WO2022240407A1

WO2022240407A1 - Delivery constructs derived from bacterial toxins and uses thereof

Info

Publication number: WO2022240407A1
Application number: PCT/US2021/032097
Authority: WO
Inventors: Randall J. Mrsny; Thomas Carl HUNTER; Geoffrey MASUYER; Keyi LIU
Original assignee: Applied Molecular Transport Inc.
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2022-11-17

Abstract

The present disclosure provides delivery constructs comprising a carrier derived from an Aeromonas exotoxin or a Chromobacterium exotoxin coupled to a heterologous payload, wherein the carrier can be capable of transporting the payload across intact polarized epithelial cells. The delivery construct can be part of a pharmaceutical composition that can be orally or nasally administered to a subject to provide for improved, effective therapies for treatment of various diseases.

Description

DELIVERY CONSTRUCTS DERIVED FROM BACTERIAL TOXINS AND USES

THEREOF

SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 12, 2021, is named 40566-731_601_SL.txt and is 245,129 bytes in size.

BACKGROUND

[0002] Epithelial layers form a barrier that can prevent effective delivery of therapeutic compounds administered through the mouth, nose, or rectum because large molecules such as proteins cannot diffuse across cell membranes or through tight junctions. Such molecules can be transported across an epithelial layer by transcytosis, a process involving endocytosis across the apical plasma membrane, vesicular transport from the apical domain to the basal domain, and exocytosis across the basal plasma membrane. However, most proteins that enter an epithelial cell by endocytosis are either recycled directly back to apical surface or directed to lysosomes, where they are degraded. This inability to be readily transported across an epithelium continues to be a limiting factor in developing commercially viable oral, nasal and rectal formulations, particularly for polypeptide-based therapeutics. A common solution is to use parenteral administration such as intravenous or subcutaneous administration, but these administration routes often result in considerable side effects, lower the therapeutic efficacy, and reduce patient convenience that can negatively affect compliance. There is a need for improved compositions and methods for transporting therapeutics into or across an epithelium.

INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

SUMMARY

[0004] The present disclosure provides carriers useful for delivering a payload across an epithelial layer. The carriers comprise a transcytosing element derived from a mono-ADP-ribosyl transferase (mART). The present disclosure further provides methods of delivering a payload across an epithelial layer.

- 1 - [0005] One aspect is a carrier-payload complex comprising: a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a transcytosing element of an

Aeromonas, Chromobacterium , Collimonas , Shewanella, Janthinobacterium , Serratia, or

Acinetobacter mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload. The carrier payload complex may comprise a carrier consisting of the transcytosing element.The transcytosing element can have at least 75%, at least 80%, at least 85%, at least 90%, at least

95%, at least 98%, at least 99% or 100% sequence identity to a transcytosing element of any one of SEQ ID NOs: 1-5, 8-15, and 18-25. The transcytosing element of an Aeromonas,

Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART can comprise or consist of a Domain I of the Aeromonas, Chromobacterium , Collimonas ,

Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. The transcytosing element of an Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or

Acinetobacter mART can comprise at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of the Aeromonas , Chromobacterium , Collimonas , Shewanella ,

Janthinobacterium , Serratia , or Acinetobacter mART.

[0006] Another aspect is a carrier-payload complex comprising: carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of SEQ ID NO: 16 or SEQ ID NO: 17. The transcytosing element of the mART can comprise or consist of a Domain I of a mART. The transcytosing element of the mART can comprise at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of a mART.

[0007] Another aspect is a carrier-payload complex comprising: a carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelium does not inhibit Vibrio cholerae cholix toxin transcytosis across the epithelium. Another aspect is a carrier-payload complex comprising: a carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause COPI to redistribute to a perinuclear region of the epithelial cell. Another aspect is a carrier-payload complex comprising: a carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause LMAN1 to redistribute to a basal domain of the epithelial cell. In some embodiments, the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of a Pseudomonas aeruginosa exotoxin. 2 [0008] In some embodiments, the carrier of any previous embodiment further comprises a

Domain III of the mART. In some embodiments, the Domain III of the mART has mono-ADP- ribosyl transferase activity. In some embodiments, the Domain III of the mART does not have mono-ADP-ribosyl transferase activity. In some embodiments, the Domain III of the mART comprises an inactivating mutation of an active site amino acid corresponding to Glutamate571 of SEQ ID NO: 1.

[0009] In some embodiments, the carrier of any previous embodiment further comprises a core 13 -stranded b-jellyroll fold. In some embodiments, the 13 -stranded b-jellyroll fold comprises a disulfide bond between cysteine residues at positions corresponding to Cysteinel 1 and Cysteinel 5 of SEQ ID NO: 18. In some embodiments, the 13 -stranded b-jellyroll fold comprises a disulfide bond between cysteine residues at positions corresponding to Cysteinel97 and Cysteine214 of SEQ ID NO: 18.

[0010] In some embodiments, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 41-48 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises GILaaSMh; wherein 1 indicates aliphatic residues I, L, and V; a indicates aromatic residues F, H, W, and Y; and h indicates hydrophobic residues A, C, F, G, H, I, K, L,

M, R, T, V, W, and Y. In some embodiments, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 98-105 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises slpWhVPl, wherein the position corresponding to 106 of SEQ ID NO: 18 is a G; wherein s indicates small residues A, C, D, G, N, P, S, T, and V; 1 indicates aliphatic residues I, L, and V; p indicates polar residues C, D, E, H, K, N, Q, R, S, and T; and h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y. In some embodiments, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 111- 117 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises spIKlfh, wherein position corresponding to 110 of SEQ ID NO: 18 is a P; wherein s indicates small residues A, C, D, G, N, P, S, T, and V; p indicates polar residues C, D, E, H, K, N, Q, R, S, and T; 1 indicates aliphatic residues I, L, and V; f indicates F and S; and h indicates hydrophobic residues A, C, F,

G, H, I, K, L, M, R, T, V, W, and Y. In some embodiments, the 13-stranded b-jellyroll fold comprises a sPlYol sequence at positions corresponding to 130-135 of SEQ ID NO: 18; wherein s indicates small residues A, C, D, G, N, P, S, T, and V; 1 indicates aliphatic residues I, L, and V; and o indicates alcohol residues S and T. In some embodiments, the 13-stranded b-jellyroll fold comprises a Rp+RWscW sequence at positions corresponding to 183-190 of SEQ ID NO: 18; wherein p indicates polar residues C, D, E, H, K, N, Q, R, S, and T; + indicates positively charged residues H, K, and R; s indicates small residues A, C, D, G, N, P, S, T, and V; and c indicates charged residues D, E, H, K, and R. In some embodiments, the 13-stranded b-jellyroll 3 fold comprises a hYNYlsQppCp sequence at positions corresponding to 205-215 of SEQ ID NO:

18; wherein h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y; 1 indicates aliphatic residues I, L, and V; and s indicates small residues A, C, D, G, N, P, S, T, and

V; and p indicates polar residues C, D, E, H, K, N, Q, R, S, and T.

[0011] In some embodiments, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 41-48 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises GILaaSMh; wherein 1 indicates aliphatic residues I, L, and V, preferably

V; a indicates aromatic residues F, H, W, and Y, preferably H and Y; and h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y, preferably L, F, T, and V. In some embodiments, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 98-105 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises slpWhVPl, wherein the position corresponding to 106 of SEQ ID NO: 18 is a G; wherein s indicates small residues A, C, D, G, N, P, S, T, and V, preferably S and T; 1 indicates aliphatic residues I, L, and V; p indicates polar residues C, D, E, H, K, N, Q, R, S, and T, preferably N and

H; and h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y, preferably

A, L, and V. In some embodiments, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 111-117 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises spIKlfh, wherein position corresponding to 110 of SEQ ID NO: 18 is a P; wherein s indicates small residues A, C, D, G, N, P, S, T, and V, preferably A, S, and T; p indicates polar residues C, D, E, H, K, N, Q, R, S, and T, preferably E, N, and S; 1 indicates aliphatic residues I, L, and V; f indicates F and S, preferably F; and h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y, preferably F, I and V. In some embodiments, the 13-stranded b-jellyroll fold comprises a sPlYol sequence at positions corresponding to 130-135 of SEQ ID NO: 18; wherein s indicates small residues A, C, D, G, N,

P, S, T, and V, preferably S and P; 1 indicates aliphatic residues I, L, and V, preferably I and L; and o indicates alcohol residues S and T. In some embodiments, the 13-stranded b-jellyroll fold comprises a Rp+RWscW sequence at positions corresponding to 183-190 of SEQ ID NO: 18; wherein p indicates polar residues C, D, E, H, K, N, Q, R, S, and T, preferably E, H, K and Q; + indicates positively charged residues H, K, and R, preferably K and R; s indicates small residues

A, C, D, G, N, P, S, T, and V, preferably A, S, and T; and c indicates charged residues D, E, H,

K, and R, preferably E and H. In some embodiments, the 13-stranded b-jellyroll fold comprises a hYNYlsQppCp sequence at positions corresponding to 205-215 of SEQ ID NO: 18; wherein h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y, preferably F, I and

V; 1 indicates aliphatic residues I, L, and V; and s indicates small residues A, C, D, G, N, P, S, T, and V, preferably A, S, and T; and p indicates polar residues C, D, E, H, K, N, Q, R, S, and T, 4 preferably H, N, Q, R, S, and T.

[0012] In a preferred embodiment, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 41-48 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises GILaaSMh; wherein 1 indicates aliphatic residue V; a indicates aromatic residues H and Y; and h indicates hydrophobic residues L, F, T, and V. In a preferred embodiment, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 98-105 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises slpWhVPl, wherein the position corresponding to 106 of SEQ ID NO: 18 is a G; wherein s indicates small residues S and T; 1 indicates aliphatic residues I, L, and V; p indicates polar residues N and H; and h indicates hydrophobic residues A, L, and V. In a preferred embodiment, the 13-stranded b-jellyroll fold comprises a beta strand comprising positions corresponding to 111-117 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises spIKlfh, wherein position corresponding to 110 of SEQ ID NO: 18 is a P; wherein s indicates small residues A, S, and T; p indicates polar residues E, N, and S; 1 indicates aliphatic residues I, L, and V; f indicates F; and h indicates hydrophobic residues F, I and V. In a preferred embodiment, the 13-stranded b-jellyroll fold comprises a sPlYol sequence at positions corresponding to 130-135 of SEQ ID NO: 18; wherein s indicates small residues S and P; 1 indicates aliphatic residues I and L; and o indicates alcohol residues S and T. In a preferred embodiment, the 13-stranded b-jellyroll fold comprises a Rp+RWscW sequence at positions corresponding to 183-190 of SEQ ID NO: 18; wherein p indicates polar residues E, H, K and Q; + indicates positively charged residues K and R; s indicates small residues A, S, and T; and c indicates charged residues E and H. In a preferred embodiment, the 13-stranded b-jellyroll fold comprises a hYNYlsQppCp sequence at positions corresponding to 205-215 of SEQ ID NO: 18; wherein h indicates hydrophobic residues F, I and V; 1 indicates aliphatic residues I, L, and V; and s indicates small residues A, S, and T; and p indicates polar residues H, N, Q, R, S, and T.

[0013] In some embodiments, the carrier of any previous embodiment is capable of transporting the heterologous payload into a polarized epithelial cell. In some embodiments, the carrier is capable of transporting the heterologous payload across the polarized epithelial cell. In some embodiments, the carrier is capable of transporting the heterologous payload from an apical surface of a polarized epithelial cell to a basal or lateral surface of the polarized epithelial cell. In some embodiments, the polarized epithelial cell is a gastrointestinal tract epithelial cell. In some embodiments, the polarized epithelial cell is a respiratory tract epithelial cell.

[0014] In some embodiments, the carrier of any previous embodiment comprises at least 70%, at least 80%, or at least 90% of a Domain I of the mART. In some embodiments, the mART is an Aeromonas mART. In some embodiments, the Aeromonas mART is an Aeromonas hydrophila 5 mART, an Aeromonas dhakensis mART. an Aeromonas salmonicida m ART, an Aeromonas piscicola mART, an Aeromonas bestiarum mART, an Aeromonas hydrophila mART, or an

Aeromonas hydrophila mART. In some embodiments, the Aeromonas mART is an Aeromonas hydrophila mART. In some embodiments, the Aeromonas hydrophila mART comprises SEQ ID

NO: 18. In some embodiments, the transcytosing element of the mART comprises at least 100,

150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 18. In some embodiments, the mART is a Chromobacterium mART. In some embodiments, the Chromobacterium mART comprises SEQ ID NO: 19. In some embodiments, the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 19. In some embodiments, the mART is a Collimonas mART. In some embodiments, the Collimonas mART comprises SEQ ID NO: 20. In some embodiments, the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID

NO: 20. In some embodiments, the mART is a Shewanella mART. In some embodiments, the

Shewanella mART comprises SEQ ID NO: 21. In some embodiments, the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ

ID NO: 21. In some embodiments, the mART is a Janthinobacterium mART. In some embodiments, the Janthinobacterium mART comprises SEQ ID NO: 22. In some embodiments, the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 22. In some embodiments, the mART is a Serratia mART. In some embodiments, the Serratia fonticola mART comprises SEQ ID NO: 23. In some embodiments, the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 23. In some embodiments, the mART is an

Acinetobacter baumannii mART. In some embodiments, the Acinetobacter baumannii mART comprises SEQ ID NO: 24. In some embodiments, the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 24. In some embodiments, the mART comprises SEQ ID NO: 25. In some embodiments, the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 25.

[0015] In some embodiments, the heterologous payload of any previous embodiment is selected from the group consisting of a macromolecule, small molecule, peptide, polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, antisense molecule, antibody, DNA, plasmid, vaccine, polymer nanoparticle, and a catalytically-active material. In some embodiments, the heterologous payload is a therapeutic payload. In some embodiments, the therapeutic payload is a hormone. In some embodiments, the therapeutic payload is a human growth hormone. In some embodiments, the hormone comprises SEQ ID NO: 49 or a sequence with at least 75%, at least 6 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 49. In some embodiments, the therapeutic payload is a modulator of inflammation in a gastrointestinal tract. In some embodiments, the heterologous payload is covalently coupled to the carrier. In some embodiments, the carrier is synthetically conjugated to the heterologous payload. In some embodiments, the carrier is genetically fused to the heterologous payload. In some embodiments, the heterologous payload is coupled to a C- terminus of the carrier. In some embodiments, the heterologous payload is coupled to an N- terminus of the carrier. In some embodiments, the carrier is indirectly coupled to the heterologous payload via a spacer. In some embodiments, the spacer is a non-cleavable spacer. In some embodiments, the spacer comprises up to 15 repeats of GS (SEQ ID NO: 54), GGS (SEQ

ID NO: 55), GGGS (SEQ ID NO: 56), GGGGS (SEQ ID NO: 57), GGGGGS (SEQ ID NO: 58), or a combination thereof. In some embodiments, the spacer comprises or consists of an amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the heterologous payload is non-covalently coupled to the carrier. In some embodiments, the carrier and heterologous payload are complexed via a nanoparticle. In some embodiments, the carrier is covalently-linked to the nanoparticle or is spray-dried on the nanoparticle.

[0016] In some embodiments, the composition is encapsulated. In some embodiments, the encapsulated composition comprises an enteric coating.

[0017] Another aspect of the disclosure is a polynucleotide encoding the carrier-payload complex of any previous embodiment. Another aspect of the disclosure is a vector comprising the polynucleotide of claim 78.

[0018] Another aspect of the disclosure is a method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier- payload complex comprising: a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella, Janthinobacterium , Serratia, or Acinetobacter mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload. In some embodiments, the carrer consists of the transcytosing element. The can comprise or consist of a Domain I of the Aeromonas, Chromobacterium , Collimonas ,

Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. The transcytosing element can comprise at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of the Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART.

[0019] Another aspect of the disclosure is a method of transporting a heterologous payload 7 into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier- payload complex comprising: a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella, Janthinobacterium ,

Serratia, or Acinetobacter mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload. In some embodiments, the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of SEQ ID NO: 16 or

SEQ ID NO: 17. In some emboidments, the transcytosing element of the mART comprises or consists of a Domain I of the mART. In some emboidments, the transcytosing element of a mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of a mART.

[0020] Another aspect of the disclosure is a method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier- payload complex comprising: a carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelium does not inhibit Vibrio cholerae cholix toxin transcytosis across the epithelium. Another aspect of the disclosure is a method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier-payload complex comprising: a carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause COPI to redistribute to a perinuclear region of the epithelial cell. Another aspect of the disclosure is a method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier-payload complex comprising: a carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause LMAN1 to redistribute to a basal domain of the epithelial.

[0021] In some embodiments of the any of the previous methods, the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of a Pseudomonas aeruginosa exotoxin. In some embodiments, the carrier further comprises Domain III of the mART. In some embodiments, the Domain III of the mART has mono-ADP-ribosyl transferase activity. In some embodiments, the Domain III of the mART does not have mono-ADP-ribosyl transferase activity. In some embodiments, the heterologous payload is released from the basal surface of the polarized epithelial cell. In some embodiments, the carrier comprises a core 13-stranded b-jellyroll fold. In some embodiments, the carrier comprises at least 70%, at least 80%, or at least 90% of a Domain I of the mART. 8 [0022] In some embodiments of the any of the previous methods, the mART is an Aeromonas mART. In some embodiments, the Aeromonas mART is an Aeromonas hydrophila mART, an

Aeromonas dhakensis mART, an Aeromonas salmonicida mART, an Aeromonas piscicola mART, or an Aeromonas bestiarum mART. In some embodiments, the Aeromonas mART is an

Aeromonas hydrophila mART. In some embodiments, the Aeromonas hydrophila mART comprises SEQ ID NO: 18 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of

SEQ ID NO: 18. In some embodiments, the mART is a Chromobacterium mART. In some embodiments, the Chromobacterium mART comprises SEQ ID NO: 19 or at least 100, 150, 180,

200, 220, or 240 contiguous amino acids of SEQ ID NO: 19. In some embodiments, the mART is a Collimonas mART. In some embodiments, the Collimonas mART comprises SEQ ID NO: 20 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 20. In some embodiments, the mART is a Shewanella mART. In some embodiments, the Shewanella mART comprises SEQ ID NO: 21 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of

SEQ ID NO: 21. In some embodiments, the mART is a Janthinobacterium mART. In some embodiments, the Janthinobacterium mART SEQ ID NO: 22 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 22. In some embodiments, the mART is a

Serratia mART. In some embodiments, the Serratia fonticola mART comprises SEQ ID NO: 23 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 23. In some embodiments, the mART is an Acinetobacter baumannii mART. In some embodiments, the

Acinetobacter baumannii mART comprises SEQ ID NO: 24 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 24. In some embodiments, the mART comprises

SEQ ID NO: 25 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID

NO: 25.

[0023] Another aspect is a method of orally delivering a heterologous payload to a subject, comprising: orally administering a carrier-payload complex to the subject and transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a transcytosing element of an Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload. In some embodiments, the carrier consists of the transcytosing element. In some embodiments, the transcytosing element of an Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises or consists of a Domain I of the Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or 9 Acinetobacter mART. In some embodiments, the transcytosing element of an Aeromonas,

Chromobacterium , Collimonas , Shewanella, Janthinobacterium , Serratia, or Acinetobacter mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of the Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or

Acinetobacter mART.

[0024] Another aspect is a method of orally delivering a heterologous payload to a subject, comprising: orally administering a carrier-payload complex to the subject; transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising: a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the transcytosing element of a mART comprises or consists of a Domain I of a mART. In some embodiments, the transcytosing element of a mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of a mART.

[0025] Another aspect is a method of orally delivering a heterologous payload to a subject, comprising: orally administering a carrier-payload complex to the subject; and transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising: a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelium does not inhibit Vibrio cholerae cholix toxin transcytosis across the epithelium.

[0026] Another aspect is a method of orally delivering a heterologous payload to a subject, comprising: orally administering a carrier-payload complex to the subject; and transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising: a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause COPI to redistribute to a perinuclear region of the epithelial cell. [0027] Another aspect is a method of orally delivering a heterologous payload to a subject, comprising: orally administering a carrier-payload complex to the subject; and transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising: a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled 10 to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause LMAN1 to redistribute to a basal domain of the epithelial cell.

[0028] In some embodiments of the previous methods, the transcytosing element has less than

80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of a Pseudomonas aeruginosa exotoxin. In some embodiments, the carrier further comprises

[0029] In some embodiments of the previous methods, the carrier comprises a core 13- stranded b-jellyroll fold. In some embodiments, the mART is an Aeromonas mART. In some embodiments, the Aeromonas mART is an Aeromonas hydrophila mART, an Aeromonas dhakensis mART, an Aeromonas salmonicida mART, an Aeromonas piscicola mART, or an Aeromonas bestiarum mART. In some embodiments, the Aeromonas mART is an Aeromonas hydrophila mART. In some embodiments, the Aeromonas hydrophila mART comprises SEQ ID NO: 18 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 18. In some embodiments, the mART is a Chromobacterium mART. In some embodiments, the Chromobacterium mART comprises of SEQ ID NO: 19 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 19. In some embodiments, the mART is a Collimonas mART. In some embodiments, the Collimonas mART comprises SEQ ID NO: 20 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 20. In some embodiments, the mART is a Shewanella mART.

[0030] The method of claim 138, wherein the Shewanella mART comprises SEQ ID NO: 21 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 21. In some embodiments, the mART is a Janthinobacterium mART. In some embodiments, the Janthinobacterium mART comprises SEQ ID NO: 22 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 22. In some embodiments, the mART is a Serratia mART. In some embodiments, the Serratia fonticola mART comprises SEQ ID NO: 23 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 23. In some embodiments, the mART is an Acinetobacter baumannii mART. In some embodiments, the Acinetobacter baumannii mART comprises SEQ ID NO: 24 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 24. In some embodiments, the mART comprises a sequence comprising SEQ ID NO: 25 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 25. 11 [0031] Another aspect is a method of treating a condition in a subject, comprising orally administering a carrier-payload complex to the subject; wherein the carrier-payload complex comprises: a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella,

Janthinobacterium , Serratia, or Acinetobacter mono-ADP-ribosyl transferase (mART), coupled to a heterologous payload; and wherein the heterologous payload is transported across a gut epithelium. In some embodiments, the transcytosing element of an Aeromonas,

Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises or consists of a Domain I of the Aeromonas, Chromobacterium, Collimonas,

Shewanella, Janthinobacterium, Serratia, or Acinetobacter mART. In some embodiments, the transcytosing element of an Aeromonas, Chromobacterium, Collimonas, Shewanella,

Janthinobacterium, Serratia, or Acinetobacter mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of the Aeromonas, Chromobacterium, Collimonas,

Shewanella, Janthinobacterium, Serratia, or Acinetobacter mART.

[0032] Another aspect is a method of treating a condition in a subject, comprising orally administering a carrier-payload complex to the subject; wherein the carrier-payload complex comprises: a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase

(mART), coupled to a heterologous payload, wherein the transcytosing element has less than

80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of

SEQ ID NO: 16 or SEQ ID NO: 17; and wherein the heterologous payload is transported across a gut epithelium. In some embodiments, the transcytosing element of a mART comprises or consists of a Domain I of a mART. In some embodiments, the transcytosing element of a mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of a mART.

[0033] Another aspect is a method of treating a condition in a subject, comprising orally administering a carrier-payload complex to the subject; wherein the carrier-payload complex comprises: carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelium does not inhibit Vibrio cholerae cholix toxin transcytosis across the epithelium; and wherein the heterologous payload is transported across a gut epithelium. Another aspect is a method of treating a condition in a subject, comprising orally administering a carrier-payload complex to the subject; wherein the carrier-payload complex comprises: a carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier- payload complex across an epithelial cell does not cause COPI to redistribute to a perinuclear 12 region of the epithelial cell; and wherein the heterologous payload is transported across a gut epithelium. Another aspect is a method of treating a condition in a subject, comprising orally administering a carrier-payload complex to the subject; wherein the carrier-payload complex comprises: a carrier comprising a transcytosing element of a mART, coupled to a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause LMAN1 to redistribute to a basal domain of the epithelial cell; and wherein the heterologous payload is transported across a gut epithelium.

[0034] In some embodiments of the previous methods, the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of a Pseudomonas aeruginosa exotoxin. In some embodiments, the carrier further comprises Domain III of the mART. In some embodiments, the Domain III of the mART has mono-ADP- ribosyl transferase activity. In some embodiments, the Domain III of the mART does not have mono-ADP-ribosyl transferase activity.

[0035] In some embodiments of the previous methods, the heterologous payload is delivered to a lamina propria of an intestinal villus. In some embodiments, the heterologous payload is delivered into systemic circulation. In some embodiments, the heterologous payload is selected from the group consisting of a macromolecule, small molecule, peptide, polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, antisense molecule, antibody, DNA, plasmid, vaccine, polymer nanoparticle, and a catalytically-active material.

[0036] In some embodiments of the previous methods, the heterologous payload is a therapeutic payload. In some embodiments, the therapeutic payload is a growth hormone. In some embodiments, the growth hormone comprises SEQ ID NO: 49 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 49.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Various features of the present disclosure are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0038] FIG. 1A shows a sequence alignment of mono-ADP-ribosyl transferases (mARTs) from multiple Aeromonas species (SEQ ID NOs: 67 and 2-5, respectively, in order of 13 appearance).

[0039] FIG. IB shows a sequence alignment of Aeromonas hydrophila exotoxin A (SEQ ID NO: 67) with Pseudomonas exotoxin A (PE) (SEQ ID NO: 7) and Cholix (SEQ ID NO: 6). The sequences are full length, and include a leader peptide that is not present in the mature polypeptide. Identical residues have a dark background with white letters. Residues with lighter characters are similar. The (a) triangles mark conserved cysteines that form intrachain disulfide bonds. The (b) triangles mark the furin cleavage site (positions 291-292). The (c) triangles mark the receptor binding site. The (d) triangles mark the eEF2 binding site. The (e) triangles mark nicotinamide adenine dinucleotide (NAD+) binding and catalytic residues. The (f) triangles mark Aeromonas exotoxin A (AE) metal ion coordination sites based on similarity with PE. Secondary structure elements (a: alpha helix; b: beta sheet; h: 3io helix) of AE are presented above the sequence. The figure was prepared with ESPript.

[0040] FIG. 2 A shows a sequence alignment of Domain I from mARTs derived from

Pseudomonas aeruginosa (SEQ ID NO: 17), Aeromonas hydrophila (SEQ ID NO: 18) Vibrio cholerae (cholix) (SEQ ID NO: 16), and Chromobacterium haemolyticum (SEQ ID NO: 19).

Ovals identify conserved cysteines that participate in disulfide bonds. The rectangle identifies a region found in mARTs isolated from Vibrio cholerae and Chromobacterium haemolyticum but not Pseudomonas aeruginosa or Aeromonas hydrophila.

[0041] FIG. 2B shows a structure of a Domain I of a Vibrio cholerae (cholix) exotoxin. A subdomain not found in Pseudomonas aeruginosa or Aeromonas hydrophila exotoxins is colored red.

[0042] FIG. 3 shows a sequence alignment of Domain III from mARTs derived from Pseudomonas aeruginosa (SEQ ID NO: 70), Aeromonas hydrophila (SEQ ID NO: 71) Vibrio cholerae (cholix) (SEQ ID NO: 68), and Chromobacterium haemolyticum (SEQ ID NO: 69). The oval indicates a conserved active site glutamic acid.

[0043] FIG. 4A is a ribbon diagram representing the crystal structure of Aeromonas exotoxin. Overall structure with domain la (15-263), red; domain II (264-383), blue; domain lb (384-413), orange; and domain III (444-626), cyan. Disulphide bridges are shown as yellow sticks, and the furin cleavage site is marked by a blue sphere. Schematic drawing of AE; the disulphide bridge between cysteines 277 and 299 of SEQ ID NO: 1 linking the two chains after furin cleavage (N) is shown in gold.

[0044] FIG. 4B shows a superposition of AE onto the structures of PE (left, grey, Protein Data Bank (PDB) 1IKQ) and cholix (right, light blue, PDB 2Q5T).

[0045] FIG. 4C shows the electrostatic surface potential of AE and PE calculated using the APBS tool in PyMOL (darker shades indicate higher surface potential: scale from .5 to + 5). AE 14 in the same orientation as (FIG. 4A); arrows mark the location of residue K57 involved in PE binding to the LRP1 receptor, and its equivalent in AE (K69).

[0046] FIG. 4D shows a superposition of Domain I only from AE (black), PE (dark grey) and cholix (light grey), with K57 in stick representation. Important secondary structure elements of AE are labelled, with star indicating the PE loop (position 60) involved in receptor-binding. [0047] FIG. 5 shows alignments of Domain I sequences of mature mARTs from Vibrio cholera (Cholix, SEQ ID NO: 16), Chromobacterium haemolyticum (SEQ ID NO: 19), Janthinobacterium lividum (SEQ ID NO: 22), Collimonas fungivorans (SEQ ID NO: 20), Serratia fonticola (SEQ ID NO: 23), Pseudomonas aeruginosa (SEQ ID NO: 17), Acinetobacter baumannii (SEQ ID NO: 24), Aeromonas hydrophila (SEQ ID NO: 18), and Shewanella putrefacians (SEQ ID NO: 21). FIG. 5A shows positions with full (*), high (:), and intermediate (.) amino acid sequence conservation. FIG. 5B shows consensus sequences computed at various thresholds of percentage identity. Consensus patterns are based on equivalence classes - sets of residues that share predefined properties. These classes are not mutually exclusive. The consensus shows the most specific class that summarizes a given column at the specified percent identity. Taylor, W.R. (1986) J. Theor. Biol. 119: 205-218. The classes are alcohol (“o”): S, T; aliphatic (“1”): I, L, V; aromatic (a): F, H, W, Y; charged (“c”): D, E, H, K, R; hydrophobic (“h”): A, C, F, G, H, I, K, L, M, R, T, V, W, Y; negative (“-“): D, E; polar (“p”): C, D, E, H, K, N, Q, R, S, T; positive (“+”)_: H, K, R; small (“s”): A, C, D, G, N, P, S, T, V; tiny (“U”): A, G, S; turnlike (“t”): A, C, D, E, G, H, K, N, Q, R, S, T; and stop (“*”): stop. Figure 5B also discloses SEQ ID NOS 16, 72, 22, 20, 23, 17, 24, 18, and 21, respectively, in order of appearance.

[0048] FIG. 6 A shows an alignment of mARTs sequences from Pseudomonas aeruginosa (SEQ ID NO: 17) and Acinetobacter baumannii (SEQ ID NO: 24).

[0049] FIG. 6B shows a phylogenetic tree of mARTs.

[0050] FIG. 7 shows an alignment of mARTs sequences that identifies predicted secondary structural features in the DI Domain. Identical residues have a dark background with white text, residues with lighter characters are similar. Secondary structure elements (a: alpha helix; h: 3io helix; b: beta sheet) are shown above the sequence. Conserved cysteines are marked with arrows. The figure was prepared with ESPript. The sequences are Domain I sequences of mature mARTs from Vibrio cholera (Cholix, SEQ ID NO: 73), Chromobacterium haemolyticum (SEQ ID NO: 74), Janthinobacterium lividum (SEQ ID NO: 75), Collimonas fungivorans (SEQ ID NO: 76), Serratia fonticola (SEQ ID NO: 77), Pseudomonas aeruginosa (SEQ ID NO: 78), Acinetobacter baumannii (SEQ ID NO: 79), Aeromonas hydrophila (SEQ ID NO: 52), and Shewanella putrefacians (SEQ ID NO: 53).

[0051] FIG. 8 shows ribbon diagrams comparing the structures of the lectin (carbohydrate 15 recognition) domain of Galectin3 (black) to Vibrio cholera (PDB 2Q5T) (FIG. 8A),

Pseudomonas aeruginosa (PDB 1IKQ) (FIG. 8B), Aeromonas hydrophila (PDB 6Z5H) (FIG.

8C) and Chromobacterium haemolyticum (FIG. 8D) mARTs and predicted structure of the

Collimonas fungivorans (FIG. 8E), Shewanella putrefacians (FIG. 8F), Janthinobacterium lividum (FIG. 8G), Serratia fonticola (FIG. 8H), and Acinetobacter baumannii (FIG. 81) mARTs (grey).

[0052] FIG. 9 shows isolated His-tagged full-length catalytically inactive mARTs.

[0053] FIG. 10 schematically shows a setup comprising an apical chamber above an epithelial cell monolayer and a basal chamber below such epithelial cell monolayer. For apical to basolateral permeability (e.g., transcytosis), test articles (e.g., carriers, delivery constructs, payloads, etc.) were applied to the apical (A) side and the amount of permeated (e.g., transcytosed) material was determined (e.g., using western blotting, chromatography, etc.) on the basolateral (B) side.

[0054] FIG. 11A shows apical to basal transcytosis across SMI-100 epithelial cells of full- length catalytically inactive mARTs, including Cholix (Chx), Pseudomonas exotoxin (PE), and mARTs from Aeromonas hydrophila (Aer) and Chromobacterium haemolyticum (Hae).

[0055] FIG. 11B shows the effect of mARTs from Pseudomonas (PE), Aeromonas hydrophila (Aer), and Chromobacterium haemolyticum (Hae) on Cholix (chx) transcytosis as determined by ELISA.

[0056] FIGs. 12A-B show sections from rat intestines injected with fusion proteins having an N-terminal mART carrier from Vibrio cholera (Chx) or Chromobacterium haemolyticum (Hmx) and a C-terminal human growth hormone cargo. Fifteen minutes after injecting the indicated fusion protein into an intestinal lumen, the intestines were fixed, embedded, sectioned, and the sections were stained with anti-hGH antibodies (green) and dapi (blue). The dotted line highlights the boundary between epithelial cells and the lamina propria. The fusion proteins were Chx-hGH (SEQ ID NO: 44 FIG. 12A) and Hmx-hGH (SEQ ID NO: 45 FIG. 12B). This figure shows that both fusion proteins are transported across the intestinal epithelia cells into the lamina propria, where they are visible as green dots within the region bordered by the dotted line. The green dots are smaller than dapi-stained nuclei in blue.

[0057] FIG. 13 shows the intracellular distribution of LMAN1 and COPI in rat intestines. Untreated intestines were fixed, embedded, sectioned, and the sections were stained with anti- LMAN1 antibodies (green), anti-COPI antibodies (red), and dapi (blue). LMAN1 is localized to small vesicles within the apical domain of the intestinal epithelial cells, where it overlaps with COPI. COPI is localized throughout the apical domain of the intestinal epithelial cells (including at the apical surface) and in lesser amounts in the basolateral domain of the intestinal epithelial 16 cells and within cells of the lamina propria. Dapi stained DNA is predominately localized in larger structures (nuclei) found in cells of both epithelial and lamina propria cells. In epithelial cells, it is localized underneath the apical region filled with COPI.

[0058] FIGs. 14A-B show the intracellular distribution of COPI in rat intestinal epithelial cells during transport of carrier-payload complexes. 50 mΐ of a 3.86 x 10⁵ M solution of Cholix- hGH (SEQ ID NO: 44) (FIG. 14A) or Hmx-hGH (SEQ ID NO: 45) (FIG. 14B) was injected into the lumen of rat intestines. Fifteen minutes after injecting the fusion protein, the intestines were fixed, embedded, sectioned, and the sections were stained with anti-COPI antibodies (red) and dapi (blue). The dotted line highlights the boundary between epithelial cells and the lamina propria. Arrows indicate the apical surface of the epithelium. Chx-hGH transport causes COPI to cluster on the apical side of the nucleus of intestinal epithelial cells; whereas COPI remains dispersed throughout the apical domain during Hmx-hGH transport.

[0059] FIGs. 15A-C show the intracellular distribution of LMAN1 in rat intestinal epithelial cells during transport of carrier-payload complexes. 50 mΐ of a 3.86 x 10⁵ M solution of Cholix- hGH (SEQ ID NO: 44) (FIG. 15A), Hmx-hGH (SEQ ID NO: 45) (FIG. 15B), or AE-hGH (SEQ ID NO: 46) (FIG. 15C) was injected into the lumen of rat intestines. Fifteen minutes after injecting the fusion protein, the intestines were fixed, embedded, sectioned, and the sections were stained with anti-hGH antibodies (green), anti-LMANl antibodies (red) and dapi (blue). The dotted line highlights the boundary between epithelial cells and the lamina propria. Arrows indicate the apical surface of the epithelium. Chx-hGH, Hmx-hGH and AE-hGH were detected in the lamina propria, indicating transcytosis across the epithelial cells. Chx-hGH, Hmx-hGH and AE-hGH were also detected in the apical and basolateral domains of the epithelial cells. During Chx-hGH transcytosis, LMAN1 colocalized with Chx-hGH within the apical and basolateral domains of the epithelial cells. In contrast, LMAN1 remained on the apical surface during Hmx- hGH transcytosis, and redistributed to the basolateral domain to a lesser extend during AE-hGH transcytosis.

DETAILED DESCRIPTION

[0060] 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 may be practiced without these details.

I. Introduction

[0061] Provided herein, in certain embodiments, are carriers, and delivery constructs (e.g., 17 carrier-payload complexes) capable of transporting one or more heterologous payload molecules

(e.g., one or more therapeutic payloads) into epithelial cells (e.g., polarized gut epithelial cells), e.g., by endocytosis, or across epithelial cells (e.g., polarized gut epithelial cells) by, e.g., by transcytosis. The delivery constructs can comprise a carrier that is coupled to the heterologous payload. The carrier can be capable of transporting the heterologous payload into or across epithelial cells using endogenous trafficking pathways. Utilization of endogenous trafficking pathways, as opposed to use of passive diffusion, can allow the carrier to shuttle the heterologous payload rapidly (e.g., at least 10⁶ cm/sec, 10⁵ cm/sec) and efficiently (e.g., at least 5%, 10%,

20%, 25%, or 50% of material applied to the apical surface) into or across epithelial cells without impairing the barrier function of these cells or the biological activity of the heterologous payload.

II. Certain Definitions

[0062] Unless the context requires otherwise, throughout the specification and claims which 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.

[0063] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody 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-expression 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 through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0064] The terms “ Vibrio cholera mART,” “cholix” and “Chx” are used interchangeably to refer to a polypeptide having at least 90% sequence identity to SEQ ID NO: 6. A Domain I of cholix refers to a polypeptide having at least 90% sequence identity to SEQ ID NO: 16.

[0065] The terms “ Pseudomonas aeruginosa mART,” “ Pseudomonas Endotoxin” and “PE” are used interchangeably to refer to a polypeptide having at least 90% sequence identity to SEQ 18 ID NO: 7. A Domain I of PE refers to a polypdptid having at least 90% sequence identity to SEQ

ID NO: 17.

[0066] The polypeptides described herein can be encoded by a nucleic acid. A nucleic acid is a type of polynucleotide comprising two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer the polypeptide encoding polynucleotide into a cell. As used herein, the term “vector” can refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrated vector, or “integrated vector,” which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an “episomal” vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Suitable vectors comprise plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors and the like. In the expression vectors regulatory elements such as promoters, enhancers, polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, which can be conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like, may be employed. Plasmid vectors can be linearized for integration into a chromosomal location. Vectors can comprise sequences that direct site-specific integration into a defined location or restricted set of sites in the genome (e.g., AttP-AttB recombination). Additionally, vectors can comprise sequences derived from transposable elements.

[0067] The nucleic acids encoding the polypeptides described herein can be used to infect, transfect, transform, or otherwise render a suitable cell transgenic for the nucleic acid, thus enabling the production of polypeptides for commercial or therapeutic uses.

III. Carriers

[0068] The carrier, or carrier portion of a delivery construct provided herein can be any molecule (e.g., small molecule, polypeptide, nucleic acid, etc.) capable of increasing the rate and/or amount of a heterologous payload (e.g., a therapeutic payload) delivered into and/or across an epithelium.

[0069] A carrier herein can have numerous attributes. In some embodiments, a carrier herein can be a mART. In some embodiments, a carrier can be a mART with a reduced (e.g., at least 50% reduced) or ablated ADP ribosylation activity (e.g., ribosylation of elongation factor 2) relative to a naturally occurring exotoxin. 19 (a) Carriers may utilize endogenous trafficking pathways

[0070] In some embodiments, a carrier herein utilizes an endogenous trafficking pathway to transport a heterologous payload coupled thereto across a polarized epithelial cell. Such carrier can be referred to herein as a transcytosing carrier. In some instances, a carrier herein can utilize an endogenous trafficking pathway to transport a heterologous payload coupled thereto into a polarized epithelial cell. Such carrier can be referred to herein as an endocytosing carrier. Within endocytosing carriers, there can be carriers that deliver a payload coupled thereto into specific regions within the polarized epithelial cells such as an apical compartment, a supranuclear compartment, or a basal compartment.

[0071] Any of the carriers herein can transport molecules coupled thereto by interacting and/or co-localizing with one or more endogenous proteins of such epithelium. The one or more endogenous proteins can be receptors or enzymes capable of moving a carrier into or across the epithelial cell. Interacting and/or co-localizing with the one or more endogenous proteins of the epithelial cell can provide a carrier with one or more functions, including endocytosis into the epithelial cell, avoidance of a lysosomal destruction pathway, trafficking from an apical compartment to a basal compartment, and/or exocytosis from the basal membrane of the epithelial cell into a submucosal compartment such as the lamina propria.

[0072] An interaction of such carrier with an endogenous protein can be a selective interaction. Such selective interaction can be a pH-dependent interaction. In instances where a carrier interacts with two or more endogenous proteins, such interactions can be sequential interactions where a first interacting protein hands the carrier off to a second interacting protein. Such sequential interactions can occur at a different pH (e.g., pH 5.5, 7.0, 7.5, etc.). An interaction between a carrier and an endogenous protein can be a covalent or non-covalent interaction. Non-covalent interactions include hydrogen bonding, van der Waals interactions, ionic bonds, p-p-interactions, etc.

[0073] In some instances, one of the endogenous proteins that a carrier can interact with can be an apical entry receptor. Interaction of a carrier with such apical entry receptor can enable the carrier to enter the epithelial cell through receptor-mediated endocytosis.

[0074] A carrier can also interact with a lysosome avoidance receptor. Such interaction with a lysosome avoidance receptor can occur inside the epithelial cell and subsequent to endocytosis. Interaction of a carrier with a lysosome avoidance receptor can enable the carrier to avoid or circumvent lysosomal degradation. Such ability can allow a carrier to significantly reduce the amount of payload coupled to the carrier reaching a lysosome of a cell, a fate that therapeutic proteins can face once taken up by the gut epithelium.

[0075] Furthermore, a carrier can interact with an apical to basal trafficking protein. Such 20 interaction can occur inside the epithelial cell and subsequent to endocytosis. Interaction of a carrier with a basal trafficking protein can enable the carrier to move from an apical compartment to a supranuclear compartment or a basal compartment.

[0076] A transcytosing carrier can also interact with a basal release protein capable of promoting exocytosis of a carrier from a basal site of an epithelial cell. Such interaction can occur at the basal site of an epithelial cell and subsequent to moving from an apical compartment to a basal compartment. Interaction of a carrier with a basal release protein can enable the carrier to access a basal recycling system that allows release of the carrier from the basal compartment into a submucosal compartment such as the lamina propria.

[0077] Thus, a transcytosing carrier herein can be a molecule that is capable of interacting with endogenous proteins, enabling such carrier to transport a payload molecule coupled thereto across a polarized epithelium, e.g., a polarized gut epithelium.

[0078] An endocytosing carrier herein can be a molecule that is capable of interacting with an apical entry receptor, allowing apical entry of such carrier. An endocytosing carrier can remain associated with an apical entry receptor after endocytosis (e.g., compared to a transcytosing carrier that can dissociate from an apical entry receptor after endocytosis to interact with, e.g., a lysosome avoidance receptor or an apical to basal trafficking protein) and within apical regions and compartments of the cell (e.g., a polarized epithelial cell). Such interactions can allow the carrier and a payload coupled thereto to avoid, or at least significantly reduce (e.g., less than about 50% compared to the payload molecule when it is not coupled to the carrier), lysosomal degradation. In some instances, an endocytosing carrier can remain in an apical compartment, and not show significant translocation to a basal compartment, for, e.g., at least about 5, 10, 15, 30, 60, or 120 minutes after apical (e.g., luminal) application of the carrier compared to a transcytosing carrier that can show complete transcytosis of nearly all apically applied molecules, e.g., about 5, 10, 15 or 30 minutes after apical (e.g., luminal) application. In some instances, at least about 50%, 75%, or 90% of carrier molecules remain in apical compartments 5 minutes after luminal application of the carrier. In some instances, at least about 50%, 75%, or 90% of carrier molecules remain in apical compartments 10 minutes after luminal application of the carrier. In some instances, at least about 50%, 75%, or 90% of carrier molecules remain in apical compartments 15 minutes after luminal application of the carrier. In some instances, at least about 50%, 75%, or 90% of carrier molecules remain in apical compartments 30 minutes after luminal application of the carrier. The percentage of carrier molecules that remain in the apical compartment of the epithelial cell can be determined by dividing the intensity of the fluorescence signal measured in a basal compartment of the cell by the intensity of the fluorescence signal measured in the apical compartment of the cell at the respective time point. 21 [0079] In other instances, an endocytosing carrier that is capable of transporting a payload to a supranuclear or basal compartment can interact with an ER Golgi Intermediate Compartment

(ERGIC) protein and/or another ER-Golgi trafficking protein complex that can allow the carrier to access such compartments inside an epithelial cell.

[0080] An endocytosing or transcytosing carrier can be a polypeptide. Such carrier can be derived from a polypeptide secreted by a bacterium, such as the mART of Aeromonas hydrophila.

[0081] A carrier can be a naturally or non-naturally occurring polypeptide of a polypeptide secreted by such bacterium.

[0082] Non-naturally occurring polypeptides can include those having a C- and/or an N- terminal modification.

[0083] In one example, a polypeptide comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid additions relative to a sequence alignment with a naturally occurring polypeptide or relative to a sequence alignment with a consensus sequence.

[0084] Examples of substitutions contemplated herein include conservative substitutions of one or more amino acids. The following six groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Serine (S), and Threonine (T); (2) Aspartic acid (D) and Glutamic acid (E); (3) Asparagine (N) and Glutamine (Q); (4) Arginine (R) and Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); and (6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

[0085] Conservative substitutions further include substitutions within the following groups: aliphatic residues (I, V, and L); aromatic residues (Y, H, W, and F); hydrophobic residues (W, F, Y, M, L, I, V, A, C, T, and H); alcohol residues (S and T); polar residues (D, E, H, K, N, Q, R, S, and T), tiny residues (A, G, C, and S); small residues (A, G, C, S, V, N, D, T, and P); bulky residues (E, F, I, K, L, M, Q, R, W, and Y); positively charged residues (K, R, and H); negatively charged residues (D and E); and charged residues (D, E, K, R, and H).

[0086] Examples of deletions include N-terminal truncations, C-terminal truncations, and internal deletions.

[0087] Examples of additions include: a signal peptide sequence, a purification peptide sequence, or other N-terminal modifications. A signal peptide sequence can comprise 1 to about 40 amino acids. In some cases, a carrier comprises an N-terminal methionine. The term “about,” as used herein in the context of a numerical value or range, generally refers to ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the numerical value or range recited or claimed, unless otherwise specified. 22 [0088] A carrier can have a substantial sequence identity (e.g., about, or greater than, 50%,

60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity, or 100% sequence identity) to a naturally occurring polypeptide (e.g., the sequence set forth in any one of SEQ ID NOs: 1-14), a consensus sequence (e.g., the sequence set forth in SEQ ID NOs: 15 or 25), or to any of the functional fragments described herein (e.g., the sequence set forth in any one of SEQ ID NOs:

16-24).

[0089] The term “sequence identity” or a percent (%) of sequence identity, as used herein can be the percentage of residues in a candidate sequence that are identical with the residues in a selected 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 in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0090] N-terminal truncations include those that remove up to 10, 20, 30, 39, or 40 amino acids at the N-terminus of a mART sequence herein (e.g., the sequence of any one of SEQ ID NOs: 1-14) or the N-terminus of Domain I of a mART sequence herein (e.g., the sequence of any one of SEQ ID NOs: 16-24). C-terminal truncations can be those described herein. Such N- and/or C-terminal truncations can result in different functions. Truncations can be described as relative to a naturally occurring sequence (e.g., the sequence of SEQ ID NO: 1), or relative to a consensus sequence (e.g., the sequence of any one of SEQ ID NOs: 15 or 25), wherein the residues are numbered from the N-terminus to the C-terminus, starting with position 1 an the N- terminus. For example, a carrier with a C-terminal truncation at position 206 relative to SEQ ID NO: 16 comprises amino acid residues 1-206 of SEQ ID NO: 16.

[0091] A carrier (e.g., an endocytosing or a transcytosing carrier) herein can be derived from a polypeptide from (e.g., secreted from) Vibrio cholerae , Pseudomonas aeruginosa , Aeromonas hydrophila , Aeromonas dhakensis , Aeromonas salmonicida , Aeromonas piscicola , Aeromonas bestiarum , Chromobacterium haemolyticum, Collimonas fungivorans , Shewanella putrefacians , Janthinobacterium lividum , Serratia fonticola , or Acinetobacter baumannii bacterium (e.g., those comprising a sequence of any one of SEQ ID NOs: 1-14). Such carrier can be referred to as a mART polypeptide. A carrier derived from a mART polypeptide can include naturally and non- naturally occurring mART polypeptide sequences, as well as those sequences that have at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a naturally occurring 23 mART polypeptide (e.g., the sequence set forth in any one of SEQ ID NOs: 1-14) described herein. A mART polypeptide derived carrier can also include endocytosing and/or transcytosing fragments (e.g., N- and/or C-terminal truncations of a mART polypeptide) of naturally and non- naturally occurring mART polypeptide sequences, wherein such endocytosing and/or transcytosing fragments can have at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any of such naturally or non-naturally occurring mART polypeptide sequences.

(b) mono-A DP-ribosyl transferases (mARTs)

[0092] A mono-ADP-ribosyl transferase (mART) is a polypeptide with at least three domains: DI, DII, and Dill. Domain I is typically located at the N-terminus of the mature protein and Domain III is typically located at the C-terminus. A mART can have a leader (or signal) peptide at its N-terminus. In its native setting, the leader peptide may target the mART for secretion. A leader peptide is removed from the mature protein by an endopeptidase. A mART may further comprise a Domain lb domain that associates with Domain I in a folded, three-dimensional structure, although it is not continuous with the DI domain in the amino acid sequence of the mART. The total size of a mART can be 600 - 650 amino acids.

[0093] Domain I (DI) of a mART can function as a carrier for endocytosis and/or transcytosis. It can comprise a core 13-stranded beta-jellyroll fold structure. Each beta-strand of DI is linked to the next by a loop region. Domain I can be about 250-270 amino acids in length. A functional Domain I can comprise at least 150 amino acids. Domain I may start after a leader peptide cleavage site (or at an N-terminus in the case where there is no leader peptide cleavage site) and may end at a HFxxx sequence or, preferably, an HFxxG or HFxxE sequence. Domain I may end at the end of a HFxxx, HFxxG or HFxxE sequence or 1, 2, or 3 amino acids from the end of a HFxxx, HFxxG or HFxxE sequence.

[0094] Domain I may comprise one or more disulfide bonds. Such disulfide bonds may link Cysteinel 1 and Cysteinel5 of SEQ ID NO: 1 and Cysteinel97 and Cysteine214 of SEQ ID NO: 18, or the cysteine pairs at the same position in an sequence alignment in a Pseudomonas mART or a Vibrio mART as shown in Fig. IB (i.e. the cysteines “corresponding” to Cysteinel 1, Cysteinel5, Cysteinel97, and Cysteine214 of SEQ ID NO: 18). Similarly, disulfide bonds may link the corresponding cysteines of a Vibrio , Pseudomonas , Shewanella, Aeromonas, Collimonas , Serratia, Jathinobacterium , or Chromobacterium mART as shown in Fig. 5B. Additionally, disulfide bonds may link the corresponding cysteines of Domain I of a mART with a sequence that differs from the sequences depected in Figs. IB and 5B.

[0095] Domain I may comprise amino acids that bind to a protein, glycoprotein, or glycolipid displayed on the surface of an epithelial cell and/or a cell sensitive to intoxication by the mART. 24 Binding of a Domain I to a cell surface protein, glycoprotein, or glycolipid can mediate entry of the mART into the cell. Domain I of a mART may enter a cell by endocytosis, micropinocytosis, or macropinocytosis and it may enter via calveolae or clathrin-coated pits. Domain I can further have transcytosis carrier activity. Domain I may comprise amino acids that interact with intracellular proteins in endosomal compartments or membranes to mediate transport within apical endosomes, to avoid trafficking to lysosomes, to direct transport to vesicles on the apical side of the nucleus, to direct transport from an apical endosome or a perinuclear vesicle to a basal or basolateral endosome, and to be released from an epithelial cell by exocytosis of a basolateral vesicle.

[0096] Domain II (DII) of a mART can have a furin cleavage site (RQPR (SEQ ID NO: 50)) surrounded by R-rich loops. Domain II can form a six alpha helix bundle. Domain II can mediate translocation of Dill of a mART across an intracellular membrane. The intracellular membrane can be an endoplasmic reticulum membrane.

[0097] Domain III (Dill) of a mART can have transferase activity. In some embodiments, transferase activity is mediated by a catalytic glutamic acid residue. The substrate for the transferase may be elongation factor Tu (EF-Tu). In some embodiments, Domain III of a mART does not comprise a catalytic glutamic acid residue, either naturally, or by design to reduce toxicity. In some embodiments, Domain III comprises a retrograde transport signal that mediates intracellular transport of the mART to the endoplasmic reticulum. The retrograde transport signal may be at or near the C-terminus of Domain III, and it may comprise the amino acids RDEL (SEQ ID NO: 51).

[0098] A catalytic glutamic acid residue may be glutamate 613 of SEQ ID NO: 6, glutamate 578 of SEQ ID NO: 7, glutamate 571 of SEQ ID NO: 1, glutamate 617 of SEQ ID NO: 8, glutamate 579 of SEQ ID NO: 12, glutamate 623 of SEQ ID NO: 11, glutamate 596 of SEQ ID NO: 10, glutamate 593 of SEQ ID NO: 9 or an active site glutamate of a mART at a corresponding location within Domain III of a mART that plays an analogous role in mono- ADP-ribosyl transferase activity.

[0099] A mART may comprise a transcytosing element. A transcytosing element is any portion of a mART that mediates transcytosis of the mART across a polarized epithelial cell. A transcytosing element may comprise all or part of Domain I of the mART. The transcytosing element may be a Domain I of a mART with an N-terminal, C-terminal, or internal deletion. The transcytosing element can mediate transcytosis across a gut epithelial cell or a pulmonary epithelial cell. The transcytosing element can mediate transcytosis across a polarized SMI-100 cell, Caco-2 cell, orMDCK cell. 25 (c) Aeromonas carrier

[00100] A carrier can be derived from an Aeromonas endotoxin (AE), i.e. the mART of an Aeromonas species, for example, Aeromonas hydrophila , Aeromonas dhakensis , Aeromonas salmonicida , Aeromonas piscicola , or Aeromonas bestiarum. Examples of AE polypeptides include the polypeptides set forth in SEQ ID NOs: 1-5 or 14. The carrier can have about, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-5 or 14.

[00101] Amino acids that have been conserved among the AE sequences identified in various Aeromonas species and between AE and other mARTs (e.g. cholix, PE, and Haemolix) may be functionally or structurally significant because they have accepted fewer mutations relative to the rest of the alignment (Capra and Singh, Bioinformatics, 23(15): 1875-1882, 2007).

[00102] A carrier can be derived from a naturally-occurring AE polypeptide by a substitution mutation (e.g. replacing the active site glutamate) and/or truncating the AE polypeptide from it N-terminus or C-terminus. A C-terminal truncation may delete all or a portion of Domain III, Domain lb, Domain II, or even Domain I. An N-terminal truncation may delete all or a portion of Domain I or Domain II. A truncated AE polypeptide may have an N-terminal deletion and a C- terminal deletion. Non-limiting examples of N-terminal truncations include deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids from the N-terminus of an AE or the N- terminus of DI of an AE. Non-limiting examples of C-terminal truncations include deletions of 10, 20, 30, 40, 50, 100, 200, 300, 350, 400, 450, 500, 550 or more amino acids from the C- terminus of an AE or the deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids from C-terminus of DI of an AE. Additional examples of C-terminal truncations delete all of Domain III except 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain III; all of Domains lb and III except 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain lb; or all of Domains II, lb and III except 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain II. Additional examples of C-terminal truncations delete Domain III plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of Domain lb; Domain III and Domain lb plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of Domain II; or Domain III, Domain lb and Domain II plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of Domain I.

(d) Chromobacterium carrier

[00103] A carrier can be derived from a mART of a Chromobacterium species. Amino acids that have been conserved among naturally occurring Chromobacteria mARTs and between Chromobacteria mARTs and related mART polypeptides isolated from other microorganisms 26 (e.g. cholix, PE, AE, and etc) may be functional iy or structurally significant because they have accepted fewer mutations relative to the rest of the alignment (Capra and Singh, Bioinformatics, 23(15): 1875-1882, 2007). An example of a Chromobacterium mART polypeptide is the polypeptide set forth in SEQ ID NO: 8 from Chromobacterium haemolyticum. The carrier can have about, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 8.

[00104] A carrier can be derived from a naturally-occurring Chromobacterium mART polypeptide by a substitution mutation (e.g. replacing the active site glutamate) and/or truncating the Chromobacterium mART polypeptide from itN-terminus or C-terminus. A C-terminal truncation may delete all or a portion of Domain III, Domain lb, Domain II, or even Domain I.

An N-terminal truncation may delete all or a portion of Domain I or Domain II. A truncated Chromobacterium mART polypeptide may have an N-terminal deletion and a C-terminal deletion. Non-limiting examples of N-terminal truncations include deletions of 1, 2, 3, 4, 5, 10,

15, 20, 25, 30, 35, 40, 45, 50 or more amino acids from the N-terminus of a Chromobacterium mART. Non-limiting examples of C-terminal truncations include deletions of 100, 200, 300, 350, 400, 450, 500, 550 or more amino acids from the C-terminus of a Chromobacterium mART and deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids from the C- terminus of a Domain I of a Chromobacterium mART. Additional examples of C-terminal truncations delete all of Domain III except 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain III; all of Domains lb and III except 1, 2, 3, 4, 5, 10,

15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain lb; or all of Domains II, lb and III except 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain II. Additional examples of C-terminal truncations delete Domain III plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of Domain lb; Domain III and Domain lb plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of Domain II; or Domain III, Domain lb and Domain II plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of Domain I.

(e) Collimonas, Shewanella, Janthinobacterium, Serratia, and Acinetobacter carriers

[00105] A carrier can be derived from a mART of a Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter species. Amino acids that have been conserved among naturally occurring Collimonas , Shewanella , Janthinobacterium , Serratia fonticola , or Acinetobacter baumannii mARTs and between Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mARTs and related mART polypeptides isolated from other microorganisms (e.g. cholix, PE, AE, and Chromobacteria etc ) may be functionally or structurally 27 significant because they have accepted fewer mutations relative to the rest of the alignment (Capra and Singh, Bioinformatics, 23(15): 1875-1882, 2007). An example of a Collimonas mART polypeptide is the polypeptide set forth in SEQ ID NO: 9 from Collimonas fungivorans. The carrier can have about, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 9. An example of a Shewanella mART polypeptide is the polypeptide set forth in SEQ ID NO: 10 from Shewanella putrefacians . The carrier can have about, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 10. An example of a Janthinobacterium mART polypeptide is the polypeptide set forth in SEQ ID NO: 11 from Janthinobacterium lividum. The carrier can have about, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 11. An example of a Serratia mART polypeptide is the polypeptide set forth in SEQ ID NO: 12 from Serratia fonticola. The carrier can have about, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 12. An example of a Acinetobacter mART polypeptide is the polypeptide set forth in SEQ ID NO: 13 from Acinetobacter baumannii. The carrier can have about, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 13.

[00106] A carrier can be derived from a naturally-occurring Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART polypeptide by a substitution mutation (e.g. replacing the active site glutamate) and/or truncating the Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART polypeptide from it N-terminus or C- terminus. A C-terminal truncation may delete all or a portion of Domain III, Domain lb, Domain II, or even Domain I. An N-terminal truncation may delete all or a portion of Domain I or Domain II. A truncated Chromobacterium mART polypeptide may have an N-terminal deletion and a C-terminal deletion. Non-limiting examples of N-terminal truncations include deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids from the N-terminus of a Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. Non-limiting examples of C-terminal truncations include deletions of 100, 200, 300, 350, 400, 450, 500, 550 or more amino acids from the C-terminus of a Collimonas , Shewanella , Janthinobacterium ,

Serratia , or Acinetobacter baumannii mART or deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids from the C-terminus of a DI domain of a Collimonas ,

Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. Additional examples of C- terminal truncations delete all of Domain III except 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain III; all of Domains lb and III except 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain lb; or all 28 of Domains II, lb and III except 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the N-terminus of Domain II. Additional examples of C-terminal truncations delete

Domain III plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of Domain lb; Domain III and Domain lb plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35,

40, 45, 50 or more amino acids of Domain II; or Domain III, Domain lb and Domain II plus an additional 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of Domain I.

IV. Payloads

[00107] In addition to the carrier polypeptide, the compositions provided herein can comprise one or more payloads, e.g., one or more heterologous payloads, or one or more biologically- active payloads, for delivery to a subject. The one or more heterologous payloads can be one or more payloads that do not have a carrier sequence, e.g., a mART sequence. The one or more payloads, e.g., one or more heterologous payloads, can be a macromolecule, small molecule, small organic molecule, peptide, polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, PNA, antisense molecule, antibody, DNA, plasmid, polysaccharide, lipid, antigen, vaccine, polymer nanoparticle, or catalytically-active material.

[00108] The one or more payloads, e.g., one or more heterologous payloads, e.g. one or more biologically active payloads, can be a macromolecule that can perform a desirable biological activity after transport across an epithelial cell and/or when introduced to the bloodstream of the subject. For example, the one or more payloads can have receptor binding activity, enzymatic activity, messenger activity (i.e., act as a hormone, cytokine, neurotransmitter, clotting factor, growth factor, or other signaling molecule), luminescent or other detectable activity, or regulatory activity, or any combination thereof. In various diagnostic embodiments, the one or more payloads can be conjugated to or can itself be a pharmaceutically acceptable gamma- emitting moiety, including but not limited to, indium and technetium, magnetic particles, radiopaque materials such as air or barium and fluorescent compounds (e.g., Alexa-488 or a red fluorescent protein). In some cases, the one or more payloads, e.g., one or more biologically active payloads, do not enter the bloodstream of the subject. In some cases, the one or more payloads, e.g., one or more biologically active payloads, enter the bloodstream of the subject. In some cases, the one or more payloads act at the lamina propria.

[00109] In various embodiments, the one or more payloads is a protein that comprises more than one polypeptide subunit. For example, the protein can be a dimer, trimer, or higher order multimer. In various embodiments, two or more subunits of the protein can be connected with a covalent bond, such as, for example, a disulfide bond. In other embodiments, the subunits of the protein can be held together with non-covalent interactions. One of skill in the art can identify such proteins and determine whether the subunits are properly associated using, for example, an 29 immunoassay.

[00110] In some cases, one or more payloads, e.g., one or more heterologous payloads, used with the methods and compositions disclosed herein can be a hormone. Examples of hormones include, but are not limited to, human growth hormone, synthetic human growth hormone, human growth hormone 2. The one or more payloads, e.g., one or more heterologous payloads, can be a polypeptide comprising, consisting of, or consisting essentially of the sequence set forth in SEQ ID NOs: 49 or a sequence with 85%, 90%, 95% or 98% identity thereto.

[00111] In various embodiments, the one or more payloads, e.g., one or more therapeutic payloads, are for example a dye, a radiopharmaceutical, a hormone, a cytokine, an anti-TNF agent, an antineoplastic compound, an agent for the treatment of hemophilia, an enzyme, a glucose lowering agent, insulin, or an insulin analog, or a derivative of insulin, or a tumor associated antigen. In some cases, the one or more therapeutic payloads is a polypeptide that is a modulator of inflammation in the GI tract. In various embodiments, the one or more payloads is a glucose-lowering agent for delivery to a subject. In various embodiments, the one or more payloads is a one or more incretins. An incretin can be glucagon-like peptide-1 (GLP-1) or Gastric inhibitory peptide (GIP).

V. Delivery Constructs

[00112] Provided herein are delivery constructs (e.g., carrier-payload complexes) that can comprise a carrier coupled to one or more heterologous payloads. A carrier can be coupled to the one or more heterologous payloads covalently or non-covalently (e.g., via ionic interactions, van der Waals interactions, p-p interactions, etc.). A carrier can be coupled directly or indirectly to the one or more heterologous payloads.

[00113] The one or more heterologous payloads can be coupled to an N- and/or C-terminus of a carrier. In some instances, the one or more heterologous payloads is directly and covalently coupled to a C-terminus of a carrier by forming a covalent amide bond between the C-terminal carboxyl group of the carrier and the N-terminal amine of a heterologous payload. In some instances, the one or more heterologous payloads is indirectly and covalently coupled to the carrier via a spacer.

[00114] Thus, in some instances, when a carrier is covalently coupled to a payload, the delivery construct can be represented according to Formula II: C-L-P or Formula III: P-L-C, wherein C is a carrier, L is a linker (spacer), or optionally a bond, and P is a heterologous payload. A delivery construct can further comprise one or more modifications on its N-terminus and/or C-terminus. Such a modification(s) can include an N-terminal methionine residue. Thus, Formula II and Formula III can also include an N-terminal methionine (e.g., M+C-L-P) or (e.g., M+P-L-C). [00115] A carrier can be coupled to one or more heterologous payloads via chemical/synthetic 30 conjugation (e.g., using amide coupling reactions) or by recombinant expression, e.g., in a bacterial (e.g., in E. coli) or mammalian (e.g., Chinese Hamster Ovary (CHO)) cell as a fusion protein.

[00116] A delivery construct, or part thereof (e.g., the carrier and/or spacer), can be a polypeptide. The term “polypeptide,” as used herein, can include both natural and unnatural amino acids.

[00117] Furthermore, a carrier can transport a payload across an intact epithelium (e.g., a polarized gut epithelium) with transport rates of at least about 10⁶ cm/sec, 10⁵ cm/sec, or 10⁴ cm/sec.

[00118] In some instances, a carrier is indirectly and non-covalently coupled to a payload. In such instances, nanoparticles (e.g., liposomes, metallic nanoparticles, polymer-based nanoparticles, etc.) can be loaded (e.g., on the inside and/or on the surface of the particle) with one or more payload molecules (e.g., IL-10, IL-22, GLP-1, etc.), and carrier molecule(s) can be coupled to such nanoparticles (e.g., onto its surface). This can allow transport of such payload- containing nanoparticles into or across polarized epithelial cells (e.g., polarized gut epithelial cells) using a carrier attached to the surface. In some cases, a nanoparticle can release the payload following transcytosis or intracellular delivery. In cases where the nanoparticle is transported across epithelial cells, the released payload can bind to receptors within submucosal tissue (e.g., lamina propria) and/or can enter the systemic circulation and thus provide a certain function (e.g., a therapeutic or diagnostic function) systemically. In other cases, where a nanoparticle releases the payload inside an epithelial cell, the payload (e.g., a nucleic acid) may provide certain intracellular functions, e.g., production of transgenes within these cells, modulation of gene expression, etc.

VI. Coupling of the payload to a carrier

[00119] In some cases, the compositions provided herein comprise a carrier coupled to a payload, e.g., a heterologous payload. The payload, e.g., heterologous payload, can be coupled to the carrier by any method known by one of skill in the art without limitation. The payload may associate with the carrier by non-covalent interactions such as ionic interactions or assembly into nano-particles. The payload may be chemically cross-linked to the carrier via covalent interactions. In some cases, the one or more payloads are fused to a carrier, resulting in a fusion molecule. In a fusion molecule the one or more payloads or one or more cations of the fusion molecule can be attached to the remainder of the fusion molecule by any method known by one of skill in the art without limitation. The payload can be introduced into any portion of the fusion molecule that does not disrupt the cell-binding or transcytosis activity of the carrier. In various embodiments, the payload is directly coupled to the N-terminus or C-terminus of the carrier. In 31 various embodiments, the payload can be connected to a side chain of an amino acid of the carrier. The payload can be indirectly coupled to the carrier via a spacer or linker. In various embodiments, the payload is coupled to the carrier with a cleavable linker such that cleavage at the cleavable linker(s) separates the payload from the remainder of the fusion molecule. In various embodiments, the payload is a polypeptide that can also comprise a short leader peptide that remains attached to the polypeptide following cleavage of the cleavable linker. For example, the payload can comprise a short leader peptide of greater than 1 amino acid, greater than 5 amino acids, greater than 10 amino acids, greater than 15 amino acids, greater than 20 amino acids, greater than 25 amino acids, greater than 30 amino acids, greater than 50 amino acids, or greater than 100 amino acids. In some cases, biological active payload can comprise a short leader peptide of less than 100 amino acids, less than 50 amino acids, less than 30 amino acids, less than 25 amino acids, less than 20 amino acids, less than 15 amino acids, less than 10 amino acids, or less than 5 amino acids. In some cases, payload can comprise a short leader peptide of between 1- 100 amino acids, between 5-10 amino acids, between 10 to 50 amino acids, or between 20 to 80 amino acids.

[00120] In embodiments where the payload is expressed together with another sequence as a fusion protein, the payload can be inserted into the fusion molecule by any method known to one of skill in the art without limitation. For example, nucleic acids coding for amino acids corresponding to the payload can be directly inserted into the nucleic acid coding for the other moiety or fusion molecule, with or without deletion of native amino acid sequences.

[00121] In embodiments where the payload is not expressed together as a fusion protein, the payload can be connected by any suitable method known by one of skill in the art, without limitation. More specifically, the exemplary methods described above for connecting a receptor binding domain to the remainder of the molecule are equally applicable for connecting the payload to the remainder of the molecule.

VII. Production of nucleic acids encoding carriers and/or payloads

[00122] In various embodiments, the carriers, payloads, and/or non-naturally occurring delivery constructs, e.g., fusion molecule of the present disclosure are prepared using the methodology described in, e.g., U.S. Patent Nos. 9,090,691 and 7,713,737, each incorporated by reference herein in their entirety.

[00123] In various embodiments, the carriers, payloads, and/or non-naturally occurring fusion molecules are synthesized using recombinant DNA methodology. Generally, this can involve creating a DNA sequence that encodes the carrier, payload, and/or fusion molecule, placing the DNA in an expression cassette under the control of a particular promoter, expressing the molecule in a host, isolating the expressed molecule and, if required, folding of the molecule into 32 an active conformational form.

[00124] DNA encoding the carrier, payload, and/or fusion molecules described herein can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth.

Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,

22: 1859-1862); the solid support method of U.S. Pat. No. 4,458,066, and the like.

[00125] Chemical synthesis can produce a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Chemical synthesis can be used to generated DNA sequences of about 100 bases. Longer sequences can be obtained by the ligation of shorter sequences.

[00126] Alternatively, subsequences can be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments can then be ligated to produce the desired DNA sequence.

[00127] In various embodiments, DNA encoding carrier, payload, and/or fusion molecules of the present disclosure can be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the gene or genes for the one or more payloads, e.g. the one or more biologically-active payloads is PCR amplified using sense and anti-sense primers having N-terminal restriction sites. This can produce one or more nucleic acids encoding the one or more payload sequences and having terminal restriction sites. A carrier having "complementary" restriction sites can similarly be cloned and then ligated to the one or more nucleic acids encoding the one or more payloads and/or to a linker attached to the one or more nucleic acids encoding the one or more payloads. Ligation of the nucleic acid sequences and insertion into a vector produces a vector encoding the one or more payloads joined to the carrier/s.

VIII. Cleavable Linkers

[00128] In various embodiments, the one or more payloads, e.g., heterologous payloads, to be delivered to the subject is coupled to the carrier using one or more cleavable linkers. The number of cleavable linkers present in the fusion molecule depends, at least in part, on the location of the one or more payloads in relation to the carrier and the nature of the biologically active payload. When the one or more payloads can be separated from the remainder of the fusion molecule with cleavage at a single linker, the fusion molecules can comprise a single cleavable linker. Further, where the one or more payloads is, e.g., a dimer or other multimer, each subunit of the one or more payloads can be separated from the remainder of the fusion molecule and/or the other subunits of the one or more payloads by cleavage at the cleavable linker. 33 [00129] In various embodiments, the cleavable linkers are cleaved by a cleaving enzyme that is present at or near the basolateral membrane of an epithelial cell. By selecting the cleavable linker to be cleaved by such enzymes, the one or more payloads can be liberated from the remainder of the fusion molecule following transcytosis across the mucous membrane and release from the epithelial cell into the cellular matrix on the basolateral side of the membrane. Further, cleaving enzymes can be used that are present inside the epithelial cell, such that the cleavable linker is cleaved prior to release of the fusion molecule from the basolateral membrane, so long as the cleaving enzyme does not cleave the fusion molecule before the fusion molecule enters the trafficking pathway in the polarized epithelial cell that results in release of the fusion molecule and one or more payloads from the basolateral membrane of the cell.

[00130] In other embodiments, a cleaving enzyme found in the plasma of the subject can be used to cleave the cleavable linker. Any cleaving enzyme known by one of skill in the art to be present in the plasma of the subject can be used to cleave the cleavable linker.

[00131] In various embodiments, the cleavable linker is cleaved by a cleaving enzyme found in the plasma of the subject. Any cleaving enzyme known by one of skill in the art to be present in the plasma of the subject can be used to cleave the cleavable linker. In some cases, plasma cleaving enzymes can be used to cleave the delivery constructs. In other embodiments, the cleavable linker comprises a nucleic acid, such as RNA or DNA. In still other embodiments, the cleavable linker comprises a carbohydrate, such as a disaccharide or a trisaccharide.

[00132] In various embodiments, the cleavable linker can be a cleavable linker that is cleaved following a change in the environment of the fusion molecule. For example, the cleavable linker can be a cleavable linker that is pH sensitive and is cleaved by a change in pH that is experienced when the fusion molecule is released from the basal-lateral membrane of a polarized epithelial cell. For instance, the intestinal lumen can be strongly alkaline, while plasma can be essentially neutral. Thus, a cleavable linker can be a moiety that is cleaved upon a shift from alkaline to neutral pH. The change in the environment of the fusion molecule that cleaves the cleavable linker can be any environmental change that that is experienced when the fusion molecule is released from the basal-lateral membrane of a polarized epithelial cell known by one of skill in the art, without limitation.

[00133] The function of the cleavable linker can generally be tested in a cleavage assay. Any suitable cleavage assay known by one of skill in the art, without limitation, can be used to test the cleavable linkers. Both cell-based and cell-free assays can be used to test the ability of an enzyme to cleave the cleavable linkers.

IX. Non-cleavable Linkers

[00134] In various embodiments, the carrier and one or more payloads can be separated by a 34 linker (spacer). When a linker is used, a linker can include one or more amino acids. Examples of linkers contemplated herein include sequences such as S, (GS)x (SEQ ID NO: 54), (GGS)x (SEQ

ID NO: 55), (GGGS)x (SEQ ID NO: 56), (GGGGS)x (SEQ ID NO: 57), or (GGGGGS)x (SEQ

ID NO: 58), wherein x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some cases, a linker can be SEQ ID NO: 48. In some cases, a linker does not include a terminal S residue, e.g., SEQ

ID NO: 47. Generally, a linker can have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. In various embodiments, however, the constituent amino acids of the linker can be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.

[00135] In various embodiments, the linker can form covalent bonds to both the carrier and to the biologically active payload. Suitable linkers include straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In various embodiments, the linker(s) can be joined to the constituent amino acids of the carrier and/or the one or more payloads through their side groups (e.g., through a disulfide linkage to cysteine). In various embodiments, the linkers are joined to the alpha carbon amino and/or carboxyl groups of the terminal amino acids of the carrier and/or the biologically active payload.

[00136] A bifunctional linker having one functional group reactive with a group on the carrier and another group reactive on the one or more payloads can be used to form the desired conjugate. Alternatively, derivatization can involve chemical treatment of the targeting moiety. Procedures for generation of, for example, free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are known (See U.S. Pat. No. 4,659,839).

X. Chemical conjugation or complexation of the payload to the carrier

[00137] In various embodiments, the payload to be delivered to the subject is chemically conjugated to the carrier. Means of chemically conjugating molecules are well known to those of skill. The procedure for conjugating two molecules can vary according to the chemical structure of the agent.

XI. Experimental Methods

[00138] Methods are provided herein for measuring endocytosis and transcytosis, identifying interaction partners for targeting signals within carrier proteins, and for observing the intracellular localization of delivery constructs and other intracellular proteins that may participate in the transcytosis of delivery constructs.

(a) Membrane Trafficking

[00139] A delivery construct may pass through layer of epithelial cells by transcytosis. During transcytosis, the delivery construct interacts with a cell surface protein displayed on the apical 35 (external) surface of the cell. The delivery construct is internalized into the epithelial cell by endocytosis, transported from the apical domain of the cell to the basal domain of the cell, and then released by exocytosis across the basal plasma membrane. A delivery construct that is transported across an epithelial cell by transcytosis must avoid being targeted to the lysosome, where it would be degraded, or being recycled back to the apical surface.

[00140] Endocytosis and transcytosis of a delivery construct can be tested by any method known by one of skill in the art, without limitation. In various embodiments, endocytosis activity can be tested by assessing the ability of a delivery construct to enter a polarized or non-polarized cell to which it binds. In cases of an mART-derived carrier, and without intending to be bound to any particular theory or mechanism of action, it is described herein that the transcytosis function that allows a delivery construct to pass through a polarized epithelial cell and the function to enter non-polarized cells can reside in the same domain or region, i.e., Domain I. Thus, the delivery construct’s ability to enter the cell can be assessed, for example, by detecting the physical presence of the construct in the interior of the cell. For example, the delivery construct can be labeled with, for example, a fluorescent marker, and the delivery construct exposed to the cell. Then, the cells can be washed, removing any delivery construct that has not entered the cell, and the amount of label remaining in the cell(s) can be determined. Detecting the label within these cells, e.g., using microscopy, can indicate that the delivery construct has entered the cell.

[00141] The delivery construct’s transcytosis ability can be tested by assessing a delivery construct’s ability to pass through a polarized epithelial cell. For example, the delivery construct can be labeled with, for example, a fluorescent marker (e.g., RFP) and contacted to the apical membranes of a layer of epithelial cells. In another example, the delivery construct can be detected using antibodies (e.g., monoclonal and/or polyclonal antibodies) directed against the delivery construct, or a portion thereof such as a mART-derived carrier or a payload.

Fluorescence detected on the basolateral side of the membrane formed by the epithelial cells

(e.g., a basolateral chamber as illustrated in FIG.10 or the lamina propria in in vivo experiments) indicates that the transcytosis capabilities of the carrier are intact.

[00142] Transcytosis can be tested in vitro using a transwell chamber as depicted in FIG. 10. Epithelial cells are grown on the upper surface of a permeable membrane. Once the cells have grown to confluence, formation of an epithelial barrier can be detected by measuring electrical resistance. Apical to Basal transcytosis (vectorial transport) can be measured by adding a delivery construct to the apical chamber and observing its appearance over time in the basolateral chamber (and vice-versa for Basal to Apical transcytosis).

[00143] In vivo transcytosis can be tested using male Wistar rats. Male Wistar rats can be housed 3-5 per cage with a 12/12 h light/dark cycle and can be 225-275 g (approximately 6-8 36 weeks old) when placed on study. Experiments can be conducted during the light phase using a non-recovery protocol that uses continuous isoflurane anesthesia. A 4-5 cm midline abdominal incision that exposes mid-jejunum regions can be conducted. Stock solutions at 3.86xl0⁵ M of test articles can be prepared in phosphate buffered saline (PBS), and 50 pL (per 250 g rat) can be administered by intraluminal injection (ILI) using a 29-gauge needle. The injection site mesentery can then be marked with a permanent marker. At study termination, a 3-5 mm region that captured the marked intestine segment can be isolated and processed for microscopic assessment.

(b) Identification of mART Carrier Interacting Partners (e.g., Proteins)

[00144] mART interacting partners (e.g., receptors, enzymes, etc.) can be identified by pull down assays. Partners that interact with mARTs in specific vesicular compartments can be identified by isolating the vesicular compartment and then performing a pull-down assay. Features of mARTs that interact with partners can be identified by comparing the partners that bind to mART variants with or without the feature. Putative interaction partners can be further analyzed using methods such as surface plasmon resonance, in vitro transcytosis studies using polarized Caco-2 human intestinal epithelial cells with a genetic knockdown of a putative interaction partner or other target, and in vivo transcytosis studies where mART polypeptide elements and specific receptors can be co-localized in established vesicular structures. A transcytosis process can involve elements that are normally restricted within specific vesicular elements of polarized intestinal epithelial cells but can be recruited or “hijacked” by, e.g., mART-derived carriers, to leave the late endosome and avoid lysosomal degradation following release from the cell into a basolateral compartment (e.g., via apical recycling mechanisms, apical receptor-mediated exocytosis, etc.).

(c) Measuring co-localization of carriers with cellular proteins

[00145] Co-localization of a carrier or carrier-payload complex or payload described herein with one or more cellular macromolecules, e.g., proteins, can be detected by fluorescence microscopy. For example, a mART-derived carrier can be applied to the apical membrane of a polarized epithelial cell(s) (e.g., Caco-2) or to intestinal epithelial tissue. Following endocytosis (e.g., receptor-mediated endocytosis), the uptake of the carrier into the cell can be determined by fluorescence microscopy, e.g., by using labeled anti-mART carrier antibodies or dye-labeled carriers, or by using anti-payload antibodies. Samples or tissue sections can further be stained with markers specific for cellular proteins such as Rab7 or Rabl 1. Various image analysis techniques can then be used to determine the relative position of the carrier to the cellular macromolecule.

XII. Polynucleotides Encoding Carriers, Payloads, and Fusion Molecules 37 [00146] In another aspect, the disclosure provides polynucleotides comprising a nucleotide sequence encoding a carrier, a payload (e.g., a heterologous payload), and non-naturally occurring fusion molecules. These polynucleotides are useful, for example, for making a carrier, a payload (e.g., a heterologous payload), and fusion molecules. In yet another aspect, the disclosure provides an expression system that comprises a recombinant polynucleotide sequence encoding a carrier, e.g., a mART-derived carrier, and a polylinker insertion site for a polynucleotide sequence encoding a payload. In various embodiments, the expression system can comprise a polynucleotide sequence that encodes a cleavable linker so that cleavage at the cleavable linker separates a payload encoded by a nucleic acid inserted into the polylinker insertion site from the remainder of the encoded fusion molecule. Thus, in embodiments where the polylinker insertion site is at an end of the encoded construct, the polynucleotide comprises one nucleotide sequence encoding a cleavable linker between the polylinker insertion site and the remainder of the polynucleotide. In embodiments where the polylinker insertion site is not at the end of the encoded construct, the polylinker insertion site can be flanked by nucleotide sequences that each encode a cleavable linker.

[00147] Various in vitro methods that can be used to prepare a polynucleotide encoding a carrier, e.g., a mART-derived carrier, payload, or fusion molecules of the disclosure include, but are not limited to, reverse transcription, the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3 SR) and the QP replicase amplification system (QB).

[00148] Guidance for using these cloning and in vitro amplification methodologies are described in, for example, U.S. Pat. No. 4,683,195; Mullis et ah, 1987, Cold Spring Harbor Symp. Quant. Biol. 51:263; and Erlich, ed., 1989, PCR Technology, Stockton Press, NY. Polynucleotides encoding a fusion molecule or a portion thereof also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the desired polynucleotide under stringent, moderately stringent, or highly stringent hybridization conditions. [00149] Construction of nucleic acids encoding the carriers, payloads, or fusion molecules of the disclosure can be facilitated by introducing an insertion site for a nucleic acid into the construct.

[00150] Further, the polynucleotides can also encode a secretory sequence at the amino terminus of the encoded carrier, payload, or fusion molecule. Such constructs are useful for producing the carriers, payloads, or fusion molecules in mammalian cells as they simplify isolation of the immunogen.

[00151] Furthermore, the polynucleotides of the disclosure also encompass derivative versions of polynucleotides encoding a carrier, payload, or fusion molecule. For example, derivatives can 38 be made by site-specific mutagenesis, including substitution, insertion, or deletion of one, two, three, five, ten or more nucleotides, of polynucleotides encoding the fusion molecule.

Alternatively, derivatives can be made by random mutagenesis.

[00152] Accordingly, in various embodiments, the disclosure provides a polynucleotide that encodes a carrier, payload, or fusion molecule. The carrier, payload, or fusion molecule can comprise a modified carrier and a payload to be delivered to a subject; and, optionally, a cleavable linker. Cleavage at the cleavable linker can separate the payload from the remainder of the fusion molecule. The cleavable linker can be cleaved by an enzyme that is present at a basal- lateral membrane of a polarized epithelial cell of the subject or in the plasma of the subject. [00153] In various embodiments, the polynucleotide hybridizes under stringent hybridization conditions to any polynucleotide of this disclosure. In further embodiments, the polynucleotide hybridizes under stringent conditions to a nucleic acid that encodes any carrier, payload, or fusion molecule of the disclosure.

[00154] In still another aspect, the disclosure provides expression vectors for expressing the carriers, payloads, or fusion molecules. Generally, expression vectors can be recombinant polynucleotide molecules comprising expression control sequences operatively linked to a nucleotide sequence encoding a polypeptide. Expression vectors can readily be adapted for function in prokaryotes or eukaryotes by inclusion of appropriate promoters, replication sequences, selectable markers, etc. to result in stable transcription and translation or mRNA. Techniques for construction of expression vectors and expression of genes in cells comprising the expression vectors are well known in the art. See, e.g., Sambrook et ah, 2001, Molecular Cloning— A Laboratory Manual, 3.sup.rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., and Ausubel et ah, eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[00155] The expression vectors can contain expression and replication signals compatible with the cell in which the carriers, payloads, or fusion molecules are expressed. The expression vectors can be introduced into the cell for expression of the carriers, payloads, or fusion molecules by any method known to one of skill in the art without limitation. The expression vectors can also contain a purification moiety that simplifies isolation of the carrier, payload, or fusion molecule.

[00156] In yet another aspect, the disclosure provides a cell comprising an expression vector for expression of the carriers, payloads, or fusion molecules, or portions thereof. The cell can be selected for its ability to express high concentrations of the carrier, payload, or fusion molecule to facilitate purification of the protein. In various embodiments, the cell is a prokaryotic cell, for example, E. coli. As described, e.g., in the examples, the carriers, payloads, and fusion molecules 39 can be properly folded and can comprise the appropriate disulfide linkages when expressed in E. coli.

[00157] In other embodiments, the cell is a eukaryotic cell. Useful eukaryotic cells include yeast and mammalian cells. Any mammalian cell known by one of skill in the art to be useful for expressing a recombinant polypeptide, without limitation, can be used to express the carriers, payloads, or fusion molecules. For example, Chinese hamster ovary (CHO) cells can be used to express the carriers, payloads, or fusion molecules.

[00158] The carrier, payloads, or fusion molecules of the disclosure can be produced by recombination, as described below. However, the carrier, payloads, or fusion molecules can also be produced by chemical synthesis using methods known to those of skill in the art.

[00159] Methods for expressing and purifying the carriers, payloads, and fusion molecules of the disclosure are described extensively herein, e.g., in the examples below. Generally, the methods can rely on introduction of an expression vector encoding the carrier, payload, and/or fusion molecule to a cell that can express the carrier, payload, and/or fusion molecule from the vector. The carrier, payload, and/or fusion molecule can then be purified for administration to a subject, e.g, in the treatment of diseases and conditions for which use of the one or more payloads contained in such formulations is indicated.

XIII. Use of the Compositions for Oral or Pulmonary Delivery

[00160] The compositions, e.g., particle, e.g., microparticle or nanoparticle compositions, e.g., microparticle pharmaceutical compositions disclosed herein can be used as drug substance in a pill or tablet for oral delivery of the payload, e.g., biologically active payload, to an individual, e.g., in the treatment of diseases and conditions for which use of the one or more payloads contained in such formulations is indicated.

[00161] The compositions, e.g., microparticle or nanoparticle pharmaceutical compositions can be formulated for oral delivery. The pharmaceutical compositions formulated for oral administration can be resistant to degradation in the digestive tract.

[00162] In some cases, pharmaceutical compositions formulated for oral administration take advantage of the carrier's ability to mediate transcytosis across the gastrointestinal (GI) epithelium. Oral administration of these pharmaceutical compositions can result in absorption of the carrier and payload (e.g. as a fusion molecule) through polarized epithelial cells of the digestive mucosa, e.g., the intestinal mucosa, followed by release of the payload, e.g., one or more payloads at the basolateral side of the mucous membrane.

[00163] Pulmonary administration of these pharmaceutical compositions can result in absorption of the carrier and payload through polarized epithelial cells of the lungs and airways. [00164] Once transported across the epithelium, the compositions, e.g., microparticle or 40 nanoparticle pharmaceutical compositions of the disclosure can exhibit extended half-life in serum, that is, the payload, e.g., biologically active payload, (e.g., of the fusion molecules) can exhibit an extended serum half-life compared to the payload, e.g., biologically active payload, in its non-fused state. The oral formulations of the pharmaceutical compositions of the present disclosure can be prepared so that they are suitable for transport to the GI epithelium and protection of the carrier, payload, or fusion molecule in the stomach. Such formulations can include carrier and dispersant components and can be in any suitable form, including aerosols

(for oral or pulmonary delivery), syrups, elixirs, tablets, including chewable tablets, hard or soft capsules, troches, lozenges, aqueous or oily suspensions, emulsions, cachets or pellets granulates, and dispersible powders. In various embodiments, the pharmaceutical compositions are employed in solid dosage forms, e.g., tablets, capsules, or the like, suitable for simple oral administration of precise dosages.

[00165] In another aspect, the present disclosure relates to methods of orally administering the pharmaceutical compositions of the disclosure. Oral administration of the microparticle pharmaceutical composition can result in absorption of the carrier, payload, or fusion molecule through polarized epithelial cells of the digestive mucosa, e.g., the intestinal mucosa, followed by (in some cases) cleavage of the fusion molecule and release of the one or more payloads at the basolateral side of the mucous membrane. The one or more payloads can then be transported directly to the liver via the hepatic portal vein. Thus, when the one or more payloads exerts a biological activity in the liver, such as, for example, activities mediated by one or more payloads binding to its cognate receptor, the one or more payloads is believed to exert an effect in excess of what would be expected based on the plasma concentrations observed in the subject, i.e., oral administration of the carrier, payload, or fusion molecule can deliver a higher effective concentration of the delivered one or more payloads to the liver of the subject than is observed in the subject's plasma.

XIV. Particles

[00166] The compositions can be in the form of particle, e.g., a microparticle or a nanoparticle and can be generated, e.g., by spraying drying and/or lyophilization. Methods provided herein include administering the compositions to a subject. In some cases, the compositions can be formulated to pass through the acidic environment of the stomach intact (e.g., the particles can be acid-resistant microparticles). Particles can penetrate through mucus samples, a thin mucus layer over cultured cells, and a healthy mucus layer over epithelial cells in vivo. The particles can become soluble at pH 5-7 and, e.g., can support binding or transport in a reconstituted mucus layer assessed in a passage study using cultured endothelial tissue layers or through intestinal tissue in either in vitro or in vivo models. In some cases, the compositions provided herein can be 41 formulated for delivery to a subject by other routes, e.g., respiratory delivery.

[00167] Also provided herein are methods and compositions comprising a carrier capable of entering a polarized epithelial cell or transcytosing across a polarized epithelial cell and a heterologous payload, wherein a molar ratio of the heterologous payload to the carrier is greater than 1:1.

[00168] In some instances, a carrier is indirectly and non-covalently coupled to a payload. In such instances, particles (e.g., liposomes, microparticles, nanoparticles, metallic nanoparticles, polymer-based nanoparticles, etc.) can be loaded (e.g., on the inside and/or on the surface of the particle) with payload molecules (e.g., IL-10, IL-22, GLP-1, etc.), and carriers, e.g., Cholix derived or PE derived carrier molecule(s) can be coupled to such nanoparticles (e.g., onto its surface).

[00169] In some cases, the particle (e.g., nanoparticle) can release the payload following transcytosis or intracellular delivery. In cases where the particle (e.g., nanoparticle) is transported across epithelial cells, the released payload can bind to receptors within submucosal tissue (e.g., lamina propria) and/or can enter the systemic circulation and thus provide a certain function (e.g., a therapeutic or diagnostic function) systemically. In other cases, where a particle (e.g., nanoparticle) releases the payload inside an epithelial cell, the payload (e.g., a nucleic acid) may provide certain intracellular functions, e.g., production of transgenes within these cells, modulation of gene expression, etc.

[00170] A particle composition, e.g., a microparticle pharmaceutical composition can be used as drug substance for pulmonary delivery of a payload, e.g., biologically active payload. Pulmonary delivery methods may include nebulization or dry powder inhalation.

[00171] Particle, e.g., microparticle or nanoparticle pharmaceutical compositions can be formulated for pulmonary delivery. Pharmaceutical compositions formulated for pulmonary administration can be readily nebulized or aerosolized. In some cases, pharmaceutical compositions formulated for pulmonary administration take advantage of the carrier's ability to mediate transcytosis across the pulmonary epithelium. Intranasal administration can be used for pulmonary delivery and can include snorting or sniffing a powder.

XV. Methods of Use

[00172] Provided herein, in some embodiments, are delivery constructs comprising a carrier coupled to a heterologous payload. The carriers provided herein can be used to transport such payload (e.g., a therapeutic payload) to various locations inside an epithelial cell such as the apical side (e.g., an apical recycling system), the basal side, and/or supranuclear compartment(s). Delivery across a polarized gut epithelium can include delivery to submucosal compartments 42 (e.g., lamina propria and/or other submucosal intestinal compartments) and/or systemic circulation (e.g., via the hepatic portal system).

[00173] The high flux transport capacities of carriers provided herein across intact epithelial barriers (e.g., a polarized gut epithelium) can be used to deliver therapeutic and/or diagnostic payload molecules to a subject in need thereof (e.g., a human or a rodent). For example, delivery of therapeutic payload to submucosal compartments, e.g., the lamina propria, can allow for treatment and/or diagnosis of diseases or conditions located at and/or originated from such locations in the GI tract, whereas systemic delivery of payload can be used to provide therapeutically effective concentrations in various cell(s), tissue(s), or organ(s) within an organism.

[00174] The methods and compositions, e.g., pharmaceutical compositions, of the present disclosure can be used to treat diseases or conditions, e.g., medical conditions. The methods and compositions can be amenable for oral and/or intra-nasal formulation and delivery. The disease or condition can be an immunologic disease, a metabolic disease, or a central nervous system (CNS) disease. “Metabolic diseases or disorders” can refer to a combination of medical disorders that, when occurring together, increase the risk of diabetes and atherosclerotic vascular disease, e.g. heart disease and stroke. Defining medical parameters for the metabolic syndrome include diabetes mellitus, impaired glucose tolerance, raised fasting glucose, insulin resistance, urinary albumin secretion, central obesity, hypertension, elevated triglycerides, elevated LDL cholesterol and reduced HDL cholesterol.

[00175] Diseases that can be treated using a delivery construct of this disclosure can include inflammatory diseases, autoimmune diseases, cancer, metabolic diseases, neurodegenerative diseases and neurological diseases, viral disease or infections, and cardiovascular disease.

[00176] Moreover, once transported across the GI epithelium, the fusion molecules of the disclosure will exhibit extended half-life in serum, that is, the one or more payloads of the fusion molecules can exhibit an extended serum half-life compared to the one or more payloads in its non-fused state, and oral administration of the fusion molecule can deliver a higher effective concentration of the delivered one or more payloads to the liver of the subject than is observed in the subject's plasma.

[00177] The constructs of the present disclosure can reduce the sensitivity of the payload to proteolytic destruction, aid in chimera refolding, and improve chimera stability during storage.

As such, the fusion molecules can be used in the preparation of a new class of pharmaceutical compositions for oral administration of biologically active therapeutic agents.

[00178] Furthermore, a delivery construct can be administered as a pharmaceutical composition to a subject in need thereof. A delivery construct herein can be formulated into a pharmaceutical 43 composition for increased therapeutic efficacy. For example, a delivery construct can be formulated such that it is being released at specific location(s) in or around the GI tract of a subject. In some instances, a delivery construct can be formulated to increase its biological activity for engaging immune cells in the various part in or around the GI tract, such as the ileum.

[00179] A delivery construct can be administered via various administration routes. In some cases, administration includes oral administration of the delivery construct. In some instances, a delivery construct is orally administered as a tablet or a capsule.

EXAMPLES

[00180] The following illustrative examples are representative of embodiments of compositions and methods described herein and are not meant to be limiting in any way.

Example 1 — Exotoxin A from Aeromonas species

[00181] Members of the diphthamide-specific class of mono-ADP-ribosyl transferases (mART), which include the diphtheria toxin (DT), Pseudomonas exotoxin A (PE) and Vibrio cholerae cholix (Chx) can be potent bacterial toxins that specifically modify the diphthamide residue of eukaryotic ribosomal elongation factor 2 (eEF2), which plays an essential role in protein synthesis. These toxins catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) onto the diphthamide, which results in inhibition of protein synthesis and thus death of the host eukaryotic cells.

[00182] PE and cholix are thought to share a similar structure and mechanism of action. They consist of 66-68 kDa A-B type toxins arranged in three functional domains (I-III) that can support receptor binding, translocation and catalytic activity, respectively. In order to reach its cytosolic destination, PE and cholix have been shown to bind to LRP1 (low density lipoprotein receptor-related protein 1) at the surface of target cells, which is followed by receptor-mediated endocytosis. The toxins can then be activated by the furin protease into di-chain molecules and be retrogradely transported to the endoplasmic reticulum (ER). The furin-generated carboxy- terminal fragment, which includes the enzymatic domain, can then be next translocated to the cytosol, where it can ADP-ribosylate eEF2.

[00183] Aeromonas exotoxin, AE, has been identified as one of the main virulence factors promoting pathogenesis of the polymicrobial infection. Aeromonas are Gram-negative, facultative anaerobic bacteria, and can be found in aquatic environments. An increased resistance to water treatment and antibiotics has made Aeromonas species into emerging human pathogens, particularly in areas hit by natural disasters such as hurricanes and tsunamis. They are thought to be responsible for a wide range of human diseases that include intestinal and extraintestinal pathologies and can be associated with acute gastroenteritis, skin and soft tissue infections but can also be observed to cause more systemic conditions such as septicaemia and meningitis. 44 Aeromonas can cause haemorrhagic septicaemia in fish, which is associated with high mortality and thus severe economic losses in aquacultures. Aeromonas pathogenesis can be promoted by an arsenal of virulence factors including several exotoxins, such as the cytotoxic enterotoxins Act and aerolysin, as well as extracellular enzymes and secretion systems.

[00184] AE is conserved across several pathogenic Aeromonas species. The NCBI database was screened for exotoxin A homologues in Aeromonas. Using PSI-BLAST, a total of 51 protein sequences were found that displayed at least 60.3% homology to PE (UniProtKB - PI 1439) with an E-value below 0.001. The dataset includes exotoxin A from several species including A. hydrophila and A. dhakensis , which share over > 95.5% identity across 40 entries. FIG. 1A. An additional closely related cluster with > 85% identity was also found in A. salmonicida , A. piscicola , and A. bestiarum. All of these species have been associated with human or zoonotic diseases.

[00185] At the primary sequence level, AE presents all the features of other mART toxins. It can be 626 residue-long (67 kDa), and pairwise sequence alignment with PE and Cholix (FIG. IB) shows conservation of key cysteines and of the furin cleavage site (RQPR (SEQ ID NO:

50)), which can be responsible for activation of the toxin and release of the catalytic fragment.

AE also possesses a KDEL (SEQ ID NO: 59)-like C-terminal sequence (RDEL (SEQ ID NO:

51)) that in PE was shown to be necessary for retrograde transport to the endoplasmic reticulum of intoxicated cells.

[00186] A difference between Cholix and AE and PE is an insert at F70-F86, which protrude from the Cholix surface. FIG. 2A - FIG. 2B. A conserved glutamic acid residue is located within the active site of the toxin domain (Dill). FIG. 3.

Example 2 — Crystal Structure of Exotoxin from Aeromonas species

[00187] The AE construct used for crystallization and structure determination corresponds to a catalytically inactive mutant (E571A). AE (NCBI WP_043170000) [residues 12-626] was codon optimized for Escherichia coli expression, synthesized and cloned into a pET-30 expression vector (GenScript, Piscataway, NJ, USA) with a N-terminal 6 x His-tag (SEQ ID NO: 60) and TEV cleavage site. Expression was carried out in E. coli K12 cells (New England Biolabs, Hitchin, UK) grown in terrific broth medium at 37 °C for approximately 3 h and induced with a 1 mM final concentration of IPTG, overnight at 16 °C. Cells were harvested and frozen at -80 °C. Cell lysis for protein extraction was performed by sonication for 15 min on ice, in 0.02 M TRIS pH 8.0 with 0.2 M NaCl and 25 mM imidazole. The protein was purified by affinity chromatography (HisTrap FF, GE Healthcare, Amersham, UK), and size exclusion (Superdex200, GE Healthcare, Amersham, UK). Sample was kept at 15 mg/mL in 0.05 M MES pH 5.5 with 0.15 M NaCl, and 5% glycerol. 45 [00188] X-ray crystallography. Crystals of AE were grown with 1 mΐ of sample mixed with 1 mΐ of reservoir solution consisting of 12% v/v polyethylene glycol 6000, 0.1 M MES pH 5.5, 0.1 M ammonium acetate, using a hanging drop set-up. Crystals grew within 2-3 days at 16 °C.

Crystals were transferred briefly into a cryo-protectant solution, consisting of the growth condition supplemented with 10% glycerol, before freezing in liquid nitrogen. Diffraction data were collected at station 103 of the Diamond Light Source (Oxon, UK), equipped with an Eiger2

XE 16M detector (Dectris, Baden, Switzerland). A Complete dataset to 2.3 A was collected from a single crystal at 100°K. Raw data images were processed and scaled with DIALS and

AIMLESS using the CCP4 suite 7.0. Molecular replacement was performed with the coordinates of the individual domains from PE (PDB code HKQ) to determine initial phases for structure solution in PHASER. The working models were refined using REFMAC5 and manually adjusted with COOT. Water molecules were added at positions where Fo-Fc electron density peaks exceeded 3s and potential hydrogen bonds could be made. Validation was performed with

MOLPROBITY. Crystallographic data statistics are summarized in Table 1. The atomic coordinates and structure factors (code 6Z5H) have been deposited in the Protein Data Bank

(http://wwpdb.org). Figures were drawn with PyMOL (Schrodinger, LLC, New York, NY,

USA).

Table 1. X-ray crystallography — data collection and refinement statistics.

46

[00189] AE crystallized in the P1211 space group (Table 2) with two identical molecules per asymmetric unit (rmsd of 0.62Ά over 601 Ca atoms). Residues 15-622 were present, with gap in the electron density map observed for segment 228-230, as well as the missing N- (tag) and C- termini (623-626), all in solvent-accessible areas.

[00190] The crystal structure of AE was solved at 2.3 A and presents a three-domain fold similar to other diphthamide-specific mART toxins. The homology with PE at structural elements suggests that AE follows a canonical mechanism of action that ends in ADP- ribosylation of eEF2. The structure of AE can be used to design compounds against diseases caused by Aeromonas and provides a tool for the design of toxin-based therapeutics.

[00191] Overall, the structure of AE presents a tri-domain architecture similar to PE and cholix with root-mean-square deviation (rmsd) of 2.5 and 2.9 A, respectively, and includes 4 conserved disulfide bridges. FIGs. 4A-D, Table 2. Individually the domains show high structural homology with their PE counterpart (rmsd < 2 A). Domain I consists of a core 13-stranded b-jellyroll fold.

It is complemented by domain lb (residues 384-413) that provides two additional b-strands, which run anti-parallel to the b-jellyroll fold and sit at the interface between the three domains. Domain II (residues 264-383), labelled as the translocation domain, presents a compact six a- 47 helices bundle. The catalytic domain (domain III) shows an a/b topology, distinct from typical nucleotide binding folds.

Table 2. Structural homology of AE with other mono-ADP-ribosyl transferases (mART) toxins. Sequence and structure alignments performed with Clustal Omega and TM-align, respectively.

PE Cholix

% ID rmsd (A) [Ca]¹ % ID rmsd (A) [Ca]¹

Overall 63.6 2.5 [593] 35.4 2.9 [575] domain la 54.4 1.9 [239] 33.0 2.3 [224] domain 11 58.8 1.6 [111] 33.6 2.3 [113] domain III 71.9 1.2 [209] 45.8 1.9 [203]

(a) Domain I and Implications for Receptor Binding

[00192] The receptor binding-domain presents a b-jellyroll fold, which is reminiscent of lectin like proteins. A structural search through the PDB database with DALI confirms a distant structural homology (Z score < 8.0) to bacterial sugar-binding proteins and human galectins. The main differences between domain I of AE and its homologues are in flexible, surface-accessible linker regions either side of the core b-sheets. In particular, helix a2 is shorter in AE and positioned in continuity to the b2 strand, compared to PE where it runs perpendicular to b2, whilst it corresponds to a simple coil in cholix. FIG. 4D.

[00193] Another difference are two extended loops in cholix that are not seen in AE and PE between strands b3-b4 and b5-b6, respectively. b4 is the central element of a slightly concave, open surface, which was shown to be implicated in binding to the LRP1 receptor in PE. This strand is conserved in AE with K69 superposing directly onto K57. FIGs. 4C, 4D. In PE, mutation at this position to glutamic acid caused a 100-fold reduction of toxicity toward mouse fibroblasts. In addition, insertion of a dipeptide (Glu-Phe) at position 60 also showed a 500-fold decrease in cytotoxicity that was associated with disruption of receptor binding. Although cholix was shown to recognize LRP1 as well, the amino acid sequence of b4 is not conserved, and the key lysine is there occupied by isoleucine (164), implying a different mechanism of LRP1- binding between PE and cholix. Here, the strong structural homology with PE suggests that AE may recognise and interact similarly with LRP1. However, further experimental work is required to confirm the role of LRP1 or identify other potential cell surface receptors for AE. 48 (b) Structural Elements Involved in Intracellular Trafficking in Target

Cells

[00194] Domain II (residues 264-383) presents a six a-helices bundle similar to that in PE, and which was shown to be involved in translocation of the toxin across membranes, although the mechanism on how this occurs is not fully understood. Importantly, this domain holds a furin protease recognition site identical to the one in PE, corresponding to sequence RQPR (SEQ ID NO: 50) (288-291), with the scissile bond between R291 and G292. This site resides on a well- ordered loop, which protrudes from domain II and is accessible at the surface of AE. FIG 4A. Furin cleavage of PE can occur at acidic pH in vitro, most likely reflecting the endosome conditions (pH < 5.5) where cleavage is believed to occur in vivo. Endoproteolytic activation of the toxin into a di-chain fragment can play a role for toxicity. In AE, furin cleavage would result in a carboxy-terminal fragment of 36kDa that holds the catalytic domain and remains associated with the rest of the toxin via a conserved disulfide bridge between C277 and C299 of SEQ ID NO: 1. This cysteine bond may be reduced downstream of the endocytic pathway with the help of protein disulfide-isomerase (PID). AE is expected to follow a similar intracellular route to PE and cholix through the endocytic pathway where it may be activated by furin and trafficked to the Golgi. In the Golgi, PE can interact with KDEL (SEQ ID NO: 59) receptors via binding of its REDL (SEQ ID NO: 61) C-terminal signal sequence, which can result in the toxin’s retrograde transport to the ER.

[00195] PE can be processed in the early stage of intoxication so that its carboxy terminal lysine (REDLK (SEQ ID NO: 62)) is removed by an extracellular carboxypeptidase that reveals the REDL (SEQ ID NO: 61) signal sequence. This variant of the canonical sequence is enough to bring PE in the ER. AE’s carboxy terminal end, RDEL (SEQ ID NO: 51), does not contain a final lysine and is also closer in sequence to the preferred KDEL motif (SEQ ID NO: 59). FIG. IB. PE engineered to have an RDEL (SEQ ID NO: 51) C-terminal sequence showed up to a 100- fold increase in cytotoxicity compared to the native REDL (SEQ ID NO: 61) sequence, which was linked to a stronger affinity for KDEL (SEQ ID NO: 59) receptors. These sequence differences with PE suggest that AE might be adapted to a more efficient intracellular trafficking. [00196] In the ER, the furin-cleaved toxin fragment undergoes partial unfolding and is exported to the cytosol by retro-translocation. PE may be able to exploit the endoplasmic-reticulum- associated protein degradation (ERAD) system, which involves the Sec61 translocon, while avoiding proteasomal degradation thanks to its low lysine content that averts poly-ubiquitination. AE is devoid of any lysine in its 36 kDa active fragment.

[00197] As it reaches the cytosol, the toxic fragment may refold with the help of host chaperones, such as Hsp90 and Hsc70, as seen with the cholera toxin. Hsp90 was shown to 49 recognize a RPPDEI (SEQ ID NO: 63)-like motif common to several ADP-ribosylating toxins, which includes the C-terminal PE sequence (PPREDL (SEQ ID NO: 64)). However, the proposed Hsp90 recognition motif can involve dual proline, which is not present in AE, and varies in cholix, suggesting both toxins may use an alternative refolding mechanism. See FIG.

2B.

(c) Conclusions

[00198] The diphtheria toxin is a member of the mono-ADP-ribosyltransferase (mART) toxin family and serves as a model to understand the mechanism of toxicity that results in inhibition of eukaryotic protein synthesis. Although they only share limited homology with DT, PE and cholix have a similar enzymatic mechanism, and all can specifically target the diphthamide of eEF2. Both DT and PE can be potent virulence factors involved in the pathophysiology resulting from the associated bacterial infections. The prevalence of toxigenic V. cholerae strains in clinical samples and the effect of the cholix toxin in animal models suggest it is involved in gastrointestinal infection. Cholix is in genomic sequences from V. cholerae samples in aquatic environments and can affect multiple animal species.

[00199] Aeromonas can be found in aquatic environments, and several species have been found to be human pathogens in a broad range of infections, as well as causing diseases in other animals. A Pseudomonas exotoxin A homologue, AE, can play a role in the virulence of Aeromonas hydrophila strains causing necrotising fasciitis, likely by causing tissue damage. [00200] AE isoforms identified in multiple Aeromonas pathogenic species and share some characteristics with other diphthamide-specific mART toxins. The tri-domain crystal structure provides evidence that AE is a fourth member of this toxin family. The structural homology with PE in certain sites of domain I suggests a similar mechanism of cell surface recognition, and future investigation should confirm if LRP1 is a main receptor. AE also has the elements required for intracellular trafficking, including the C-terminal KDEL (SEQ ID NO: 59)-like signal peptide that allows retrograde transport to the ER. The exposed furin cleavage site should promote activation of the toxin in its di-chain form, which is held together by a conserved cysteine bridge that is later reduced to free the active fragment for translocation in the cytosol. Finally, domain III is a conserved element across mART toxins, and comparison with PE indicates that the structure of AE is compatible with eEF2-binding for presentation of the diphthamide to the toxin catalytic site. The NAD+ binding site and catalytic pocket match the mechanism of ADP- ribosylation described previously for diphthamide-specific mART toxins. Altogether, these results suggest that AE inhibits protein synthesis and cell death, thereby promoting bacterial infection.

[00201] The unique features observed in the structure of AE are the presence of a metal- 50 binding site in a non-conserved, negatively charged pocket centrally located between the three domains, and a clear negatively charged cleft that runs between domains I and II. The distinct electrostatic surface potential of these areas suggest that AE may be a useful tool to study biophysical pH-mediated modifications in mART toxins.

Example 3 — Identification of Additional mono-ADP-ribosyl transferase toxins

[00202] mARTs sequences were identified from Chromobacterium haemolyticum (SEQ ID NO: 8), Collimonas fungivorans (SEQ ID NO: 9), Shewanella putrefacians (SEQ ID NO: 10), Janthinobacterium lividum (SEQ ID NO: 11), Serratia fonticola (SEQ ID NO: 12), and Acinetobacter baumannii (SEQ ID NO: 13). Alignments of these sequences are presented in FIG. 5A and 5B. Although the additional mARTs were initially identified by searching for homologs of the DI domains of cholix, PE and AE; the Dill domains of the additional mARTs are more similar to each other and to the Dill domains of cholix, PE and AE than the DI domains. The coverage (%) and percentage of sequence identity of each mART to cholix is shown in Table 3.

Table 3. Homology of mARTs to cholix.

[00203] The three mART domains were originally defined in the structure of Pseudomonas aeruginosa Exotoxin A (PE, PDB 1IKQ, McKay et al (2001) J. Mol. Biol. 314: 823-837). PE has a 25 aa N-terminal leader peptide that is cleaved by signal peptidase, leaving a mature polypeptide with an N-terminal AEEA (SEQ ID NO: 65) sequence that defines the N-terminus of Domain I of PE (Gray et al (1984) PNAS 81 : 2645-2649). A methionine is appended to the N- terminal for recombinant expression by E. coli. This N-terminal methionine is expected to be cotranslationally cleaved by methionine aminopeptidase (Wingfield (2017) Current Protocols in Protein Science 88: 6.14.1-6.14.3). The C-terminus of Domain I of PE ends with HFPE (SEQ ID NO: 66). The domain boundaries of cholix were defined by aligning the cholix sequence and structure to PE. The domain boundaries of the mARTs discovered according to their sequence similarity to PE and cholix were defined by aligning their sequences to cholix and PE (see FIG. 5), as shown in Table 4. 51 Table 4. mART domains

[00204] The mARTs sequences of Pseudomonas aeruginosa and Acinetobacter baumannii are highly similar to each other, with the notable exception that the Acinetobacter baumannii does not have an active site glutamate residue. FIG. 6A. Domain I of PE can efficiently carry heterologous cargo across airway (or kidney) epithelia, suggesting a similar function for the Acinetobacter mART. A phylogenetic tree comparing Domain I of the mARTs revealed that the mARTs of Chromobacterium and Janthinobacterium are highly similar to each other and more distantly related to Cholix, suggesting that they can mediate transcytosis across gastric epithelia. FIG. 6B.

[00205] Alignments of the mARTs yielded consensus sequences for the full-length polypeptides (SEQ ID NO: 15) and Domain I (SEQ ID NO: 25). The alignments also show conserved features in Domain I, including a two pairs of cysteines that may form disulfide bonds, a GVLHYSM motif (residues 43-49 of SEQ ID NO: 25), an WLVPIG motif (residues 119-124 of SEQ ID NO: 25), and a furin cleavage motif RQKRWSEW (residues 201-208 of SEQ ID NO:

25). Additional conserved features shown in FIG. 5B include beta strands comprising GILaaSMh, slpWhVPl followed by a P, and spIKlfh preceeded by G, at positions corresponding to 40-49, 98-105, and 111-117, respectively, of SEQ ID NO: 18; and sequence motifs comprising sPlYol, Rp+RWscW, and hYNYlsQppCp at positions corresponding to 130-135, 183-190, and 205-215, respectively, of SEQ ID NO: 18.

[00206] As previously noted for AE, cholix and PE, although the mART Domain I sequences from different genera have relatively low sequence homology, they share common structural features including conserved patterns of predicted secondary structures. FIG. 7. The Domain I structures of the mARTs all have, or are predicted by homology modeling to have, a core 13- stranded b-jellyroll fold, similar to the structure of galectin 3 (PDB 2NN8). FIGs. 8A-8I.

Example 4 — Expression and Purification of catalytically inactive mARTs

[00207] The catalytically inactive mARTs have a Glutamate to Alanine mutation analogous to 52 a previously described catalytically inactive cholix mutant (Jorgensen et al, J Biol Chem (2008)

283(16): 10671-8). SEQ ID NOs: 27, 29, 31, 33, 35, 37, 39, 41, and 43. Nucleotide sequences encoding mature mARTs were codon-optimized for expression in Esherichia coli. SEQ ID NOs:

26, 28, 30, 32, 34, 36, 38, 40, and 42. The nucleotide sequences were synthesized and cloned into a pET-30 expression vector (GenScript, USA) with sequences encoding N-terminal His₆-tag

(SEQ ID NO: 60) and TEV cleavage sites. The mARTs were expressed in T7 Express Competent

E. coli (New England Biolabs, UK) grown in terrific broth medium at 37 °C for approximately 3 h (until OD600 reached 0.8-1.0 density) and induced with a 1 mM final concentration of IPTG, for overnight expression at 16 °C. Cells were harvested and frozen at -80 °C. Cell lysis for protein extraction was performed by sonication for 15 min on ice, in 0.02 M TRIS pH 8.0 with

0.2 M NaCl and 25 mM imidazole. The proteins were purified by affinity chromatography

(HisTrap FF, GE Healthcare, UK) with elution in 0.02 M TRIS pH 8.0 with 0.2 M NaCl and 250 mM imidazole, then dialised or buffer exchanged in 0.025 M TRIS pH 8.0, with 0.15 M NaCl, and a final size exclusion purification (Superdex200, GE Healthcare, UK) in 0.025 M TRIS pH

8.0, with 0.15 MNaCl, 5% glycerol, 0.5mM TCEP. Samples were concentrated (Vivaspin,

Sartorius, UK) and kept between 10-25 mg/mL for storage at -80 °C. The yields are shown in

Table 5

Table 5. Yields of recombinant, catalytically inactive, mARTs with an N-terminal His₆ tag (SEQ ID NO: 60) and TEV cleavage site.

Example 5 — Transcytosis of full-length mARTs from Aeromonas and Chromobacterium

[00208] Primary intestinal epithelial cells SMI-100 were grown to confluence on transwell filters to assay for transcytosis in vitro according to the method illustrated in FIG. 10. The medium was aspirated and replaced with 100 mΐ of each indicated His-tagged mART (20 pg/ml) in the apical chamber and 500 mΐ of PBS in the basal chamber. After 1 hour at 37° C, the apical 53 and basal solutions were collected and analyzed by Western blotting. The blot was probed with an anti-His tag mAb. FIG. 11A. The results demonstrate apical to basolateral transcytosis of the mARTs from Vibrio cholera (cholix, chx), Pseudomonas aeruginosa (PE), Aeromonas hydrophila (Aer), and Chromobacterium haemolyticum (Hae) across the epithelial layer formed by the SMI- 100 cells.

[00209] Cholix transcytosis was quantified by immunoprecipitating His-tagged cholix from the basal medium with an anti-cholix polyclonal antibody followed by measuring the amount of His- tagged cholix in each immuno-precipitate by ELISA using the anti-His tag mAb. FIG. 11B. The results demonstrate that the mARTs from Pseudomonas , Aeromonas and Chromobacterium did not affect the amount of cholix transcytosis, suggesting that these mARTs homologs do not compete with each other during transcytosis because they interact with different transport proteins as they pass through the cells.

Example 6 — Carriers derived from Domain I of the mART

[00210] The carrier function of Domain I of the mARTs was tested by fusing them to the N- terminus of human growth hormone (hGH) and assaying for transcytosis in rat intestines. In one such experiment, 50 mΐ of a 3.86 x 10⁵ M solution of Cholix266-hGH (Chx-hGH) or Haemolix- hGH (Hmx-hGH) was injected into the lumen intestinal of a rat intestine. After 15 minutes, intestinal tissue was excised, fixed, embedded, stained for hGH, and counterstained with Dapi to identify nuclei. The results demonstrated that the Chx and Hmx carriers were both capable of transporting an hGH payload from the apical surface of intestinal villi to the lamina propria (i.e. within the dotted lines of FIGs. 12A-B). Indeed, the extent of transport mediated by the Chx and Hmx DI domains was comparable. Similar results were observed for Domain I of AE fused to hGH.

Example 7 — Transcytosis of Cholix, Haemolix, and Aeromonas Exotoxin have distinct effects on the redistribution of ER-Golgi trafficking proteins

[00211] COPI is a coat protein primarily used for retrograde vesicular transport from the cis- Golgi to the ER. LMAN1 (ERGIC-53) is a lectin localized to the ER-Golgi intermediate compartment. In resting intestinal epithelial cells, COPI is observed on vesicles throughout the apical domain, including vesicles at the apical surface, and LMAN1 is concentrated in vesicles adjacent to the nucleus (FIG. 13).

[00212] Intestinal epithelial cells were injected with Chx-hGH, Hmx-hGH or AE-hGH to investigate the effect of transcytosis of a cohort of proteins on COPI and LMAN1 localization. Chx-hGH transcytosis caused COPI to redistribute to a tight organized band in the supranuclear region (FIG. 14A), whereas LMAN1 colocalized with Chx-hGH and redistributed to the basal surface of the cell (FIG. 15A). Dramatically different results were observed during Hmx-hGH 54 transcytosis. COPI was no-longer detected at the apical surface, but remained localized throughout the apical domain (FIG. 13B). LMAN1 did not colocalize with Hmx-hGH and its intracellular localization was unaffected by Hmx-hGH transcytosis (FIG. 14B). AE-hGH had an intermediate phenotype, causing redistribution of LMAN1 (FIG. 15C) but not COPI.

[00213] Thus, pulses of cholix, haemolix and Aeromonas exotoxin transcytosis have distinct effects on COPI and LMAN1 localization.

Table 6. Sequences

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

Claims

WHAT IS CLAIMED IS:

1. A carrier-payload complex comprising: a) a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella, Janthinobacterium , Serratia, or Acinetobacter mono- ADP-ribosyl transferase (mART), coupled to b) a heterologous payload.

2. The carrier-payload complex of claim 1, wherein the carrier consists of the transcytosing element.

3. The carrier-payload complex of claim 1 or claim 2, wherein the transcytosing element has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a transcytosing element of any one of SEQ ID NOs: 1-5, 8-15, and 18- 25.

4. The carrier-payload complex of claim 1, wherein the transcytosing element of an Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises or consists of a Domain I of the Aeromonas, Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART.

5. The carrier-payload complex of claim 1, wherein the transcytosing element of an Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of the Aeromonas , Chromobacterium , Collimonas , Shewanella ,

Janthinobacterium , Serratia , or Acinetobacter mART.

6. A carrier-payload complex comprising: a) a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to b) a heterologous payload, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of SEQ ID NO: 16 or SEQ ID NO: 17.

7. The carrier-payload complex of claim 6, wherein the transcytosing element of the mART comprises or consists of a Domain I of a mART. 73

8. The carrier-payload complex of claim 6, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of a mART.

9. A carrier-payload complex comprising: a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelium does not inhibit Vibrio cholerae cholix toxin transcytosis across the epithelium.

10. A carrier-payload complex comprising: a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause COPI to redistribute to a perinuclear region of the epithelial cell.

11. A carrier-payload complex comprising: a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause LMAN1 to redistribute to a basal domain of the epithelial cell.

12. The carrier-payload complex of any one of claims 9-11, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of a Pseudomonas aeruginosa exotoxin.

13. The carrier-payload complex of any one of claims 1-12, wherein the carrier further comprises a Domain III of the mART.

14. The carrier-payload complex of claim 13, wherein the Domain III of the mART has mono-ADP-ribosyl transferase activity.

15. The carrier-payload complex of claim 13, wherein the Domain III of the mART does not have mono-ADP-ribosyl transferase activity.

16. The carrier-payload complex of claim 15, wherein the Domain III of the mART comprises an inactivating mutation of an active site amino acid corresponding to Glutamate571 of SEQ ID NO: 1.

17. The carrier-payload complex of any one of claims 1-16, wherein the carrier comprises a core 13 -stranded b-jellyroll fold.

18. The carrier-payload complex of claim 17, wherein the 13 -stranded b-jellyroll fold comprises a disulfide bond between cysteine residues at positions corresponding to Cysteinel 1 and Cysteinel5 of SEQ ID NO: 18. 74 The carrier-payload complex of any one of claims 17-18, wherein the 13-stranded b- jellyroll fold comprises a disulfide bond between cysteine residues at positions corresponding to Cysteine 197 and Cysteine214 of SEQ ID NO: 18. The carrier-payload complex of any one of claims 17-19, wherein the 13-stranded b- jellyroll fold comprises a beta strand comprising positions corresponding to 41-48 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises GILaaSMh, wherein

1 indicates aliphatic residues I, L, and V, a indicates aromatic residues F, H, W, and Y, and h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y. The carrier-payload complex of any one of claims 17-20, wherein the 13-stranded b- jellyroll fold comprises a beta strand comprising positions corresponding to 98-105 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises slpWhVPl, wherein the position corresponding to 106 of SEQ ID NO: 18 is a G, wherein s indicates small residues A, C, D, G, N, P, S, T, and V,

1 indicates aliphatic residues I, L, and V, p indicates polar residues C, D, E, H, K, N, Q, R, S, and T, and h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y. The carrier-payload complex of any one of claims 17-21, wherein the 13-stranded b- jellyroll fold comprises a beta strand comprising positions corresponding to 111-117 of SEQ ID NO: 18, wherein the sequence of the beta strand comprises spIKlfh, wherein position corresponding to 110 of SEQ ID NO: 18 is a P, wherein s indicates small residues A, C, D, G, N, P, S, T, and V, p indicates polar residues C, D, E, H, K, N, Q, R, S, and T,

1 indicates aliphatic residues I, L, and V, f indicates F and S, and h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y. The carrier-payload complex of any one of claims 17-22, wherein the 13-stranded b- jellyroll fold comprises a sequence at positions corresponding to 130-135 of SEQ ID NO: 18, 75 wherein the sequence comprises sPlYol, wherein s indicates small residues A, C, D, G, N, P, S, T, and V,

1 indicates aliphatic residues I, L, and V, and o indicates alcohol residues S and T. The carrier-payload complex of any one of claims 17-23, wherein the 13-stranded b- jellyroll fold comprises a sequence at positions corresponding to 183-190 of SEQ ID NO: 18, wherein the sequence comprises Rp+RWscW, wherein p indicates polar residues C, D, E, H, K, N, Q, R, S, and T,

+ indicates positively charged residues H, K, and R, s indicates small residues A, C, D, G, N, P, S, T, and V, c indicates charged residues D, E, H, K, and R. The carrier-payload complex of any one of claims 17-24, wherein the 13-stranded b- jellyroll fold comprises a sequence at positions corresponding to 205-215 of SEQ ID NO: 18, wherein the sequence comprises hYNYlsQppCp, wherein h indicates hydrophobic residues A, C, F, G, H, I, K, L, M, R, T, V, W, and Y,

1 indicates aliphatic residues I, L, and V, and s indicates small residues A, C, D, G, N, P, S, T, and V, and p indicates polar residues C, D, E, H, K, N, Q, R, S, and T. The carrier-payload complex of any one of claims 1-25, wherein the carrier is capable of transporting the heterologous payload into a polarized epithelial cell. The carrier-payload complex of claim 26, wherein the carrier is capable of transporting the heterologous payload across the polarized epithelial cell. The carrier-payload complex of claim 27, wherein the carrier is capable of transporting the heterologous payload from an apical surface of a polarized epithelial cell to a basal or lateral surface of the polarized epithelial cell. The carrier-payload complex of any one of claims 26-28, wherein the polarized epithelial cell is a gastrointestinal tract epithelial cell. The carrier-payload complex of any one of claims 26-28, wherein the polarized epithelial cell is a respiratory tract epithelial cell. 76 The carrier-payload complex of any one of claims 1-30, wherein the carrier comprises at least 70%, at least 80%, or at least 90% of a Domain I of the mART. The carrier-payload complex of any one of claims 1-31, wherein the mART is an Aeromonas mART. The carrier-payload complex of claim 32, wherein the Aeromonas mART is an Aeromonas hydrophila mART, an Aeromonas dhakensis mART. an Aeromonas salmonicida mART, an Aeromonas piscicola mART, an Aeromonas bestiarum mART, an Aeromonas hydrophila mART, or an Aeromonas hydrophila mART. The carrier-payload complex of claim 32, wherein the Aeromonas mART is an Aeromonas hydrophila mART. The carrier-payload complex of claim 34, wherein the Aeromonas hydrophila mART comprises SEQ ID NO: 18. The carrier-payload complex of claim 34, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 18. The carrier-payload complex of any one of claims 1-31, wherein the mART is a Chromobacterium mART. The carrier-payload complex of claim 37, wherein the Chromobacterium mART comprises SEQ ID NO: 19. The carrier-payload complex of claim 37, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 19. The carrier-payload complex of any one of claims 1-31, wherein the mART is a Collimonas mART. The carrier-payload complex of claim 40, wherein the Collimonas mART comprises SEQ ID NO: 20. The carrier-payload complex of claim 40, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 20. The carrier-payload complex of any one of claims 1-31, wherein the mART is a Shewanella mART.

The carrier-payload complex of claim 43, wherein the Shewanella mART comprises SEQ ID NO: 21. 77 The carrier-payload complex of claim 43, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 21. The carrier-payload complex of any one of claims 1-31, wherein the mART is a Janthinobacterium mART. The carrier-payload complex of claim 46, wherein the Janthinobacterium mART comprises SEQ ID NO: 22. The carrier-payload complex of claim 46, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 22. The carrier-payload complex of any one of claims 1-31, wherein the mART is a Serratia mART. The carrier-payload complex of claim 49, wherein the Serratia fonticola mART comprises SEQ ID NO: 23. The carrier-payload complex of claim 49, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 23. The carrier-payload complex of any one of claims 1-31, wherein the mART is an Acinetobacter baumannii mART. The carrier-payload complex of claim 52, wherein the Acinetobacter baumannii mART comprises SEQ ID NO: 24. The carrier-payload complex of claim 52, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 24. The carrier-payload complex of any one of claims 1-31, wherein the mART comprises SEQ ID NO: 25. The carrier-payload complex of any one of claims 1-31, wherein the transcytosing element of the mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 25. The carrier-payload complex of any one of claims 1-56 wherein the heterologous payload is selected from the group consisting of a macromolecule, small molecule, peptide, polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, antisense molecule, antibody, DNA, plasmid, vaccine, polymer nanoparticle, and a catalytically-active material. 78 The carrier-payload complex of claim 57, wherein the heterologous payload is a therapeutic payload. The carrier-payload complex of claim 58, wherein the therapeutic payload is a hormone. The carrier-payload complex of claim 59, wherein the therapeutic payload is a human growth hormone. The carrier-payload complex of claim 59, wherein the hormone comprises SEQ ID NO:

49 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 49. The carrier-payload complex of claim 58, wherein the therapeutic payload is a modulator of inflammation in a gastrointestinal tract. The carrier-payload complex of any one of claims 1-62, wherein the heterologous payload is covalently coupled to the carrier. The carrier-payload complex of claim 63, wherein the carrier is synthetically conjugated to the heterologous payload. The carrier-payload complex of claim 63, wherein the carrier is genetically fused to the heterologous payload. The carrier-payload complex of claim 63, wherein the heterologous payload is coupled to a C-terminus of the carrier. The carrier-payload complex of claim 63, wherein the heterologous payload is coupled to an N-terminus of the carrier. The carrier-payload complex of any one of claims 63-67, wherein the carrier is indirectly coupled to the heterologous payload via a spacer. The carrier-payload complex of claim 68, wherein the spacer is a non-cleavable spacer. The carrier-payload complex of claim 69, wherein the spacer comprises up to 15 repeats of GS (SEQ ID NO: 54), GGS (SEQ ID NO: 55), GGGS (SEQ ID NO: 56), GGGGS (SEQ ID NO: 57), GGGGGS (SEQ ID NO: 58), or a combination thereof. The carrier-payload complex of claim 68, wherein the spacer comprises or consists of an amino acid sequence set forth in SEQ ID NO: 47. The carrier-payload complex of claim 68, wherein the spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 48. The carrier-payload complex of any one of claims 1-72, wherein the heterologous payload is non-covalently coupled to the carrier. The carrier-payload complex of any one of claims 1-73, wherein the carrier and heterologous payload are complexed via a nanoparticle. 79 The carrier-payload complex of claim 74, wherein the carrier is covalently-linked to the nanoparticle or is spray-dried on the nanoparticle. The carrier-payload complex of any of claims 1-75, wherein the composition is encapsulated. The carrier-payload complex of claim 76, wherein the encapsulated composition comprises an enteric coating. A polynucleotide encoding the carrier-payload complex of any one of claims 1-77. A vector comprising the polynucleotide of claim 78. A method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier-payload complex comprising: a) a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella, Janthinobacterium , Serratia, or Acinetobacter mono-ADP-ribosyl transferase (mART), coupled to b) a heterologous payload. The method of claim 80, wherein the carrer consists of the transcytosing element. The method of claim 80, wherein the transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises or consists of a Domain I of the Aeromonas, Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. The method of claim 80, wherein the transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of the Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. A method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier-payload complex comprising: a) a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a transcytosing element of an Aeromonas , Chromobacterium , Collimonas , Shewanella , 80 Janthinobacterium , Serratia, or Acinetobacter mono-ADP-ribosyl transferase

(mART), coupled to b) a heterologous payload. wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of SEQ ID NO: 16 or SEQ ID NO: 17. The method of claim 84, wherein the transcytosing element of the mART comprises or consists of a Domain I of the mART. The method of claim 84, wherein the transcytosing element of a mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of a mART. A method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier-payload complex comprising: a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelium does not inhibit Vibrio cholerae cholix toxin transcytosis across the epithelium. A method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier-payload complex comprising: a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause COPI to redistribute to a perinuclear region of the epithelial cell. A method of transporting a heterologous payload into a polarized epithelial cell, comprising contacting the polarized epithelial cell with a carrier-payload complex comprising: a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause LMAN1 to redistribute to a basal domain of the epithelial. The method of any one of claims 87-89, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of a Pseudomonas aeruginosa exotoxin. 81 The method of any one of claims 80-90, wherein the carrier further comprises Domain III of the mART. The method of claim 91, wherein the Domain III of the mART has mono-ADP-ribosyl transferase activity. The method of claim 91, wherein the Domain III of the mART does not have mono-ADP- ribosyl transferase activity. The method of any one of claims 80-93, wherein the heterologous payload is released from the basal surface of the polarized epithelial cell. The method of any one of claims 80-94, wherein the carrier comprises a core 13-stranded b-jellyroll fold. The method of any one of claims 80-95, wherein the carrier comprises at least 70%, at least 80%, or at least 90% of a Domain I of the mART. The method of any one of claims 80-96, wherein the mART is an Aeromonas mART. The method of claim 97, wherein the Aeromonas mART is an Aeromonas hydrophila mART, an Aeromonas dhakensis mART, an Aeromonas salmonicida mART, an Aeromonas piscicola mART, or an Aeromonas bestiarum mART. The method of claim 98, wherein the Aeromonas mART is an Aeromonas hydrophila mART. The method of claim 99, wherein the Aeromonas hydrophila mART comprises SEQ ID NO: 18 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 18. The method of any one of claims 80-96, wherein the mART is a Chromobacterium mART. The method of claim 101, wherein the Chromobacterium mART comprises SEQ ID NO: 19 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 19. The method of any one of claims 80-96, wherein the mART is a Collimonas mART. The method of claim 103, wherein the Collimonas mART comprises SEQ ID NO: 20 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 20. The method of any one of claims 80-96, wherein the mART is a Shewanella mART. The method of claim 105, wherein the Shewanella mART comprises SEQ ID NO: 21 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 21. The method of any one of claims 80-96, wherein the mART is a Janthinobacterium mART. The method of claim 107, wherein the Janthinobacterium mART SEQ ID NO: 22 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 22. 82 The method of any one of claims 80-96, wherein the mART is a Serratia mART. The method of claim 109, wherein the Serratia fonticola mART comprises SEQ ID NO: 23 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 23. The method of any one of claims 80-96, wherein the mART is an Acinetobacter baumannii mART. The method of claim 111, wherein the Acinetobacter baumannii mART comprises SEQ ID NO: 24 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 24. The method of any one of claims 80-96, wherein the mART comprises SEQ ID NO: 25 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 25. A method of orally delivering a heterologous payload to a subject, comprising:

(a) orally administering a carrier-payload complex to the subject;

(b) transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a) a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella, Janthinobacterium , Serratia , or Acinetobacter mono- ADP-ribosyl transferase (mART), coupled to b) a heterologous payload. The method of claim 114, wherein the carrier consists of the transcytosing element. The method of claim 114, wherein the transcytosing element of an Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises or consists of a Domain I of the Aeromonas, Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. The method of claim 114, wherein the transcytosing element of an Aeromonas , Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of the Aeromonas , Chromobacterium , Collimonas ,

Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. A method of orally delivering a heterologous payload to a subject, comprising:

(a) orally administering a carrier-payload complex to the subject; and

(b) transcytosing the carrier-payload complex across a polarized 83 epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising: a) a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to b) a heterologous payload, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of SEQ ID NO: 16 or SEQ ID NO: 17.

119. The method of claim 118, wherein the transcytosing element of a mART comprises or consists of a Domain I of a mART.

120. The method of claim 118, wherein the transcytosing element of a mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of a mART.

121. A method of orally delivering a heterologous payload to a subject, comprising:

(a) orally administering a carrier-payload complex to the subject; and

(b) transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising: a) a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelium does not inhibit Vibrio cholerae cholix toxin transcytosis across the epithelium.

122. A method of orally delivering a heterologous payload to a subject, comprising:

(a) orally administering a carrier-payload complex to the subject; and

(b) transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising: a) a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause COPI to redistribute to a perinuclear region of the epithelial cell. 84 A method of orally delivering a heterologous payload to a subject, comprising:

(a) orally administering a carrier-payload complex to the subject; and

(b) transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, wherein the carrier-payload complex comprises: a carrier comprising: a) a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause LMAN1 to redistribute to a basal domain of the epithelial cell. The method of any one of claims 121-123, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of a Pseudomonas aeruginosa exotoxin. The method of any one of claims 114-124, wherein the carrier further comprises Domain III of the mART. The method of claim 125, wherein the Domain III of the mART has mono-ADP-ribosyl transferase activity. The method of claim 125, wherein the Domain III of the mART does not have mono- ADP-ribosyl transferase activity. The method of claim 127, wherein the Domain III of the mART comprises an inactivating mutation of an active site amino acid corresponding to Glutamate571 of SEQ ID NO: 1. The method of any one of claims 114-128, wherein the carrier comprises a core 13- stranded b-jellyroll fold. The method of any one of claims 114-129, wherein the mART is an Aeromonas mART. The method of claim 130, wherein the Aeromonas mART is an Aeromonas hydrophila mART, an Aeromonas dhakensis mART, an Aeromonas salmonicida mART, an Aeromonas piscicola mART, or an Aeromonas bestiarum mART. The method of claim 131, wherein the Aeromonas mART is an Aeromonas hydrophila mART. The method of claim 132, wherein the Aeromonas hydrophila mART comprises SEQ ID NO: 18 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 18. The method of any one of claims 114- 129, wherein the mART is a Chromobacterium mART. 85 The method of claim 134, wherein the Chromobacterium mART comprises of SEQ ID

NO: 19 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 19. The method of any one of claims 114-129, wherein the mART is a Collimonas mART. The method of claim 136, wherein the Collimonas mART comprises SEQ ID NO: 20 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 20. The method of any one of claims 114- 129, wherein the mART is a Shewanella mART. The method of claim 138, wherein the Shewanella mART comprises SEQ ID NO: 21 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 21. The method of any one of claims 114-129, wherein the mART is a Janthinobacterium mART. The method of claim 140, wherein the Janthinobacterium mART comprises SEQ ID NO:

22 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 22. The method of any one of claims 114-129, wherein the mART is a Serratia mART. The method of claim 142, wherein the Serratia fonticola mART comprises SEQ ID NO:

23 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 23. The method of any one of claims 114-129, wherein the mART is an Acinetobacter baumannii mART. The method of claim 144, wherein the Acinetobacter baumannii mART comprises SEQ ID NO: 24 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 24. The method of any one of claims 114-145, wherein the mART comprises a sequence comprising SEQ ID NO: 25 or at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of SEQ ID NO: 25. A method of treating a condition in a subject, comprising orally administering a carrier- payload complex to the subject; wherein the carrier-payload complex comprises: a) a carrier comprising a transcytosing element having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mono- ADP-ribosyl transferase (mART), coupled to b) a heterologous payload; and wherein the heterologous payload is transported across a gut epithelium. 86 The method of claim 147, wherein the transcytosing element of an Aeromonas,

Chromobacterium , Collimonas , Shewanella, Janthinobacterium , Serratia, or Acinetobacter mART comprises or consists of a Domain I of the Aeromonas, Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. The method of claim 147, wherein the transcytosing element of an Aeromonas, Chromobacterium , Collimonas , Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of the Aeromonas , Chromobacterium , Collimonas ,

Shewanella , Janthinobacterium , Serratia , or Acinetobacter mART. A method of treating a condition in a subject, comprising orally administering a carrier- payload complex to the subject; wherein the carrier-payload complex comprises: a) a carrier comprising a transcytosing element of a mono-ADP-ribosyl transferase (mART), coupled to b) a heterologous payload, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of SEQ ID NO: 16 or SEQ ID NO: 17; and wherein the heterologous payload is transported across a gut epithelium. The method of claim 150, wherein the transcytosing element of a mART comprises or consists of a Domain I of a mART. The method of claim 150, wherein the transcytosing element of a mART comprises at least 100, 150, 180, 200, 220, or 240 contiguous amino acids of a Domain I of a mART. A method of treating a condition in a subject, comprising orally administering a carrier- payload complex to the subject; wherein the carrier-payload complex comprises: a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelium does not inhibit Vibrio cholerae cholix toxin transcytosis across the epithelium; and wherein the heterologous payload is transported across a gut epithelium. A method of treating a condition in a subject, comprising orally administering a carrier- payload complex to the subject; wherein the carrier-payload complex comprises: 87 a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause COPI to redistribute to a perinuclear region of the epithelial cell; and wherein the heterologous payload is transported across a gut epithelium. A method of treating a condition in a subject, comprising orally administering a carrier- payload complex to the subject; wherein the carrier-payload complex comprises: a) a carrier comprising a transcytosing element of a mART, coupled to b) a heterologous payload, wherein transcytosis of the carrier-payload complex across an epithelial cell does not cause LMAN1 to redistribute to a basal domain of the epithelial cell; and wherein the heterologous payload is transported across a gut epithelium. The method of any one of claims 153-155, wherein the transcytosing element has less than 80%, less than 90% or less than 95% amino acid sequence identity to a transcytosing element of a Pseudomonas aeruginosa exotoxin. The method of any one of claims 147-156, wherein the carrier further comprises Domain III of the mART. The method of claim 157, wherein the Domain III of the mART has mono-ADP-ribosyl transferase activity. The method of claim 157, wherein the Domain III of the mART does not have mono- ADP-ribosyl transferase activity. The method of any one of claims 147-159, wherein the heterologous payload is delivered to a lamina propria of an intestinal villus. The method of any one of claims 147-159, wherein the heterologous payload is delivered into systemic circulation. The method of any one of claims 147-161, wherein the heterologous payload is selected from the group consisting of a macromolecule, small molecule, peptide, polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, antisense molecule, antibody, DNA, plasmid, vaccine, polymer nanoparticle, and a catalytically-active material. The method of claim 162, wherein the heterologous payload is a therapeutic payload. The method of claim 163, wherein the therapeutic payload is a growth hormone. The method of claim 164, wherein the growth hormone comprises SEQ ID NO: 49 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 49. 88