Polypeptide conjugates for intracellular delivery of nucleic acids

Abstract

The present disclosure provides for polypeptide conjugates. The polypeptide conjugates disclosed herein comprise a polyarginine peptide and a cyclic cell-penetrating peptide (cCPP) conjugated, directly or indirectly, to the polyarginine peptide. The present disclosure demonstrates that cCPPs conjugated to polyarginine peptides can be used to deliver nucleic acids to the cytosol of cells.

Description

(19) *EP004717776A2* (11) EP 4 717 776 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 01.04.2026 Bulletin 2026 / 14 (21) Application number: 26155045.3 (22) Date of filing: 02.07.2019 (51) International Patent Classification (IPC): C12N 15 / 88 (2006.01) (52) Cooperative Patent Classification (CPC): C12N 15 / 111; A61K 47 / 645; A61K 47 / 6455; C07K 7 / 02; C07K 7 / 06; C07K 7 / 08; C07K 7 / 64; C12N 15 / 88; C07K 2319 / 10; C12N 2310 / 14; C12N 2310 / 3513; C12N 2320 / 32 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 02.07.2018 US 201862692939 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 19831072.4 / 3 817 776 (71) Applicant: Ohio State Innovation Foundation Columbus, OH 43201 (US) (72) Inventors: • PEI, Dehua Columbus, Ohio, 43220 (US) • BUYANOVA, Marina Columbus, Ohio, 43212 (US) • QIAN, Ziqing Wellesley, Massachusetts, 02482 (US) (74) Representative: Berggren Oy P.O. Box 16 Fabianinkatu 21 00101 Helsinki (FI) Remarks: •The complete document including Reference Table(s) and the Sequence Listing(s) can be downloaded from the EPO website •This application was filed on 29‑01‑2026 as a divisional application to the application mentioned under INID code 62. (54) POLYPEPTIDE CONJUGATES FOR INTRACELLULAR DELIVERY OF NUCLEIC ACIDS (57) The present disclosure provides for polypeptide conjugates. The polypeptide conjugates disclosed here- in comprise a polyarginine peptide and a cyclic cell- penetrating peptide (cCPP) conjugated, directly or indir- ectly, to the polyarginine peptide. The present disclosure demonstrates that cCPPs conjugated to polyarginine peptides can be used to deliver nucleic acids to the cytosol of cells. EP 4 71 7 77 6 A 2 Processed by Luminess, 75001 PARIS (FR) Description STATEMENT REGARDING FEDERAL FUNDING

[0001] This inventionwasmadewithgovernment support underAgreementNos.GM1100208andGM122459awarded by the NIH. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Thisapplication claims thebenefit of priority toU.S.ProvisionalApplicationNo. 62 / 692,939, filedJuly 2, 2018, the entire contents of which are incorporated herein by reference in its entirety for all purposes. STATEMENT REGARDING SEQUENCE LISTING

[0003] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing filename: CYPT_013_01WO_SeqList_ST25.txt date created, June 25, 2019, file size ≈ 49 kilobytes. FIELD OF THE INVENTION

[0004] Thepresent disclosure relates to polypeptide conjugates comprisinga cell-penetratingpeptide (CPP), directly or indirectly attached toagroup that binds to a nucleic acid sequencebyelectrostatic interactions. Thegroup that binds to the nucleic acid sequence by electrostatic interactions comprises at least one peptide or polyamine, such as polyarginine peptide. BACKGROUND

[0005] Nucleic acids and their synthetic analogs hold enormous potential as therapeutic agents, especially against targets that are challenging for conventional drug modalities (e.g., intracellular protein-protein interactions and mis- sing / defective proteins caused by genetic mutations). In the classical gene therapy approach, a gene of interest is incorporated into a viral vector or bacterial plasmid anddelivered to tissues / cells to restore normal or correct for pathologic gene expression. More recently, a wide variety of strategies exploiting short oligonucleotides have been explored. For example, antisense oligonucleotides and small interferingRNAs (siRNAs) can be used to specifically knockdown virtually anysingleorgroupofgenes.Splice-switchingoligonucleotides,microRNAs,andanti-microRNAscanalsoenhance target gene expression or modulate / switch mRNA splicing to express the desired gene products. Another potentially very powerful approach is the use of gene-editing platforms (e.g., TALENs and CRISPR / CAS9) to alter the genomic DNA and provideapermanent cureof thedisease.Finally, chemically stabilizedmRNAscanbedirectly delivered intodiseasedcells and tissues and used as templates for protein synthesis. Collectively, these nucleic acid-based approaches greatly expand the space of pharmacologic targets which are otherwise undruggable with conventional drugs.

[0006] Despite their undoubted potential, clinical translation of nucleic acid-based drugs is limited by their poor bioavailability in the target tissues / cells. Becauseof their highmolecularweights andnegative charges (with the exception of a few oligonucleotide analogs), nucleic acids cannot cross the cellular membranes to reach the cell interior. Therefore, these nucleic acid-basedmoleculesmust be delivered into the target tissues / cells by an appropriate delivery system. The present disclosure is directed towards a novel nucleic acid delivery system which can effectively penetrate through the cellular membranes to reach the interior of the cells. SUMMARY OF THE INVENTION

[0007] In various embodiments, the present disclosure provides for polypeptide conjugates comprising: a) a group that binds to a nucleic acid sequence by electrostatic interactions (P) comprising at least one peptide or polyamine; and b) at least one cell-penetrating peptide (CPP); wherein each peptide comprises at least three monomers selected from arginine, arginine-analog, lysine, lysine- analog, histidine, or histidine-analog; wherein the P is conjugated to theCPP through a bond or at least one linker (L); and wherein the polypeptide conjugate is optionally charged. 2 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0008] In some embodiments, the polypeptide conjugate as disclosed herein has a molar ratio of P:CPP ranging from about 30:1 to about 1:2.

[0009] In some embodiments, the polypeptide conjugate as disclosed herein has an averagemolecular weight ranging from about 3 kDa to about 100 kDa.

[0010] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: CPP-L‑[P]n‑L-CPP (I) wherein n is an integer from 1 to 50; and wherein P at each occurrence is same or different.

[0011] In some embodiments of the polypeptide conjugate as disclosed herein, P comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog.

[0012] In some embodiments of the polypeptide conjugate as disclosed herein, P comprises a polyamine selected from a spermidine polymer or a spermine polymer.

[0013] In some embodiments of the polypeptide conjugate as disclosed herein, at least one of the CPP is a cyclic CPP (cCPP). In some embodiments, each CPP is, independently, a cyclic CPP (cCPP). In some embodiments, the cCPP comprises from4 to 14aminoacidmonomers. In other embodiments, each cCPP is, independently, selected fromTable 4.

[0014] In some embodiments of the polypeptide conjugate as disclosed herein, the cCPP is a cyclo(fΦRrRrQ) (SEQ ID NO: 118) peptide or a cyclo(FfΦRrRrQ) (SEQ ID NO: 16), wherein: F is a L-phenylalanine; f is a D-phenylalanine; Φ is an L‑2-naphthylalanine; R is a L-arginine; r is a D-arginine; and Q is a L-glutamine.

[0015] In someembodiments of the polypeptide conjugate as disclosed herein, the pArg comprises at least five arginine monomers or arginine-analog monomers. In some embodiments, the pArg further comprises at least one cysteine monomer.

[0016] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: CPP-L‑[pArg]n‑L-CPP (II) wherein the [pArg]n (SEQ ID NO: 130) is or a charged species thereof.

[0017] In some embodiments of the polypeptide conjugate as disclosed herein, n is an integer 1 to 40.

[0018] In some embodiments of the polypeptide conjugate as disclosed herein, at least one L comprises a divalent optionally substituted group selected from amino acid, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1‑X-R2)z‑, or combinations thereof; wherein each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; 3 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 each X is independently N, S, and O; and z is an integer selected from 1 to 20.

[0019] In some embodiments of the polypeptide conjugate as disclosed herein, at least one L comprises an optionally substituted ‑(O-CH2CH2)z‑ or an optionally substituted ‑(CH2CH2‑O)z‑. In someembodiments, at least one L comprises a divalent 8-amino‑3,6-dioxaoctanoic acid residue. In other embodiments, at least one L comprises a divalent 8-ami- no‑3,6,9-trioxaundecanoic acid residue.

[0020] In some embodiments of the polypeptide conjugate as disclosed herein, at least one L comprises a physiological cleavable group (PCG). In some embodiments, each PCG is, independently, selected from ‑S-S‑, carbonate, thiocarbo- nate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. In other embodiments, each PCG comprises at least one ‑S-S‑.

[0021] In some embodiments of the polypeptide conjugate of formula (I), at least one of the "‑" between L and [P]n represents a bond between two sulfur atoms (disulfide bond). In other embodiments, the "‑" between each L and [P]n represents a bond between two sulfur atoms (disulfide bond).

[0022] In someembodimentsof thepolypeptide conjugatedisclosedherein, eachP, independently, further comprisesat least one group selected from: or wherein the bond to the hydrogen on at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP (or cCPP)

[0023] In someembodimentsof thepolypeptide conjugatedisclosedherein, eachP, independently, further comprisesat least one group selected from: 4 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0024] In some embodiments of the polypeptide conjugate disclosed herein, at least one of the P further comprises In other embodiments, at least one of the P further comprises at least two groups selected form

[0025] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: CPP-L‑([P1]p‑L1)t‑[P]n‑(L2‑[P2]q)t‑L-CPP (III) wherein n, p, and q are each independently an integer from 1 to 50; 5 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 t is each independently 0 or 1; P1 and P2 each comprises at least one peptide or polyamine, wherein each peptide comprises at least three monomers selected fromarginine, arginine-analog, lysine, lysine-analog, histidine, or histidine-analog, wherein P, P1 and P2, at each occurrence, are same or different; and L1 and L2 are each independently absent or L as defined in claim 1, wherein L, L1, and L2, at each occurrence, are same or different.

[0026] In some embodiments of the polypeptide conjugate of formula (III), P, P1 or P2 comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog. In other embodiments, P, P1 or P2 comprises a polyamine selected from a spermidine polymer or a spermine polymer.

[0027] In someembodimentsof thepolypeptideconjugateof formula (III), at least oneof theCPP isacyclicCPP(cCPP). In other embodiments, eachCPP is, independently, a cyclic CPP (cCPP). In one embodiment, the cCPPcomprises from4 to 14 amino acid monomers. In another embodiment, each cCPP is, independently, selected from Table 4.

[0028] In some embodiments of the polypeptide conjugate of formula (III) as disclosed herein, the cCPP is a cyclo(fΦRrRrQ) (SEQ ID NO: 118) peptide or a cyclo(FfΦRrRrQ) (SEQ ID NO: 16) , wherein: F is a L-phenylalanine; f is a D-phenylalanine; Φ is an L‑2-naphthylalanine; R is a L-arginine; r is a D-arginine; and Q is a L-glutamine.

[0029] In some embodiments of the polypeptide conjugate of formula (III), the pArg comprises at least five arginine monomers or arginine-analogmonomers. In one embodiment, the pArg further comprises at least one cysteinemonomer.

[0030] In some embodiments of the polypeptide conjugate of formula (III), n is an integer 1 to 40. In some embodiments of the polypeptide conjugate of formula (III), n is an integer 5 to 40.

[0031] In someembodimentsof thepolypeptide conjugateof formula (III), at least oneof L, L1, or L2 comprisesadivalent optionally substituted group selected from amino acid, polyethylene glycol, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1‑X-R2)z‑, or combinations thereof; each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20.

[0032] In some embodiments of the polypeptide conjugate of formula (III), at least one of L, L1, or L2 comprises an optionally substituted ‑(O-CH2CH2)z‑ or an optionally substituted - (CH2CH2‑O)z‑.

[0033] In someembodimentsof thepolypeptide conjugateof formula (III), at least oneof L, L1, or L2 comprisesadivalent 8-amino‑3,6-dioxaoctanoic acid residue. In some embodiments, at least one of L, L1, or L2 comprises a divalent 8- amino‑3,6,9-trioxaundecanoic acid residue. In other embodiments, at least one of L, L1, or L2 comprises a physiological cleavable group (PCG).

[0034] In some embodiments of the polypeptide conjugate of formula (III) as disclosed herein, each PCG is, indepen- dently, selected from ‑S-S‑, carbonate, thiocarbonate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. In some embodiments, each PCG comprises at least one ‑S-S‑.

[0035] In some embodiments of the polypeptide conjugate of formula (III), at least one of the "‑" between L and ([P1]p‑L1)t, L and [P]n, or L and (L2‑[P2]q)t represents a bond between two sulfur atoms (disulfide bond).

[0036] In someembodiments of the polypeptide conjugate of formula (III), at least one of P, P1 or P2 further comprises at least one group selected from: 6 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or wherein the bond to the hydrogen on at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP.

[0037] In someembodiments of the polypeptide conjugate of formula (III), at least one of P, P1 or P2 further comprises at least one group selected from: 7 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0038] In some embodiments of the polypeptide conjugate of formula (III), at least one of P, P1 or P2 further comprises In other embodiments, at least two of P, P1 or P2 further comprises a group selected form

[0039] In some embodiments of the polypeptide conjugate of formula (III), the pArg further comprises one or more β- alanine monomers. In some embodiments, each pArg comprises, independently, 3, 4, 5, 6, 7, or 8 arginine monomers. In otherembodiments, eachpArgcomprises5argininemonomers. Inoneembodiment, at least onepArg further comprisesa cysteine monomer.

[0040] In some embodiments of the polypeptide conjugate of formula (I), (II), or (III), the pArg is each independently selected from: ‑Cys‑(Arg)x-Cys‑ (SEQ ID NO: 131), ‑Cys-βAla‑(Arg)X‑Cys‑ (SEQ ID NO: 132), ‑Cys‑(Arg)X-βAla-Cys‑ (SEQ ID NO: 133), or ‑Cys-βAla‑(Arg)X-βAla-Cys‑ (SEQ ID NO: 134), wherein x = 3, 4, 5, 6, 7, or 8.

[0041] In some embodiments of the polypeptide conjugate of formula (I), (II), or (III), P is each independently selected from: ‑Cys‑(Arg)x-Cys‑ (SEQ IDNO: 131), ‑Cys-βAla‑(Arg)X‑Cys‑ (SEQ IDNO: 132), ‑Cys‑(Arg)X-βAla-Cys‑ (SEQ IDNO: 133), or ‑Cys-βAla‑(Arg)X-βAla-Cys‑ (SEQ ID NO: 134), ‑Cys‑(polyamine)x-Cys‑, ‑Cys-βAla‑(polyamine)X‑Cys‑, - Cy- s‑(polyamine)X-βAla-Cys‑, ‑Cys-βAla‑(polyamine)X-βAla-Cys‑ (SEQ IDNO: 135), ‑S‑(polyamine)X‑S‑, wherein x =3, 4, 5, 6, 7, or 8.

[0042] In some embodiments, the polypeptide conjugate of formula (I) as disclosed herein has the structure 8 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 , or a charged species thereof.

[0043] In some embodiments, the polypeptide conjugate of formula (I) as disclosed herein has the structure , or a charged species thereof.

[0044] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: CPP-L‑([P]m)s (IV) wherein: s is an integer from 1 to 10; each m is, independently, an integer from 1 to 50; and wherein P at each occurrence is same or different.

[0045] In some embodiments of the polypeptide conjugate of formula (IV), P comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog. In some embodiments, P comprises a polyamine selected from a spermidine polymer or a spermine polymer.

[0046] In some embodiments of the polypeptide conjugate of formula (IV), the CPP is a cyclic CPP (cCPP). In some embodiments, the cCPP comprises from 4 to 14 amino acidmonomers. In other embodiments, the cCPP is selected from Table 4.

[0047] In some embodiments of the polypeptide conjugate of formula (IV), the pArg comprises at least five arginine monomers or arginine-analog monomers. In some embodiments, the pArg further comprises at least one cysteine monomer.

[0048] In some embodiments of the polypeptide conjugate of formula (IV), L comprises a divalent optionally substituted group selected from amino acid, polyethylene glycol, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1-X-R2)z‑, or combinations thereof; each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20.

[0049] In some embodiments of the polypeptide conjugate of formula (IV), L comprises an optionally substituted ‑(O- CH2CH2)z‑ or an optionally substituted ‑(CH2CH2‑O)z‑. In some embodiments, L comprises a divalent 8-amino‑3,6- dioxaoctanoic acid residue. In other embodiments, L comprises a divalent 8-amino‑3,6,9-trioxaundecanoic acid residue. In one embodiment, L comprises a physiological cleavable group (PCG).

[0050] In some embodiments of the polypeptide conjugate of formula (IV), each PCG is, independently, selected from ‑S-S‑, carbonate, thiocarbonate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. In some embodi- ments, each PCG comprises at least one ‑S-S‑.

[0051] In some embodiments of the polypeptide conjugate of formula (IV), L comprises a polythiolamine or a 3,5- bis(mercaptomethyl)benzoyl (Bmb) amide.

[0052] In some embodiments of the polypeptide conjugate of formula (IV), L comprises two or more physiological cleavable groups.

[0053] In some embodiments of the polypeptide conjugate of formula (IV), the "‑" between L and ([P]m)s represents a bond between two sulfur atoms (disulfide bond).

[0054] In some embodiments of the polypeptide conjugate of formula (IV), each P, independently, further comprises at least one group selected from: 9 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or wherein the bond to the hydrogen on one at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP.

[0055] In some embodiments of the polypeptide conjugate of formula (IV), each P, independently, further comprises at least one group selected from: 10 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0056] In some embodiments of the polypeptide conjugate of formula (IV), at least one of the P further comprises In other embodiments, at least one of the P further comprises at least two groups selected from

[0057] In some embodiments of the polypeptide conjugate of formula (IV), s is 2, 3, 4, or 5.

[0058] In some embodiments, the polypeptide conjugate of formula (IV) as disclosed herein has the structure 11 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 , or a charged species thereof.

[0059] In some embodiments, the polypeptide conjugate of formula (IV) as disclosed herein has the structure or a charged species thereof.

[0060] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: cCPP-L‑[P]n‑[P]m‑(L-cCPP)v (V) wherein, cCPP is a cyclic CPP; and n is an integer selected from 1 to 50; m is an integer selected from 0 to 49 provided that the sum of n and m is 50 or less; v is 0 or 1; wherein P at each occurrence is same or different; and wherein when v is 0, the last [P] in [P]m is monovalent.

[0061] In some embodiments of the polypeptide conjugate of formula (V), the "‑" between [P]n and [P]m represents a 12 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 bond between two sulfur atoms (a disulfide bond).

[0062] In some embodiments, the polypeptide conjugate of formula (IV) has the following structure: or a charged species thereof.

[0063] In some embodiments of the polypeptide conjugate of formula (VI), the "‑" between [P] and [P] represents a bond between two sulfur atoms (a disulfide bond).

[0064] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: cCPP-L‑[P]n‑[P]m‑[P]o‑(L-cCPP)v (VII) wherein, cCPP is a cyclic CPP; and n is an integer selected from 1 to 50; m and o, are each independently, an integer from 0 to 49 provided that the sum of n, m, and o is 50 or less; v is 0 or 1; wherein P at each occurrence is same or different; and wherein when v is 0, the last [P] in [P]o is monovalent.

[0065] In some embodiments of the polypeptide conjugate of formula (VII) as disclosed herein, at least one of the "‑" between [P]n and [P]m or [P]m and [P]o represents a bond between two sulfur atoms (a disulfide bond). In other embodiments, the "‑" between [P]n and [P]m and between [P]m and [P]o represents a bond between two sulfur atoms (a disulfide bond).

[0066] In someembodiments, the polypeptide conjugate of formula (VI) as disclosedherein, has the following structure: 13 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 , or a charged species thereof

[0067] In some embodiments of the polypeptide conjugate of formula (VIII) as disclosed herein, at least one of the "‑" between [P] and [P] represents abondbetween twosulfur atoms (adisulfidebond). Inother embodiment, each "‑" between [P] and [P] represents a bond between two sulfur atoms (a disulfide bond).

[0068] The present disclosure also relates to a complex comprising any one of the polypeptide conjugates as disclosed herein and at least one nucleic acid sequence.

[0069] The present disclosure also relates to a cell comprising any one of the polypeptide conjugates as disclosed herein.

[0070] The present disclosure also relates to a cell comprising a complex comprising any one of the polypeptide conjugates as disclosed herein and a nucleic acid sequence.

[0071] The present disclosure also relates to a method of delivering a nucleic acid sequence to a cell, comprising contacting the cell with any one of the complexes as disclosed herein.

[0072] Thepresent disclosure also relates to amethod of delivering anucleic acid sequence to a cell of a subject in need thereof, comprising administering any one of the complexes as disclosed herein.

[0073] The present disclosure also relates to a method of treating a disease or condition in a patient in need thereof, comprising administering any one of the complexed as disclosed herein to the patient. BRIEF DESCRIPTION OF FIGURES

[0074] Figure 1 shows a design concept of polypeptide conjugate of the disclosure. Figure 2 shows an analytical HPLC (high-performance liquid chromatography) trace of polypeptide conjugate synthesized in Example 1. The arrows to CRC and cyclo(fΦRrRrQ) (SEQ ID NO: 118) points to HPLC trace of conjugate after reduction with 10 mM DTT (dithiothreitol). Figure 3 shows 1H NMR (nuclear magnetic resonance) spectrum of the polypeptide conjugate synthesized in Example 1. Figure 4A shows gel-filtration chromatography traces of poly-L-lysine standard. Figure4Bshowsgel-filtrationchromatography tracesof thepolypeptide conjugate synthesized inExample1 (CRC5 - top line at t = 9 min; CRC10 bottom line at t = 9 min). Figure 5A shows the effect of CRC5polymer synthesized in Example 1 onHeLa cells as assayed by theMTT (3‑(4,5- dimethylthiazol‑2-yl)‑2,5-diphenyltetrazolium bromide) test. Figure 5B shows the effect of lipofectamine on HeLa cells as assayed by the MTT Figure 6 shows live-cell confocal microscope images of HeLa cells treated with the CRC5 / 5’-FAM-siLuc complex (Example 1) at 3 µM siRNA in OptiMEM (top) or at 1 µM siRNA in DMEM with 1% FBS, 1% Abs (bottom). Left, GFP channel; Center, DIC; and right, overlap of the above. Figure 7 shows knockdown of luciferase expression in HeLa-Luc cells by CRC5 / siLuc complexes (Example 1, 75 nM), the individual components, and lipofectamine / siLuc complex at 24 h and 48 h after treatment. Figure 8 showsMALDI-TOFMS spectra of CPP12‑(R5)2 (top), CPP12‑(R10)2 (middle), and CPP12‑(R15)2 (bottom). 14 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 Figure 9 shows live-cell confocal microscope images of HeLa cells treated with the CPP9‑(R5)2 / 5’-FAM-siLuc complex (Example 2) at 3 µM siRNA. Center, GFP channel; Right, DIC; and left, overlap of the above. Figure 10 shows knockdown of luciferase expression in HeLa-Luc cells by CPP12‑(Rn)2 / siLuc complexes (Example 2, 75 nM) and lipofectamine / siLuc complex at 48 h after treatment. Figure 11 shows MALDI-TOF MS spectra of (CPP9-R5)2 (top) and (CPP9-R8)2 (bottom). Figure 12 shows live-cell confocal microscope images of HeLa cells treated with the (CPP9-R5)2 / 5’-FAM-siLuc complex (Example 3) at 3 µM siRNA. Center, GFP channel; Right, DIC; and left, overlap of the above. Figure 13 shows knockdown of luciferase expression in HeLa-Luc cells by CPP9-Rn)2 / siLuc complexes (Example 3, 75 nM) and lipofectamine / siLuc complex at 48 h after treatment. Figure 14 shows MALDI-TOF MS spectra of the polypeptide conjugate synthesized according to Example 4. Figure 15 shows the effect of the polypeptide conjugate synthesized according to Example 4 on HeLa cells as assayed by the MTT test. Figure 16 shows live-cell confocal microscope images of HeLa cells treated with the polypeptide conjugate synthesized according to Example 4 / 5’-FAM-siLuc complex at 1 µM siRNA in DMEM with 1% FBS (top) or 3 µM siRNA in OptiMEM (bottom). Center, GFP channel; Right, DIC; and left, overlap of the above. Figure 17 shows knockdown of luciferase expression in HeLa-Luc cells by the polypeptide conjugate of Example 4 / siLuc complexes (Example 4, 75 nM) and lipofectamine / siLuc complex at 48 h after treatment. Figure 18 is a graph of gene expression (%) with cells treated with SiLuc and either Lipo2000 or CRC5. Figure 19 is a graph of gene expression (%) with cells treated with SiGFP and either Lipo2000 or CRC5. DETAILED DESCRIPTION

[0075] All publications, patents and patent applications, including any drawings and appendices therein are incorpo- rated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Definitions

[0076] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0077] Throughout the present specification, the terms "about" and / or "approximately" may be used in conjunction with numerical valuesand / or ranges.The term"about" isunderstood tomean thosevaluesnear toa recitedvalue,aswell as the recited value.

[0078] Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise all values and subranges therein. Thus, the range "from 50 to 80" includes all possible values therein (e.g., 50, 51, 52, 53, 54, 55, 56, etc.) andall possible ranges therein (e.g., 51‑79, 52‑78, 53‑77, 54‑76, 55‑75, 60‑70, etc.). Furthermore, all valueswithin a given rangemaybeanendpoint for the rangeencompassed thereby (e.g., the range 50‑80 includes the ranges with endpoints such as 55‑80, 50‑75, etc.).

[0079] The term "a" or "an" refers to one or more of that entity; for example, "a polypeptide conjugate" refers to one or more polypeptide conjugates or at least one polypeptide conjugate. As such, the terms "a" (or "an"), "one ormore" and "at least one" are used interchangeably herein. In addition, reference to "a polypeptide conjugate" by the indefinite article "a" or "an" does not exclude the possibility that more than one of the polypeptide conjugates is present, unless the context clearly requires that there is one and only one of the polypeptide conjugates.

[0080] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0081] Asusedherein, "treat," "treating," "treatment" andvariants thereof, refers toanyadministrationof thepolypeptide conjugate of the present disclosure that partially or completely alleviates, ameliorates, prevents, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one ormore symptoms or features of a disease or a condition as described herein.

[0082] As used herein, "therapeutically effective" refers to an amount of the polypeptide conjugate or the complex thereof of the present disclosure that can deliver anamount of a therapeutic nucleic acidwhich confers a therapeutic effect on a patient.

[0083] As used herein, "cell penetrating peptide" or "CPP" refers to any peptide which is capable of penetrating a cell membrane. As used herein, "cyclic cell penetrating peptide" or "cCPP" refers to any cyclic peptide which is capable of penetrating a cell membrane.

[0084] As used herein, "linker" or "L" refers to a moiety that covalently attaches two or more components of the 15 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 polypeptide conjugatesdisclosedherein (e.g., a linkermaycovalently attachaCPPandagroup that binds toanucleic acid sequence by electrostatic interactions [i.e., P]). In some embodiments, the linker can be natural or non-natural amino acid or polypeptide. In other embodiments, the linker is a synthetic compound containing two or more appropriate functional groups suitable to bind, e.g., theCPPand, independently, P. In someembodiments, the linker is about 3 to about 100 (e.g., about 3 to about 20) atoms in linear length (not counting the branched atoms or substituents). In some embodiments, the linker provides about 1 Å to about 400 Å in distance of the two groups to which it connects.

[0085] As used herein, "polypeptide" refers to a string of at least two amino acids attached to one another by a peptide bond. There is noupper limit to the number of aminoacids that canbe included in apolypeptide. Further, polypeptidesmay include non-natural amino acids, amino acid analogs, or other synthetic molecules that are capable of integrating into a polypeptide.

[0086] As used herein, "polyarginine peptide" refers to a string of at least two arginine amino acids (independently D or L). In some embodiments, the polyarginine peptide has the following repeating units where n is an integer selected from 1 to 100 and the polyarginine peptide can have one additional arginine monomer to make an odd-numbered chain.

[0087] As used herein, a "monomer" refers to an amino acid residue in a polypeptide. In some embodiments, an amino acid monomer is divalent. In other embodiments, an amino acid monomer may be trivalent if the monomer is further substituted. For example, a cysteinemonomer can independently formpeptidebondsat theNandC termini, andalso form a disulfide bond.

[0088] As used herein, an "amino acid-analog" or "analog" (e.g., "arginine-analog", "lysine-analog" or "histidine- analog") refers to a variant of an amino acid that retains at least one function of the amino acid, such as the ability to bindanoligonucleotide throughelectrostatic interactions.Suchvariantsmayhaveanelongatedor shorter side chain (e.g., by one or more ‑CH2‑ groups that retains the ability to bind an oligonucleotide through electrostatic interactions, or alternatively, the modification can improve the ability to bind an oligonucleotide through electrostatic interactions. For example, an arginine analog may include an additional methylene or ethylene between the backbone and guanidine / - guanidinium group. Other examples include amino acids with one or more additional substituents (e.g., Me, Et, halogen, thiol, methoxy, ethoxy, C1-haloalkyl, C2-haloalkyl, amine, guanidine, etc). The amino acid-analog can be monovalent, divalent, or trivalent.

[0089] Throughout the present specification, peptides and amino acid monomers are depicted as charge neutral species. It is to be understood that such speciesmay bear a positive or negative charge depending on the conditions. For example, at pH7, theN-terminusof anaminoacid is protonatedandbearsapositive charge (-NH3 +), and theC-terminusof an amino acid is deprotonated and bears a negative charge (-CO2 -). Similarly, the side chains of certain amino acids may bear a positive or negative charge.

[0090] Asused herein, a "charged species" refers to amoiety bearing either a positive or negative charge. For example, when the CPP or cCPP contains arginine or arginine analog monomers, the guanidine group under certain conditions, can be protonated to form a guanidinium group 16 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 i.e., the charged species. In someembodiments, the between about 5%and about 100%of themoieties in the conjugates described herein that are capable of bearing a charge, are changed, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, inclusive of all values and subranges therebetween.

[0091] As used herein, "divalent" refers to moiety having two points of attachment to the rest of the molecule. For example, a divalent linker groupwas two points of attachment in a polypeptide conjugate, the first point of attachment is to the cell-penetrating peptide (CPP) and the secondpoint of attachment is to the group that binds to a nucleic acid sequence by electrostatic interactions (P).

[0092] "Alkyl" or "alkyl group" refers to a fully saturated, straight or branched hydrocarbon chain radical having fromone to twelve carbonatoms, andwhich is attached to the rest of themoleculebyasingle bond.Alkyls comprisinganynumber of carbonatoms from1 to 12are included. Analkyl comprising up to 12 carbonatoms is aC1‑C12alkyl, an alkyl comprisingup to10carbonatoms is aC1‑C10alkyl, an alkyl comprisingup to6 carbonatoms is aC1‑C6alkyl andanalkyl comprisingup to 5 carbon atoms is aC1‑C5alkyl. AC1‑C5alkyl includesC5 alkyls, C4 alkyls, C3 alkyls, C2 alkyls andC1 alkyl (i.e.,methyl). A C1‑C6 alkyl includes all moieties described above for C1‑C5 alkyls but also includes C6 alkyls. A C1‑C10 alkyl includes all moieties described above forC1‑C5alkyls andC1‑C6alkyls, but also includesC7,C8,C9 andC10 alkyls. Similarly, aC1‑C12 alkyl includesall the foregoingmoieties, but also includesC11andC12alkyls.Non-limitingexamplesofC1‑C12alkyl include methyl, ethyl,n-propyl, i-propyl, sec-propyl,n-butyl, i-butyl, sec-butyl, t-butyl,n-pentyl, t-amyl,n-hexyl,n-heptyl,n-octyl,n- nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0093] "Alkylene" or "alkylene chain" refers to a fully saturated, straight or branched divalent hydrocarbon chain radical, having fromone to forty carbon atoms.Non-limiting examples ofC2‑C40 alkylene include ethylene, propylene, n-butylene, pentylene, and the like. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted as described herein.

[0094] "Alkenyl" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms, and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl group comprising any number of carbon atoms from 2 to 12 are included. An alkenyl group comprising up to 12 carbon atoms is a C2‑C12 alkenyl, an alkenyl comprising up to 10 carbon atoms is a C2‑C10 alkenyl, an alkenyl group comprising up to 6 carbon atoms is a C2‑C6 alkenyl and an alkenyl comprising up to 5 carbon atoms is aC2‑C5 alkenyl. AC2‑C5 alkenyl includesC5 alkenyls, C4 alkenyls, C3 alkenyls, andC2 alkenyls. AC2‑C6 alkenyl includes all moieties described above for C2‑C5 alkenyls but also includes C6 alkenyls. A C2‑C10 alkenyl includes all moieties describedabove forC2‑C5alkenyls andC2‑C6alkenyls, but also includesC7,C8,C9andC10alkenyls. Similarly, a C2‑C12alkenyl includesall the foregoingmoieties, but also includesC11andC12alkenyls.Non-limitingexamplesofC2‑C12 alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl‑1-propenyl, 1-butenyl, 2-butenyl, 3- butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1- heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-dece- nyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dode- cenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10- dodecenyl, and 11-dodecenyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0095] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain radical, having from two to forty carbon atoms, and having one or more carbon-carbon double bonds. Non-limiting examples of C2‑C40 alkenylene include ethenylene (-CH=CH‑), propenylene, butenylene, and the like. Unless stated otherwise specifically in the specification, an alkenylene chain can be optionally substituted.

[0096] "Alkynyl" or "alkynyl group" refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms, and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl group comprising any number of carbon atoms from 2 to 12 are included. An alkynyl group comprising up to 12 carbon atoms is a C2‑C12 alkynyl, an alkynyl comprising up to 10 carbon atoms is a C2‑C10 alkynyl, an alkynyl group comprising up to 6 carbon atoms is a C2‑C6 alkynyl and an alkynyl comprising up to 5 carbon atoms is a C2‑C5 alkynyl. A C2‑C5 alkynyl includes C5 alkynyls, C4 alkynyls, C3 alkynyls, and C2 alkynyls. A C2‑C6 alkynyl includes all moieties described above for C2‑C5 alkynyls but also includes C6 alkynyls. A C2‑ClO alkynyl includes all 17 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 moieties described above forC2‑C5alkynyls andC2‑C6alkynyls, but also includesC7,C8,C9 andC10 alkynyls. Similarly, a C2‑C12alkynyl includesall the foregoingmoieties, but also includesC11andC12alkynyls.Non-limiting examplesofC2‑C12 alkenyl include ethynyl, propynyl, butynyl, pentynyl and the like. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0097] "Alkynylene" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain radical, having from two to forty carbon atoms, and having one or more carbon-carbon triple bonds. Non-limiting examples of C2‑C40 alkynylene includeethynylene (-C≡C‑), propargyleneand the like.Unless statedotherwise specifically in thespecification, an alkynylene chain can be optionally substituted.

[0098] "Aryl" refers to a hydrocarbon ring system comprising hydrogen, 6 to 40 carbon atoms and at least one aromatic ring. For purposes of this disclosure, the aryl can be amonovalent or a divalent radical (not counting substituents), which can be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, andwhich can include fused or bridged ring systems. Aryl radicals include, but are not limited to, radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene,as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. In some embodiments, the aryl radical can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, an aryl group can be optionally substituted.

[0099] "Carbocyclyl," "carbocyclic ring" or "carbocycle" refers to a rings structure,wherein the atomswhich form the ring are each carbon.Carbocyclic rings can comprise from3 to 20 carbon atoms in the ring. Carbocyclic rings include aryls and cycloalkyl and rings that are fully unsaturated, partially unsaturated, and fully saturated. In some embodiments, the carbocyclyl can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, a carbocyclyl group can be optionally substituted.

[0100] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon radical having from3 to 40 carbon atoms and at least one ring, wherein the ring consists solely of carbon and hydrogen atoms, which can include fused or bridged ring systems. For purposes of this disclosure, the cycloalkyl can be a monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclo- pentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. In some embodiments, the cycloalkyl radical can be divalent whenusedasa linker or asapart of a linker.Unlessotherwisestated specifically in the specification, a cycloalkyl groupcan be optionally substituted.

[0101] "Cycloalkenyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having from 3 to 40 carbon atoms, at least one ring having, and one or more carbon-carbon double bonds, wherein the ring consists solely of carbon and hydrogen atoms, which can include fused or bridged ring systems. For purposes of this invention, the cycloalkenyl can be a monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkenyl radicals include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloctenyl, and the like. Polycyclic cycloalkenyl radicals include, for example, bicyclo[2.2.1]hept‑2-enyl and the like. In some embodiments, the cycloalkenyl radical can be divalent when used as a linker or as a part of a linker. Unless otherwise stated specifically in the specification, a cycloalkenyl group can be optionally substituted.

[0102] "Cycloalkynyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having from 3 to 40 carbonatoms,at least one ring, andoneormorecarbon-carbon triplebonds,wherein the ringconsists solelyof carbonand hydrogenatoms,which can include fusedor bridged ring systems.For purposesof this invention, the cycloalkynyl canbea monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkynyl radicals include, for example, cycloheptynyl, cyclooctynyl, and the like. In some embodiments, the cycloalkynyl radical can be divalent when used as a linker or asapart of a linker.Unlessotherwise stated specifically in the specification, a cycloalkynyl groupcanbeoptionally substituted.

[0103] "Heterocyclyl," "heterocyclic ring" or "heterocycle" refers to a stable 3‑ to 20-membered aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this invention, the heterocyclyl radical can be a monovalent or a divalent radical (not counting substituents).Heterocyclycl or heterocyclic rings includeheteroaryls asdefinedbelow.Unless stated otherwise specifically in the specification, the heterocyclyl radical can be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3] dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindo- lyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4- piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomor- pholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. In some embodiments, the heterocyclyl radical canbedivalentwhenusedasa linkeror asapart of a linker.Unless statedotherwisespecifically in thespecification, a heterocyclyl group can be optionally substituted. 18 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0104] "Heteroaryl" refers to a 5‑ to 20-membered ring system radical comprising hydrogen atoms, one to fourteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of this invention, the heteroaryl radical can be amonovalent or a divalent radical (not counting substituents) and can be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems;and thenitrogen, carbonor sulfuratoms in theheteroaryl radical canbeoptionallyoxidized; thenitrogenatomcan be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, car- bazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl‑1H-pyrrolyl, phe- nazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). In some embodiments, the heteroaryl radical can be divalent whenusedasa linkerorasapart of a linker.Unlessstatedotherwisespecifically in thespecification,aheteroaryl groupcan be optionally substituted.

[0105] The term "ether" used herein refers to a straight or branched divalent radical moiety ‑[(CH2)m‑O‑(CH2)n]z‑ wherein each ofm, n, and z are independently selected from1 to 40. Examples include, but are not limited to, polyethylene glycol. Unless stated otherwise specifically in the specification, the ether can be optionally substituted.

[0106] The term "substituted" used herein means any of the above groups (i.e., alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, and / or ether) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfonegroups, sulfonyl groups, andsulfoxidegroups; anitrogenatom ingroups suchasamines, amides,alkylamines, dialkylamines, arylamines,alkylarylamines, diarylamines,N-oxides, imides, andenamines;asilicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also means any of the above groups in which one or more hydrogen atomsare replacedbyahigher-order bond (e.g., a double‑or triple-bond) toaheteroatomsuchasoxygen inoxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" includes any of the above groups in which one or more hydrogen atoms are replaced with ‑NRgRh, ‑NRgC(=O)Rh, ‑NRgC(=O)NRgRh, ‑NRgC(=O)ORh, ‑NRgSO2Rh, ‑OC(=O)NRg Rh, ‑ORg, ‑SRg, ‑SORg, ‑SO2Rg, ‑OSO2Rg, ‑SO2ORg, =NSO2Rg, and ‑SO2NRgRh. "Substituted also means any of the above groups in which one or more hydrogen atoms are replaced with ‑C(=O)Rg, ‑C(=O)ORg, ‑C(=O)NRgRh, ‑CH2SO2Rg, ‑CH2SO2NRgRh. In the foregoing, Rg and Rh are the same or different and independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, hetero- cyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and / or heteroarylalkyl. "Substituted" further means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, hetero- aryl, N-heteroaryl and / or heteroarylalkyl group. In addition, each of the foregoing substituents can also be optionally substitutedwith one ormore of the above substituents. Further, those skilled in the art will recognize that "substituted" also encompasses instances inwhich one ormore atomson any of the above groups are replaced by a substituent listed in this paragraph, and the substituent forms a covalent bond with the CPP, P, or L. For example, in certain embodiments, any of the above groups can be substituted at a first position with a carboxylic acid (i.e., ‑C(=O)OH) which forms an amide bond with a lysine in the CPP, or a group can be substituted at a second position with a thiol group which forms a disulfide bond with a cysteine (or amino acid analog having a thiol group). Polypeptide Conjugates

[0107] Nucleic acid delivery systems can be divided into two main strategies: 1) viral delivery and 2) non-viral delivery. Viral vectors have the advantage of high efficacy, but can result in immunogenicity and tumorigenicity. Additionally, viral delivery is limited to biologically synthesized nucleic acids and incompatible with short synthetic oligonucleotides or their analogs. Non-viral delivery vectors include various cationic lipids, polymers, carbohydrate analogs, and cell-penetrating peptides (CPPs). These vectors are usually mixed with nucleic acids to form complexes such as nanoparticles or liposomes. The complexes / conjugates are taken up by cells through various endocytic pathways, including macropino- cytosis, clathrin‑ and caveolae-mediated endocytosis. The main limitation of the non-viral delivery systems has been the poor endosomal escape efficiency, resulting in the entrapment of the vast majority of the cargoes inside the endoso- 19 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 mal / lysosomal compartments. The nanoparticle-based systems are also limited to distribution into organs / tissues with good blood access and / or relatively large blood vessel fenestrations, such as liver, spleen, or kidney.

[0108] The present disclosure relates to a discovery that some CPPs, including cyclic CPPs (cCPPs), are highly active with cytosolic deliveryefficienciesofup to120%(compared to2%forTat). TheCPPsbinddirectly to theplasmamembrane phospholipidsandare internalizedbyvariousendocyticmechanisms.The inventorsdiscovered that cCPPsareespecially remarkably efficient in endosomal escape, by binding to the early endosomal membrane and inducing budding of small vesicles enriched with the CPPs from the endosomal membrane. Subsequent collapse of the budded vesicles releases the CPPs (and CPP-cargo conjugates) into the cytosol. Moreover, the cCPPs have proven highly effective for cytosolic delivery of a wide variety of cargos, including small molecules, linear peptides, cyclic peptides, and proteins.

[0109] The present disclosure relates to a discovery of novel biodegradable polypeptide conjugate comprising at least one CPP, which form non-covalent complexes with nucleic acids (e.g., siRNA). Without being bound by theory, the polypeptide conjugate / nucleic acid complex can effectively enter the cytosol of mammalian cells, where the complex undergoes spontaneous degradation in the reducing environment, releasing the nucleic acid cargo for modulation of the biological activity of a specific target(s).

[0110] In various embodiments, the polypeptide conjugates described herein comprise: a) a group that binds to a nucleic acid sequence by electrostatic interactions (P) comprising at least one peptide or polyamine; and b) at least one cell-penetrating peptide (CPP); wherein each peptide comprises at least three monomers selected from arginine, arginine-analog, lysine, lysine- analog, histidine, or histidine-analog; wherein the P is conjugated to theCPP through a bond or at least one linker (L); and wherein the polypeptide conjugate is optionally charged.

[0111] In some embodiments, the polypeptide conjugate as disclosed herein has a molar ratio of P:CPP ranging from about 30:1 to about 1:2. In someembodiments, themolar ratio of P:CPP is about 30:1, about 29:1, about 28:1, about 27:1, about 26:1, about 25:1, about 24:1, about 23:1, about 22:1, about 21:1, about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, or about 1:2, inclusiveof all values and subranges therebetween.

[0112] In some embodiments, the polypeptide conjugate as disclosed herein has an averagemolecular weight ranging from about 1 kDa to about 100 kDa. In some embodiments, the averagemolecular weight of the polypeptide conjugate is about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33k Da, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kDa, about 65 kDa, about 66 kDa, about 67 kDa, about 68 kDa, about 69 kDa, about 70 kDa, about 71 kDa, about 72 kDa, about 73 kDa, about 74 kDa, about 75 kDa, about 76 kDa, about 77 kDa, about 78 kDa, about 79 kDa, about 80 kDa, about 81 kDa, about 82 kDa, about 83 kDa, about 84 kDa, about 85 kDa, about 86 kDa, about 87 kDa, about 88 kDa, about 89 kDa, about 90 kDa, about 91 kDa, about 92 kDa, about 93 kDa, about 94 kDa, about 95 kDa, about 96 kDa, about 97 kDa, about 98 kDa, about 99 kDa, or about 100 kDa, inclusive of all ranges and subranges therebetween. In some embodiments, the polypeptide conjugate as disclosed herein has an average molecular weight ranging from about 3 kDa to about 100 kDa.

[0113] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: CPP-L‑[P]n‑L-CPP (I) wherein n is an integer from 1 to 50; and wherein P at each occurrence is same or different.

[0114] In some embodiments of the polypeptide conjugate as disclosed herein, P comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog.

[0115] In some embodiments of the polypeptide conjugate as disclosed herein, P comprises a polyamine selected from a spermidine polymer or a spermine polymer.

[0116] In some embodiments of the polypeptide conjugate as disclosed herein, at least one of the CPP is a cyclic CPP 20 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (cCPP). In some embodiments, the CPP is, each independently, a cyclic CPP (cCPP). In some embodiments, cCPP comprises from4 to 14aminoacidmonomers. In other embodiments, cCPP is, each independently, selected fromTable 4.

[0117] In someembodiments of thepolypeptide conjugate asdisclosedherein, the cCPP is a cyclo(fΦRrRrQ) peptide or a cyclo(FfΦRrRrQ), wherein: F is a L-phenylalanine; f is a D-phenylalanine; Φ is an L‑2-naphthylalanine; R is a L-arginine; r is a D-arginine; and Q is a L-glutamine.

[0118] In someembodiments of the polypeptide conjugate as disclosed herein, the pArg comprises at least five arginine monomers or arginine-analog monomers. In some embodiments, the pArg further comprises at least one cysteine monomer.

[0119] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: CPP-L‑[pArg]n‑L-CPP (II) wherein the [pArg]n is or a charged species thereof.

[0120] In some embodiments of the polypeptide conjugate as disclosed herein, n is an integer 1 to 40. In some embodiments of the polypeptide conjugate as disclosed herein, n is an integer 2 to 40. In other embodiments of the polypeptide conjugate as disclosed herein, n is an integer selected from 2 to 30. In other embodiments of the polypeptide conjugate as disclosed herein, n is an integer selected from2 to 20. In other embodiments of the polypeptide conjugate as disclosed herein, n is an integer selected from 2 to 10.

[0121] In some embodiments of the polypeptide conjugate as disclosed herein, at least one L comprises a divalent optionally substituted group selected from amino acid, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1‑X-R2)z‑, or combinations thereof; wherein each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20.

[0122] In some embodiments of the polypeptide conjugate as disclosed herein, at least one L comprises an optionally substituted ‑(O-CH2CH2)z‑or anoptionally substituted ‑ (CH2CH2‑O)z‑. In someembodiments, at least oneL comprises a divalent 8-amino‑3,6-dioxaoctanoic acid residue. In other embodiments, at least one L comprises a divalent 8-ami- no‑3,6,9-trioxaundecanoic acid residue.

[0123] In some embodiments of the polypeptide conjugate as disclosed herein, at least one L comprises a physiological cleavable group (PCG). In some embodiments, each PCG is, independently, selected from ‑S-S‑, carbonate, thiocarbo- nate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. In other embodiments, each PCG comprises at least one ‑S-S‑.

[0124] In some embodiments of the polypeptide conjugate of formula (I) as disclosed herein, at least one of the "‑" 21 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 between L and [P]n represents a bond between two sulfur atoms (disulfide bond). In other embodiments, the "‑" between L and [P]n each represents a bond between two sulfur atoms (disulfide bond).

[0125] In some embodiments of the polypeptide conjugate as disclosed herein, each P, independently, further comprises at least one group selected from: or wherein the bond to the hydrogen on at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP.

[0126] In some embodiments of the polypeptide conjugate as disclosed herein, each P, independently, further comprises at least one group selected from: 22 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0127] In some embodiments of the polypeptide conjugate as disclosed herein, at least one of the P further comprises In other embodiments, at least one of the P further comprises at least two groups selected from

[0128] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: CPP-L‑([P1]p‑L1)t‑[P]n‑(L2‑[P2]q)t‑L-CPP (III) wherein n, p, and q are each independently an integer from 1 to 50; t is each independently 0 or 1; P1 and P2 each comprises at least one peptide or polyamine, wherein each peptide comprises at least three monomers selected fromarginine, arginine-analog, lysine, lysine-analog, histidine, or histidine-analog, wherein P, P1 and P2, at each occurrence, are same or different; and L1 and L2 are each independently absent or L as defined in claim 1, wherein L, L1, and L2, at each occurrence, are same or different.

[0129] In some embodiments of the polypeptide conjugate of formula (III) as disclosed herein, P, P1 or P2 comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog. In some embodimentsof thepolypeptide conjugateof formula (III) asdisclosedherein,P,P1orP2comprisesapolyargininepeptide 23 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (pArg) comprising three monomers selected from arginine or arginine-analog. In other embodiments, P, P1 or P2 comprises a polyamine selected from a spermidine polymer or a spermine polymer.

[0130] In some embodiments of the polypeptide conjugate of formula (III) as disclosed herein, P, P1 or P2 comprises mixtures of monomers selected from arginine, arginine-analog, lysine, lysine-analog, histidine, or histidine-analog (e.g., ‑Arg-Lys-Arg‑; ‑Arg-Arg-His‑; etc). In some embodiments of the polypeptide conjugate of formula (III) as disclosed herein, P, P1 or P2 further comprises at least one amino acid monomer (e.g., such as those shown in Table 1).

[0131] In someembodimentsof thepolypeptideconjugateof formula (III) asdisclosedherein, at least oneof theCPP isa cyclic CPP (cCPP). In other embodiments, the CPP is, each independently, a cyclic CPP (cCPP). In one embodiment, the cCPP comprises from 4 to 14 amino acid monomers. In another embodiment, the cCPP is, each independently, selected from Table 4.

[0132] In some embodiments of the polypeptide conjugate of formula (III), the cCPP is a cyclo(fΦRrRrQ) (SEQ ID NO: 118) peptide or a cyclo(FfΦRrRrQ) (SEQ ID NO: 16) , wherein: F is a L-phenylalanine; f is a D-phenylalanine; Φ is an L‑2-naphthylalanine; R is a L-arginine; r is a D-arginine; and Q is a L-glutamine.

[0133] In some embodiments of the polypeptide conjugate of formula (III), the pArg comprises at least three arginine monomers or arginine-analog monomers. In some embodiments of the polypeptide conjugate of formula (III), the pArg comprises three argininemonomers or arginine-analogmonomers. In someembodiments of the polypeptide conjugate of formula (III), thepArg comprisesat least fiveargininemonomersor arginine-analogmonomers. In other embodiments, the pArg further comprises at least one amino acid monomer (e.g., such as those shown in Table 4). In one embodiment, the pArg further comprisesat least one cysteinemonomer. In oneembodiment, at least onepArg further comprisesa thioether moiety (-S‑).

[0134] In some embodiments of the polypeptide conjugate of formula (III), n is an integer 1 to 40. In some embodiments of the polypeptide conjugate of formula (III), n is an integer 5 to 40.

[0135] In someembodimentsof thepolypeptide conjugateof formula (III), at least oneof L, L1, or L2 comprisesadivalent optionally substituted group selected from amino acid, polyethylene glycol, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1‑X-R2)z‑, or combinations thereof; each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20.

[0136] In some embodiments of the polypeptide conjugate of formula (III), at least one of L, L1, or L2 comprises an optionally substituted ‑(O-CH2CH2)z‑ or an optionally substituted ‑(CH2CH2‑O)z‑.

[0137] In someembodimentsof thepolypeptide conjugateof formula (III), at least oneof L, L1, or L2 comprisesadivalent 8-amino‑3,6-dioxaoctanoic acid residue. In some embodiments, at least one of L, L1, or L2 comprises a divalent 8- amino‑3,6,9-trioxaundecanoic acid residue. In other embodiments, at least one of L, L1, or L2 comprises a physiological cleavable group (PCG).

[0138] In someembodimentsof thepolypeptideconjugateof formula (III), eachPCGis, independently, selected from ‑S- S‑, carbonate, thiocarbonate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. In some embodi- ments, each PCG comprises at least one -S-S-.

[0139] In some embodiments of the polypeptide conjugate of formula (III), at least one of the "‑" between L and ([P1]p‑L1)t, L and [P]n, or L and (L2‑[P2]q)t represents a bond between two sulfur atoms (disulfide bond).

[0140] In someembodiments of the polypeptide conjugate of formula (III), at least one of P, P1 or P2 further comprises at least one group selected from: 24 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or wherein the bond to the hydrogen on at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP.

[0141] In someembodiments of the polypeptide conjugate of formula (III), at least one of P, P1 or P2 further comprises at least one group selected from: 25 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0142] In some embodiments of the polypeptide conjugate of formula (III), at least one of P, P1 or P2 further comprises In other embodiments, at least at least two of P, P1 or P2 further comprises a group selected form

[0143] In some embodiments of the polypeptide conjugate of formula (III), the pArg further comprises one or more β- alanine monomers. In some embodiments, each pArg comprises, independently, 3, 4, 5, 6, 7, or 8 arginine monomers. In other embodiments, each pArg comprises 3 arginine monomers. In other embodiments, each pArg comprises 5 arginine monomers. In other embodiments, at least one pArg further comprises at least one amino acid monomer (e.g., such as thoseshown inTable4). In oneembodiment, at least onepArg further comprisesacysteinemonomer. Inoneembodiment, at least one pArg further comprises a thioether moiety (-S-).

[0144] In some embodiments of the polypeptide conjugate of formula (III), n, p, and q are each independently an integer from1 to 40. In other embodiments, n, p, and q are each independently an integer from1 to 30. In other embodiments, n, p, and q are each independently an integer from1 to 20. In other embodiments, n, p, and qare each independently an integer from 1 to 10. In other embodiments, n, p, and q are each independently an integer from 1 to 5.

[0145] In some embodiments of the polypeptide conjugate of formula (I), (II), or (III) as disclosed herein, P is each independently selected from: ‑Cys‑(Arg)x-Cys‑ (SEQ ID NO: 131) , ‑Cys-βAla‑(Arg)X‑Cys‑ (SEQ ID NO: 132), ‑Cy- s‑(Arg)X-βAla-Cys‑ (SEQ ID NO: 133), or ‑Cys-βAla‑(Arg)X-βAla-Cys‑ (SEQ ID NO: 134), ‑Cys‑(polyamine)X‑Cys‑, ‑Cys- βAla‑(polyamine)X‑Cys‑, ‑Cys‑(polyamine)X-βAla-Cys‑, ‑Cys-βAla‑(polyamine)X-βAla-Cys‑ (SEQ ID NO: 135), or ‑S‑(polyamine)X‑S‑, wherein x = 3, 4, 5, 6, 7, or 8.

[0146] In some embodiments, the polypeptide conjugate of formula (I) as disclosed herein has the structure 26 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 , or a charged species thereof.

[0147] In some embodiments, the polypeptide conjugate of formula (I) as disclosed herein has the structure , or a charged species thereof.

[0148] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: CPP-L‑([P]m)s (IV) wherein: s is an integer from 1 to 10; each m is, independently, an integer from 1 to 50; and wherein P at each occurrence is same or different.

[0149] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, P comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog. In some embodiments, P comprises a polyamine selected from a spermidine polymer or a spermine polymer.

[0150] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, at least one of theCPP is a cyclic CPP (cCPP). In some embodiments, the cCPP comprises from 4 to 14 amino acid monomers. In other embodiments, the cCPP is, each independently, selected from Table 4.

[0151] In someembodiments of the polypeptide conjugate of formula (IV) as disclosed herein, the pArg comprises three arginine monomers or arginine-analog monomers. In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, the pArg comprises at least five arginine monomers or arginine-analog monomers. In other embodi- ments, the pArg further comprises at least one amino acid monomer (e.g., such as those shown in Table 4). In some embodiments, the pArg further comprises at least one cysteinemonomer. In one embodiment, the pArg further comprises a cysteine monomer. In one embodiment, at least one pArg further comprises a thioether moiety (-S‑).

[0152] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, L comprises a divalent optionally substituted group selected from amino acid, polyethylene glycol, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1‑X-R2)z‑, or combinations thereof; each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20.

[0153] In someembodiments of thepolypeptide conjugate of formula (IV) as disclosedherein, L comprises anoptionally 27 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 substituted ‑(O-CH2CH2)z‑ or an optionally substituted - (CH2CH2‑O)z‑. In some embodiments, L comprises a divalent 8- amino‑3,6-dioxaoctanoic acid residue. In other embodiments, L comprises a divalent 8-amino‑3,6,9-trioxaundecanoic acid residue. In one embodiment, L comprises a physiological cleavable group (PCG).

[0154] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, each PCG is, indepen- dently, selected from ‑S-S‑, carbonate, thiocarbonate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. In some embodiments, each PCG comprises at least one ‑S-S‑.

[0155] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, L comprises a polythiolamine or a 3,5-bis(mercaptomethyl)benzoyl (Bmb) amide.

[0156] In some embodiments of the polypeptide conjugate of formula (IV), L comprises two or more physiological cleavable groups.

[0157] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, the "‑" between L and ([P]m)s represents a bond between two sulfur atoms (disulfide bond).

[0158] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, each P, independently, further comprises at least one group selected from: or wherein the bond to the hydrogen on one at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP.

[0159] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, each P, independently, further comprises at least one group selected from: 28 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0160] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, at least one of the P further comprises In other embodiments, at least one of the P further comprises at least two groups selected form

[0161] In some embodiments of the polypeptide conjugate of formula (IV) as disclosed herein, s is 2, 3, 4, or 5.

[0162] In some embodiments, the polypeptide conjugate of formula (IV) as disclosed herein has the structure 29 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or charged species thereof.

[0163] In some embodiments, the polypeptide conjugate of formula (IV) as disclosed herein has the structure or charged species thereof.

[0164] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: cCPP-L‑[P]n‑[P]m‑(L-cCPP)v (V) wherein, cCPP is a cyclic CPP; and n is an integer selected from 1 to 50; m is an integer selected from 0 to 49 provided that the sum of n and m is 50 or less; v is 0 or 1; wherein P at each occurrence is same or different; and wherein when v is 0, the last [P] in [P]m is monovalent.

[0165] In some embodiments of the polypeptide conjugate of formula (V), v is 1.

[0166] In some embodiments of the polypeptide conjugate of formula (V) as disclosed herein, the "‑" between [P]n and 30 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 [P]m represents a bond between two sulfur atoms (a disulfide bond).

[0167] In someembodiments, the polypeptide conjugate of formula (IV) as disclosedherein, has the following structure:

[0168] In someembodimentsof thepolypeptideconjugateof formula (VI) asdisclosedherein, the "‑" between [P] and [P] represents a bond between two sulfur atoms (a disulfide bond).

[0169] In some embodiments, the polypeptide conjugate as disclosed herein has the following structure: cCPP-L‑[P]n‑[P]m‑[P]o‑(L-cCPP)v (VII) wherein, cCPP is a cyclic CPP; and n is an integer selected from 1 to 50; m and o, are each independently, an integer from 0 to 49 provided that the sum of n, m, and o is 50 or less; v is 0 or 1; wherein P at each occurrence is same or different; and wherein when v is 0, the last [P] in [P]o is monovalent.

[0170] In some embodiments of the polypeptide conjugate of formula (VII), v is 1.

[0171] In some embodiments of the polypeptide conjugate of formula (VII) as disclosed herein, at least one of the "‑" between [P]n and [P]m or [P]m and [P]o represents a bond between two sulfur atoms (a disulfide bond). In other embodiments, the "‑" between [P]n and [P]m and between [P]m and [P]o represents a bond between two sulfur atoms (a disulfide bond).

[0172] In someembodiments, the polypeptide conjugate of formula (VI) as disclosedherein, has the following structure: 31 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0173] In some embodiments of the polypeptide conjugate of formula (VIII) as disclosed herein, at least one of the "‑" between [P] and [P] represents abondbetween twosulfur atoms (adisulfidebond). Inother embodiment, each "‑" between [P] and [P] represents a bond between two sulfur atoms (a disulfide bond).

[0174] In some embodiment, the dash "‑" between P (including P1 and P2), L (including L1 and L2), and CPP (including cCPP) in formula (I), (II), (III), (IV), (V), (VI), (VII), and (VIII) represents each component’s spatial orientation. Tobeclear, "‑" not strictlyasaC-Cbond,although insomeembodiments, itmaybe. Insomeembodiments,eachPcanbediscussedas its own component having a chemical group necessary to covalently attach to L. In some embodiments, each P can be discussed as its own component having a chemical group necessary to covalently attach to another group of P. In some embodiments, theCPPcanbediscussedas its owncomponent havinga chemical groupnecessary to covalently attach to L.One skilled in the artwould readily understandhoweach component, described separately, can covalently attach to one another to provide the polypeptide conjugate as disclosed herein.

[0175] In someembodiments, any of L (including L1 andL2) disclosedherein canbe the linker that is covalently attached to P and / or to the CPP. In some embodiments, any of L disclosed herein can describe the linker moiety before covalently attaching it to Pand / or toCPP. In a non-limited example, L can comprise a chemical group (e.g., ‑SH, ‑NH2, ‑OHetc)which canbe reactedwithanother chemical grouponor attached toPorCPP inorder to formacovalent bond, e.g., disulfidebond (-S-S‑), amine bond (-NH‑), ether bond (-O‑), amide bond (-NH(O)‑), ester bond (-C(O)O‑), etc. In one embodiment, a chemical group already present in L as described herein can be used to covalently attach L to theP and / or to theCPP. The chemistry used to covalently attach P to L and L to CPP can be readily understood by one skilled in the art.

[0176] In one embodiment, any of L disclosed herein can further comprise a chemical group useful in covalently attaching L to the P and / or to the CPP. Cell-Penetrating Peptides (CPP)

[0177] As discussed above, the polypeptide conjugates disclosed herein comprise cell-penetrating peptides (CPPs). CPPsare peptides that facilitate cellular intakeor uptakeof variousmolecular equipment, often refers to as a "cargo" (e.g., nucleic acid or siRNA). The "cargo" is associated with the peptides through non-covalent interactions, such as by electrostatic interactions. The function of the CPPs are to deliver the cargo into cells, a process that commonly occurs through endocytosis and subsequently released into cytosol of mammalian cells. (i) CPPs typically havean amino acid composition that either contains a high relative abundance of positively charged amino acids such as arginine or has sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids. Some CPPs comprise hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake. Non-limiting examples of linear CPPs include Polyarginine (e.g., R9 or R11), Antennapedia sequences, HIV-TAT, Penetratin, Antp‑3A (Antp mutant), Buforin II. Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep‑1, SynB1, Pep‑7, HN‑1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol, and BGTC (Bis-Guanidinium-Tren-Cholesterol).

[0178] In someembodiments,CPPsare cyclicCPPs (cCPPs). ThecCPPmaybeor includeanyamino sequence,which facilitates cellular uptake of the polypeptide conjugates disclosed herein. Suitable cCPPs for use in the polypeptide conjugates andmethods described herein can include naturally occurring sequences,modified sequences, and synthetic sequences. In embodiments, the total number of amino acids in the cCPPmay be in the range of from 4 to about 20 amino 32 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 acids, e.g., about5, about6, about7, about8, about9, about10,about11,about12,about13,about14, about15, about16, about17, about18, andabout 19aminoacids, inclusiveofall rangesandsubranges therebetween. Insomeembodiments, the cCPPs disclosed herein comprise about 4 to about to about 13 amino acids. In particular embodiments, the CPPs disclosed herein comprise about 6 to about 10 amino acids, or about 6 to about 8 amino acids.

[0179] Each amino acid in the CPP or cCPPmay be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modifiedaminoacid, and / or aminoacidanalog, that isnot oneof the20commonnaturally occurringaminoacidsor the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be theD-isomer of the natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamicacid, glycine, histidine, isoleucine, leucine, lysine,methionine,napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative, or combinations thereof. These, and others, are listed in the Table 1 along with their abbreviations used herein. Table 1. Amino Acid Abbreviations Amino Acid Abbreviations* L-amino acid Abbreviations* D-amino acid Alanine Ala (A) ala (a) Allosoleucine AIle aile Arginine Arg (R) arg (r) Asparagine Asn (N) asn (n) aspartic acid Asp (D) asp (d) Cysteine Cys (C) cys (c) Cyclohexylalanine Cha cha 2,3-diaminopropionic acid Dap dap 4-fluorophenylalanine Fpa (Σ) pfa glutamic acid Glu (E) glu (e) glutamine Gln (Q) gin (q) glycine Gly (G) gly (g) histidine His (H) his (h) Homoproline (aka pipecolic acid) Pip (Θ) pip (θ) isoleucine Ile (I) ile (i) leucine Leu (L) leu (1) lysine Lys (K) lys (k) methionine Met (M) met (m) napthylalanine Nal (Φ) nal (ϕ) norleucine Nle (Ω) nle phenylalanine Phe (F) phe (F) phenylglycine Phg (Ψ) phg 4‑(phosphonodifluoromethyl)phenylalanine F2Pmp (Λ) f2pmp proline Pro (P) pro (p) sarcosine Sar (Ξ) sar selenocysteine Sec (U) sec (u) serine Ser (S) ser (s) threonine Thr (T) thr (y) tyrosine Tyr (Y) tyr (y) 33 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Amino Acid Abbreviations* L-amino acid Abbreviations* D-amino acid tryptophan Trp (W) trp (w) valine Val (V) val (v) * single letter abbreviations: when shown in capital letters herein it indicates the L-amino acid form, when shown in lower case herein it indicates the D-amino acid form.

[0180] In some embodiments, the cCPPs may include any combination of at least two arginines and at least two hydrophobic amino acids. In someembodiments, the cCPPsmay include any combination of two to three arginines and at least two hydrophobic amino acids.

[0181] In some embodiments, the CPP used in polypeptide conjugates described herein has a structure comprising Formula 1: (AAu)m‑AAq-AA2‑AA3‑AA4‑(AAz)n 1 wherein: each of AA1, AA2, AA3, and AA4, are independently selected from a D or L amino acid, each of AAu and AAz, at each instance and when present, are independently selected from a D or L amino acid, and m and n are independently selected from a number from 0 to 6.

[0182] In some embodiments of the Formula 1, at least two of AAu (when present), AA1, AA2, AA3, AA4, and AAz (when present), are independently arginine. In some embodiments, at least two of AAu (when present), AA1, AA2, AA3, AA4, and AAz (whenpresent), are independently anaminoacidhavingahydrophobic sidechain. In someembodiments, at least two of AAu (when present), AA1, AA2, AA3, AA4, and AAz (when present), are independently arginine and at least two of AAu (when present), AA1, AA2, AA3, AA4, and AAz (when present), are independently an having a hydrophobic side chain.

[0183] In someembodiments, eachamino acid having ahydrophobic side chain is independently selected fromglycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine‑2-carboxylic acid, or norleucine, each of which is optionally substitutedwith one ormore substituents. In particular embodiments, each amino acid having a hydrophobic side chain is independently an amino acid having a hydrophobic aromatic side chain. In some embodiments, the amino acid having a hydrophobic aromatic side chain is naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. In particular embodiments, the amino acid having a hydrophobic side chain is piperidine‑2-carboxylic acid, naphthylalanine, tryptophan, or phenylalanine, each of which is optionally substituted with one or more substituents.

[0184] The optional substituent can be any atom or group which does not significantly reduce the cytosolic delivery efficiency of theCPPor cCPP, e.g., a substituent that does not reduce relative cytosolic delivery efficiency to less than that of c(FΦRRRRQ) (SEQ ID NO: 9) . In some embodiments, the optional substituent can be a hydrophobic substituent or a hydrophilic substituent. In certain embodiments, the optional substituent is a hydrophobic substituent. In some embodi- ments, the substituent increases the solvent-accessible surfacearea (as definedherein) of the hydrophobic aminoacid. In some embodiments, the substituent can be a halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio. In some embodiments, the substituent is a halogen.

[0185] Amino acids having higher hydrophobicity values can be selected to improve cytosolic delivery efficiency of a CPP relative to amino acids having a lower hydrophobicity value. In some embodiments, each hydrophobic amino acid independently has a hydrophobicity value which is greater than that of glycine. In other embodiments, each hydrophobic amino acid independently is a hydrophobic amino acid having a hydrophobicity valuewhich is greater than that of alanine. In still other embodiments, each hydrophobic amino acid independently has a hydrophobicity value which is greater or equal to phenylalanine.Hydrophobicitymaybemeasuredusing hydrophobicity scales known in theart. Table 2 below lists hydrophobicity values for various amino acids as reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. U. S. A. 1984;81(1):140‑144), Engleman, et al. (Ann. Rev. of Biophys. Biophys. Chem. 1986;1986(15):321‑53), Kyte andDoolittle (J. Mol. Biol. 1982;157(1):105‑132), Hoop andWoods (Proc. Natl. Acad. Sci. U. S. A. 1981;78(6):3824‑3828), and Janin (Nature. 1979;277(5696):491‑492), the entirety of each of which is herein incorporated by reference in its entirety. In particular embodiments, hydrophobicity is measured using the hydrophobicity scale reported in Engleman, et al. 34 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 Table 2. Hydrophobicity Values of Amino Acids Amino Acid Group Eisenberg and Weiss Englemanet al. Kyrie and Doolittle Hoop and Woods Janin Ile Nonpolar 0.73 3.1 4.5 ‑1.8 0.7 Phe Nonpolar 0.61 3.7 2.8 ‑2.5 0.5 Val Nonpolar 0.54 2.6 4.2 ‑1.5 0.6 Leu Nonpolar 0.53 2.8 3.8 ‑1.8 0.5 Trp Nonpolar 0.37 1.9 ‑0.9 ‑3.4 0.3 Met Nonpolar 0.26 3.4 1.9 ‑1.3 0.4 Ala Nonpolar 0.25 1.6 1.8 ‑0.5 0.3 Gly Nonpolar 0.16 1.0 ‑0.4 0.0 0.3 Cys Unch / Polar 0.04 2.0 2.5 ‑1.0 0.9 Tyr Unch / Polar 0.02 ‑0.7 ‑1.3 ‑2.3 ‑0.4 Pro Nonpolar ‑0.07 ‑0.2 ‑1.6 0.0 ‑0.3 Thr Unch / Polar ‑0.18 1.2 ‑0.7 ‑0.4 ‑0.2 Ser Unch / Polar ‑0.26 0.6 ‑0.8 0.3 ‑0.1 His Charged ‑0.40 ‑3.0 ‑3.2 ‑0.5 ‑0.1 Glu Charged ‑0.62 ‑8.2 ‑3.5 3.0 ‑0.7 Asn Unch / Polar ‑0.64 ‑4.8 ‑3.5 0.2 ‑0.5 Gln Unch / Polar ‑0.69 ‑4.1 ‑3.5 0.2 ‑0.7 Asp Charged ‑0.72 ‑9.2 ‑3.5 3.0 ‑0.6 Lys Charged ‑1.10 ‑8.8 ‑3.9 3.0 ‑1.8 Arg Charged ‑1.80 ‑12.3 ‑4.5 3.0 ‑1.4

[0186] The chirality of the amino acids can be selected to improve cytosolic uptake efficiency. In some embodiments, at least two of the amino acids have the opposite chirality. In some embodiments, the at least two amino acids having the opposite chirality can be adjacent to each other. In some embodiments, at least three amino acids have alternating stereochemistry relative toeachother. In someembodiments, theat least threeaminoacidshaving thealternating chirality relative to each other can be adjacent to each other. In some embodiments, at least two of the amino acids have the same chirality. In some embodiments, the at least two amino acids having the same chirality can be adjacent to each other. In some embodiments, at least two amino acids have the same chirality and at least two amino acids have the opposite chirality. In someembodiments, theat least twoaminoacidshaving theopposite chirality canbeadjacent to theat least two amino acids having the same chirality. Accordingly, in some embodiments, adjacent amino acids in theCPP can have any of the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D.

[0187] In some embodiments, an arginine is adjacent to an amino acid having a hydrophobic side chain. In some embodiments, the arginine has the same chirality as the amino acid having a hydrophobic side chain. In some embodiments, at least two arginines are adjacent to each other. In still other embodiments, three arginines are adjacent to each other. In some embodiments, at least two amino acids each having a hydrophobic side chain are adjacent to each other. Inotherembodiments, at least threeaminoacidseachhavingahydrophobicsidechainareadjacent toeachother. In otherembodiments, theCPPsdescribedherein compriseat least twoconsecutiveaminoacidseachhavingahydrophobic side chain and at least two consecutive arginines. In further embodiments, one hydrophobic amino acid is adjacent to one of the arginines. In still other embodiments, the CPPs described herein comprise at least three consecutive amino acids each having a hydrophobic side chain and there consecutive arginines. In further embodiments, one hydrophobic amino acid is adjacent to one of the arginines. These various combinations of amino acids can have any arrangement of D and L aminoacids, e.g., the sequencesdescribedabove.Asusedherein, adjacent refers to aminoacids that are coupled to each other through a peptide bond.

[0188] In someembodiments, any four adjacent aminoacids in theCPPsdescribed herein canhaveoneof the following sequences: AAH2‑AAH1‑R-r, AAH2‑AAH1-r-R, R-r-AAH1‑AAH2, or r-R-AAH1‑AAH2, wherein each of AAH1 and AAH2 are independently an amino acid having a hydrophobic side chain. Accordingly, in some embodiments, the CPPs used in the 35 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 polypeptide conjugates described herein have a structure according any of Formula 2A‑2D: and wherein: each of AAH1 and AAH2 are independently an amino acid having a hydrophobic side chain; at each instance and when present, each of AAU and AAZ are independently any amino acid; and m and n are independently selected from a number from 0 to 6.

[0189] In someembodiments, the total number of aminoacids (including r,R,AAH1,AAH2), in theCPPsof Formula 2A to 2D are in the range of 6 to 10. In some embodiments, the total number of amino acids is 6. In some embodiments, the total number of amino acids is 7. In some embodiments, the total number of amino acids is 8. In some embodiments, the total number of amino acids is 9. In some embodiments, the total number of amino acids is 10.

[0190] In some embodiments of Formula 2A‑2D, the sum of m and n is from 2 to 6. In some embodiments, the sum of m and n is 2. In some embodiments, the sum of m and n is 3. In some embodiments, the sum of m and n is 4. In some embodiments, the sum of m and n is 5. In some embodiments, the sum of m and n is 6. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0191] In someembodiments, eachamino acid having ahydrophobic side chain is independently selected fromglycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine‑2-carboxylic acid, or norleucine, each of which is optionally substitutedwith one ormore substituents. In particular embodiments, each amino acid having a hydrophobic side chain is independently an amino acid having a hydrophobic side chain. In some embodiments, the aromatic hydrophobic amino acid is naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substitutedwith one ormore substituents. In particular embodiments, the amino acid having a hydrophobic side chain is piperidine‑2-carboxylic acid, naphthylalanine, tryptophan, or phenylalanine, each of which is optionally sub- stituted with one or more substituents.

[0192] In some embodiments of Formula 2A‑2D, each of AAH1 and AAH2 are independently an amino acid having a hydrophobic side chain with a hydrophobicity value which is greater than that of glycine. In other embodiments, each of AAH1 and AAH2 are independently an amino acid having a hydrophobic side chain with a hydrophobicity value which is greater than that of alanine. In still other embodiments, each of AAH1 and AAH2 are independently an amino acid having a hydrophobicsidechainwithahydrophobicity valuewhich isgreater than thatof phenylalanine, e.g., asmeasuredusing the hydrophobicity scales described above, including Eisenberg and Weiss (Proc. Natl. Acad. Sci. U. S. A. 1984;81(1):140‑144), Engleman, et al. (Ann. Rev. of Biophys. Biophys. Chem. 1986;1986(15):321‑53), Kyte andDoolittle (J. Mol. Biol. 1982;157(1):105‑132), Hoop andWoods (Proc. Natl. Acad. Sci. U. S. A. 1981;78(6):3824‑3828), and Janin (Nature. 1979;277(5696):491‑492), (seeTable1above). Inparticular embodiments, hydrophobicity ismeasuredusing the hydrophobicity scale reported in Engleman, et al.

[0193] The presence of an amino acid having a hydrophobic side chain on the N‑ or C-terminal of a D-Arg or L-Arg, or a combination thereof, has also found to improve the cytosolic uptake of the CPP (and the attached cargo). For example, in some embodiments, the CPPs (including cCPPs) disclosed herein may include AAH1‑D-Arg or D-Arg-AAH1. In other embodiments, the CPPs disclosed herein may include AAH1‑L-Arg or L-Arg-AAH1.

[0194] The size of the hydrophobic chain of the amino acid on the N‑ or C-terminal of the D-Arg or an L-Arg, or a combination thereof (i.e., AAH1),may be selected to improve cytosolic delivery efficiency of theCPP. For example, a larger hydrophobic chain on an amino acid on the N‑ or C-terminal of a D-Arg or L-Arg, or a combination thereof, improves 36 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 cytosolic delivery efficiency compared to an otherwise identical sequence having a smaller hydrophobic amino acid. The sizeof the hydrophobic aminoacid canbemeasured in termsofmolecularweight of the hydrophobic aminoacid, the steric effects of the hydrophobic amino acid, the solvent-accessible surface area (SASA) of the side chain, or combinations thereof. In someembodiments, the size of the hydrophobic amino acid ismeasured in terms of themolecular weight of the hydrophobic amino acid, and the larger hydrophobic amino acid has a side chain with amolecular weight of at least about 90 g / mol, or at least about 130 g / mol, or at least about 141 g / mol. In other embodiments, the size of the amino acid is measured in termsof theSASAof the hydrophobic side chain, and the larger hydrophobic aminoacid has a side chainwith a SASA greater than alanine, or greater than glycine.

[0195] In other embodiments, AAH1 has a hydrophobic side chain with a SASA greater than or equal to about piperidine‑2-carboxylic acid, greater than or equal to about tryptophan, greater than or equal to about phenylalanine, or equal toorgreater thanaboutnaphthylalanine. In someembodiments,AAH1hasasidechainsidewithaSASAofat least about200Å2,at least about210Å2,at least about220Å2, at leastabout240Å2, at leastabout250Å2, at leastabout260Å2, at least about 270Å2, at least about 280Å2, at least about 290Å2, at least about 300Å2, at least about 310Å2, at least about 320Å2,or at least about 330Å2. In someembodiments, AAH2 has a side chain sidewith aSASAof at least about 200Å2, at least about 210 Å2, at least about 220 Å2, at least about 240 Å2, at least about 250 Å2, at least about 260 Å2, at least about 270Å2, at least about 280Å2, at least about 290Å2, at least about 300Å2, at least about 310Å2, at least about 320Å2,or at least about 330Å2. In someembodiments, the side chains of AAH1andAAH2havea combinedSASAof at least about 350 Å2, at least about 360 Å2, at least about 370 Å2, at least about 380 Å2, at least about 390 Å2, at least about 400 Å2, at least about410Å2, at least about420Å2, at least about 430Å2, at least about440Å2, at least about 450Å2, at least about460Å2, at least about 470Å2, at least about 480Å2, at least about 490Å2, greater than about 500Å2, at least about 510Å2, at least about520Å2, at least about530Å2, at least about 540Å2, at least about550Å2, at least about 560Å2, at least about570Å2, at least about 580Å2, at least about 590Å2, at least about 600Å2, at least about 610Å2, at least about 620Å2, at least about 630Å2, at least about 640Å2, greater than about 650Å2, at least about 660Å2, at least about 670Å2, at least about 680Å2, at least about 690 Å2, or at least about 700 Å2. In some embodiments, AAH2 is a hydrophobic amino acid with a side chain having a SASA that is less than or equal to the SASA of the hydrophobic side chain of AAH1.

[0196] By way of example, and not by limitation, a cCPP having a Nal-Arg motif exhibits improved cytosolic delivery efficiency compared to an otherwise identical CPP having a Phe-Arg motif; a cCPP having a Phe-Nal-Arg motif exhibits improved cytosolic delivery efficiency compared to an otherwise identical cCPP having a Nal-Phe-Arg motif; and a phe- Nal-Arg motif exhibits improved cytosolic delivery efficiency compared to an otherwise identical cCPP having a nal-Phe- Arg motif.

[0197] Asusedherein, "hydrophobic surfacearea" or "SASA" refers to the surface area (reported as squareAngstroms; Å2) of an amino acid side chain that is accessible to a solvent. In particular embodiments, SASA is calculated using the ’rollingball’ algorithmdevelopedbyShrake&Rupley (JMolBiol. 79 (2): 351‑71),which is herein incorporatedby reference in its entirety for all purposes. This algorithm uses a "sphere" of solvent of a particular radius to probe the surface of the molecule. A typical value of the sphere is 1.4 Å, which approximates to the radius of a water molecule.

[0198] SASA values for certain side chains are shown below in Table 3. In certain embodiments, the SASA values described herein are based on the theoretical values listed in Table 3 below, as reported by Tien, et al. (PLOSONE 8(11): e80635. https: / / doi.org / 10.1371 / journal.pone.0080635, which is herein incorporated by reference in its entirety for all purposes. Table 3. SASA Values of Amino Acid Side Chains Residue Theoretical Empirical Miller et al. (1987) Rose et al. (1985) Alanine 129.0 121.0 113.0 118.1 Arginine 274.0 265.0 241.0 256.0 Asparagine 195.0 187.0 158.0 165.5 Aspartate 193.0 187.0 151.0 158.7 Cysteine 167.0 148.0 140.0 146.1 Glutamate 223.0 214.0 183.0 186.2 Glutamine 225.0 214.0 189.0 193.2 Glycine 104.0 97.0 85.0 88.1 Histidine 224.0 216.0 194.0 202.5 Isoleucine 197.0 195.0 182.0 181.0 Leucine 201.0 191.0 180.0 193.1 37 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Residue Theoretical Empirical Miller et al. (1987) Rose et al. (1985) Lysine 236.0 230.0 211.0 225.8 Methionine 224.0 203.0 204.0 203.4 Phenylalanine 240.0 228.0 218.0 222.8 Proline 159.0 154.0 143.0 146.8 Serine 155.0 143.0 122.0 129.8 Threonine 172.0 163.0 146.0 152.5 Tryptophan 285.0 264.0 259.0 266.3 Tyrosine 263.0 255.0 229.0 236.8 Valine 174.0 165.0 160.0 164.5

[0199] In someembodiments, theCPPdoesnot includeanaminoacidwith ahydrophobic sidechain on theN‑and / orC- terminal of AAH2‑AAH1‑R-r, AAH2‑AAH1-r-R, R-r-AAH1‑AAH2, or r-R-AAH1‑AAH2. In alternative embodiments, the CPP does not include an amino acid having a hydrophobic side chain which is larger (as described herein) than at least one of AAH1 or AAH2. In further embodiments, the CPP does not include an amino acid with a hydrophobic side chain having a surface area greater than AAH1. For example, in embodiments in which at least one of AAH1 or AAH2 is phenylalanine, the cPPdoes not further include a naphthylalanine (although theCPP include at least one amino acid with a hydrophobic side chain that is smaller than AAH1 and AAH2, e.g., leucine). In still other embodiments, the CPP does not include a naphthylalanine in addition to the hydrophobic amino acids in AAH2‑AAH1‑R-r, AAH2‑AAH1-r-R, R-r-AAH1‑AAH2, or r-R- AAH1‑AAH2.

[0200] Thechirality of theaminoacids (i.e.,Dor Laminoacids) canbeselected to improvecytosolic deliveryefficiencyof the CPP (and the attached cargo as described below). In some embodiments, the hydrophobic amino acid on theN‑ or C- terminal of an arginine (e.g., AAH1) has the same or opposite chirality as the adjacent arginine. In some embodiments, AAH1 has the opposite chirality as the adjacent arginine. For example, when the arginine is D-Arg (i.e. "r"), AAH1 is a D- AAH1, andwhen the arginine is L-Arg (i.e., "R"), AAH1 is a L-AAH1. Accordingly, in someembodiments, theCPPsdisclosed herein may include at least one of the following motifs: D-AAH1‑D-arg, D-arg-D-AAH1, L-AAH1‑L-Arg, or L-Arg-LAAH1. In particular embodiments,whenarginine isD-arg, AAHcanbeD-nal,D-trp, orD-phe. In another non-limiting example,when arginine is L-Arg, AAH can be L-Nal, L-Trp, or L-Phe.

[0201] In some embodiments, the CPPs (including cCPPs) described herein include three arginines. Accordingly, in some embodiments, the CPPs described herein include one of the following sequences: AAH2‑AAH1‑R-r-R, AAH2‑AAH1‑R-r-r, AAH2‑AAH1-r-R-R, AAH2‑AAH1-r-R-r, R-R-r-AAH1‑AAH2, r-R-r-AAH1‑AAH2, r-r-R-AAH1‑AAH2, or, R-r- R-AAH1‑AAH2. In particular embodiments, theCPPShaveoneof the following sequencesAAH2‑AAH1‑R-r-R, AAH2‑AAH1- r-R-r, r-R-r-AAH1‑AAH2, or R-r-R-AAH1‑AAH2. In some embodiments, the chirality of AAH1 and AAH2 can be selected to improve cytosolic uptake efficiency, e.g., as described above,whereAAH1has the samechirality as the adjacent arginine, and AAH1 and AAH2 have the opposite chirality.

[0202] In some embodiments, the CPPs described herein include at least three amino acids having a hydrophobic side chain. Accordingly, in some embodiments, the CPPs described herein include one of the following sequences: AAH3‑AAH2‑AAH1‑R-r, AAH3‑AAH2‑AAH1‑R-r, AAH3‑AAH2‑AAH1-r-R, AAH3‑AAH2‑AAH1-r-R, R-r-AAH1‑AAH2‑AAH3, R-r- AAH1‑AAH2‑AAH3, r-R-AAH1‑AAH2‑AAH3, or, r-R-AAH1‑AAH2‑AAH3,whereinAAH3 is any aminoacid having ahydrophobic side chain described above, e.g., piperidine‑2-carboxylic acid, naphthylalanine, tryptophan, or phenylalanine. In some embodiments, the chirality of AAH1, AAH2, and AAH3 can be selected to improve cytosolic uptake efficiency, e.g., as described above, where AAH1 has the same chirality as the adjacent arginine, and AAH1 and AAH2 have the opposite chirality. In other embodiments, the size of AAH1, AAH2, and AAH3 can be selected to improve cytosolic uptake efficiency, e.g., as described above, where AAH3 has a SASA of less than or equal to AAH1 and / or AAH2.

[0203] In some embodiments, AAH1 and AAH2 have the same or opposite chirality. In certain embodiments, AAH1 and AAH2have the opposite chirality. Accordingly, in someembodiments, theCPPsdisclosedherein include at least oneof the following sequences: D-AAH2‑L-AAH1‑R-r; L-AAH2‑D-AAH1-r-R; R-r-D-AAH1‑L-AAH2; or r-R‑ L-AAH1‑D-AAH1, wherein each of D-AAH1 and D-AAH2 is a hydrophobic amino acid having a D configuration, and each of L-AAH1 and L-AAH2 is a hydrophobic amino acid having an L configuration. In some embodiments, each of D-AAH1 and D-AAH2 is independently selected from the group consisting of D-pip, D-nal, D-trp, and D-phe. In particular embodiments, D-AAH1 or D-AAH2 is D- nal. Inotherparticular embodiments,D-AAH1 isD-nal. In someembodiments, eachofL-AAH1andL-AAH2 is independently selected from the group consisting of L-Pip, L-Nal, L-Trp, and L-Phe. In particular embodiments, each of L-AAH1 and L- 38 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 AAH2 is L-Nal. In other particular embodiments, L-AAH1 is L-Nal.

[0204] As discussed above, the disclosure provides for various modifications to a cyclic peptide sequence which may improve cytosolic delivery efficiency. In some embodiments, improved cytosolic uptake efficiency can be measured by comparing the cytosolic delivery efficiency of the polypeptide conjugate or complex of the present disclosure having the modified sequence to a proper control sequence. In some embodiments, the control sequence does not include a particularmodification (e.g.,matching chirality of R andAAH1) but is otherwise identical to themodified sequence. In other embodiments, the control has the following sequence: cyclic(FΦRRRRQ)

[0205] As used herein cytosolic delivery efficiency refers to the ability of a polypeptide conjugate or complex of the present disclosure to traverse a cell membrane and enter the cytosol. In embodiments, cytosolic delivery efficiency of the CPP is not dependent on a receptor or a cell type. Cytosolic delivery efficiency can refer to absolute cytosolic delivery efficiency or relative cytosolic delivery efficiency.

[0206] Absolute cytosolic delivery efficiency is the ratio of cytosolic concentration of a polypeptide conjugate or complex of the present disclosure over the concentration of the polypeptide conjugate or complex of the present disclosure in the growthmedium. Relative cytosolic delivery efficiency refers to the concentration of a polypeptide conjugate or complex of the present disclosure in the cytosol compared to the concentration of a control polypeptide conjugate or complex of the present disclosure in the cytosol. Quantification can be achieved by fluorescently labeling the polypeptide conjugate or complex of the present disclosure (e.g., with a FTIC dye) andmeasuring the fluorescence intensity using techniqueswell- known in the art.

[0207] In particular embodiments, relative cytosolic delivery efficiency is determined by comparing (i) the amount of a polypeptide conjugate or complex of the present disclosure internalized bya cell type (e.g., HeLa cells) to (ii) the amount of the polypeptide conjugate or complex of the present disclosure internalized by the same cell type. To measure relative cytosolic delivery efficiency, the cell typemaybe incubated in thepresenceof a cell-penetrating peptide of the invention for a specified period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.) after which the amount of the polypeptide conjugate or complex of the present disclosure internalized by the cell is quantified using methods known in the art, e.g., fluorescence microscopy. Separately, the same concentration of the control is incubated in the presence of the cell type over the same period of time, and the amount of the control internalized by the cell is quantified.

[0208] Non-limiting examples of suitable cyclic cell penetrating peptide are provided in Table 4. Table 4. Examples of Cyclic Cell-Penetrating Peptides (cCPPs) ID cCPP Sequence SEQ ID NO PCT 1 cyclo(FΦRRRQ) 1 PCT 2 cyclo(FΦRRRC) 2 PCT 3 cyclo(FΦRRRU) 3 PCT 4 cyclo(RRRΦFQ) 4 PCT 5 cyclo(RRRRΦF) 5 PCT 6 cyclo(FΦRRRR) 6 PCT 7 cyclo(FϕrRrRq) 7 PCT 8 cyclo(FϕrRrRQ) 8 PCT 9 cyclo(FΦRRRRQ) 9 PCT 10 cyclo(fΦRrRrQ) 10 PCT 11 cyclo(RRFRΦRQ) 11 PCT 12 cyclo(FRRRRΦQ) 12 PCT 13 cyclo(rRFRΦRQ) 13 PCT 14 cyclo(RRΦFRRQ) 14 PCT 15 cyclo(CRRRRFWQ) 15 PCT 16 cyclo(FfΦRrRrQ) 16 PCT 17 cyclo(FFΦRRRRQ) 17 PCT 18 cyclo(RFRFRΦRQ) 18 PCT 19 cyclo(URRRRFWQ) 19 39 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) ID cCPP Sequence SEQ ID NO PCT 20 cyclo(CRRRRFWQ) 20 PCT 21 cyclo(FΦRRRRQK) 21 PCT 22 cyclo(FΦRRRRQC) 22 PCT 23 cyclo(fΦRrRrRQ) 23 PCT 24 cyclo(FΦRRRRRQ) 24 PCT 25 cyclo(RRRRΦFDΩC) 25 PCT 26 cyclo(FΦRRR) 26 PCT 27 cyclo(FWRRR) 27 PCT 28 cyclo(RRRΦF) 28 PCT 29 cyclo(RRRWF) 29 SAR 1 cyclo(FΦRRRRQ) 30 SAR 19 cyclo(FFRRRQ) 31 SAR 20 cyclo(FFrRrQ) 32 SAR 21 cyclo(FFRrRQ) 33 SAR 22 cyclo(FRFRRQ) 34 SAR 23 cyclo(FRRFRQ) 35 SAR 24 cyclo(FRRRFQ) 36 SAR 25 cyclo(GΦRRRQ) 37 SAR 26 cyclo(FFFRAQ) 38 SAR 27 cyclo(FFFRRQ) 39 SAR 28 cyclo(FFRRRRQ) 40 SAR 29 cyclo(FRRFRRQ) 41 SAR 30 cyclo(FRRRFRQ) 42 SAR 31 cyclo(RFFRRRQ) 43 SAR 32 cyclo(RFRRFRQ) 44 SAR 33 cyclo(FRFRRRQ) 45 SAR 34 cyclo(FFFRRRQ) 46 SAR 35 cyclo(FFRRRFQ) 47 SAR 36 cyclo(FRFFRRQ) 48 SAR 37 cyclo(RRFFFRQ) 49 SAR 38 cyclo(FFRFRRQ) 50 SAR 39 cyclo(FFRRFRQ) 51 SAR 40 cyclo(FRRFFRQ) 52 SAR 41 cyclo(FRRFRFQ) 53 SAR 42 cyclo(FRFRFRQ) 54 SAR 43 cyclo(RFFRFRQ) 55 SAR 44 cyclo(GΦRRRRQ) 56 SAR 45 cyclo(FFFRRRRQ) 57 SAR 46 cyclo(RFFRRRRQ) 58 40 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) ID cCPP Sequence SEQ ID NO SAR 47 cyclo(RRFFRRRQ) 59 SAR 48 cyclo(RFFFRRRQ) 60 SAR 49 cyclo(RRFFFRRQ) 61 SAR 50 cyclo(FFRRFRRQ) 62 SAR 51 cyclo(FFRRRRFQ) 63 SAR 52 cyclo(FRRFFRRQ) 64 SAR 53 cyclo(FFFRRRRRQ) 65 SAR 54 cyclo(FFFRRRRRRQ) 66 SAR 55 cyclo(FΦRrRrQ) 67 SAR 56 cyclo(XXRRRRQ) 68 SAR 57 cyclo(FfFRrRQ) 69 SAR 58 cyclo(fFfrRrQ) 70 SAR 59 cyclo(fFfRrRQ) 71 SAR 60 cyclo(FfFrRrQ) 72 SAR 61 cyclo(tFϕrRrQ) 73 SAR 62 cyclo(fΦfrRrQ) 74 SAR 63 cyclo(ϕFfrRrQ) 75 SAR 64 cyclo(FΦrRrQ) 76 SAR 65 cyclo(fΦrRrQ) 77 SAR 66 Ac‑(Lys-fFRrRrD) 78 SAR 67 Ac‑(Dap-fFRrRrD) 79 SAR 68 80 SAR 69 81 SAR 70 82 SAR 71 83 Pin1 15 cyclo(Pip-Nal-Arg-Glu-arg-arg-glu) 84 Pin1 16 cyclo(Pip-Nal-Arg-Arg-arg-arg-glu) 85 Pin1 17 cyclo(Pip-Nal-Nal-Arg-arg-arg-glu) 86 Pin1 18 cyclo(Pip-Nal-Nal-Arg-arg-arg-Glu) 87 Pin1 19 cyclo(Pip-Nal-Phe-Arg-arg-arg-glu) 88 Pin1 20 cyclo(Pip-Nal-Phe-Arg-arg-arg‑ Glu) 89 Pin1 21 cyclo(Pip-Nal-phe-Arg-arg-arg‑ glu) 90 Pin1 22 cyclo(Pip-Nal-phe-Arg-arg-arg‑ Glu) 91 Pin1 23 cyclo(Pip-Nal-nal-Arg-arg-arg‑ Glu) 92 41 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) ID cCPP Sequence SEQ ID NO Pin1 24 cyclo(Pip-Nal-nal-Arg-arg-arg‑ glu) 93 Rev‑13 [Pim-RQRR-Nlys]GRRRb 94 hLF 95 cTat [KrRrGrKkRrE]c 96 cR10 [KrRrRrRrRrRE]c 97 L‑50 [RVRTRGKRRIRRpP] 98 L‑51 [RTRTRGKRRIRVpP] 99 [WR]4 [WRWRWRWR] 100 MCoTI-II 101 Rotstein et al. Chem. Eur. J. 2011 [P-Cha-r-Cha-r-Cha-r-Cha-r-G]d 102 Lian et al. J. Am. Chem. Soc. 2014 Tm(SvP-F2Pmp-H)‑Dap‑(FΦRRRR-Dap)]f 103 Lian et al. J. Am. Chem. Soc. 2014 [Tm(a-Sar-D-pThr-Pip-ΦRAa)‑Dap‑(FΦRRRR-Dap)]f 104 IA8b [CRRSRRGCGRRSRRCG]g 105 Dod‑[R5] [K(Dod)RRRR] 106 LK‑3 LKKLCKLLKKLCKLAG LKKLCKLLKKLCKLAG 107 RRRR‑[KRRRE]c 108 RRR‑[KRRRRE]c 109 RR‑[KRRRRRE]c 110 R‑[KRRRRRRE]c 111 [CR]4 [CRCRCRCR] 112 cyc3 [Pra-LRKRLRKFRN-AzK]h 113 PMB T-Dap‑[Dap-Dap-f-L-Dap-Dap-T] 114 GPMB T-Agp‑[Dap-Agp-f-L-Agp-Agp-T] 115 cCPP1 cyclo(FΦRRRRQ) 116 cCPP12 cyclo(FfΦRrRrQ) 117 cCPP9 cyclo(fΦRrRrQ) 118 cCPP11 cyclo(fΦRrRrRQ) 119 cCPP18 cyclo(FϕrRrRq) 120 cCPP13 cyclo(FϕrRrRQ) 121 cCPP6 cyclo(FΦRRRRRQ) 122 cCPP3 cyclo(RRFRΦRQ) 123 cCPP7 cyclo(FFΦRRRRQ) 124 cCPP8 cyclo(RFRFRΦRQ) 125 cCPP5 cyclo(FΦRRRQ) 126 42 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) ID cCPP Sequence SEQ ID NO cCPP4 cyclo(FRRRRΦQ) 127 cCPP10 cyclo(rRFRΦRQ) 128 cCPP2 cyclo(RRΦFRRQ) 129 Φ, L‑2-naphthylalanine; ϕ, D‑2-naphthylalanine; Pim, pimelic acid; Nlys, lysine peptoid residue; D-pThr, D-phos- phothreonine; Pip, L-piperidine‑2-carboxylic acid; Cha, L‑3-cyclohexyl-alanine; Tm, trimesic acid; Dap, L‑2,3-diami- nopropionic acid; Sar, sarcosine; F2Pmp, L-difluorophosphonomethyl phenylalanine; Dod, dodecanoyl; Pra, L-pro- pargylglycine; AzK, L‑6-Azido‑2-amino-hexanoic; Agp, L‑2-amino‑3-guanidinylpropionic acid; bCyclization between Pim and Nlys; cCyclization between Lys and Glu; dMacrocyclization by multicomponent reaction with aziridine alde- hyde and isocyanide; eCyclization between the main-chain of Gln residue; fN-terminal amine and side chains of two Dap residues bicyclized with Tm; gThree Cys side chains bicyclized with tris(bromomethyl)benzene; hCyclization by the click reaction between Pra and Azk.

[0209] Additionally, the cCPP used in the polypeptide conjugates and methods described herein can include any sequencedisclosed in:U.S.App.No. 15 / 312,878;U.S.App.No. 15 / 360,719;U.S.App.No.62 / 438,141, andU.S.App.No. 62 / 507,483, each of which is incorporated by reference in its entirety for all purposes. Group that Binds to the Cargo by Electrostatic Interactions (P)

[0210] The polypeptide conjugate of the present disclosure comprises a) a group that binds to a nucleic acid sequence ("cargo") by electrostatic interactions (P) comprising at least one peptide or polyamine; and b) at least one cell-penetrating peptide (CPP); wherein each peptide comprises at least three monomers selected from arginine, arginine-analog, lysine, lysine- analog, histidine, or histidine-analog; wherein the P is conjugated to theCPP through a bond or at least one linker (L); and wherein the polypeptide conjugate is optionally charged.

[0211] In one embodiment, the group that electrostatically interacts with the cargo (P) comprises a polyarginine peptide (pArg). In some embodiments, the polyarginine peptide comprises three arginine monomers or arginine-analog mono- mers. In some embodiments, the polyarginine peptide comprises four argininemonomers or arginine-analogmonomers. In some embodiments, the polyarginine peptide comprises five arginine monomers or arginine-analog monomers. In some embodiments, the polyarginine peptide comprises 3 to 50 arginine monomers or arginine-analog monomers.

[0212] In some embodiments, P comprises polyarginine peptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ariginine monomers or arginine-analog monomers.

[0213] In some embodiments, P comprises a polyarginine peptide having the following repeating units where n is an integer selected from 1 to 100 and the polyarginine peptide can have one additional arginine monomer to make an odd- numbered chain. 43 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0214] In some embodiments, P comprises a polyarginine peptide having the following repeating units where n is an integer selected from 1 to 50. In some embodiment, n is 1, 2, 3, 4, or 5.

[0215] In one embodiment, the group that electrostatically interacts with the cargo (P) comprises a polylysine peptide (pLys). In some embodiments, the polylysine peptide comprises three lysine monomers or lysine-analog monomers. In some embodiments, the polylysine peptide comprises four lysine monomers or lysine-analog monomers. In some embodiments, thepolylysinepeptidecomprisesfive lysinemonomersor lysine-analogmonomers. In someembodiments, the polylysine peptide comprises 3 to 50 lysine monomers or lysine-analog monomers.

[0216] In someembodiments, P comprises polylysine peptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 lysine monomers or lysine-analog monomers.

[0217] In one embodiment, the group that electrostatically interactswith the cargo (P) comprises a polyhistidine peptide (pHis). In some embodiments, the polyhistidine peptide comprises three histidine monomers or histidine-analog mono- mers. In someembodiments, the polyhistidine peptide comprises four histidinemonomers or histidine-analogmonomers. In some embodiments, the polyhistidine peptide comprises five histidine monomers or histidine-analog monomers. In some embodiments, the polyhistidine peptide comprises 3 to 50 histidine monomers or histidine-analog monomers.

[0218] In some embodiments, P comprises polyhistidine peptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 histidine monomers or histidine-analog monomers.

[0219] In one embodiment, the group that electrostatically interacts with the cargo (P) comprises a peptide comprises monomers selected from arginine, arginine-analog, lysine, lysine-analog, histidine, histidine-analog, or mixtures thereof. In some embodiments, the peptide comprises three monomers selected from arginine, arginine-analog, lysine, lysine- analog, histidine, or histidine-analog. In some embodiments, the peptide comprises four monomers selected from arginine, arginine-analog, lysine, lysine-analog, histidine, or histidine-analog. In some embodiments, the peptide comprises five monomers selected from arginine, arginine-analog, lysine, lysine-analog, histidine, or histidine-analog. In some embodiments, the peptide comprises 3 to 50 monomers selected from arginine, arginine-analog, lysine, lysine- analog, histidine, or histidine-analog.

[0220] In some embodiments, P comprises a peptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 monomers selected from arginine, arginine-analog, lysine, lysine-analog, histidine, or histidine-analog. In some embodi- ments, arginine,arginine-analog, lysine, lysine-analog, histidine, or histidine-analogmonomerscanbe inanyorder,DorL, 44 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or permutation thereof. In some embodiment, the monomers alternate D and L in the peptide sequence.

[0221] In some embodiments, P comprises a polyamine selected from a spermidine polymer or a spermine polymer. In some embodiments, P comprises a polyamine structure: or wherein t is an integer selected from 1‑50, or a charged species thereof.

[0222] In some embodiments, P further comprises at least one cysteine monomer or a cysteine-analog monomer. In some embodiments, P further comprises at least two cysteine monomers or cysteine-analog monomers.

[0223] In some embodiments, P further comprises at least one group selected from: or wherein the bond to the hydrogen on one at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP.

[0224] In some embodiments, P further comprises at least one group selected from: 45 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0225] In someembodiments, at least oneof theP (includingP1andP2) in thepolypeptideconjugateasdisclosedherein further comprises In other embodiments, at least one of the P in the polypeptide conjugate as disclosed herein further comprises at least two groups selected form 46 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0226] In some embodiments, P (including P1 and P2) further comprises one or more β-alanine monomers. In one embodiment, P (including P1 and P2) further comprises at least one amino acid monomer (e.g., such as those shown in Table4). Inoneembodiment,P (includingP1andP2) further comprisesa thioethermoiety (-S‑). In someembodiments,P is pArg further comprising one or more β-alanine monomers.

[0227] In some embodiments, P (including P1 and P2) comprises ‑Cys‑(Arg)x-Cys‑(SEQ ID NO: 131), ‑Cys-βA- la‑(Arg)X‑Cys‑ (SEQ ID NO: 132), ‑Cys‑(Arg)X-βAla-Cys‑(SEQ ID NO: 133), ‑Cys-βAla‑(Arg)X-βAla-Cys‑ (SEQ ID NO: 134), ‑Cys‑(polyamine)X‑Cys‑, ‑Cys-βAla‑(polyamine)X‑Cys‑, ‑Cys‑(polyamine)X-βAla-Cys‑, ‑Cys-βAla‑(polyamine)X- βAla-Cys‑ (SEQ ID NO: 135), ‑S‑(polyamine)X‑S‑, wherein x = 3, 4, 5, 6, 7, or 8.

[0228] In someembodiments, "‑" betweendifferent groupsofP (including [P]n, [P]mand [P]o) representsabondbetween two sulfur atoms (a disulfide bond).

[0229] In some embodiments, P, P1, or P2, at each occurrence, are same or different.

[0230] In some embodiments, P is charged. In some embodiments, P is positively charged. In some embodiments, P is positively chargedand interactwith negatively chargednucleic acids by non-covalent interactions. In someembodiments, P is positively charged and interact with negatively charged nucleic acids by electrostatic interactions.

[0231] In some embodiments, P binds (by electrostatic interactions) to a cargo to form 1:1 (mol:mol) complex. Without being bound to any theory, the complex formed between P and the cargo (e.g., nucleic acid) can protect the cargo and P from enzymatic degradation during storage or in vivo circulation.

[0232] In some embodiments, P is biodegradable. In some embodiments, P can undergo reductive cleavage inside the cytosol. In some embodiments, the reductive cleavage is by cleavage of the disulfide bonds. In some embodiment, the degradation is by proteolytic degradation. In some embodiments, P can release the cargo inside the cytosol. Linkers (L)

[0233] In some embodiments, the polypeptide conjugate comprises a linker (L) that connects the CPP to the P. In some embodiment of the polypeptide conjugate as disclosed herein, at least one L comprises a divalent optionally substituted group selected from amino acid, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, hetero- cyclyl, heteroaryl, ‑(R1-X-R2)z‑, or combinations thereof; wherein each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20.

[0234] In some embodiments of the polypeptide conjugate as disclosed herein, at least one L comprises a divalent polyethyleneglycolmoiety. In someembodiments, at least oneLcomprisesanoptionally substituted ‑(O-CH2CH2)z‑or an optionally substituted ‑(CH2CH2‑O)z‑. In some embodiments, at least one L comprises a divalent 8-amino‑3,6-dioxaoc- tanoic acid residue. In other embodiments, at least one L comprises a divalent 8-amino‑3,6,9-trioxaundecanoic acid residue.

[0235] In some embodiments, L comprises one or more groups selected from: 47 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0236] In some embodiments, at least one L comprises a physiological cleavable group (PCG). In some embodiments, eachPCG is, independently, selected from ‑S-S‑, carbonate (-O-C(O)O‑), thiocarbonate (-O-C(S)O‑), thioester (-C(S)O‑), sulfoxide (-S(O)‑), hydrazine (-H2N-NH2‑), or protease-cleavable dipeptide linker. In other embodiments, each PCG comprises at least one ‑S-S‑.

[0237] In some embodiments, L comprises two or more PCGs. Non-limiting examples of such L include dendrimers (e.g., cyclotriphosphazene, polypropylenimine, polylysine, and polyamidoamine dendrimers having appropriate terminal groups to form a PCG), dendrons (e.g., 2-bis(hydroxymethyl)propanyl-based dendrons having appropriate terminal groups to form a PCG), and hyperbranched polymers (e.g., hyperbranched bis-MPA polyester ving appropriate terminal groups to form a PCG).

[0238] In someembodiments of anyoneof thepolypeptide conjugateasdisclosedherein, at least oneof the "‑" between L (including L1 and L2) andP (including [P]n [P1]p, [P2]q and ([P]m)s) represents a bond between two sulfur atoms (disulfide bond). In other embodiments, the "‑" between L and P each represents a bond between two sulfur atoms (disulfide bond).

[0239] In some embodiments, L, L1, or L2, at each occurrence, are same or different. Complex

[0240] The present disclosure also relates to a complex comprising any one of the polypeptide conjugate as disclosed herein and a nucleic acid sequence (also referred to as cargo).

[0241] In some embodiments, the cargo is charged. In some embodiments, the cargo is negatively charged. In some embodiments, the negatively charged cargo interacts with positively charged P by non-covalent interactions. In some embodiments, the negatively charged cargo interacts with positively charged P by electrostatic interactions.

[0242] In someembodiments, the cargo is anucleic acid. In someembodiments, the cargo is anucleic acid sequence. In some embodiment, the nucleic acid or the nucleic acid sequence is therapeutically active or therapeutically effective.

[0243] In some embodiments, the cargo comprises therapeutically active agent for a gene therapy. In some embodi- ments, the cargo comprises splice-switching oligonucleotides, microRNAs, anti-microRNAs, antisense oligonucleotides, small interfering DNAs, plasmid DNAs, small interfering RNAs and / or mRNAs.

[0244] In some embodiments, the cargo comprises therapeutically active agent for a gene-editing.

[0245] In some embodiments, the cargo can enhance target gene expression or modulate / or switch mRNA splicing to express desired gene products.

[0246] Thepresent disclosurealso relates to a cell comprisinganyoneof thepolypeptide conjugateasdisclosedherein. The present disclosure also relates to a cell comprising a complex comprising any one of the polypeptide conjugate as disclosed herein and a nucleic acid sequence. Polypeptide Conjugate as a Delivery Device

[0247] The present disclosure also relates to a method of delivering the cargo (e.g., a nucleic acid sequence) to a cell, comprising contacting the cell with any one of the polypeptide conjugate complex as disclosed herein.

[0248] The present disclosure also relates to a method of delivering the cargo to a cell of a subject in need thereof, comprising administering any one of the polypeptide conjugate complex as disclosed herein.

[0249] The present disclosure also relates to a method of treating a disease or condition in a patient in need thereof, comprising administering any one of the polypeptide conjugate complex as disclosed herein to the patient. The disease or conditionmaybeanydiseaseor condition that canbe treated bygene replacement or gene therapy. In someembodiment, the disease or condition is cancer, genetic diseases, autoimmunity, inflammatory diseases, neurodegenerative diseases, or infectious diseases. 48 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 Exemplary Embodiments

[0250] Exemplary Embodiments of the present invention include, but are not limited to, the following. Embodiment 1. A polypeptide conjugate comprising: a) a group that binds to a nucleic acid sequencebyelectrostatic interactions (P) comprisingat least onepeptide or polyamine; and b) at least one cell-penetrating peptide (CPP); wherein each peptide comprises at least three monomers selected from arginine, arginine-analog, lysine, lysine- analog, histidine, or histidine-analog; wherein the P is conjugated to theCPP through a bond or at least one linker (L); and wherein the polypeptide conjugate is optionally charged. Embodiment2.ThepolypeptideconjugateofEmbodiment1,wherein themolar ratio ofP:CPP ranges fromabout 30:1 to about 1:2. Embodiment3.ThepolypeptideconjugateofEmbodiment1orEmbodiment2,wherein thepolypeptideconjugatehas an average molecular weight ranging from about 3 kDa to about 100 kDa. Embodiment 4. The polypeptide conjugate of any one of Embodiments 1‑3 having the following structure: CPP-L‑[P]n‑L-CPP wherein n is an integer from 1 to 50; and wherein P at each occurrence is same or different. Embodiment 5. The polypeptide conjugate of any one of Embodiments 1‑4, wherein P comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog. Embodiment 6. The polypeptide conjugate of any one of Embodiments 1‑5, wherein P comprises a polyamine selected from a spermidine polymer or a spermine polymer. Embodiment 7. The polypeptide conjugate of any one of Embodiments 1‑6, wherein at least one of theCPP is a cyclic CPP (cCPP). Embodiment 8. The polypeptide conjugate of any one of Embodiments 1‑7, wherein each CPP is, independently, a cyclic CPP (cCPP). Embodiment 9. The polypeptide conjugate of Embodiment 7 or 8, wherein each cCPP independently comprises from 4 to 14 amino acid monomers. Embodiment 10. The polypeptide conjugate of Embodiment 9, wherein each cCPP is, independently, selected from Table 4. Embodiment 11. The polypeptide conjugate of Embodiment 9, wherein the cCPP is a cyclo(fΦRrRrQ) peptide or a cyclo(FfΦRrRrQ), wherein: F is a L-phenylalanine; f is a D-phenylalanine; Φ is an L‑2-naphthylalanine; R is a L-arginine; r is a D-arginine; and Q is a L-glutamine. Embodiment 12. Thepolypeptide conjugateof anyoneofEmbodiments 5‑11,wherein thepArgcomprisesat least five arginine monomers or arginine-analog monomers. Embodiment 13. The polypeptide conjugate of any one of Embodiments 5‑12, wherein the pArg further comprises at least one cysteine monomer. Embodiment 14. The polypeptide conjugate of Embodiment 5 having the following structure: CPP-L‑[pArg]n‑L-CPP wherein the [pArg]n is 49 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or a charged species thereof. Embodiment 15. The polypeptide conjugate of any one of Embodiments 4‑14, wherein n is an integer 1 to 40. Embodiment 16. The polypeptide conjugate of any one of Embodiments 4‑15, wherein at least one L comprises a divalent optionally substituted group selected from amino acid, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1-X-R2)z‑, or combinations thereof; wherein each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20. Embodiment 17. The polypeptide conjugate of any one of Embodiments 4‑16, wherein at least one L comprises an optionally substituted ‑(O-CH2CH2)z‑ or an optionally substituted - (CH2CH2‑O)z‑. Embodiment 18. The polypeptide conjugate of any one of Embodiments 4‑17, wherein at least one L comprises a divalent 8-amino‑3,6-dioxaoctanoic acid residue. Embodiment 19. The polypeptide conjugate of any one of Embodiments 4‑17, wherein at least one L comprises a divalent 8-amino‑3,6,9-trioxaundecanoic acid residue. Embodiment 20. The polypeptide conjugate of any one of Embodiments 4‑19, wherein at least one L comprises a physiological cleavable group (PCG). Embodiment 21. The polypeptide conjugate of Embodiment 20, wherein each PCG is, independently, selected from ‑S-S‑, carbonate, thiocarbonate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. Embodiment22.ThepolypeptideconjugateofEmbodiment20or21,whereineachPCGcomprisesat least one ‑S-S‑. Embodiment 23. The polypeptide conjugate of any one of Embodiment 4‑22, wherein at least one of the "‑" between L and [P]n represents a bond between two sulfur atoms (disulfide bond). Embodiment 24. Thepolypeptide conjugateof anyoneofEmbodiments 4‑22,wherein the "‑" betweeneachLand [P]n represents a bond between two sulfur atoms (disulfide bond). Embodiment 25. The polypeptide conjugate of any one of Embodiments 1‑24, wherein each P, independently, further comprises at least one group selected from: 50 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or wherein the bond to the hydrogen on at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP. Embodiment 26. The polypeptide conjugate of Embodiment 25, wherein each P, independently, further comprises at least one group selected from: Embodiment 27. The polypeptide conjugate of any one of Embodiments 1‑24, wherein at least one of the P further comprises 51 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 Embodiment 28. The polypeptide conjugate of any one of Embodiments 1‑24, wherein at least one of the P further comprises at least two groups selected from Embodiment 29. The polypeptide conjugate of Embodiment 1 having the following structure: CPP-L‑([P1]p‑L1)t‑[P]n‑(L2‑[P2]q)t‑L-CPP wherein n, p, and q are each independently an integer from 1 to 50; t is each independently 0 or 1; P1 and P2 each comprises at least one peptide or polyamine, wherein each peptide comprises at least three monomers selected from arginine, arginine-analog, lysine, lysine-analog, histidine, or histidine-analog, wherein P, P1 and P2, at each occurrence, are same or different; and L1 and L2 are each independently absent or L as defined in claim 1,wherein L, L1, and L2, at each occurrence, are same or different. Embodiment 30. The polypeptide conjugate of Embodiment 29, wherein P, P1 or P2 comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog. Embodiment 31. The polypeptide conjugate of Embodiment 29 or 30, wherein P, P1 or P2 comprises a polyamine selected from a spermidine polymer or a spermine polymer. Embodiment 32. The polypeptide conjugate of any one of Embodiments 29‑31, wherein at least one of the CPP is a cyclic CPP (cCPP). Embodiment 33. The polypeptide conjugate of any one of Embodiments 29‑32, wherein the CPP is, each indepen- dently, a cyclic CPP (cCPP). Embodiment 34. Thepolypeptide conjugate ofEmbodiment 32or 33,wherein the cCPPcomprises from4 to14amino acid monomers. Embodiment 35. The polypeptide conjugate of Embodiment 34, wherein the cCPP is, each independently, selected from Table 4. Embodiment 36. The polypeptide conjugate of Embodiment 35, wherein the cCPP is a cyclo(fΦRrRrQ) peptide or a cyclo(FfΦRrRrQ), wherein: F is a L-phenylalanine; f is a D-phenylalanine; Φ is an L‑2-naphthylalanine; R is a L-arginine; r is a D-arginine; and Q is a L-glutamine. Embodiment 37. The polypeptide conjugate of any one of Embodiments 30‑35, wherein the pArg comprises at least five arginine monomers or arginine-analog monomers. Embodiment 38. The polypeptide conjugate of any one of Embodiments 30‑37,wherein the pArg further comprises at least one cysteine monomer. Embodiment 39. The polypeptide conjugate of any of Embodiments 29‑38, wherein n is an integer 5 to 40. Embodiment 40. The polypeptide conjugate of any one of Embodiments 29‑39, wherein at least one of L, L1, or L2 comprisesadivalent optionally substitutedgroupselected fromaminoacid, polyethyleneglycol, alkylene, alkenylene, 52 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1-X-R2)z‑, or combinations there- of; wherein each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20. Embodiment 41. The polypeptide conjugate of any one of Embodiments 29‑40, wherein at least one of L, L1, or L2 comprises an optionally substituted ‑(O-CH2CH2)z‑ or an optionally substituted ‑(CH2CH2‑O)z‑. Embodiment 42. The polypeptide conjugate of any one of Embodiments 29‑40, wherein at least one of L, L1, or L2 comprises a divalent 8-amino‑3,6-dioxaoctanoic acid residue. Embodiment 43. The polypeptide conjugate of any of Embodiments 29‑40, wherein at least one of L, L1, or L2 comprises a divalent 8-amino‑3,6,9-trioxaundecanoic acid residue. Embodiment 44. The polypeptide conjugate of any of Embodiments 29‑43, wherein at least one of L, L1, or L2 comprises a physiological cleavable group (PCG). Embodiment 45. The polypeptide conjugate of Embodiment 44, wherein each PCG is, independently, selected from ‑S-S‑, carbonate, thiocarbonate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. Embodiment46.ThepolypeptideconjugateofEmbodiment44or45,whereineachPCGcomprisesat least one ‑S-S‑. Embodiment 47. The polypeptide conjugate of any of Embodiments 29‑46, wherein at least one of the "‑" between L and ([P1]p‑L1)t, L and [P]n, or L and (L2‑[P2]q)t represents a bond between two sulfur atoms (disulfide bond). Embodiment 48. The polypeptide conjugate of any of Embodiments 1‑47, wherein at least one of P, P1 or P2 further comprises at least one group selected from: or wherein the bond to the hydrogen on at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP Embodiment 49. Thepolypeptide conjugateofEmbodiment 48,wherein at least oneofP,P1orP2 further comprisesat least one group selected from: 53 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 Embodiment 50. The polypeptide conjugate of any one of Embodiments 29‑49, wherein at least one of P, P1 or P2 further comprises Embodiment 51. The polypeptide conjugate of any one of Embodiments 29‑49, wherein at least two of P, P1 or P2 further comprises 54 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 Embodiment 52. The polypeptide conjugate of Embodiment 5 or 30, wherein the pArg further comprises one or more β-alanine monomers. Embodiment 53. The polypeptide conjugate of Embodiment 5 or 30, wherein each pArg comprises, independently, 3, 4, 5, 6, 7, or 8 arginine monomers. Embodiment 54. The polypeptide conjugate of Embodiment 5 or 30, wherein each pArg comprises 5 arginine monomers. Embodiment 55. The polypeptide conjugate of any one of Embodiments 52‑54, wherein at least one pArg further comprises a cysteine monomer. Embodiment56.ThepolypeptideconjugateofEmbodiments5or30,wherein thepArg iseach independently selected from: -Cys‑(Arg)X‑Cys‑, ‑Cys-βAla‑(Arg)X‑Cys‑, ‑Cys‑(Arg)X-βAla-Cys‑, or ‑Cys-βAla‑(Arg)X-βAla-Cys‑, ‑Cys‑(Arg)X‑Cys‑, ‑Cys-βAla‑(Arg)X‑Cys‑, ‑Cys‑(Arg)X-βAla-Cys‑, or ‑Cys-βAla‑(Arg)X-βAla-Cys‑, wherein x = 3, 4, 5, 6, 7, or 8. Embodiment 57. The polypeptide conjugate of Embodiment 4 or 29, wherein the P is each independently selected from: -Cys‑(Arg)X‑Cys‑, ‑Cys-βAla‑(Arg)X‑Cys‑, ‑Cys‑(Arg)X-βAla-Cys‑, or ‑Cys-βAla‑(Arg)X-βAla-Cys‑, ‑Cys‑(polyami- ne)X‑Cys‑, ‑Cys-βAla‑(polyamine)X‑Cys‑, ‑Cys‑(polyamine)X-βAla-Cys‑, ‑Cys-βAla‑(polyamine)X-βAla-Cys‑, ‑S‑(polyamine)X‑S‑, wherein x = 3, 4, 5, 6, 7, or 8. Embodiment 58. The polypeptide conjugate of Embodiment 1 having the structure , or a charged species thereof. Embodiment 59. The polypeptide conjugate of Embodiment 1 having the structure , or a charged species thereof. Embodiment 60. The polypeptide of Embodiment 1 having the following structure: CPP-L‑([P]m)s, wherein: s is an integer from 1 to 10; each m is, independently, an integer from 1 to 50; and wherein P at each occurrence is same or different. Embodiment 61. The polypeptide conjugate of Embodiment 60, wherein P comprises a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog. Embodiment 62. Thepolypeptide conjugateofEmbodiment 60or 61,whereinPcomprisesapolyamine selected from 55 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 a spermidine polymer or a spermine polymer. Embodiment 63. The polypeptide conjugate of any of Embodiments 60‑62, wherein the CPP is a cyclic CPP (cCPP). Embodiment 64. The polypeptide conjugate of Embodiment 63, wherein the cCPPcomprises from4 to 14 amino acid monomers. Embodiment 65. The polypeptide conjugate of Embodiment 64, wherein the cCPP is selected from Table 4. Embodiment 66. The polypeptide conjugate of any of Embodiments 61‑65, wherein the pArg comprises at least five arginine monomers or arginine-analog monomers. Embodiment67.ThepolypeptideconjugateofanyofEmbodiments61‑65,wherein thepArg further comprisesat least one cysteine monomer. Embodiment 68. The polypeptide conjugate of Embodiment 60‑67, wherein L comprises a divalent optionally substituted group selected from amino acid, polyethylene glycol, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ‑(R1-X-R2)z‑, or combinations thereof; wherein each of R1 and R2 are independently selected from a bond, alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, wherein R1 and R2 are not both a bond; each X is independently N, S, and O; and z is an integer selected from 1 to 20. Embodiment 69. The polypeptide conjugate of any one of Embodiments 60‑68, wherein L comprises an optionally substituted ‑(O-CH2CH2)z‑ or an optionally substituted ‑(CH2CH2‑O)z‑. Embodiment 70. The polypeptide conjugate of any one of Embodiments 60‑68, wherein at least one L comprises a divalent 8-amino‑3,6-dioxaoctanoic acid residue. Embodiment 71. The polypeptide conjugate of any of Embodiments 60‑68, wherein L comprises a divalent 8- amino‑3,6,9-trioxaundecanoic acid residue. Embodiment 72. The polypeptide conjugate of any of Embodiments 60‑71, wherein L comprises a physiological cleavable group (PCG). Embodiment 73. The polypeptide conjugate of Embodiment 72, wherein each PCG is, independently, selected from ‑S-S‑, carbonate, thiocarbonate, thioester, sulfoxide, hydrazine, or protease-cleavable dipeptide linker. Embodiment74.ThepolypeptideconjugateofEmbodiment72or73,whereineachPCGcomprisesat least one ‑S-S‑. Embodiment 75. The polypeptide conjugate of Embodiment 60‑67, wherein L comprises a polythiolamine or a 3,5- bis(mercaptomethyl)benzoyl (Bmb) amide. Embodiment 76. The polypeptide conjugate of Embodiment 60‑67, wherein L comprises comprises two or more a physiological cleavable group (PCG). Embodiment 77. The polypeptide conjugate of any of Embodiments 60‑76, wherein the "‑" between L and ([P]m)s represents a bond between two sulfur atoms (disulfide bond). Embodiment 78. The polypeptide conjugate of any of Embodiments 60‑77, wherein each P, independently, further comprises at least one group selected from: or 56 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 wherein the bond to the hydrogen on at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the CPP Embodiment 79. The polypeptide conjugate of Embodiment 78, wherein each P, independently, further comprises at least one group selected from: Embodiment 80. The polypeptide conjugate of any one of Embodiments 60‑78, wherein at least one of the P further comprises 57 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 Embodiment 81. The polypeptide conjugate of any one of Embodiments 60‑80, wherein at least one of the P further comprises at least two groups selected form Embodiment 82. The polypeptide conjugate of Embodiment 60‑81, wherein s is 2, 3, 4, or 5. Embodiment 83. The polypeptide conjugate of any one of Embodiments 61‑67 having the structure , or a charged species thereof. Embodiment 84. The polypeptide conjugate of Embodiment 60 having the structure 58 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or a charged species thereof. Embodiment 85. The polypeptide conjugate of Embodiment 4 having the following structure: cCPP-L‑[P]n‑[P]m‑(L-cCPP)v wherein, cCPP is a cyclic CPP; and n is an integer selected from 1 to 50; m is an integer selected from 0 to 49 provided that the sum of n and m is 50 or less; v is 0 or 1; wherein P at each occurrence is same or different; and wherein when v is 0, the last [P] in [P]m is monovalent. Embodiment 86. The polypeptide conjugate of Embodiment 85, wherein the "‑" between [P]n and [P]m represents a bond between two sulfur atoms (a disulfide bond). Embodiment 87. The polypeptide conjugate of Embodiment 85 having the following structure: , or a charged species thereof. Embodiment 88. Thepolypeptide conjugate of Embodiment 87,wherein the "‑" between [P] and [P] represents a bond between two sulfur atoms (a disulfide bond). Embodiment 89. The polypeptide of Embodiment 1 having the following structure: cCPP-L‑[P]n‑[P]m‑[P]o‑(L-cCPP)v wherein, cCPP is a cyclic CPP; and n is an integer selected from 1 to 50; m and o, are each independently, an integer from 0 to 49 provided that the sum of n, m, and o is 50 or less; v is 0 or 1; wherein P at each occurrence is same or different; and wherein when v is 0, the last [P] in [P]o is monovalent. Embodiment 90.Thepolypeptide conjugateofEmbodiment 89,wherein at least oneof the "‑" between [P]n and [P]mor [P]m and [P]o represents a bond between two sulfur atoms (a disulfide bond). Embodiment 91. The polypeptide conjugate of Embodiment 89, wherein the "‑" between [P]n and [P]m and between [P]m and [P]o represents a bond between two sulfur atoms (a disulfide bond). Embodiment 92. The polypeptide conjugate of Embodiment 89 having the following structure: 59 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 , or a charged species thereof. Embodiment 93. The polypeptide conjugate of Embodiment 92, wherein at least one of the "‑" between [P] and [P] represents a bond between two sulfur atoms (a disulfide bond). Embodiment 94. The polypeptide conjugate of Embodiment 92, wherein each "‑" between [P] and [P] represents a bond between two sulfur atoms (a disulfide bond). Embodiment 95. A complex comprising the polypeptide conjugate of any one of Embodiments 1‑94 and at least one nucleic acid sequence. Embodiment 96. A cell comprising the complex of Embodiment 95. Embodiment 97. A cell comprising the polypeptide conjugate of any one of Embodiments 1‑94. Embodiment 98. A method of delivering a nucleic acid sequence to a cell, comprising contacting the cell with the complex of Embodiment 95. Embodiment 99. A method of delivering a nucleic acid sequence to a cell of a subject in need thereof, comprising administering the complex of Embodiment 95. Embodiment100.Amethodof treatingadiseaseorcondition inapatient inneed thereof, comprisingadministering the complex of Embodiment 95 to said patient. EXAMPLES Example 1. Polypeptide conjugate with two terminal cCPPs

[0251] Design and Synthesis. A non-covalent complex between cyclic CPP-based delivery vector and a nucleic acid (NA) cargo of interest instead of covalent attachment was selected for a number of reasons. First, in a covalent CPP- nucleic acid conjugate, the negatively charged nucleic acid may interact with the positively charged CPP (either intramolecularly or intermolecularly) and mutually interfere with each other’s function. Second, in a covalent adduct, the nucleic acid cargo is unprotected from nuclease action and may have limited in vivo stability. Third, production of a covalent conjugate is more complex, requiring chemical synthesis of each component followed by biorthogonal con- jugation, whereas a non-covalent complex can be formed by simply mixing the two components. However, it was also desired to avoid the formation of nanoparticles, which are largely limited to biodistribution into the liver and kidney (see Juliano, R. L. Nucleic Acids Res., 2016, 44(14): 6518‑6548; Shi, B. et al., J HistochemCytochem., 2011, 59(8): 727‑740). With these considerations in mind, a novel NA delivery system was designed consisting of a biodegradable polyarginine sequenceflankedby twocyclicCPPs,whicharecopolymerized through the formationof aseriesof disulfidebonds (Figure 1). The polyarginine moiety was expected to bind tightly to a NA cargo (e.g., siRNA) through electrostatic interactions to form a 1:1 (mol / mol) complex, while the two terminal CPPs would be exposed for binding to the cell membrane and ensuring cellular entry of the complex. Non-covalent complexation could allow the same vector to deliver different siRNA sequences (and potentially other NAs). 60 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55

[0252] CPP9 (seeTable 4), which has a cytosolic delivery efficiency of 62%,was selected for this study. Tominimize any mutual interference between the CPP and the cargo, a long, flexible linker (miniPEG) was attached to the Gln side chain and a cysteine was added to the other end of the linker. The polyarginine sequencewas formed by polymerization of Cys- Arg-Arg-Arg-Arg-Arg-Cys (SEQ ID NO: 136) (CR5C) through the formation of disulfide bonds. A pentaarginine was selectedbecause longer polyarginines (e.g.,R10),without bound toany theory,wouldbindmore tightly toNAsandmaynot effectively release the cargo inside the cell.

[0253] Peptides CR5C (SEQ ID NO: 136) and cyclo(fΦRrRrQ)‑miniPEG-Cys (SEQ ID NO: 141) were synthesized manually onRink amide resin (0.54mmol / g) using standard Fmoc chemistry. The typical coupling reaction contained 5 eq of Fmoc-amino acid, 5 eqHATUand 10 eqDIPEA inDMFwithmixing for 1 h. TheN-terminal Fmoc groupwas removed by 20%piperidine in DMF. For cyclo(fΦRrRrQ) peptide (SEQ IDNO: 118), after the entire sequence was completed but prior to Fmoc deprotection, the allyl group on the C-terminal Glu was removed by treatment with 0.1 eq Pd(PPh3)4 and 10 eq phenylsilane inDCM (3 x 15min). TheN-terminal Fmocwas then removed and the peptidewas cyclized by treatment with 5 eq PyBOP, 5 eq HOBt and 10 eq DIPEA in DMF for 3 h. The peptides were cleaved from the resin and side chain deprotected by treatment with 90 / 2.5 / 2.5 / 2.5 / 2.5 (v / v) TFA / TIPS / DMB / DCM / EDT / water for 3 h. The peptides were triturated with cold ethyl ether and purified by reversed-phase HPLC. The purity of peptides (>95%) was confirmed with an analytical reversed-phase HPLC and the identity of peptides was confirmed by MALDI-TOF mass spectrometry.

[0254] Three different polymerization reactions were carried out by mixing 5, 10 or 20 equivalents of CR5C and 1 equivalent of CPP9-miniPEG-Cys in PBS (pH 7.4) containing 30% DMSO and stirring the mixture for 24 h. The reaction products were dialyzed against a semipermeable membrane (MWCO: 10 kDa) to remove DMSO and low-MW species. The resulting polymers were lyophilized. The expected MWs of the polymers are listed in Table 5. Table 5. Theoretical molecular weight (MW) of CRC polymers Polymer Mole ratio of CR5C per CPP9 Theoretical MW (kDa) CRC5 5 12.7 CRC10 10 26.9 CRC15 20 42.8

[0255] Polymer Characterization by Analytical HPLC. Analytical HPLC of the CRC5 polymer (from 5:1 CR5C and CPP9) revealed amixture ofmany species, with themost abundant species having a retention time of 43.3min (Figure 2). Treatment of the CRC5 polymer with 10mMDTTconverted the polymer into amixture of predominantly two species, with retention times corresponding to those of CR5C andCPP9-miniPEG-Cys, suggesting that the CRC5 polymer was indeed formed, but contained a mixture of polymers of different numbers of CR5C units.

[0256] Estimation of MW by 1H-NMR. The 1H-NMR spectra of CRC5 and CRC10 polymers (5 mM concentration in 100%D2O)were recordedat 600MHz (Bruker,MA,USA).Figure3shows the 1H-NMRspectrumofCRC5.Resonancesat δ1.4‑1.6 (signal a), δ1.6‑1.9 (signal b) andδ3.1‑3.3 (signal d)were assigned to the ‑CH2‑groups in thearginine side chain. The signals at δ2.8‑3.1 (signal c) were assigned to the ‑CH2‑ group of cysteine. The signals at δ3.6‑3.9 (signal e) were derived from theminiPEG linker. The signals at δ4.0‑4.4 (signal f) were from theCα‑H’s of amino acids. Signals at δ6.7‑8.4 (g) are assigned to the aromatic protons from phenylalanine and naphthylalanine. The average number of repeating units (CR5C) in thepolymers (n) and theMWof thepolymerswereestimatedbycomparing the integratedsignal intensitiesof the arginine side chains (signals a, b, c, d) and the aromatic protons (signal g). For the CRC5 polymer, n was found to be 8, corresponding to a MW of 10.6 kDa. For the CRC10 polymer, n was found to be 20 and the average MW was 22.6 kDa. These values are similar to the theoretical values (Table 5).

[0257] Estimation of MW by Gel-Filtration Chromatography (GPC). The MW distribution of the polymers (in 0.1 M NaCl) was assessed byGPCon aWaters 600HPLC system (Waters,MA, USA) equippedwith a TSKgel G3000PWxl-CP column (TosohBiosciences,CA,USA)andUVdetectionat 214nm.Poly-L-lysine (PLL) of varyingMWs (4.8, 12, 24and61 61 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 kDa) were used as MW standards (Figure 4A). Comparison of the GPC chromatogram of CRC5 to PLL suggested that 1‑2% of the reaction mixture corresponded to a species with MW ~60 kDa (peak at ~6.2 min). The predominant species, however, had retention timesof 8.3 and9.0min, corresponding toMWof 4‑5kDaand2‑4 kDa, respectively (Figure 4B, top line at t = 9 min). A similar MW distribution was observed for the CRC10 polymer. However, the ~60 kDa species (peak at ~6.2 min) was present at a higher percentage (~15% of the total) than in CRC5 (Figure 4B, bottom line at t = 9 min). A species with very high MW (retention time = 3.2 min) was also observed. It should be noted that neither the NMRmethod described above nor the GPC analysis can provide an accurate measurement of the MWs. More accurate MW determination will require prior separation of the polymer mixture into individual species and NMR analysis of the individual species.

[0258] Cytotoxicity.The cytotoxicity of CRC5polymer onHeLa cellswas evaluated using theMTTassay. HeLa cells (3 x103cellsperwell) in100µLofDMEMcontaining10%FBSwereseeded in96-well plateand incubated for24h.Thencells were treated with varying concentrations (0‑40 µM) of CRC5 and incubated at 37 °C with 5%CO2 for 72 h. An MTTstock solution (10 µL; 5 mg / mL) was added into each well. The plate was incubated at 37 °C for 4 h. Then 100 µL of SDS-HCl solubilizing buffer was added into each well, and the resulting solution was mixed thoroughly. The plate was incubated at 37 °Covernight. Theabsorbanceof the formazanproductwasmeasuredat 570nmonaTecanM1000plate reader.CRC5 was relatively non-toxic to HeLa cells, causing ≤15% reduction in viability over the concentration range of 0 to 40 µM (or 0‑500µg / mL;Figure5A). In contrast, additionof 2µLof thecommercial lipofectaminesolution (which is the recommended amount for transfection experiments) reduced the viability of HeLa cells by 35% (Figure 5B).

[0259] siRNA Binding. The ability of CRC5 and CRC10 to bind siRNA targeting the firefly luciferase gene (siLuc) was tested by the gel retardation method using various N / P ratios, which correspond to the ratio between positively charged amino groups in the polymer (N stands for nitrogen in amino group) to negatively charged phosphate groups in the nucleic acid (P stands for phosphorous in phosphate group). The CRC polymer / siRNA complexes were formed with a fixed concentrationof siRNAand increasingconcentrationsofCRCpolymer toN / P ratiosof1:1 to50:1.FreesiRNAwasusedas a control. The samples were separated by electrophoresis on 1% agarose gel containing ethidium bromide at 70 V for 30 min inTris-acetate-EDTA(TAE)buffer.While freesiLucRNAgaveadiscretebandon thegel, additionof theCRCpolymers progressively decreased the intensity of the free RNA band and resulted in the formation of apparently an RNA / polymer complex that failed to migrate out of the sample loading well. Complete conversion of free siRNA into the RNA / polymer complex was observed at an N / P ratio of 10 for CRC5, while the corresponding ratio was 5 for CRC10. As expected, treatment of a preformed siLuc / CRC5 complex with 10 mMDTT resulted in a discrete band with the samemobility as the free RNA.

[0260] Cellular Uptake. To monitor the intracellular uptake of the CRC5 / siLuc complex, 5’-fluorescein (FAM)‑labelled siLuc (1 or 3 µM) was mixed with CRC5 to give an N / P ratio of 10 in OptiMEM or DMEMwith 1% FBS and 1% Abs. HeLa cells were seeded in a 35mm glass-bottomedmicrowell dish at a density of 3 x 104 cells / mL and cultured overnight. Cells werewashed twicewithDPBSand treatedwith theCRC5 / 5’-FAM-siRNAcomplex for 2h.After removal of themedium, the cellsweregentlywashedwithDPBS twiceand imagedonaNikonA1R live-cell confocalmicroscopeequippedwitha100× oil objective or aVisitech Infinity 3Hawk2D-array live cell confocalmicroscopeequippedwith 60×oil objective.Datawere analyzedusingNISElemenetsARorMetaMorphPremier. Amixof diffuseandpunctate fluorescencewasobserved inside the cytoplasm of all treated cells, indicating that the CRC5 / siLuc complex was able to enter the cells and at least partially escaped from the endosomes into the cytosol (Figure 6). In Figure 6, live-cell confocal microscopic images of HeLa cells treated with the CRC5 / 5’-FAM-siLuc complex at 3 µM siRNA in OptiMEM (top) or at 1 µM siRNA in DMEMwith 1% FBS, 1% Abs (bottom). Left, GFP channel; Center, DIC; and right, overlap of the above.

[0261] Knockdownof LuciferaseExpression.The ability of CRCpolymers to deliver siRNA intracellularly was tested on aHeLa cell line stably transfectedwith a firefly luciferase gene (HeLa-Luc). The siLuc sequences usedwere: sense 5’- CUUACGCUGAGUACUUCGAdTdT‑3’ (SEQ ID NO: 137) and antisense 5’-UCGAAGUACUCAGCGUAAGdTdT‑3’ (SEQ ID NO: 138). Hela-Luc cells were seeded onto 96-well plate at a density of 1.5 x 104 cells / well in 100 µL of DMEM containing 10% FBS and cultured overnight. CRC5 / siLuc complexes were formed by mixing CRC5 with siLuc in 20 mM HEPES,pH7.4 followedby15min incubation.Complexeswere thenmixedwithOptiMEM,added to cells and incubatedat 37 °C for 24 or 48 h. Free siLuc, CRC5, Lipofectamine2000 / siLuc, and pCRC / siLuc were used as controls. pCRC is a polymer of CR5C without CPP9 at the two termini. Lipofectamine2000 was complexed with siRNA according to the manufacturer’s protocol. A luciferase assay kit (OZBiosciences)wasused to quantitate the luciferasegene silencing level according to manufacturer’s protocol. Luminescence was measured on a Tecan Infinite M1000 plate reader.

[0262] Treatment of HeLa-Luc cells with free siLuc, CRC5 alone, or the pCRC / siLuc complex did not reduce the luciferase expression (Figure 9). On the other hand, the CRC5 / siLuc complex substantially decreased the luciferase activity (50‑75%)at all N / P ratios tested,with the highest silencing efficiency (75%) observedat anN / P ratio of 10 ([siLuc] = 75 nM or 1 µg / mL and 6.6 µg / mL CRC5) and after 48 h of incubation. In fact, the gene silencing efficiency of the CRC5 / siLuc complex was comparable to that of Lipofectamine2000 / siLuc. However, as described above, CRC5 has much lower cytotoxicity than Lipofectamine. At their effective concentrations for siRNAdelivery, lipofectamine 2000 (2µL, the actual concentration of which is undisclosed by the commercial supplier) reduced the viability of HeLa-Luc cells by 62 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 35%, whereas CRC5 (6.6 µg / mL or ~0.5 µM) showed no detectable cytotoxicity (Figure 7). Example 2. Polypeptide Conjugate with one cCPP terminal

[0263] Design and Synthesis.While the CRC polymers described in Example 1 are effective for siRNA delivery and simple to prepare, they are mixtures of different species, the precise MWs and structures of which can bemore difficult to characterize.

[0264] In this example, a cyclicCPPwascovalently linked to a3,5-bis(mercaptomethyl)benzoyl (Bmb)moiety througha long, flexible linker,miniPEG-lysine (Scheme1). CPP12,which has cytosolic delivery efficiency of 121%,was selected for this design (see Qian, 2016). Two polyarginine peptides (R5, R10, or R15) are conjugated to the Bmb scaffold through disulfide bonds. We envisioned that the two polyarginine peptides would bind tightly to double-stranded siRNA via electrostatic interactions, leaving the cyclic CPP exposed for cellular uptake. Upon entering the cytosol, the disulfide bonds would be cleaved by GSH, releasing the siRNA for biological function.

[0265] The cCPP-polyarginine conjugate was prepared by first synthesizing CPP12-miniPEG-Lys(Mtt)‑NH2 on Rink amide resin. The Mtt group on the lysine side chain was selectively removed by treatment with 2% TFA and bis(tri- tyl)‑protected Bmb was coupled to the lysine side chain with Oxyma / DIC / DIPEA as coupling reagents. The resulting 63 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 peptide, CPP12-miniPEG-Lys(Bmb)‑NH2, was cleaved from the resin and side chain deprotected using the standard conditions and purified by reversed-phase HPLC. The freshly eluted peptide was treated with an excess of 2,2’- dithiodipyridine to protect (and activate) the two thiol groups of Bmb. Meanwhile, the polyarginine peptides CRn, where n = 5, 10 or 15, were synthesized on the solid phase with a cysteine residue added at the N-terminus. After deprotection, cleavage, and HPLC purification, the CRn peptide was mixed with the thiopyridylated CPP12 at neutral pH to afford the conjugatesCPP12‑(Rn)2. The identity of thevectorswasconfirmedbyMALDI-TOFmassspectrometry (Table6, Figure8). In Figure 8: MALDI-TOF MS spectra of CPP12‑(R5)2 (top), CPP12‑(R10)2 (middle), and CPP12‑(R15)2 (bottom). Table 6. MW of CPP12‑(Rn)2 polymers Polymer MW calculated observed CPP12‑(R5)2 3510 3508 CPP12‑(R10)2 5076 5079 CPP12‑(R15)2 6636 6638

[0266] siRNA Binding. CPP12‑(R5)2, CPP12‑(R10)2 and CPP12‑(R15)2 were tested for binding to siLuc by the gel retardationmethod using various N / P ratios as described above. TheCPP12‑(Rn)2 / siRNA complexeswere formedwith a fixed concentration of siRNAand increasing concentrations ofCPP12‑(Rn)2 to giveN / P ratios of 0.5:1 to 10:1. Free siRNA wasusedasacontrol.Complete conversionof freesiRNA into theRNA / polymer complexwasobservedat anN / P ratio of 5 for CPP12‑(R5)2 and N / P ratio of 3 for CPP12‑(R10)2 and CPP12‑(R15)2. Treatment of the preformed siLuc / CPP12‑(R5)2 complex with 10mMDTT resulted in a discrete band with the samemobility as the free RNA (Figure 12, top right). On the other hand, similar treatment of the siLuc / CPP 12‑(R10)2 and siLuc / CPP12‑(R15)2 complexes with 10 mM DTT did not result in adiscreteRNAband (Figure 12,middle andbottom right), indicating thatR10 andR15 remain bound to siRNAafter DTT treatment.

[0267] Cellular Uptake. 5’-FAM-labelled siLuc (3 µM) was mixed with CPP12‑(R5)2 at an N / P ratio of 5 in OptiMEM medium.HeLa cellswere treatedwith theCPP12‑(R5)2 / 5’-FAM-siRNAcomplex for 2 h.Cellswerewashed and imagedby confocal microscopy as described above. Interestingly, CPP12‑(R5)2 and 5’-FAM-siRNA formed large, insoluble particles of nanometer sizes (Figure9). InFigure9: center,GFPchannel; right,DIC; and left, overlapof theabove.Althoughsomeof the labeled siRNA was internalized by the cells, the intracellular fluorescence level was lower than that obtained with the copolymer of Example 1.

[0268] Knockdownof LuciferaseExpression.Theability ofCPP12‑(Rn)2 to deliver siLuc intracellularlywas tested on HeLa-Luc cells as described above. CPP12‑(Rn)2 / siLuc complexes were formed at various N:P ratios by mixing CPP12‑(Rn)2 with siLuc in 20 mM HEPES, pH 7.4 followed by 15 min incubation. The complexes were then mixed with OptiMEM medium, added to cells, and incubated at 37 °C for 48 h. Lipofectamine2000 / siLuc was used as a positive control. As shown in Figure 10, treatment of HeLa-Luc cells with the CPP12‑(Rn)2 / siLuc complexes (75 nM siLuc) at N:P ratios of 1 to 5 only slightly reduced the expression of the luciferase (≤15%), in agreement with their inefficient cellular uptake (Figure 9). Example 3. Modification of peptide conjugate in Example 2

[0269] Design and Synthesis. In modifying the peptide conjugate of Example 2, cyclic CPP (e.g., CPP9) was directly attached to a polyarginine peptide (Rn) through aminiPEG linker. A cysteine was added to the C-terminus of the peptide. The entire molecule was readily synthesized by standard solid-phase peptide chemistry, cleaved off the solid support, deprotected, and purified by HPLC. Exposure of the peptide to an oxidant (e.g., DMSO) resulted in homodimerization through the formation of a disulfide between the C-terminal cysteines. Again, the (Rn)2 moiety (where n = 5 or 8) in the central section is expected to bind to siRNA with high affinity in the oxidizing extracellular environment, whereas the two terminalCPPswouldmediateendocytic uptake.Once inside the cytosol, thedisulfidebondwouldbe,without bound toany theory, reducedand the siRNAwouldbe released.Oneadvantageof thisdesignwas thought tobe that thepresenceof two CPPs should enhance the cellular uptake efficiency of the vector (relative to Example 2). The identity of the synthesized peptide conjugate was determined byMALDI-TOFmass spectrometry (Table 7 and Figure 11). In Figure 11, MALDI-TOF mass spectra of (CPP9-R5)2 (top) and (CPP9-R8)2 (bottom). 64 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 Table 7. MW of CPP9-Rn)2 polymers Polymer MW calculated observed (CPP9-R5)2 4250 4247 (CPP9-R8)2 5190 5192

[0270] siRNABinding. (CPP9-R5)2 and (CPP9-R8)2 were evaluated for binding to siLuc by the gel retardation method at various N / P ratios as described above. The (CPP9-Rn)2 / siRNA complexes were formed with a fixed concentration of siRNAand increasing concentrationsofCPP12‑(Rn)2 to giveN / P ratios of 0.5:1 to 20:1. Free siRNAwasusedasacontrol. Complete conversion of free siRNA into the RNA / polymer complex was observed at an N / P ratio of 3 for (CPP9-R5)2 and N / P ratio of 1 for (CPP9-R8)2.

[0271] Cellular Entry by Confocal Microscopy. 5’-FAM-labelled siLuc (3 µM) was mixed with (CPP9-R5)2 at an N / P ratio of 2 in OptiMEM. HeLa cells were seeded andwashed as described above and treated with the (CPP9-R5)2 / 5’-FAM- siRNA complex for 2 h. Cells were washed and imaged as described above. The complex formed insoluble aggregates which are clearly visible under the microscope. Internalization of the complex by HeLa cells was also evident and somewhatmoreefficient than the1st-generationvector (Figure12). InFigure12,Center,GFPchannel;Right,DIC;and left, overlap of the above.

[0272] Knockdown of Luciferase Expression. The ability of (CPP9-Rn)2 vectors to deliver siLuc intracellularly was tested onHeLa-Luc cells as described above. (CPP9-Rn)2 / siLuc complexes were formed at different N:P ratios bymixing (CPP9-Rn)2 with siLuc in 20 mM HEPES, pH 7.4 followed by 15 min incubation. Complexes were then mixed with OptiMEM, added to the cells and incubated at 37 °C for 48 h. Lipofectamine2000 / siLuc was used as a positive control. Treatment of HeLa-Luc cells with the (CPP9-R5)2 / siLuc and (CPP9-R8)2 / siLuc complexes atN:P ratio of 1:1 (75 nMsiLuc) decreased the luciferase activity by 30‑35% (Figure 13). Interestingly, the (CPP9-R8)2 / siLuc complex was slightly less effective than the (CPP9-R5)2 / siLuc complex, likely because of the less efficient release of siRNA from the former. Example 4. Modification of peptide conjugate in Example 2

[0273] DesignandSynthesis.TheaboveExamplessuggest that for optimal siRNAdeliveryefficiency, thepolyarginine moiety shouldhavesufficient length to bind to siRNAwithhighaffinityand interactwithentire lengthof thesiRNA toprevent it fromnuclease action.Once inside the cell, the polymermust be broken down into small fragments that readily dissociate from the siRNA. The polypeptide conjugate of this Example has the general structure of CPP9-R5‑S-S-R6‑S-S-R5‑CPP9 (SEQ IDNO: 144), which consists of two cyclic CPP9-miniPEG-R5 (SEQ IDNO: 142) units covalently linked to an internal R6 (SEQ ID NO: 143) unit through two disulfide bonds. The polypeptide conjugate of this Example is structurally very similar to the copolymers fromExample 1, but can be readily synthesized as a single species. Briefly, theCPP9-miniPEG- R5‑Cys (SEQ ID NO: 145) unit was synthesized by standard solid-phase peptide chemistry and reacted with dithiodipyr- idine to give the thiopyrodylated form,which is stable upon storage. The internal R6 unit has aCys-β-Ala dipeptide on each 65 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 side to give some flexibility to the polymer structure. Simplymixing a 2:1 ratio (mol / mol) of theCPP9-miniPEG-R5 (SEQ ID NO: 142) and R6 (SEQ ID NO: 143) units at the physiological pH gave the desired polymer as the predominant species, which was purified to homogeneity by reversed-phase HPLC. The identity of polypeptide conjugate of this Example was confirmed by MALDI-TOF mass spectrometry (Figure 14).

[0274] Cytotoxicity.The polypeptide conjugate of this Examplewas tested for cytotoxicity against HeLa cells using the MTTassay. HeLa cells were treated with varying concentrations (0‑40 µM) of the 3rd-generation vector and incubated at 37 °C with 5% CO2 for 72 h. Cells were treated with MTT stock solution and SDS-HCl solubilizing buffer as described above. The absorbance of the formazan product was measured at 570 nm on a Tecan M1000 plate reader. In contrast to the copolymers, which caused ≤20% reduction in viability up to 40 µM concentration (Figure 5A), the polypeptide conjugate of this Example reduced the viability of HeLa cells in a dose-dependent manner, by 70% at 40 µM (Figure 15).

[0275] siRNABinding.The polypeptide conjugate of this Examplewas able to bind siLuc. Complete conversion of free siRNA into the siRNA / vector complexwas observed at anN / P ratio of 2. Treatment of the preformed siLuc / vector complex with 10mMDTTdid not regenerate the free siRNAband; instead, a broad, smeared siRNAbandwas observed, indicating that siRNA remained bound to the polyarginine species even after reduction of the disulfide bonds.

[0276] CellularUptakebyConfocalMicroscopy.5’-FAM-labelledsiLuc (1or3µM)wasmixedwith the3rd-generation vectorat anN / P ratioof 2 inOptiMEMorDMEMwith1%FBS.HeLacellswere treatedwith thepolypeptideconjugateof this Example / 5’-FAM-siRNAcomplex for 2 h.Cellswerewashedand imagedby live-cell confocalmicroscopy.Cellular entry of the polypeptide conjugate of this Example / 5’-FAM-siRNA complex was more efficient than the Examples 2 or 3, and the diffuse fluorescence throughout the entire cell volume indicate that the complex had escaped from the endosome into the cytosol and nucleus (Figure 16). Formation of insoluble aggregates was still observed. In Figure 16, at 1 µM siRNA in DMEMwith 1% FBS (top) or 3 µM siRNA in OptiMEM (bottom). Center, GFP channel; Right, DIC; and left, overlap of the above.

[0277] Knockdown of Luciferase Expression. Treatment of HeLa-Luc cells with the 3polypeptide conjugate of this Example / siLuc complexes at N:P ratios of 1:1 and 3:1 (both at 75 nM siLuc RNA) for 48 h at 37 °C reduced the luciferase expression by 22%and 29%, respectively. Under the same conditions, the lipofectamine2000 / siLuc complex reduced the luciferase expression by 84% (Figure 17).

[0278] Knockdown of Firefly Luciferase Expression Normalized by Renilla Luciferase Expression. The ability of CRC polymers to deliver siRNA intracellularly was tested on a HeLa cell line stably transfected with Firefly and Renilla luciferase genes (Dual-HeLa). Firefly luciferase serves as an experimental reporter, while Renilla luciferase serves as a control reporter allowing to normalize the specific gene silencing of an experimental reporter. The siLuc sequences used were: sense 5’-AAmCGmCmUGGGmCGmUmUAAmUmCAAdTdT‑3’ (SEQ ID NO: 139) and antisense 5’-UUGAU- mUAACGCCmCAGCGUUdTdT‑3’ (SEQ IDNO:140).Dual-HeLa cellswere seededontowhite 96-well plates at a density of 1.0 x 104 cells / well in 100 µL of DMEM containing 10% FBS and cultured overnight. CRC5 / siLuc complexes were formed bymixing CRC5 with siLuc in 20mMHEPES, pH 7.4 followed by 15 min incubation. Complexes were then mixed 66 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 with OptiMEM, added to cells and incubated at 37 °C for 48 h. Lipofectamine 2000 / siLuc was used as a positive control. Lipofectamine 2000 was complexed with siRNA according to the manufacturer’s protocol. Dual-Glo Luciferase Assay System (Promega, USA) was used to quantitate the luciferase gene silencing level according tomanufacturer’s protocol. Luminescence was measured on a Tecan Infinite M1000 plate reader.

[0279] Treatment of HeLa-Luc cells with the CRC5 / siLuc complex decreased the Firefly luciferase activity by 49%at an N:P ratio = 10 ([siLuc] = 75 nM and 6.6 µg / mL CRC5) after 48 h of incubation after normalization by Renilla luciferase expression (Figure 18). Under similar conditions, Lipofactamine 2000 resulted in 86% reduction.

[0280] KnockdownofGFPExpression.Theability ofCRCpolymers todeliver siRNA intracellularlywasalso testedon a HeLa cell line stably transfected with GFP gene (HeLa-GFP). The siGFP used in this experiment was Silencer™ GFP (eGFP) siRNA (#AM4626, Thermo Fisher, USA). HeLa-GFP cells were seeded onto 96-well plate at a density of 1.0 x 104 cells / well in 100µL of DMEMcontaining 10%FBSand cultured overnight. CRC5 / siLuc complexeswere formed bymixing CRC5 with siLuc in 20 mM HEPES, pH 7.4 at N:P = 10 followed by 15 min incubation. Complexes were then mixed with OptiMEM, added to cells and incubated at 37 °C for 48 h. Lipofectamine 2000 / siLuc was used as a positive control. After incubation cellswere lysed on ice for 30min in IP lysis buffer supplementedwith proteaseandphosphatase inhibitors. Cell lysates were centrifuged and 15000 rpm for 10min, and the extracted proteins were collected. Protein concentration was measured using a BCAProtein Assay Kit (Thermo Fisher, USA) and equal amount of total proteins (~200 ng) were added to a black 384-well plate. Fluorescence intensity was measured at 510 nm on a Tecan Infinite M1000 plate reader.

[0281] Treatment of HeLa-GFP cells with the CRC5 / siGFP complex at N:P ratio = 10 ([siLuc] = 75 nM and 6.6 µg / mL CRC5) decreased the GFP expression by 31% after 48 h of incubation, which was comparable to the efficiency of Lipofectamine 2000 (46%; Figure 19). Discussion (Examples 1‑4)

[0282] Without bound to any theory, the ideal siRNA delivery vector (polypeptide conjugates) should bind to siRNAwith high affinity and interact with the entire siRNA molecule to prevent the latter from nuclease action. Once inside the cell, however, the vector shouldbe readily brokendown into small fragments that readily dissociate from the siRNA. In addition, because liposome‑ and nanoparticle-based siRNA delivery systems often resulted in accumulation of siRNA in well- vascularized tissues such as the liver, spleen, and kidney, soluble, oligomeric vector / siRNA complexes (ideally 1:1 vector / siRNA complex) which should have better tissue penetration and potentially broader distribution in vivo are desirable. All four siRNAdelivery vectors described in theExamples are capable of delivering siRNA intomammalian cells and knocking down the expression of luciferase gene, but meet the above design criteria to different extents. Overall, the polypeptide of Example 1 demonstrated better performance than Examples 2‑3. It very effectively delivers siRNA into the cytosol of mammalian cells (as evidenced by confocal microscopy) and most efficiently knocks down luciferase expres- sion. It exhibitedminimal cytotoxicity to HeLa cells at up to 40µMconcentration. It did not form any insoluble species (i.e., nanoparticles). It is also operationally very simple to prepare. The challengewith Example 1’s polypeptide conjugatemay be its structural heterogeneity (i.e., different number of R5 units in the copolymer), which may requires extra attention during their preparation in order to produce copolymers of consistent compositions.

[0283] The threevectorsaspreparedaccording toExamples2‑4,on theotherhand,havewell-definedstructures (single species) and are readily prepared in pure forms. However, the challenge was the formation of insoluble aggregates and generally having lower siRNAdeliveryefficienciesandhigher cytotoxicities.Presumably,without bound toany theory, their smaller sizes (compared to the conjugate of Example 1) result inweaker binding to siRNAwhenoutside the cell and during endocyticuptake.Because their polyargininesectionsmaybe tooshort to cover theentire siRNAsurface, thecCPPsat the termini may bind to the siRNA as well, potentially interfering with their membrane binding and CPP function. At the same time, binding of multiple polyarginine peptides to one siRNA molecule (and / or multiple siRNA molecules to the same polyarginine peptide) could condense nucleic acids into large, insoluble nanoparticles, as was commonly observed for other arginine-richCPPs such as Tat (seeArthanari, Y. et al., J. ControlledRelease 2010, 145, 272‑280) andR9 (see Law, M. et al., Biotechnol Prog,, 2008, 24, 957‑963). After cytosolic entry and reduction of the disulfide bonds, the fragments derived from thevectors inExamples2‑4contain9ormorearginine residues [except forCPP9‑(R5)2]. Asdemonstratedby gel electrophoresis in Examples 2 and 4, these fragments remain tightly associated with the siRNA and reduce the knockdown efficiency of the siRNA. Additionally, the polyarginine fragmentsmay bind to endogenous nucleic acids inside the cell, causing cytotoxicity. In comparison, reduction of the copolymer of Example 1 produces fragments with 5 or less arginine residues, which without bound to any theory do not bind to siRNA or other nucleic acids with high affinity as demonstrated by gel electrophoresis in Examples 1.

[0284] The Examples demonstrate that the copolymerization of cCPPs and pentaarginine peptides through disulfide bondinggenerateda family of cationic copolymers thatbindsiRNAwithhighaffinity, effectivelydeliver them into thecytosol ofmammalian cells, and then release them for functional knockdown of specificmRNA levels. This novel deliverymethod would be useful in delivering nucleic acids to target cells.

[0285] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present 67 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0286] While the invention has been described in connection with proposed specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosureascomewithin knownor customarypracticewithin theart towhich the inventionpertainsandasmaybe applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. Claims 1. A polypeptide conjugate comprising (a) a group P, P1 or P2 that binds to a nucleic acid sequence by electrostatic interactions comprising at least one peptide or polyamine, wherein each peptide comprises three to fifty monomers selected from arginine, arginine- analog, lysine, lysine-analog, histidine, or histidine-analog; (b) at least one cyclic cell-penetratingpeptide (cCPP), comprising from4 to 14aminoacidmonomers, comprising at least two arginines and at least two amino acids with hydrophobic side chains; and (c) at least one linker (L) that conjugates P, P1 or P2 to the cCPP; wherein the polypeptide conjugate has a structure selected from: (i) cCPP-L‑[P]n‑L-cCPP wherein: n is an integer from 1 to 50; and P at each occurrence is same or different; (ii) cCPP-L‑([P1]p‑L1)t‑[P]n‑(L2‑[P2]q)t‑L-cCPP wherein: n, p, and q are each independently an integer from 1 to 50; each t is independently 0 or 1; and P, P1, and P2, at each occurrence, are same or different; and L, L1, and L2, at each occurrence, are same or different; (iii) cCPP-L‑([P]m)s, wherein: s is an integer from 1 to 10; each m is, independently, an integer from 1 to 50; and P at each occurrence is same or different; (iv) cCPP-L‑[P]n‑[P]m‑(L-cCPP)v wherein: n is an integer from 1 to 50; m is an integer from 0 to 49 provided that the sum of n and m is 50 or less; 68 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 v is 0 or 1; P at each occurrence is same or different; and when v is 0, the last [P] in [P]m is monovalent; and (v) cCPP-L‑[P]n‑[P]m‑[P]o‑(L-cCPP)v wherein: n is an integer from 1 to 50; m and o, are each independently, an integer from 0 to 49 provided that the sum of n, m, and o is 50 or less; v is 0 or 1; P at each occurrence is same or different; and when v is 0, the last [P] in [P]o is monovalent. 2. The polypeptide conjugate of claim 1, wherein P, P1, and / or P2 comprises (i) a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog or (ii) a polyamine selected from a spermidine polymer or a spermine polymer. 3. The polypeptide conjugate of claims 1 or 2, wherein the cCPP comprises two to three arginines and at least two hydrophobic amino acids. 4. The polypeptide conjugate of any one of claims 1‑3, wherein the cCPP has the structure of Formula 1: (AAU)m‑AA1‑AA2‑AA3‑AA4‑(AAz)n wherein: each of AA1, AA2, AA3, and AA4, are independently a D or L amino acid, each of AAU and AAZ, at each instance and when present, are independently a D or L amino acid, and m and n are independently a number from 0 to 6, at least two of AAU (when present), AA1, AA2, AA3, AA4, and AAZ (when present), are independently arginine and at least two of AAU (when present), AA1, AA2, AA3, AA4, andAAZ (when present), are independently an amino acid having a hydrophobic side chain. 5. The polypeptide conjugate of any of claims 1‑4, wherein the cCPP comprises at least two consecutive amino acids each having a hydrophobic side chain and at least two consecutive arginines. 6. The polypeptide conjugate of any of claims 1‑5, wherein the cCPP comprises at least three consecutive amino acids each having a hydrophobic side chain and three consecutive arginines. 7. Thepolypeptide conjugate of any one of claims 1‑5,wherein any four adjacent amino acids in the cCPPhasone of the following sequences: AAH2‑AAH1‑R-r, AAH2‑AAH1-r-R, R-r-AAH1‑AAHZ, or r-R-AAH1‑AAHZ, wherein each of AAH1 and AAH2 are independently an amino acid having a hydrophobic side chain. 8. The polypeptide conjugate of any of claims 1‑5, wherein the cCPP has a structure according any of Formula 2A‑2D: (AAu)m‑AAH2‑AAH1‑R-r‑(AAz)n 2A (AAu)m-r-R-AAH1‑AAH2‑(AAz)n 2B (AAu)m‑AAH2‑AAH1-r-R‑(AAz)n 2C and 69 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 (AAu)m‑R-r-AAH1‑AAH2‑(AAz)n 2D; wherein: each of AAH1 and AAH2 are independently an amino acid having a hydrophobic side chain; at each instance and when present, each of AAu and AAz are independently any amino acid; and mandnare independently selected fromanumber from0 to6,wherein the total number of aminoacids (including r, R, AAH1, AAH2) are in the range of 6 to 10. 9. The polypeptide conjugate of any of claims 1‑8, wherein each amino acid having a hydrophobic side chain is independentlyanaminoacidhavingahydrophobicaromatic sidechainselected fromnaphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. 10. The polypeptide conjugate of any of claims 1‑9, wherein the amino acid having a hydrophobic side chain is selected from piperidine‑2-carboxylic acid, naphthylalanine, tryptophan, or phenylalanine, each of which is optionally sub- stituted with one or more substituents. 11. The polypeptide conjugate of any one of claims 1‑10, wherein the CPP is a cyclo(fΦRrRrQ) peptide or a cyclo(FfΦRrRrQ), wherein: F is a L-phenylalanine; f is a D-phenylalanine; Φ is an L‑2-naphthylalanine; R is a L-arginine; r is a D-arginine; and Q is a L-glutamine. 12. The polypeptide conjugate of claim 2 having the following structure: cCPP-L‑[pArg]n‑L-cCPP wherein the [pArg]n is or a charged species thereof. 13. The polypeptide conjugate of any one of claims 1‑12, wherein at least one L comprises: 70 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 14. The polypeptide conjugate of any one of claims 1‑13, wherein each P, independently, further comprises at least one group selected from: or wherein the bond to the hydrogen on at least one of the N‑ or C‑ termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the cCPP. 15. The polypeptide conjugate of claim 1 having a structure selected from: 71 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 72 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 73 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 or a charged species thereof. 16. A complex comprising the polypeptide conjugate of any one of claims 1‑15 and at least one nucleic acid sequence. 17. A complex according to claim 16 for use in delivering a nucleic acid sequence to a cell of a subject in need thereof. 74 EP 4 717 776 A2 5 10 15 20 25 30 35 40 45 50 55 75 EP 4 717 776 A2 76 EP 4 717 776 A2 77 EP 4 717 776 A2 78 EP 4 717 776 A2 79 EP 4 717 776 A2 80 EP 4 717 776 A2 81 EP 4 717 776 A2 82 EP 4 717 776 A2 83 EP 4 717 776 A2 84 EP 4 717 776 A2 85 EP 4 717 776 A2 86 EP 4 717 776 A2 87 EP 4 717 776 A2 88 EP 4 717 776 A2 89 EP 4 717 776 A2 90 EP 4 717 776 A2 91 EP 4 717 776 A2 92 EP 4 717 776 A2 93 EP 4 717 776 A2 94 EP 4 717 776 A2 95 EP 4 717 776 A2 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. 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[0283] (19) *EP004717776A3* (11) EP 4 717 776 A3 (12) EUROPEAN PATENT APPLICATION (88) Date of publication A3: 29.04.2026 Bulletin 2026 / 18 (43) Date of publication A2: 01.04.2026 Bulletin 2026 / 14 (21) Application number: 26155045.3 (22) Date of filing: 02.07.2019 (51) International Patent Classification (IPC): A61K 47 / 64 (2017.01) C07K 19 / 00 (2006.01) C07K 7 / 08 (2006.01) C07K 7 / 06 (2006.01) C07K 7 / 02 (2006.01) C07K 7 / 64 (2006.01) C12N 15 / 11 (2006.01) C12N 15 / 88 (2006.01) (52) Cooperative Patent Classification (CPC): C12N 15 / 111; A61K 47 / 645; A61K 47 / 6455; C07K 7 / 02; C07K 7 / 06; C07K 7 / 08; C07K 7 / 64; C12N 15 / 88; C07K 2319 / 10; C07K 2319 / 80; C12N 2310 / 14; C12N 2310 / 3513; C12N 2320 / 32 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 02.07.2018 US 201862692939 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 19831072.4 / 3 817 776 (71) Applicant: Ohio State Innovation Foundation Columbus, OH 43201 (US) (72) Inventors: • PEI, Dehua Columbus, Ohio, 43220 (US) • BUYANOVA, Marina Columbus, Ohio, 43212 (US) • QIAN, Ziqing Wellesley, Massachusetts, 02482 (US) (74) Representative: Berggren Oy P.O. Box 16 Fabianinkatu 21 00101 Helsinki (FI) (54) POLYPEPTIDE CONJUGATES FOR INTRACELLULAR DELIVERY OF NUCLEIC ACIDS (57) The present disclosure provides for polypeptide conjugates. The polypeptide conjugates disclosed here- in comprise a polyarginine peptide and a cyclic cell- penetrating peptide (cCPP) conjugated, directly or indir- ectly, to the polyarginine peptide. The present disclosure demonstrates that cCPPs conjugated to polyarginine peptides can be used to deliver nucleic acids to the cytosol of cells. EP 4 71 7 77 6 A 3 Processed by Luminess, 75001 PARIS (FR) 2 EP 4 717 776 A3 5 10 15 20 25 30 35 40 45 50 55 3 EP 4 717 776 A3 5 10 15 20 25 30 35 40 45 50 55 摘要 本公开提供多肽缀合物。本文公开的多肽缀合物包含多聚精氨酸肽以及直接或间接缀 合至该多聚精氨酸肽的环状细胞穿透肽（cCPP）。本发明公开表明，缀合至多聚精氨酸肽的 cCPP可用于将核酸递送至细胞的细胞质。

Claims

1. A polypeptide conjugate comprising (a) a group P, P1 or P2 that binds to a nucleic acid sequence by electrostatic interactions comprising at least one peptide or polyamine, wherein each peptide comprises three to fifty monomers selected from arginine, arginine-analog, lysine, lysine-analog, histidine, or histidine-analog; (b) at least one cyclic cell-penetrating peptide (cCPP), comprising from 4 to 14 amino acid monomers, comprising at least two arginines and at least two amino acids with hydrophobic side chains; and (c) at least one linker (L) that conjugates P, P1 or P2 to the cCPP; wherein the polypeptide conjugate has a structure selected from: (i)         cCPP-L-[P]n-L-cCPP wherein: n is an integer from 1 to 50; and P at each occurrence is same or different; (ii)         cCPP-L-([P1]p-L1)t-[P]n-(L2-[P2]q)t-L-cCPP wherein: n, p, and q are each independently an integer from 1 to 50; each t is independently 0 or 1; and P, P1, and P2, at each occurrence, are same or different; and L, L1, and L2, at each occurrence, are same or different; (iii)         cCPP-L-([P]m)s, wherein: s is an integer from 1 to 10; each m is, independently, an integer from 1 to 50; and P at each occurrence is same or different; (iv)         cCPP-L-[P]n-[P]m-(L-cCPP)v wherein: n is an integer from 1 to 50; m is an integer from 0 to 49 provided that the sum of n and m is 50 or less; v is 0 or 1; P at each occurrence is same or different; and when v is 0, the last [P] in [P]m is monovalent; and (v)         cCPP-L-[P]n-[P]m-[P]o-(L-cCPP)v wherein: n is an integer from 1 to 50; m and o, are each independently, an integer from 0 to 49 provided that the sum of n, m, and o is 50 or less; v is 0 or 1; P at each occurrence is same or different; and when v is 0, the last [P] in [P]o is monovalent.

2. The polypeptide conjugate of claim 1, wherein P, P1, and / or P2 comprises (i) a polyarginine peptide (pArg) comprising at least three monomers selected from arginine or arginine-analog or (ii) a polyamine selected from a spermidine polymer or a spermine polymer.

3. The polypeptide conjugate of claims 1 or 2, wherein the cCPP comprises two to three arginines and at least two hydrophobic amino acids.

4. The polypeptide conjugate of any one of claims 1-3, wherein the cCPP has the structure of Formula 1:         (AAU)m-AA1-AA2-AA3-AA4-(AAz)n wherein: each of AA1, AA2, AA3, and AA4, are independently a D or L amino acid, each of AAU and AAZ, at each instance and when present, are independently a D or L amino acid, and m and n are independently a number from 0 to 6, at least two of AAU (when present), AA1, AA2, AA3, AA4, and AAZ (when present), are independently arginine and at least two of AAU (when present), AA1, AA2, AA3, AA4, and AAZ (when present), are independently an amino acid having a hydrophobic side chain.

5. The polypeptide conjugate of any of claims 1-4, wherein the cCPP comprises at least two consecutive amino acids each having a hydrophobic side chain and at least two consecutive arginines.

6. The polypeptide conjugate of any of claims 1-5, wherein the cCPP comprises at least three consecutive amino acids each having a hydrophobic side chain and three consecutive arginines.

7. The polypeptide conjugate of any one of claims 1-5, wherein any four adjacent amino acids in the cCPP has one of the following sequences:         AAH2-AAH1-R-r, AAH2-AAH1-r-R, R-r-AAH1-AAHZ, or r-R-AAH1-AAHZ, wherein each of AAH1 and AAH2 are independently an amino acid having a hydrophobic side chain.

8. The polypeptide conjugate of any of claims 1-5, wherein the cCPP has a structure according any of Formula 2A-2D:         (AAu)m-AAH2-AAH1-R-r-(AAz)n     2A         (AAu)m-r-R-AAH1-AAH2-(AAz)n     2B         (AAu)m-AAH2-AAH1-r-R-(AAz)n     2C and         (AAu)m-R-r-AAH1-AAH2-(AAz)n     2D; wherein: each of AAH1 and AAH2 are independently an amino acid having a hydrophobic side chain; at each instance and when present, each of AAu and AAz are independently any amino acid; and m and n are independently selected from a number from 0 to 6, wherein the total number of amino acids (including r, R, AAH1, AAH2) are in the range of 6 to 10.

9. The polypeptide conjugate of any of claims 1-8, wherein each amino acid having a hydrophobic side chain is independently an amino acid having a hydrophobic aromatic side chain selected from naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents.

10. The polypeptide conjugate of any of claims 1-9, wherein the amino acid having a hydrophobic side chain is selected from piperidine-2-carboxylic acid, naphthylalanine, tryptophan, or phenylalanine, each of which is optionally substituted with one or more substituents.

11. The polypeptide conjugate of any one of claims 1-10, wherein the CPP is a cyclo(fΦRrRrQ) peptide or a cyclo(FfΦRrRrQ), wherein: F is a L-phenylalanine; f is a D-phenylalanine; Φ is an L-2-naphthylalanine; R is a L-arginine; r is a D-arginine; and Q is a L-glutamine.

12. The polypeptide conjugate of claim 2 having the following structure:         cCPP-L-[pArg]n-L-cCPP wherein the [pArg]n is or a charged species thereof.

13. The polypeptide conjugate of any one of claims 1-12, wherein at least one L comprises:

14. The polypeptide conjugate of any one of claims 1-13, wherein each P, independently, further comprises at least one group selected from: or wherein the bond to the hydrogen on at least one of the N- or C- termini is replaced by a bond to the peptide or polyamine; and wherein the bond to the hydrogen on the thiol group is replaced by a bond to the cCPP.

15. The polypeptide conjugate of claim 1 having a structure selected from: or a charged species thereof.

16. A complex comprising the polypeptide conjugate of any one of claims 1-15 and at least one nucleic acid sequence.

17. A complex according to claim 16 for use in delivering a nucleic acid sequence to a cell of a subject in need thereof.

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