001300612.1)

[0078] GGCFPAGAQVRLENGERVALSAVKPGDRVLAMGEDGTPTFSDVLIFLDREPN RLRAFQVIETQDPPRRLALTPAHLLFIADNHTEPAAHFRATFASHVQPGQYVLVSGVPGL- 11 -4223116.V10399.2076001QPARVAAVSTHVALGSYAPLTRHGTLVVEDVVASCFAAVADHHLAQLAFWPLRLFPSL AWGSWTPSEGVHWYPQMLYRLGRLLLEESTFHPLGMSGAGS (SEQ ID NO: 15)

[0079] Mouse DHH-derived Hint domain (NP 031883.1)

[0080] GGCFPGNATVRLRSGERKGLRELHRGDWVLAADAAGRVVPTPVLLFLDRDL QRRASFVAVETERPPRKLLLTPWHLVFAARGPAPAPGDFAPVFARRLRAGDSVLAPGGD ALQPARVARVAREEAVGVFAPLTAHGTLLVNDVLASCYAVLESHQWAHRAFAPLRLLHALGALLPGGAVQPTGMHWYSRLLYRLAEELM (SEQ ID NO: 16)Internal Palmitoleoylation

[0081] In some embodiments, a polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a polypeptide sufficient for palmitoylation by a membranebound O-acyltransferase enzyme.

[0082] In some embodiments, a membrane-bound O-acyltransferase enzyme is Porcupine.

[0083] In some embodiments, a membrane-bound O-acyltransferase enzyme is a human protein-serine O-palmitoleoyltransferase porcupine e.g., NP 001269096.1 (SEQ ID NO: 17)).

[0084] MVWVVLLSLLCYLVLFLCRHSSHRGVFLSVTILIYLLMGEMHMVDTVTWHK MRGAQMIVAMKAVSLGFDLDRGEVGTVPSPVEFMGYLYFVGTIVFGPWISFHSYLQAV QGRPLSCRWLQKVARSLAL ALLCL VLSTCVGPYLFPYFIPLNGDRLLRKWLRAYESAVS FHFSNYFVGFLSEATATLAGAGFTEEKDHLEWDLTVSKPLNVELPRSMVEVVTSWNLP MSYWLNNYVFKNALRLGTFSAVLVTYAASALLHGFSFHLAAVLLSLAFITYVEHVLRK RL ARILS AC VLSKRCPPDCSHQHRLGLGVRALNLLF GAL AIFHLAYLGSLFDVDVDDTTEEQGYGMAYTVHKWSELSWASHWVTFGCWIFYRLIG (SEQ ID NO: 17)

[0085] In some embodiments, a membrane-bound O-acyltransferase enzyme is a mouse protein-serine O-palmitoleoyltransferase porcupine (e.g., NP 001295403.1 (SEQ ID NO: 18)).

[0086] MAQDARWLRAYESAVSFHFSNYFVGFLSEATATLAGAGFTEEKDHLEWDLT VSRPLNVELPRSMVEVVTSWNLPMSYWLNNYVFKNALRLGTFSAVLVTYAASALLHGF SFHLAAVLLSLAFITYVEHVLRKRLAQILSACILSKRCLPDCSHRHRLGLGVRALNLLFG ALAIFHLSYLGSLFDVDVDDTTEEQGYGMAYTVHKWSELSWASHWVTFGCWIFYRLIG (SEQ ID NO: 18)

[0087] In some embodiments, a polypeptide sufficient for palmitoylation by the membranebound O-acyltransferase enzyme comprises MRQECKCHGMSGSCTVRTCWM (SEQ ID NO: 19).- 12 -4223116.V10399.2076001Vectors

[0088] The disclosure also provides, among other things, a vector (e.g., a plasmid) comprising any one or more of the polynucleotides disclosed herein.

[0089] The term “expression vector” refers to a replicable nucleic acid from which one or more proteins can be expressed when the expression vector is transformed into a suitable expression host cell.

[0090] In some embodiments, a vector (e.g., expression vector) further comprises an expression control polynucleotide sequence operably linked to the polynucleotide, a polynucleotide sequence encoding a selectable marker, or both.

[0091] In some embodiments, an expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. The term “promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.

[0092] In some embodiments, an expression control polynucleotide sequence comprises a constitutive promoter sequence.

[0093] In some embodiments, an expression control polynucleotide sequence comprises an inducible promoter sequence. In some embodiments, an inducible promoter is a chemically inducible promoter. In some embodiments, a chemically inducible promoter is positively inducible (e.g., tetracycline on (Tet-on), inducible by tetracycline (Tet) and / or doxycycline (Dox)). In some embodiments, an inducible promoter comprises a Dox-inducible promoter. In some embodiments, an inducible promoter comprises a Tet-inducible promoter. In some embodiments, an inducible promoter comprises a lecdysone-inducible promoter. In some embodiments, a chemically inducible promoter is negatively inducible (e.g., tetracycline off (Tet-on), suppressible by Tet and / or Dox). In some embodiments, an inducible promoter comprises a Dox-suppressible promoter. In some embodiments, an inducible promoter comprises a Tet-suppressible promoter.

[0094] The term “operably linked” means that the nucleic acid is positioned in the recombinant polynucleotide, e.g., vector, in such a way that enables expression of the nucleic acid under control of the element (e.g., promoter) to which it is linked.

[0095] The term “selectable marker element” is an element that confers a trait suitable for artificial selection. Selectable marker elements can be negative or positive selection markers.Cells

[0096] The disclosure also provides, among other things, a cell comprising any one or more of the polynucleotides or vectors disclosed herein.- 13 -4223116.V10399.2076001

[0097] In some embodiments, a cell is in vitro or ex vivo. In some embodiments, a cell is in vivo.

[0098] A cell may reside in or be obtained (e.g., isolated) from a biological entity containing expressed genetic materials. The biological entity may be a plant, animal, or microorganism. In some embodiments, a cell resides in a biological entity or a tissue. In some embodiments, a cell or its progeny was obtained (e.g., isolated) from a biological entity in vivo. In some embodiments, a cell or its progeny is cultured in vitro.

[0099] In some embodiments, a cell comprises or is an animal cell, or a plant cell.

[0100] In some embodiments, a cell comprises or is an animal cell. In some embodiments, a cell comprises or is a mammalian cell. In some embodiments, a mammalian cell is selected from a dog cell, a cat cell, a mouse cell, a rat cell, a hamster cell, a guinea pig cell, a horse cell, a pig cell, a sheep cell, a cow cell, a chimpanzee cell, a macaque cell, a cynomolgus monkey cell, or a human cell. In some embodiments, a cell is derived from a primate (e.g., a human or a nonhuman primate). In some embodiments, a cell comprises or is a human cell.

[0101] In some embodiments, a cell comprises or is a chimeric antigen receptor (CAR) cell. Non-limiting examples of CAR cells include CAR-T, CAR-Macrophages (CAR-M), and CAR- natural killer cells (CAR-NK). In some embodiments, a CAR cell comprises or is a CAR-T cell.

[0102] In some embodiments, a cell comprises or is a plant cell.

[0103] In some embodiments, a polynucleotide is integrated into the genomic DNA of a cell.In some embodiments, a polynucleotide is extrachromosomal.

[0104] In some embodiments, a cell is a single cell.Kits

[0105] Also provided herein, among other things, is a kit comprising a container and, optionally, an instruction for use, wherein the container comprises any one or more of the polynucleotides, vectors, or cells disclosed herein, or any combination of the foregoing.Methods of Modification In a Cell

[0106] Also provided herein, among other things, is a method of modifying a non-Hedgehog therapeutic protein in a cell, wherein the method comprises expressing one or more polynucleotides or vectors disclosed herein in a cell described herein.- 14 -4223116.V10399.2076001N-terminal Palmitoylation

[0107] In some embodiments, a method of modifying a non-Hedgehog therapeutic protein comprises expressing a polynucleotide or vector in a cell expressing a Hedgehog acyltransferase, wherein the polynucleotide or vector comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a polypeptide sufficient for palmitoylation by the Hedgehog acyltransferase.

[0108] In some embodiments, a polypeptide is N-terminal to a non-Hedgehog therapeutic protein. In some embodiments, a polypeptide is at the N-terminus of a fusion protein.

[0109] In some embodiments, a Hedgehog acyltransferase is a mammalian Hedgehog acyltransferase. In some embodiments, a Hedgehog acyltransferase is a mouse Hedgehog acyltransferase (e.g., NCBI ID NP 659130.2 (SEQ ID NO: 1), translated from NCBI Gene ID 226861).

[0110] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 1 (e.g., NCBI ID NP 001116306.1 (SEQ ID NO:2)).

[0111] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 2 (e.g., NCBI ID NP_001164035.1 (SEQ ID NO:3)).

[0112] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 3 (e.g., NCBI ID NP_001164059.1 (SEQ ID NO:4)).

[0113] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 4 (e.g., NCBI ID NP_001164058.1 (SEQ ID NO:5)).

[0114] In some embodiments, a Hedgehog acyltransferase is an insect Hedgehog acyltransferase, rasp (e.g., NP_523898.1 (SEQ ID NO:6)).

[0115] In some embodiments, a Hedgehog acyltransferase is selected from SEQ ID NOs: l-6. In some embodiments, a Hedgehog acyltransferase is selected from SEQ ID NOs:l-5. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:1. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:2. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:3. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NON. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:5. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:6.

[0116] In some embodiments, a polypeptide comprises an N-terminal sequence of a preproprotein or proprotein (e.g., a proprotein comprising CGPGRG at the N-terminus) of a vertebrate Hedgehog protein (e.g., a vertebrate sonic, desert, or Indian Hedgehog protein). In- 15 -4223116.V10399.2076001 some embodiments, a polypeptide comprises an N-terminal sequence of a vertebrate Sonic Hedgehog preproprotein. In some embodiments, a polypeptide comprises an N-terminal sequence of a vertebrate Sonic Hedgehog proprotein.

[0117] In some embodiments, a polypeptide comprises an N-terminal sequence of a human sonic hedgehog protein isoform 1 preproprotein (e.g., NP 000184.1 (SEQ ID NO:7)).

[0118] In some embodiments, a polypeptide comprises an N-terminal sequence of a human Indian hedgehog protein isoform 1 preproprotein (e.g., NP 002172.2 (SEQ ID NO:8)).

[0119] In some embodiments, a polypeptide comprises an N-terminal sequence of a human desert hedgehog protein isoform 1 preproprotein (e.g., NP 066382.1 (SEQ ID NO:9)).

[0120] In some embodiments, an N-terminal sequence is at least 6 amino acids in length, for example, at least: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, an N-terminal sequence is at least 8 amino acids in length. In some embodiments, an N-terminal sequence is at least 13 amino acids in length.

[0121] In some embodiments, an N-terminal sequence is about: 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 amino acids in length, for example, about: 6-20, 6-19, 6-18, 6-17, 6-16,6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13,7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13- 16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 amino acids in length.

[0122] In some embodiments, an N-terminal sequence (e.g, of a proprotein comprising CGPGRG at the N-terminus) is about 6 amino acids in length. In some embodiments, an N- terminal sequence is about 7 amino acids in length. In some embodiments, an N-terminal sequence is about 8 amino acids in length. In some embodiments, an N-terminal sequence is about 9 amino acids in length. In some embodiments, an N-terminal sequence is about 10 amino acids in length. In some embodiments, an N-terminal sequence is about 11 amino acids in length. In some embodiments, an N-terminal sequence is about 12 amino acids in length. In some embodiments, an N-terminal sequence is about 13 amino acids in length. In some embodiments, an N-terminal sequence is about 14 amino acids in length. In some embodiments, an N-terminal sequence is about 15 amino acids in length. In some embodiments, an N-terminal sequence is about 16 amino acids in length. In some embodiments, an N-terminal sequence is about 174223116.V10399.2076001 amino acids in length. In some embodiments, an N-terminal sequence is about 18 amino acids in length. In some embodiments, an N-terminal sequence is about 19 amino acids in length. In some embodiments, an N-terminal sequence is about 20 amino acids in length.

[0123] In some embodiments, an N-terminal sequence comprises at least 1 polar residue, for example, at least: 2, 3, 4, or 5 polar residues. In some embodiments, an N-terminal sequence comprises about: 1, 2, 3, 4, or 5 polar residues. In some embodiments, an N-terminal sequence comprises about: 1-5, 1-4, 1-5, 1-6, 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 polar residues.

[0124] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog proprotein of X1X2X3X4RG (SEQ ID NO: 10), wherein: Xi is C or S; X2 is G, A, S, Q, or P; X3 is P or A; and X4 is G or A.

[0125] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog proprotein of CGPGRG (SEQ ID NO: 11).

[0126] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog proprotein of CGPGRGFG (SEQ ID NO: 12).Auto-Processing Domain-Mediated Modification

[0127] In some embodiments, a method comprises expressing a polynucleotide or vector in a cell, wherein the polynucleotide or vector comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a Hedgehog auto-processing domain having lipophilic modification activity.

[0128] In some embodiments, a lipophilic modification is a cholesterol modification.

[0129] In some embodiments, a method comprises expressing a polynucleotide or vector in a cell, wherein the polynucleotide or vector comprises a sequence encoding a Hedgehog autoprocessing domain having sterol modification activity.

[0130] In some embodiments, a sterol modification is a cholesterol modification.

[0131] In some embodiments, a polypeptide is C-terminal to a non-Hedgehog therapeutic protein. In some embodiments, a polypeptide is at the C-terminus of a fusion protein.

[0132] In some embodiments, a Hedgehog auto-processing domain is a mammalian SHH- derived Hint domain. In some embodiments, a Hedgehog auto-processing domain is a primate (e.g., human) SHH-derived Hint domain. In some embodiments, a Hedgehog auto-processing domain is a rodent e.g., mouse) SHH-derived Hint domain.

[0133] In some embodiments, a Hedgehog auto-processing domain comprises any one of SEQ ID NOs: 13-16. In some embodiments, a Hedgehog auto-processing domain comprises SEQ- 17 -4223116.V10399.2076001ID NO: 13. In some embodiments, a Hedgehog auto-processing domain comprises SEQ ID NO: 14. In some embodiments, a Hedgehog auto-processing domain comprises SEQ ID NO: 15. In some embodiments, a Hedgehog auto-processing domain comprises SEQ ID NO: 16.Internal Palmitoleoylation

[0134] In some embodiments, a method comprises expressing a polynucleotide or vector in a cell expressing a membrane-bound O-acyltransferase enzyme, wherein the polynucleotide or vector comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) a sequence encoding a polypeptide sufficient for palmitoylation by the membrane-bound O-acyltransferase enzyme.

[0135] In some embodiments, a membrane-bound O-acyltransferase enzyme is Porcupine.

[0136] In some embodiments, a membrane-bound O-acyltransferase enzyme is a human protein-serine O-palmitoleoyltransferase porcupine (e.g., NP 001269096.1 (SEQ ID NO: 17)).

[0137] In some embodiments, a membrane-bound O-acyltransferase enzyme is a mouse protein-serine O-palmitoleoyltransferase porcupine (e.g., NP 001295403.1 (SEQ ID NO: 18)).

[0138] In some embodiments, a polypeptide sufficient for palmitoylation by the membranebound O-acyltransferase enzyme comprises MRQECKCHGMSGSCTVRTCWM (SEQ ID NO: 19).

[0139] In some embodiments, a method further comprises isolating a modified nonHedgehog therapeutic protein from a cell.

[0140] In some embodiments, a method further comprises characterizing a non-Hedgehog therapeutic protein.

[0141] In some embodiments, a method further comprises measuring serum half-life of a non-Hedgehog therapeutic protein.

[0142] In some embodiments, a non-Hedgehog therapeutic protein e.g., a peptide hormone or an anti-inflammatory cytokine) has a short serum half-life.

[0143] In some embodiments, a method increases serum half-life of a non-Hedgehog therapeutic protein.

[0144] In some embodiments, a non-Hedgehog therapeutic protein e.g., a pro-inflammatory cytokine) is secreted by a cell e.g., a CAR cell such as a CAR-T cell). In some embodiments, a method further comprises measuring spatial distribution of secreted non-Hedgehog therapeutic proteins or activities thereof.- 18 -4223116.V10399.2076001

[0145] In some embodiments, a method further comprises administering a non-Hedgehog therapeutic protein to a subject in need thereof. In some embodiments, a subject is a human patient.Methods of Modification of Purified Proteins

[0146] Also provided herein, among other things, is a method of modifying a purified nonHedgehog therapeutic protein.N-terminal Palmitoylation

[0147] In some embodiments, a method comprises: a) expressing a polynucleotide in a host cell, wherein the polynucleotide comprises: i. a protein coding sequence encoding a non-Hedgehog therapeutic protein, and ii. a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase, b) purifying the non-Hedgehog therapeutic protein, and c) contacting the purified non-Hedgehog therapeutic protein with the Hedgehog acyltransferase and palmityl-CoA.

[0148] In some embodiments, a polypeptide is N-terminal to a non-Hedgehog therapeutic protein. In some embodiments, a polypeptide is at the N-terminus of a fusion protein.

[0149] In some embodiments, a Hedgehog acyltransferase is a mammalian Hedgehog acyltransferase. In some embodiments, a Hedgehog acyltransferase is a mouse Hedgehog acyltransferase (e.g., NCBI ID NP 659130.2 (SEQ ID NO: 1), translated from NCBI Gene ID 226861).

[0150] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 1 (e.g., NCBI ID NP 001116306.1 (SEQ ID NO:2)).

[0151] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 2 (e.g, NCBI ID NP_001164035.1 (SEQ ID NO:3)).

[0152] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 3 (e.g, NCBI ID NP_001164059.1 (SEQ ID NO:4)).

[0153] In some embodiments, a Hedgehog acyltransferase is a human Hedgehog acyltransferase isoform 4 (e.g., NCBI ID NP_001164058.1 (SEQ ID NO:5)).

[0154] In some embodiments, a Hedgehog acyltransferase is an insect Hedgehog acyltransferase, rasp (e.g., NP_523898.1 (SEQ ID NO:6)).- 19 -4223116.V10399.2076001

[0155] In some embodiments, a Hedgehog acyltransferase is selected from SEQ ID NOs: l-6. In some embodiments, a Hedgehog acyltransferase is selected from SEQ ID NOs:l-5. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:1. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:2. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:3. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:4. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:5. In some embodiments, a Hedgehog acyltransferase is of SEQ ID NO:6.

[0156] In some embodiments, a polypeptide comprises an N-terminal sequence of a preproprotein or proprotein (e.g., comprising CGPGRG at the N-terminus) of a vertebrate Hedgehog protein (e.g., a vertebrate sonic, desert, or Indian Hedgehog protein).

[0157] In some embodiments, a polypeptide comprises an N-terminal sequence of a vertebrate Sonic Hedgehog preproprotein. In some embodiments, a polypeptide comprises an N- terminal sequence of a vertebrate Sonic Hedgehog proprotein.

[0158] In some embodiments, a polypeptide comprises an N-terminal sequence of a human sonic hedgehog protein isoform 1 preproprotein (e.g., NP 000184.1 (SEQ ID NO:7)).

[0159] In some embodiments, a polypeptide comprises an N-terminal sequence of a human Indian hedgehog protein isoform 1 preproprotein (e.g., NP 002172.2 (SEQ ID NO:8)).

[0160] In some embodiments, a polypeptide comprises an N-terminal sequence of a human desert hedgehog protein isoform 1 preproprotein (e.g., NP 066382.1 (SEQ ID NO:9)).

[0161] In some embodiments, an N-terminal sequence (e.g, comprising CGPGRG at the N- terminus) is at least 6 amino acids in length, for example, at least: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, an N-terminal sequence is at least 8 amino acids in length. In some embodiments, an N-terminal sequence is at least 13 amino acids in length.

[0162] In some embodiments, an N-terminal sequence is about: 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 amino acids in length, for example, about: 6-20, 6-19, 6-18, 6-17, 6-16,6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13,7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13- 16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 amino acids in length.4223116.V10399.2076001

[0163] In some embodiments, an N-terminal sequence is about 6 amino acids in length. In some embodiments, an N-terminal sequence is about 7 amino acids in length. In some embodiments, an N-terminal sequence is about 8 amino acids in length. In some embodiments, an N-terminal sequence is about 9 amino acids in length. In some embodiments, an N-terminal sequence is about 10 amino acids in length. In some embodiments, an N-terminal sequence is about 11 amino acids in length. In some embodiments, an N-terminal sequence is about 12 amino acids in length. In some embodiments, an N-terminal sequence is about 13 amino acids in length. In some embodiments, an N-terminal sequence is about 14 amino acids in length. In some embodiments, an N-terminal sequence is about 15 amino acids in length. In some embodiments, an N-terminal sequence is about 16 amino acids in length. In some embodiments, an N-terminal sequence is about 17 amino acids in length. In some embodiments, an N-terminal sequence is about 18 amino acids in length. In some embodiments, an N-terminal sequence is about 19 amino acids in length. In some embodiments, an N-terminal sequence is about 20 amino acids in length.

[0164] In some embodiments, an N-terminal sequence comprises at least 1 polar residue, for example, at least: 2, 3, 4, or 5 polar residues. In some embodiments, an N-terminal sequence comprises about: 1, 2, 3, 4, or 5 polar residues. In some embodiments, an N-terminal sequence comprises about: 1-5, 1-4, 1-5, 1-6, 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 polar residues.

[0165] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog protein proprotein of X1X2X3X4RG (SEQ ID NO: 10), wherein: Xi is C or S; X2 is G, A, S, Q, or P; X3 is P or A; and X4 is G or A.

[0166] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog protein proprotein of CGPGRG (SEQ ID NO: 11).

[0167] In some embodiments, a polypeptide comprises an N-terminal sequence of a hedgehog protein proprotein of CGPGRGFG (SEQ ID NO: 12).Internal Palmitoleoylation

[0168] In some embodiments, a method comprises: a) expressing a polynucleotide in a host cell, wherein the polynucleotide comprises: i. a protein coding sequence encoding a non-Hedgehog therapeutic protein, and ii. a sequence encoding a polypeptide sufficient for palmitoylation by a membrane-bound O-acyltransferase enzyme, b) purifying the non-Hedgehog therapeutic protein, and- 21 -4223116.V10399.2076001 c) contacting the purified non-Hedgehog therapeutic protein with the membranebound O-acyltransferase enzyme and palmityl-CoA.

[0169] In some embodiments, a membrane-bound O-acyltransferase enzyme is Porcupine.

[0170] In some embodiments, a membrane-bound O-acyltransferase enzyme is a human protein-serine O-palmitoleoyltransferase porcupine (e.g., NP 001269096.1 (SEQ ID NO: 17)).

[0171] In some embodiments, a membrane-bound O-acyltransferase enzyme is a mouse protein-serine O-palmitoleoyltransferase porcupine (e.g., NP 001295403.1 (SEQ ID NO: 18)).

[0172] In some embodiments, a polypeptide sufficient for palmitoylation by the membranebound O-acyltransferase enzyme comprises MRQECKCHGMSGSCTVRTCWM (SEQ ID NO: 19).

[0173] In some embodiments, a method further comprises characterizing a non-Hedgehog therapeutic protein.

[0174] In some embodiments, a method further comprises measuring serum half-life of a non-Hedgehog therapeutic protein.

[0175] In some embodiments, a non-Hedgehog therapeutic protein e.g., a peptide hormone or an anti-inflammatory cytokine) has a short serum half-life.

[0176] In some embodiments, a method disclosed herein increases serum half-life of a nonHedgehog therapeutic protein.

[0177] In some embodiments, a method further comprises administering a non-Hedgehog therapeutic protein to a subject in need thereof. In some embodiments, a subject is a human patient.EXEMPLIFICATIONExample 1. MethodsPalmitoylation

[0178] The C-terminal palmitoylation motif was derived from the coding sequence from Sonic Hedgehog, including the Hedgehog signal sequence and the first 8 amino acids of the mouse Sonic Hedgehog coding sequence. This tag was developed based on prior characterization (Hardy & Resh (2012)), which suggested that the palmitate molecule is transiently attached to the cystine immediately after the cleavage signal (cleavage site denoted asA) but is ultimately transferred to the N-terminal amino group in the fully processed protein. This palmitoylation reaction is performed by HHAT (Hedgehog Acyltransferase) using Palmityl-CoA as a cofactor.

[0179] Start - (SEQ ID NO:20) N-terminal fusion to target protein- 22 -4223116.V10399.2076001

[0180] MLLLLARCFLVILASSLLVCPGLAACGPGRGFG (SEQ ID NO:20)Sterol Modification

[0181] The C-terminal cholesterol modification was applied using the Hedgehog Intein (Hint) domain. A fusion protein that attached the previously described Hedgehog Intein (Porter (1996)) to the C-terminal of the nanobody fusion protein was generated. Prior characterization revealed that after splicing of the intein, cholesterol is applied to the terminal glycine immediately before the cleavage site (denoted asA). That the Hint domain was autocatalytically processed was verified using polyacrylamide gel electrophoresis, which showed a band consistent with the processed form of the fusion protein. The C-terminal cholesterol modification is auto catalytic and does not require expression of any additional enzymes to achieve correct modification.

[0182] C-term of target protein (SEQ ID NO: 13)

[0183] GGACFPGSATVHLEQGGTKLVKDLRPGDRVLAADDQGRLLYSDFLTFLDRDE GAKKVFYVIETLEPRERLLLTAAHLLFVAPHNDSGPTPGPSALFASRVRPGQRVYVVAE RGGDRRLLPAAVHSVTLREEEAGAYAPLTAHGTILINRVLASCYAVIEEHSWAHRAFAP FRLAHALLAALAPARTDGGGGGSIPAAQSATEARGAEPTAGIHWYSQLLYHIGTWLLDS ETMHPLGMAVKSS*[STOP] (SEQ ID NO: 13)Internal Palmitoleoylation

[0184] The internal palmitoleoylation modification was derived from the sequence adjacent to the known palmitoleoylation site of the Wntl ligand. This motif was based on prior characterization of the natural sequence requirements for Wnt palmitoleoylation (Miranda (2014)), but the tag described here was further truncated to achieve the minimal tag necessary for palmitoleoylation. The Wnt palmitoleoylation reaction requires the enzyme Porcupine and the co-factor palmitoleoyl-CoA to attach palmitoleate to an internal serine (denoted with [S]).

[0185] N-terminal fusionC-terminal fusion

[0186] MRQECKCHGM[S]GSCTVRTCWM (SEQ ID NO: 19)Generation of sender cell lines

[0187] All sender cell lines were generated using piggyBac (PB) transposase to generate chromosomal insertions of the relevant transgenes, driven by a 4-Hydroxytam oxifen (4-OHT; CAS No. 68392-35-8)-inducible promoter (although a broad range of possible gene expression strategies would work). Modified fusion proteins can be generated in any mammalian cell line- 23 -4223116.V10399.2076001 without further modification, and possibly in insect cells. Here, all proteins were expressed either in NIH3T3 (e.g., American Type Culture Collection (ATCC) CRL-1658) mouse fibroblasts, which endogenously express Hedgehog acyltransferase (Hhat) and Porcupine, or in human HEK cells, which also endogenously expressed HHat and Porcupine.Constructs

[0188] Example construct components:4223116.V10399.2076001X, N or SA, cleavage site[S], site to which palmitoleate is attached

[0189] For single-molecule tracking:4223116.vl0399.2076001-26-4223116.vl0399.2076001-27 -4223116.vl0399.2076001A, cleavage position, where cholesterol is ligated *, stop

[0190] For mouse PK / PD experiments:A, cholesterol insertion site*, stopSingle-Particle Tracking

[0191] To measure the effect of the modifications, “sender” cells expressing the nanobody fusion allele were co-plated with “receiver” cells (wild-type NIH3T3 cells) at a ratio of approximately 1 sender : 100 receivers, and the culture was allowed to grow to confluence. The senders were induced to express the nanobodies for approximately 18 hours, the HALO tag was labelled with 50pM JF549i for 5 minutes, then washed 3 times with clean media, and diffusion of each fusion protein was measured using TIRF microscopy (as described in Schlissel (2024)).- 28 -4223116.vl0399.2076001SlowSPT to Identify Matrix Interactions

[0192] To measure the lifetime of interactions between each fusion protein and the extracellular matrix, TIRF microscopy was performed under low illumination power using 5- second exposures to specifically image long-term protein-matrix binding events. The “survival probability” of each particle is an estimate of its dissociation rate constant (koir) for the extracellular matrix.Purification of V5-tagged fusion proteins

[0193] Confluent cultures of NIH3T3 sender cells were induced with 50nM 4-OHT to secrete each fusion protein, then the fusion proteins were labelled for 5 minutes with 50pM JANELIA FLUOR® cell-impermeable dye (JF549i). The fusion proteins were purified from conditioned using anti-V5 conjugated magnetic beads, eluted with V5 peptide, and then used either for single-molecule microscopy or for poly acrylamide gel electrophoresis.Purification of His-Tagged Fusion Proteins

[0194] Human embryonic kidney SV-40 T-antigen-containing HEK239T sender cells were transfected to constitutively express interleukin 10 (IL 10) fusion proteins and grown to confluence. IL10 fusion proteins were purified with Agarose-nitrilotriacetic acid (NTA) resins and eluted with 250mM imidazole.Example 2. Re-purposing Hedgehog biology to Covalently Modify Proteins with Useful Hydrophobic Groups

[0195] Aspects of Hedgehog biology were re-purposed to covalently modify proteins with useful hydrophobic groups, including palmitate or cholesterol. Briefly, a minimal (8-amino acid) N-terminal tag (Hhat seq.), modified by an endogenously expressed Hedgehog Acyltransferase (Hhat), was used to generate palmitate-fused nanobodies (FIG. IB). Palmitate-fused versions of a nanobody and cytokines interleukin 4 (IL4) and interleukin 13 (IL 13) were generated and tested.

[0196] In parallel, the Hedgehog C-terminal HINT domain was fused to a nanobody resulting in the formation of a nanobody that is C-terminally tagged with a cholesterol group (FIG. 1C). Cholesterol-tagged IL4 and IL13 were also generated.

[0197] Effects of tagging proteins with palmitate or cholesterol were studied by generating (i) histograms of instantaneous diffusion rates (FIGs. 2A & 2B) and (ii) survival curves for each fusion protein in the slowest (extracellular matrix-associated) subpopulation (FIGs. 3 A & 3B).- 29 -4223116.V10399.2076001

[0198] Sender cells were plated with receiver cells of the indicated genotypes and induced to secrete a nanobody -HALO fusion protein for 24 hours. The HALO tag was labelled with JF549i, and single molecules were visualized and tracked using total internal reflection fluorescence (TIRF) microscopy. Each molecule’s trajectory was split into 5-step sub-trajectories, the diffusion rate of each sub -trajectory was calculated, and the rate histogram was plotted (FIGs. 2A & 2B). A subpopulation in this diffusion rate histogram is a biochemically distinct subpopulation of nanobody molecules. From left to right, the major subpopulations are thought to be: (i) extracellular matrix-associated, (ii) receptor-associated, (iii) membrane-embedded, and (iv) freely diffusive.

[0199] Addition of cholesterol clearly increased the receptor-associated and membrane- embedded subpopulations of nanobody molecules, regardless of whether the nanobody’s target Patched (Pte) is expressed (FIGs. 2A & 2B, bottom panels). The results suggest that tagging proteins with cholesterol caused them to interact with cell membranes more so than their untagged counterparts.

[0200] Addition of palmitate also increased the matrix-associated subpopulation of nanobody molecules, regardless of whether Pte is expressed (FIGs. 2A & 2B, middle panels). The results suggest that tagging proteins with palmitate caused them to interact with the extracellular matrix more than their un-tagged counterparts.

[0201] Very slow microscopy (5-second exposures under minimal excitation power) was used to measure the binding lifetime (residence time) of the slowest molecules. Addition of palmitate caused nanobodies to bind much more stably to the extracellular matrix, and addition of cholesterol had no discernable effect on the same property (FIGs. 3 A & 3B). Without being bound by theory, the data suggest that the mechanism of palmitate binding to the extracellular matrix reflects the mechanism of palmitate binding albumin.

[0202] Both modifications increased a nanobody’s ability to form a signaling gradient in cell culture, likely by restricting their diffusion and promoting their interaction with receptors. As shown in FIG. 4, addition of palmitate or cholesterol increased the ability of nanobody signaling agonists to form a signaling gradient. Without being bound by theory, the observations are thought to reflect confinement of the nanobodies to the vicinity of the sender cells upon modification with cholesterol. Accumulation of the nanobodies to a sufficient level in turn activates their receptors.

[0203] Both modifications also enhanced the potency of the nanobody ligands. Briefly, conditioned media containing each modified nanobody was used to stimulate Hedgehog-- 30 -4223116.V10399.2076001 responsive receiver cells for 48 hours. Nanobodies simulated Hedgehog signaling by trapping the "active" state of the Hedgehog receptor, and lipophilic modifications to the nanobodies enhanced the potency of the nanobody ligands (FIG. 5). To perform the experiment, sender cells were induced to express either an unmodified nanobody, a palmitate-modified nanobody, or a cholesterol-modified nanobody. Conditioned media from each sender cell culture was collected and the HALO-tagged ligands were labelled with an excess of HALO-ligand JF549i. The relative concentration of each ligand in each sample of conditioned growth medium was calculated by performing poly-acrylamide gel electrophoresis, using a fluorescence scanner to quantify the fluorescently tagged nanobody ligand, and then the conditioned media was applied to continently cultured Hedgehog-sensitive reporter cells. After 48h, receiver cells were trypsinized, resuspended and measured using flow cytometry to count the fraction of cells that had induced a fluorescent reporter in response to the conditioned medium.Example 3. Manufacturing Routes

[0204] In one set of experiments, proteins will be expressed in mammalian cell lines that naturally express Hhat (for N-terminal palmitoylation (z.e., palmitate modification)) or Porcn (for internal palmitoleoylation (z.e., palmitoleate modification)).

[0205] In one set of experiments, proteins will be expressed in insect cells that naturally express insect Hhat homolog (for N-terminal palmitoylation) or insect Porcn homolog (for internal palmitoleoylation).

[0206] In one set of experiments, proteins will be expressed and purified, and recombinant Hhat will be added (for N-terminal palmitoylation) or recombinant Porcn will be added (for internal palmitoleoylation).REFERENCES1. Hardy & Resh, Identification of N-terminal residues of Sonic Hedgehog important for palmitoylation by Hedgehog acyltransferase, J Biol Chem. 287(51):42881-89 (2012).2. Porter el al., Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain, Cell 86(l):21-34 (1996).3. Miranda el al., Identification of the WNT1 residues required for palmitoylation by Porcupine, FEBS Lett. 588(24):4815-24 (2014).4. Schlissel el al., Diffusion barriers imposed by tissue topology shape Hedgehog morphogen gradients, Proc Natl Acad Sci U S A. 121(36):e2400677121 (2024).- 31 -4223116.V10399.2076001ADDITIONAL REFERENCEGrimm JB, Brown TA, English BP, Lionnet T, Lavis LD. Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments. Methods Mol Biol. 2017;1663: 179-188. doi: 10.1007 / 978-l-4939-7265-4_15. PMID: 28924668.

[0207] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0208] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.- 32 -4223116.V1

Claims

0399.2076001CLAIMSWhat is claimed is:

1. A polynucleotide encoding a fusion protein, wherein the polynucleotide comprises: a) a protein coding sequence encoding a non-Hedgehog therapeutic protein, and b) at least one of the following: i) a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase, wherein the polypeptide is N-terminal to the non-Hedgehog therapeutic protein, ii) a sequence encoding a Hedgehog auto-processing domain having sterol modification activity, wherein the domain is C-terminal to the nonHedgehog therapeutic protein, and iii) a sequence encoding a polypeptide sufficient for palmitoylation by a membrane-bound O-acyltransferase enzyme.

2. The polynucleotide of claim 1, comprising a sequence encoding a polypeptide sufficient for palmitoylation by a Hedgehog acyltransferase.

3. The polynucleotide of claim 1 or 2, wherein the Hedgehog acyltransferase is a mammalian Hedgehog acyltransferase.

4. The polynucleotide of claim 3, wherein the Hedgehog acyltransferase is a mouse Hedgehog acyltransferase.

5. The polynucleotide of claim 1 or 2, wherein the Hedgehog acyltransferase is an insect Hedgehog acyltransferase.

6. The polynucleotide of any one of claims 2-5, wherein the polypeptide comprises an N- terminal sequence of a vertebrate Hedgehog protein preproprotein or proprotein.

7. The polynucleotide of any one of claims 2-6, wherein the polypeptide comprises an N- terminal sequence of a human sonic hedgehog protein isoform 1 preproprotein or proprotein.

8. The polynucleotide of any one of claims 2-7, wherein the polypeptide comprises SEQ ID NO: 10.- 33 -4223116.V10399.20760019. The polynucleotide of any one of claims 2-8, wherein the polypeptide comprises SEQ ID NO:11.

10. The polynucleotide of any one of claims 2-9, wherein the polypeptide comprises SEQ ID NO: 12.

11. The polynucleotide of claim 1, comprising a sequence encoding a Hedgehog autoprocessing domain having sterol modification activity.

12. The polynucleotide of claim 11, wherein the sterol modification is a cholesterol modification.

13. The polynucleotide of claim 11 or 12, wherein the Hedgehog auto-processing domain is a human SHH-derived Hint domain.

14. The polynucleotide of claim 11 or 12, wherein the Hedgehog auto-processing domain comprises any one of SEQ ID NOs: 13-16.

15. The polynucleotide of claim 1, comprising a sequence encoding a polypeptide sufficient for palmitoylation by a membrane-bound O-acyltransferase enzyme.

16. The polynucleotide of claim 15, wherein the membrane-bound O-acyltransferase enzyme is a protein-serine O-palmitoleoyltransferase porcupine.

17. The polynucleotide of claim 16, wherein the membrane-bound O-acyltransferase enzyme is a human protein-serine O-palmitoleoyltransferase porcupine.

18. The polynucleotide of claim 16, wherein the membrane-bound O-acyltransferase enzyme is a mouse protein-serine O-palmitoleoyltransferase porcupine.

19. The polynucleotide of any one of claims 15-18, wherein the polypeptide sufficient for palmitoylation by the membrane-bound O-acyltransferase enzyme comprises SEQ ID NO:19.

20. The polynucleotide of any one of claims 1-19, wherein the non-Hedgehog therapeutic protein is a therapeutic antibody.

21. The polynucleotide of claim 20, wherein the therapeutic antibody is a nanobody.- 34 -4223116.V10399.207600122. The polynucleotide of any one of claims 1-21, further comprising a tag coding sequence encoding a protein tag.

23. The polynucleotide of claim 22, wherein the protein tag comprises an epitope tag, a selflabeling protein tag, or both.

24. The polynucleotide of claim 23, wherein the epitope tag comprises a V5 tag, a His tag, or both.

25. A vector comprising the polynucleotide of any one of claims 1-24.

26. A cell comprising the polynucleotide of any one of claims 1-24 or the vector of claim 25.

27. The cell of claim 26, wherein the polynucleotide or vector is integrated into the genomic DNA.

28. The cell of claim 26, wherein the polynucleotide or vector is extrachromosomal.

29. The cell of any one of claims 26-28, wherein the cell is in vitro or ex vivo.

30. The cell of any one of claims 26-28, wherein the cell is in vivo.

31. The cell of any one of claims 26-28, wherein the cell is an animal cell.

32. The cell of any one of claims 26-31, wherein the cell is a mammalian cell.

33. The cell of any one of claims 26-32, wherein the cell is a human cell.

34. The cell of any one of claims 26-33, wherein the cell is a chimeric antigen receptor(CAR) cell.

35. The cell of claim 34, wherein the CAR cell is a CAR-T cell.

36. The cell of any one of claims 26-30, wherein the cell is a plant cell.

37. The cell of any one of claims 26-36, wherein the cell is a single cell.

38. A kit comprising the polynucleotide of any one of claims 1-24, the vector of claim 25, or the cell of any one of claims 26-37.- 35 -4223116.V10399.207600139. A method of modifying a non-Hedgehog therapeutic protein, comprising expressing the polynucleotide of any one of claims 1-10 and 15-24 in a cell expressing the Hedgehog acyltransferase.

40. A method of modifying a non-Hedgehog therapeutic protein, comprising expressing the polynucleotide of any one of claims 1, 11-14, and 20-24 in a cell.

41. A method of modifying a non-Hedgehog therapeutic protein, comprising expressing the polynucleotide of any one of claims 1 and 15-24 in a cell expressing the membranebound O-acyltransferase enzyme.

42. The method of any one of claims 39-41, wherein the cell is in vitro or ex vivo.

43. The method of any one of claims 39-41, wherein the cell is in vivo.

44. The method of any one of claims 39-43, wherein the cell is an animal cell.

45. The method of any one of claims 39-44, wherein the cell is a mammalian cell.

46. The method of any one of claims 39-45, wherein the cell is a human cell.

47. The method of any one of claims 39-46, wherein the cell is a CAR cell.

48. The method of claim 47, wherein the CAR cell is a CAR-T cell.

49. The method of any one of claims 39-43, wherein the cell is a plant cell.

50. The method of any one of claims 39-49, wherein the cell is a single cell.

51. The method of any one of claims 39-50, further comprising isolating the modified nonHedgehog therapeutic protein from the cell.

52. A method of modifying a non-Hedgehog therapeutic protein, comprising: a) expressing the polynucleotide of any one of claims 1-10 and 19-24 in a host cell, b) purifying the non-Hedgehog therapeutic protein, and c) contacting the purified non-Hedgehog therapeutic protein with the Hedgehog acyltransferase and palmityl-CoA.

53. A method of modifying a non-Hedgehog therapeutic protein, comprising:- 36 -4223116.V10399.2076001 a) expressing the polynucleotide of any one of claims 1 and 15-24 in a host cell, b) purifying the non-Hedgehog therapeutic protein, and c) contacting the purified non-Hedgehog therapeutic protein with the membranebound O-acyltransferase enzyme and palmityl-CoA.

54. The method of claim 52 or 53, wherein the host cell is a bacterium.

55. The method of claim 52 or 53, wherein the host cell is an insect cell.

56. The method of any one of claims 39-55, further comprising characterizing the non¬Hedgehog therapeutic protein.

57. The method of any one of claims 39-56, further comprising measuring serum half-life of the non-Hedgehog therapeutic protein.

58. The method of any one of claims 39-57, further comprising administering the nonHedgehog therapeutic protein to a subject in need thereof.

59. The method of claim 58, wherein the subject is a human patient.

60. A protein produced by the method of any one of claims 39-55.- 37 -4223116.V1

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