Peptides and nanoparticles for intracellular delivery of molecules

Abstract

The present application is directed to a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a first peptide comprising a cell-penetrating peptide (CPP), a second peptide comprising a cell-penetrating peptide, and a cargo molecule. The second peptide comprises a polyethylene glycol (PEG) moiety linked to the second CPP, and the first peptide does not have a PEG moiety. The present application is also directed to a cargo delivery complex comprising a CPP and a cargo molecule, wherein the CPP is a retro-inverso peptide. The present application is also directed to a cargo delivery complex comprising a CPP and a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR. Methods of making and using the cargo delivery complex are also disclosed.

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority of French Patent Application No. FR1904115, filed on April 17, 2019, the entire content of which is incorporated herein by reference for all purposes.

[0002] The present invention relates to a complex / nanoparticle containing a peptide useful for delivering cargo molecules into cells.

Background Art

[0003] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated herein by reference in their entirety. Small molecules remain the main drugs often used in clinics, but their therapeutic effects have reached their limits, for example, due to insufficient ability to reach the target, lack of specificity, the need for high doses, which in turn lead to toxicity and major side effects. Over the past decade, we have witnessed a dramatic acceleration in the discovery of larger therapeutic molecules such as proteins, peptides, and nucleic acids that exhibit high specificity for the target but do not follow Lipinski's rule to avoid the limitations of small molecule and gene-based therapies. The pharmaceutical efficacy of these molecules remains limited by low stability in vivo and low cellular uptake. Therefore, "delivery" has become a central piece of the puzzle regarding therapy, and new milestones have been established to validate delivery strategies: (a) being non-toxic, (b) efficiency in vivo at low doses, (c) ease of handling for therapeutic applications, (d) rapid endosomal release, and (e) being able to reach the target. Great expectations are placed on virus-based delivery strategies for gene and cell therapy, but their clinical applications are troubled by side effects and toxic effects (Ibraheem et al. (2014) Int J Pharm 459, 70-83). Research has mainly focused on the development of virus-free strategies, and various methods have been proposed, including formulations based on lipids, polycationic nanoparticles, and peptides, but only a few of these technologies are efficient in vivo and have reached the clinic (Yin et al. (2014) Nat Rev Genet 15, 541-555). Therefore, improved methods are needed to efficiently deliver mRNA or RNAi into target cells.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0005] This application provides a cargo delivery complex and nanoparticles useful for intracellular delivery of cargo molecules. In some embodiments, a cargo delivery complex for intracellular delivery of a cargo molecule comprises: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, and the first peptide does not have a PEG moiety. In some embodiments, a cargo delivery complex for intracellular delivery of a cargo molecule comprises a cell-penetrating peptide and a cargo molecule, and the cell-penetrating peptide is a retro-inverso peptide. In some embodiments, a cargo delivery complex for intracellular delivery of a cargo molecule comprises: a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, and the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, viruses, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule does not include a virus. In some embodiments, a cell-penetrating peptide, such as the first cell-penetrating peptide or the second cell-penetrating peptide, is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide.

[0006] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, and the first peptide has no PEG moiety. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-9 peptide. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128.In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are the same. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, viruses, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmids, oligonucleotides, and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises or further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm. In some embodiments, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is about 50 to 1. In some embodiments, the PEG moiety is linear PEG. In some embodiments, the PEG moiety is branched PEG. In some embodiments, the molecular weight of the PEG moiety is about 5 kDa to about 10 kDa. In some embodiments, the PEG moiety consists of about 1 to 10 ethylene glycol units. In some embodiments, the PEG moiety is conjugated to the N-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to the C-terminus of the second cell-penetrating peptide. In some embodiments, the first peptide and / or the second peptide further comprises one or more moieties selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, cholesterol, a nuclear localization signal, a nuclear export signal, an antibody or an antibody fragment thereof, a peptide, a polysaccharide, and a targeting sequence, and the one or more moieties are covalently linked to the N-terminus of the first cell-penetrating peptide or the second cell-penetrating peptide, or to the PEG moiety.In some embodiments, one or more moieties comprise a targeting sequence. In some embodiments, the targeting sequence is selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, and IGSR. In some embodiments, the targeting sequence is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR. In some embodiments, the targeting sequence is covalently linked via a linker to a first cell-penetrating peptide or a second cell-penetrating peptide. In some embodiments, one or more moieties comprise an acetyl group and / or a stearyl group. In some embodiments, the first peptide and / or the second peptide further comprises one or more moieties selected from the group consisting of cysteamide, cysteine, thiol, amide, optionally substituted nitrilotriacetic acid, carboxyl, optionally substituted linear or branched C1-C6 alkyl, primary or secondary amine, oside derivative, lipid, phospholipid, fatty acid, cholesterol, nuclear localization signal, nuclear export signal, antibody, polysaccharide, and a targeting sequence, and the one or more moieties are covalently linked to the C-terminus of the first cell-penetrating peptide, the C-terminus of the second cell-penetrating peptide, or a PEG moiety. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a retro-inverso peptide.

[0007] In some embodiments, a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide, and the cell-penetrating peptide is a retro-inverso peptide, a cargo delivery complex is provided. In some embodiments, the retro-inverso peptide comprises the sequence of SEQ ID NO: 85 or 86. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, and protein complexes. In some embodiments, the cargo molecule comprises a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide and analogs thereof. In some embodiments, the cargo molecule comprises mRNA. In some embodiments, the cargo molecule comprises RNAi. In some embodiments, the cargo molecule does not contain a virus.

[0008] In some embodiments, provided is a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the cell-penetrating peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide, and the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR. In some embodiments, the targeting sequence is covalently linked via a linker to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, and protein complexes. In some embodiments, the cargo molecule comprises a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the cargo molecule comprises mRNA. In some embodiments, the cargo molecule comprises RNAi. In some embodiments, the cargo molecule does not contain a virus.

[0009] The present application also provides a nanoparticle comprising a core that includes the above-described cargo delivery complex. In some embodiments, the core is coated with a shell comprising a peripherally cell-penetrating peptide. In some embodiments, the peripherally cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide.

[0010] The present application also provides a pharmaceutical composition comprising any one of the above-described cargo delivery complexes or nanoparticles and a pharmaceutically acceptable carrier.

[0011] This application also provides a method for preparing the above cargo delivery complex, comprising: a) combining a first peptide and a second peptide, thereby forming a peptide mixture; and b) combining the peptide mixture with a cargo, thereby forming a cargo delivery complex.

[0012] This application also provides a method for preparing the above cargo delivery complex, comprising combining a peptide with a cargo molecule, thereby forming a cargo delivery complex.

[0013] In some embodiments according to any of the above methods for preparing a cargo delivery complex, the peptide or peptide mixture and the cargo molecule are combined at a molar ratio of from about 1:1 to about 100:1, respectively. In some embodiments, the method comprises mixing a first solution containing a cargo molecule with a second solution containing a peptide or peptide mixture to form a third solution, wherein the third solution is adjusted to contain or comprise: i) from about 0 to 5% sucrose; ii) from about 0 to 5% glucose; iii) from about 0 to 50% DMEM; iv) from about 0 to 80 mM NaCl; or v) from about 0 to 20% PBS, and incubating the third solution to form a cargo delivery complex. In some embodiments, the first solution contains the cargo in sterile water and / or the second solution contains the peptide or peptide mixture in sterile water. In some embodiments, the third solution is adjusted to contain: i) from about 0 to 5% sucrose; ii) from about 0 to 5% glucose; iii) from about 0 to 50% DMEM; iv) from about 0 to 80 mM NaCl; or v) from about 0 to 20% PBS after incubating to form a cargo delivery complex. In some embodiments, the method further comprises a filtration process of filtering the cargo delivery complex through a membrane with a pore size. In some embodiments, the diameter of the pores is at least about 0.1 μm.

[0014] This application also provides a method for delivering one or more cargos into cells, comprising contacting the cells with the above cargo delivery complex or nanoparticles, wherein the cargo delivery complex contains one or more cargos.

[0015] The present application also provides a method for delivering one or more cargos into the tissue or organ of an individual, the method comprising the step of administering to the individual an effective amount of the above cargo delivery complex, nanoparticle, or pharmaceutical composition, wherein the tissue or organ is selected from the group consisting of liver, lung, kidney, brain, intestine, spleen, heart, muscle, and lymph node. In some embodiments, the cargo delivery complex is administered intravenously. In some embodiments, the individual is a human.

[0016] The present application also provides a method for treating a disease or condition in an individual, the method comprising the step of administering to the individual an effective amount of the above cargo delivery complex, nanoparticle, or pharmaceutical composition. In some embodiments, the disease or condition is associated with diseased cells in an organ or tissue selected from the group consisting of liver, lung, kidney, brain, intestine, spleen, heart, muscle, and lymph node. In some embodiments, the disease or condition is selected from the group consisting of cancer, diabetes, autoimmune diseases, blood diseases, heart diseases, vascular diseases, inflammatory diseases, fibrotic diseases, viral infectious diseases, genetic diseases, eye diseases, liver diseases, lung diseases, muscle diseases, protein deficiency diseases, lysosomal storage diseases, neurological diseases, kidney diseases, aging and degenerative diseases, and diseases characterized by abnormal cholesterol levels. In some embodiments, the cargo delivery complex is administered intravenously. In some embodiments, the individual is a human.

[0017] The present application also provides a kit comprising the above cargo delivery complex, nanoparticle, or pharmaceutical composition. In certain embodiments, for example, the following items are provided. (Item 1) a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo delivery complex for delivering a cargo molecule into a cell, the cargo molecule comprising a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, and the first peptide has no PEG moiety. (Item 2) The cargo delivery complex according to item 1, wherein the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. (Item 3) The cargo delivery complex according to item 1 or 2, wherein the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. (Item 4) The cargo delivery complex according to any one of items 1 to 3, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-3 peptide. (Item 5) The cargo delivery complex according to item 4, wherein the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14, 75, and 76. (Item 6) The cargo delivery complex according to any one of items 1 to 5, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-6 peptide. (Item 7) The cargo delivery complex according to item 6, wherein the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 40 and 77. (Item 8) The cargo delivery complex according to any one of items 1 to 7, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-9 peptide. (Item 9) The cargo delivery complex according to item 8, wherein the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52 and 78. (Item 10) The cargo delivery complex according to any one of items 1 to 9, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is an ADGN-100 peptide. (Item 11) The cargo delivery complex according to item 10, wherein the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 70, 79 and 80. (Item 12) The cargo delivery complex according to any one of items 1 to 11, wherein the first cell-penetrating peptide and the second cell-penetrating peptide are the same. (Item 13) The cargo delivery complex according to any one of items 1 to 12, wherein the cargo molecule is selected from the group consisting of nucleic acids, viruses, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. (Item 14) The cargo delivery complex according to item 13, wherein the cargo molecule is a nucleic acid. (Item 15) The cargo delivery complex according to item 14, wherein the nucleic acid is selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide and its analogs. (Item 16) The cargo delivery complex according to item 15, wherein the nucleic acid comprises mRNA. (Item 17) The cargo delivery complex according to item 15 or 16, wherein the nucleic acid comprises RNAi. (Item 18) The cargo delivery complex according to item 16 or 17, wherein the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets RNA, and the expression of the RNA is associated with the disease or condition. (Item 19) The cargo delivery complex according to any one of items 14 to 18, wherein the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1. (Item 20) The cargo delivery complex according to any one of items 14 to 19, wherein the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm. (Item 21) The cargo delivery complex according to any one of items 1 to 20, wherein the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is about 20:1 to about 1:1. (Item 22) The cargo delivery complex according to any one of items 1 to 21, wherein the PEG moiety is linear PEG. (Item 23) The cargo delivery complex according to any one of items 1 to 21, wherein the PEG moiety is a branched PEG. (Item 24) The cargo delivery complex according to any one of items 1 to 23, wherein the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. (Item 25) The cargo delivery complex according to any one of items 1 to 24, wherein the PEG moiety consists of about 1 to 10 ethylene glycol units. (Item 26) The cargo delivery complex according to any one of items 1 to 25, wherein the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. (Item 27) The cargo delivery complex according to any one of items 1 to 25, wherein the PEG moiety is conjugated to a site within the second cell-penetrating peptide. (Item 28) The first peptide and / or the second peptide further comprises one or more moieties selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, cholesterol, a nuclear localization signal, a nuclear export signal, an antibody or an antibody fragment thereof, a peptide, a polysaccharide, and a targeting sequence, and the one or more moieties are covalently linked to the N-terminus of the first cell-penetrating peptide or the second cell-penetrating peptide, or to the PEG moiety. The cargo delivery complex according to any one of items 1 to 27. (Item 29) The cargo delivery complex according to item 28, wherein the one or more moieties are covalently linked to the N-terminus of the first cell-penetrating peptide, the N-terminus of the second cell-penetrating peptide or the PEG moiety via a linker. (Item 30) The cargo delivery complex according to item 28, wherein the one or more moieties comprise a targeting sequence. (Item 31) The cargo delivery complex according to item 30, wherein the targeting sequence is selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, and IGSR. (Item 32) The cargo delivery complex according to item 31, wherein the targeting sequence is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR. (Item 33) The cargo delivery complex according to any one of items 30 to 32, wherein the targeting array is covalently linked to the first cell-penetrating peptide or the second cell-penetrating peptide via a linker. (Item 34) The cargo delivery complex according to any one of items 28 to 33, wherein the one or more moieties comprise an acetyl group and / or a stearyl group. (Item 35) The first peptide and / or the second peptide further comprises one or more moieties selected from the group consisting of cysteamide, cysteine, thiol, amide, nitrilotriacetic acid optionally substituted, carboxyl, linear or branched C optionally substituted 1 ~C 6 alkyl, primary or secondary amine, oside derivative, lipid, phospholipid, fatty acid, cholesterol, nuclear localization signal, nuclear export signal, antibody, polysaccharide, and targeting array, and the one or more moieties are covalently linked to the C-terminus of the first cell-penetrating peptide, the C-terminus of the second cell-penetrating peptide, or the PEG moiety. The cargo delivery complex according to any one of items 1 to 34. (Item 36) The cargo delivery complex according to item 35, wherein the one or more moieties are covalently linked to the C-terminus of the first cell-penetrating peptide, the C-terminus of the second cell-penetrating peptide, or the PEG moiety via a linker. (Item 37) The cargo delivery complex according to any one of items 1 to 36, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is a retro-inverso peptide. (Item 38) A cargo delivery complex for intracellular delivery of a cargo molecule, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide, and the cell-penetrating peptide is a retro-inverso peptide. (Item 39) The cargo delivery complex according to item 37 or 38, wherein the retro-inverso peptide comprises the sequence of SEQ ID NO: 85 or 86. (Item 40) A cargo delivery complex for delivering a cargo molecule, comprising: a) a peptide comprising a cell-permeable peptide; and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR. (Item 41) The cargo delivery complex according to item 40, wherein the cell-permeable peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. (Item 42) The cargo delivery complex according to item 40 or 41, wherein the targeting sequence is covalently linked via a linker to the N-terminus of the cell-permeable peptide. (Item 43) The cargo delivery complex according to item 42, wherein the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group. (Item 44) The cargo delivery complex according to any one of items 38 to 43, wherein the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. (Item 45) The cargo delivery complex according to item 44, wherein the cargo molecule comprises a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. (Item 46) The cargo delivery complex according to item 45, wherein the cargo molecule comprises mRNA. (Item 47) The cargo delivery complex according to item 45 or 46, wherein the cargo molecule comprises RNAi. (Item 48) The cargo delivery complex according to any one of items 38 to 47, wherein the cargo molecule does not contain a virus. (Item 49) A nanoparticle comprising a core containing the cargo delivery complex according to any one of items 1 to 48. (Item 50) The nanoparticle according to item 49, wherein the core is coated with a shell containing a peripheral cell-permeable peptide. (Item 51) The nanoparticle according to item 50, wherein the peripheral cell-permeable peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. (Item 52) A pharmaceutical composition comprising a cargo delivery complex according to any one of Items 1 to 48 or a nanoparticle according to any one of Items 49 to 51, and a pharmaceutically acceptable carrier. (Item 53) A method for preparing a cargo delivery complex according to any one of Items 1 to 37, comprising: a) combining the first peptide and the second peptide, thereby forming a peptide mixture; and b) combining the peptide mixture with the cargo, thereby forming the cargo delivery complex. (Item 54) A method for preparing a cargo delivery complex according to any one of Items 38 to 48, comprising combining the peptide with the cargo molecule, thereby forming the cargo delivery complex. (Item 55) The method according to Item 53 or 54, wherein the peptide or the peptide mixture and the cargo molecule are combined in a molar ratio of from about 1:1 to about 100:1. (Item 56) A method according to any one of Items 53 to 55, comprising mixing a first solution containing the cargo molecule with a second solution containing the peptide or peptide mixture to form a third solution, wherein the third solution is adjusted to contain or be adjusted to contain: i) from about 0 to 5% sucrose; ii) from about 0 to 5% glucose; iii) from about 0 to 50% DMEM; iv) from about 0 to 80 mM NaCl; or v) from about 0 to 20% PBS, and incubating the third solution to form the cargo delivery complex. (Item 57) The method according to Item 56, wherein the first solution contains the cargo in sterile water and / or the second solution contains the peptide or peptide mixture in sterile water. (Item 58) The method according to Item 56 or 57, wherein after incubating the third solution to form the cargo delivery complex, the third solution is adjusted to contain: i) from about 0 to 5% sucrose; ii) from about 0 to 5% glucose; iii) from about 0 to 50% DMEM; iv) from about 0 to 80 mM NaCl; or v) from about 0 to 20% PBS. (Item 59) The method according to any one of Items 53 to 58, further comprising a filtration process of filtering the cargo delivery complex through a membrane with a pore size. (Item 60) The method according to Item 59, wherein the diameter of the pores is at least about 0.1 μm. (Item 61) A method for delivering one or more cargos into cells, comprising the step of contacting the cells with a cargo delivery complex according to any one of items 1 to 48 or a nanoparticle according to any one of items 49 to 51, wherein the cargo delivery complex contains one or more cargos. (Item 62) A method for delivering one or more cargos into a tissue or organ of an individual, comprising the step of administering to the individual an effective amount of a cargo delivery complex according to any one of items 1 to 48, a nanoparticle according to any one of items 49 to 51, or a pharmaceutical composition according to item 52, wherein the tissue or organ is selected from the group consisting of liver, lung, kidney, brain, intestine, spleen, heart, muscle, and lymph node. (Item 63) A method for treating a disease or condition in an individual, comprising the step of administering to the individual an effective amount of a cargo delivery complex according to any one of items 1 to 48, a nanoparticle according to any one of items 49 to 51, or a pharmaceutical composition according to item 52. (Item 64) The method according to item 63, wherein the disease or condition is related to diseased cells in an organ or tissue selected from the group consisting of liver, lung, kidney, brain, intestine, spleen, heart, muscle, and lymph node. (Item 65) The method according to item 63 or 64, wherein the disease or condition is selected from the group consisting of cancer, diabetes, autoimmune diseases, blood diseases, heart diseases, vascular diseases, inflammatory diseases, fibrotic diseases, viral infectious diseases, genetic diseases, eye diseases, liver diseases, lung diseases, muscle diseases, protein deficiency diseases, lysosomal storage diseases, neurological diseases, kidney diseases, aging and degenerative diseases, and diseases characterized by abnormal cholesterol levels. (Item 66) The method according to any one of items 62 to 65, wherein the cargo delivery complex is administered intravenously or intramuscularly. (Item 67) The method according to any one of items 62 to 66, wherein the individual is a human. (Item 68) A kit containing a cargo delivery complex according to any one of items 1 to 48, a nanoparticle according to any one of items 49 to 51, or a pharmaceutical composition according to item 52.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0029] The present application provides a complex and a nanoparticle comprising a cell - penetrating peptide (CPP) and one or more mRNAs, wherein the CPP is suitable for delivering one or more mRNAs (e.g., mRNAs encoding therapeutic products such as tumor suppressors) into cells. The complex and the nanoparticle may contain multiple mRNAs. The mRNA may include, for example, mRNAs encoding therapeutic proteins (e.g., tumor suppressors, immunomodulators, etc.). In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR). In some embodiments, the complex and the nanoparticle are preferentially localized to a target tissue such as diseased tissue, e.g., a tumor. In some embodiments, the complex and the nanoparticle further comprise an RNAi such as an RNAi targeting an endogenous gene. In some embodiments, the RNAi targets an endogenous gene associated with a disease, e.g., an oncogene. In some embodiments, the RNAi targets an exogenous gene.

[0030] Accordingly, in one aspect, the present application provides novel cargo - delivery complexes and nanoparticles, which are described in more detail below.

[0031] In another aspect, methods are provided for delivering mRNA into cells using cell - permeable peptides. In another aspect, methods are provided for delivering a complex or nanoparticle comprising mRNA and a cell - permeable peptide into a local tissue, organ, or cell. In another aspect, methods are provided for treating a disease or disorder by administering to a subject a complex or nanoparticle as described herein comprising mRNA and a cell - permeable peptide.

[0032] Also provided are pharmaceutical compositions (e.g., in the form of complexes and nanoparticles) comprising a cell - permeable peptide and one or more mRNAs and their use for treating diseases. Definitions

[0033] As used herein, the term "retro - inverso peptide" refers to a peptide composed of D - amino acids in a reverse sequence, which, when extended, is assumed to have a side - chain topology similar to its parent molecule, but with amide peptide bonds inverted.

[0034] As used herein, the term "wild - type" is a term understood by those skilled in the art and means the typical, naturally - occurring form of an organism, strain, gene, or characteristic, as distinguished from mutant or variant forms.

[0035] The term "variant" as used herein should be construed to indicate a quality having a pattern that deviates from that which occurs naturally.

[0036] The terms "non - naturally occurring" or "engineered" are used interchangeably and indicate the involvement of human hands. When referring to a nucleic acid molecule or polypeptide, this term means that the nucleic acid molecule or polypeptide does not contain at least substantially one other component that is naturally associated and found in nature.

[0037] "Complementary" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence, either in the form of traditional Watson-Crick base pairing or other non-traditional forms. Percent complementarity indicates the percentage of residues within a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5 out of 10, 6 out of 10, 7 out of 10, 8 out of 10, 9 out of 10, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Fully complementary" means that all of the consecutive residues of a nucleic acid sequence hydrogen bond with the same number of consecutive residues within a second nucleic acid sequence. "Substantially complementary", as used herein, refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids hybridizing under stringent conditions.

[0038] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may collectively be referred to as a "gene product". When a polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells.

[0039] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0040] The terms "therapeutic agent", "therapeutic capable agent", or "treatment agent" are used interchangeably and refer to a molecule or compound that, when administered to a subject, confers some beneficial effect. Beneficial effects include the ability to perform a diagnostic determination; amelioration of a disease, symptom, disorder, or pathological condition; reduction or prevention of the onset of a disease, symptom, disorder, or condition; and generally, counteracting a disease, symptom, disorder, or pathological condition.

[0041] As used herein, "treatment" or "treating" refers to a procedure for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit. A therapeutic benefit means any improvement or effect relevant to the treatment of one or more diseases, conditions, or symptoms being treated.

[0042] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent that is sufficient to produce a beneficial or desired result. A therapeutically effective amount can vary depending on one or more of the subject being treated and the disease state, the subject's weight and age, the severity of the disease state, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. This term also applies to the dosage that results in an image being provided for detection by any one of the imaging methods described herein. The specific dosage can vary depending on one or more of the specific agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue in which the imaging is performed, and the physical delivery system employed.

[0043] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0044] References to "about" values or parameters include (and describe) embodiments that are directed to the value or parameter itself. For example, a reference to "about X" includes a description of "X".

[0045] The compositions and methods of the present invention can include, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful.

[0046] Unless otherwise noted, technical terms are used in accordance with their conventional usage. Complexes and Nanoparticles Complexes

[0047] In some embodiments, a cargo delivery complex is provided that includes a cell - penetrating peptide for delivering one or more cargo molecules intracellularly. A cargo delivery complex comprising a peptide mixture

[0048] In some embodiments, a cargo delivery complex for intracellular delivery of a cargo molecule, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, and the first peptide has no PEG moiety, is provided. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, viruses, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of iRNA (e.g., siRNA, miRNA, or shRNA, etc.), gRNA, mRNA, DNA, DNA, DNA plasmid, oligonucleotides and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the cargo molecule (e.g., nucleic acid, etc.) is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.).In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0049] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, and the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.). In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP peptide (e.g., PEP-1, PEP-2, or PEP-3 peptide, etc.), or a VEPEP peptide (e.g., ADGN-100, VEPEP-3, VEPEP-4, VEPEP-5, VEPEP-6, or VEPEP-9 peptide, etc.). In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., from about 20 to about 500 nm, from about 50 to about 400 nm, from about 60 to about 300 nm, from about 80 to about 200 nm, or from about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of from about 1 to 10 (e.g., from about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., from about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0050] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, and the first cell-penetrating peptide and / or the second cell-penetrating peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is selected from the group consisting of VEPEP-6 peptide and ADGN-100 peptide. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, viruses, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of iRNA (e.g., siRNA, miRNA, or shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, wherein the mRNA encodes a therapeutic protein for treating a disease or condition, and the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the cargo molecule (e.g., nucleic acid, etc.) is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.). In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 - about 500 nm, about 50 - about 400 nm, about 60 - about 300 nm, about 80 - about 200 nm, or about 100 - about 160 nm, etc.).In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-permeable peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-permeable peptide.

[0051] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, and the cargo molecule is selected from the group consisting of nucleic acids, viruses, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid or comprises a nucleic acid. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-74 and 81. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-9 peptide.In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are the same. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are different. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0052] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0053] In some embodiments, a cargo delivery complex for intracellular delivery of a cargo molecule, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., from about 20 to about 500 nm, from about 50 to about 400 nm, from about 60 to about 300 nm, from about 80 to about 200 nm, or from about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., from about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0054] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising: a) a first peptide comprising a first cell-permeable peptide; b) a second peptide comprising a second cell-permeable peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-permeable peptide, the first peptide has no PEG moiety, the ratio of the first cell-permeable peptide to the second cell-permeable peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the first cell-permeable peptide and / or the second cell-permeable peptide is a VEPEP-9 peptide. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-permeable peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-permeable peptide.

[0055] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the first cell-penetrating peptide and / or the second cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., from about 20 to about 500 nm, from about 50 to about 400 nm, from about 60 to about 300 nm, from about 80 to about 200 nm, or from about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., from about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0056] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-74 and 81. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 to 10 ethylene glycol units (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.). In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0057] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the cargo molecule is a nucleic acid or comprises a nucleic acid, and the cargo delivery complex is provided. In some embodiments, the nucleic acid is selected from the group consisting of iRNA (e.g., siRNA, miRNA, or shRNA, etc.), gRNA, mRNA, DNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., between about 1:1 and about 50:1, or between about 20:1, etc.). In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-74 and 81. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide.In some embodiments, the first cell-permeable peptide and / or the second cell-permeable peptide is the VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the first cell-permeable peptide and / or the second cell-permeable peptide is the VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the first cell-permeable peptide and / or the second cell-permeable peptide is the VEPEP-9 peptide. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the first cell-permeable peptide and / or the second cell-permeable peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the first cell-permeable peptide and the second cell-permeable peptide are the same. In some embodiments, the first cell-permeable peptide and the second cell-permeable peptide are different. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 - about 500 nm, about 50 - about 400 nm, about 60 - about 300 nm, about 80 - about 200 nm, or about 100 - about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 - 10 (e.g., about 1 - 8, 2 - 7, 1 - 5, or 6 - 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa - about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa - about 0.5 kDa (e.g., about 0.05 - 0.1 kDa, 0.05 - 0.4 kDa, 0.1 - 0.3 kDa, 0.05 - 0.25 kDa, 0.25 - 0.5 kDa, etc.).In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0058] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the cargo molecule is a virus or comprises a virus, is provided. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-74 and 81. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-9 peptide.In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are the same. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are different. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 - about 500 nm, about 50 - about 400 nm, about 60 - about 300 nm, about 80 - about 200 nm, or about 100 - about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 - 10 (e.g., about 1 - 8, 2 - 7, 1 - 5, or 6 - 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa - about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa - about 0.5 kDa (e.g., about 0.05 - 0.1 kDa, 0.05 - 0.4 kDa, 0.1 - 0.3 kDa, 0.05 - 0.25 kDa, 0.25 - 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0059] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the cargo molecule is a polypeptide or comprises a polypeptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-74 and 81. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-9 peptide.In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are the same. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are different. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 - about 500 nm, about 50 - about 400 nm, about 60 - about 300 nm, about 80 - about 200 nm, or about 100 - about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 - 10 (e.g., about 1 - 8, 2 - 7, 1 - 5, or 6 - 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa - about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa - about 0.5 kDa (e.g., about 0.05 - 0.1 kDa, 0.05 - 0.4 kDa, 0.1 - 0.3 kDa,  0.05 - 0.25 kDa, 0.25 - 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0060] In some embodiments, a cargo delivery complex for delivering a cargo molecule into a cell, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the cargo molecule is a protein / nucleic acid complex or comprises a protein / nucleic acid complex, and a cargo delivery complex is provided. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-74 and 81. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-9 peptide.In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are the same. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are different. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0061] In some embodiments, a cargo delivery complex for intracellular delivery of a cargo molecule, comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the cargo molecule is a virus-like particle or comprises a virus-like particle, a cargo delivery complex is provided. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-74 and 81. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-9 peptide.In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are the same. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are different. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20-about 500 nm, about 50-about 400 nm, about 60-about 300 nm, about 80-about 200 nm, or about 100-about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1-10 (e.g., about 1-8, 2-7, 1-5, or 6-10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa to about 0.5 kDa (e.g., about 0.05-0.1 kDa, 0.05-0.4 kDa, 0.1-0.3 kDa, 0.05-0.25 kDa, 0.25-0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0062] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from about 20:1 to about 1:1 (e.g., from about 15:1 to about 2:1, from about 10:1 to about 4:1, etc.), and the cargo molecule is a protein complex or comprises a protein complex. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-74 and 81. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-3 peptide. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-6 peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the VEPEP-9 peptide.In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are the same. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are different. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 - about 500 nm, about 50 - about 400 nm, about 60 - about 300 nm, about 80 - about 200 nm, or about 100 - about 160 nm, etc.). In some embodiments, the PEG moiety consists of about 1 - 10 (e.g., about 1 - 8, 2 - 7, 1 - 5, or 6 - 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa - about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa - about 0.5 kDa (e.g., about 0.05 - 0.1 kDa, 0.05 - 0.4 kDa, 0.1 - 0.3 kDa, 0.05 - 0.25 kDa, 0.25 - 0.5 kDa, etc.). In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0063] In some embodiments, the PEG moiety is linear PEG. In some embodiments, the PEG moiety is branched PEG.

[0064] In some embodiments, the first peptide and / or the second peptide further comprises one or more moieties selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, cholesterol, a nuclear localization signal, a nuclear export signal, an antibody or an antibody fragment thereof, a peptide, a polysaccharide, and a targeting sequence, and the one or more moieties are covalently linked to the N-terminus of the first cell-penetrating peptide or the second cell-penetrating peptide, or to the PEG moiety. In some embodiments, the one or more moieties are covalently linked to the N-terminus of the first cell-penetrating peptide, the N-terminus of the second cell-penetrating peptide or the PEG moiety via a linker. In some embodiments, the one or more moieties comprise an acetyl group and / or a stearyl group. In some embodiments, the one or more moieties comprise a targeting sequence. In some embodiments, the targeting sequence is covalently linked to the first cell-penetrating peptide or the second cell-penetrating peptide via a linker. In some embodiments, the targeting sequence is covalently linked to the first cell-penetrating peptide or the second cell-penetrating peptide without a linker.

[0065] In some embodiments, the first peptide and / or the second peptide further comprises one or more moieties selected from the group consisting of cysteamide, cysteine, thiol, amide, nitrilotriacetic acid optionally substituted, carboxyl, linear or branched (ramified) C1-C6 alkyl optionally substituted, primary or secondary amine, oxide derivative, lipid, phospholipid, fatty acid, cholesterol, nuclear localization signal, nuclear export signal, antibody, polysaccharide, and targeting sequence, and the one or more moieties are covalently linked to the C-terminus of the first cell-penetrating peptide, the C-terminus of the second cell-penetrating peptide, or the PEG moiety. In some embodiments, the one or more moieties are covalently linked to the C-terminus of the first cell-penetrating peptide, the C-terminus of the second cell-penetrating peptide, or the PEG moiety via a linker. In some embodiments, the one or more moieties comprise an acetyl group and / or a stearyl group. In some embodiments, the one or more moieties comprise a targeting sequence. In some embodiments, the targeting sequence is covalently linked to the first cell-penetrating peptide or the second cell-penetrating peptide via a linker. In some embodiments, the targeting sequence is covalently linked to the first cell-penetrating peptide or the second cell-penetrating peptide without a linker.

[0066] In some embodiments, the targeting sequence is selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, and IGSR. In some embodiments, the targeting sequence is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.

[0067] In some embodiments, the linker described herein includes a polyglycine linker. In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker includes a PEG linker moiety. In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG linker moiety is a linear PEG. In some embodiments, the PEG linker moiety is a branched PEG. In some embodiments, the linker includes beta-alanine. In some embodiments, the linker includes at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further includes serine. In some embodiments, the linker includes the GGGGS or SGGGG sequence. In some embodiments, the linker includes a glycine-beta-alanine motif.

[0068] In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a retro-inverso peptide. In some embodiments, the retro-inverso peptide includes the sequence of SEQ ID NO: 85 or 86.

[0069] In some embodiments, the first peptide and / or the second peptide includes the sequence of SEQ ID NO: 1-112. Cargo delivery complex comprising a retro-inverso cell-penetrating peptide

[0070] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide. In some embodiments, the cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a PEP peptide (e.g., PEP-1, PEP-2, or PEP-3 peptide, etc.), or a VEPEP peptide (e.g., ADGN-100, VEPEP-3, VEPEP-4, VEPEP-5, VEPEP-6, or VEPEP-9 peptide, etc.). In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes.

[0071] In some embodiments, provided is a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is selected from the group consisting of PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide, and the cell-penetrating peptide is a retro-inverso peptide. In some embodiments, the cell-penetrating peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule does not contain a virus. In some embodiments, the cargo molecule comprises a nucleic acid selected from the group consisting of RNAi (e.g., siRNA, miRNA, shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the cargo molecule comprises mRNA. In some embodiments, the cargo molecule comprises or further comprises RNAi (e.g., siRNA, miRNA, shRNA, etc.). In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the cargo molecule (e.g., nucleic acid, etc.) is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.).

[0072] In some embodiments, provided is a cargo delivery complex for intracellular delivery of a cargo molecule, the complex comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide and the cargo molecule is virus-free. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule comprises a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of RNAi (e.g., siRNA, miRNA, shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotides, and analogs thereof. In some embodiments, the cargo molecule comprises mRNA. In some embodiments, the cargo molecule comprises or further comprises RNAi (e.g., siRNA, miRNA, shRNA, etc.). In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the cargo molecule (e.g., nucleic acid, etc.) is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.). In some embodiments, the cell-penetrating peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide.

[0073] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, the cargo delivery complex comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide, the cell-penetrating peptide is an ADGN-100 peptide, and the cargo molecule does not contain a virus. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, the cargo delivery complex comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide, the cell-penetrating peptide is an ADGN-100 peptide, and the cargo molecule contains a nucleic acid. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the nucleic acid is selected from the group consisting of RNAi (e.g., siRNA, miRNA, shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the cargo molecule contains mRNA. In some embodiments, the cargo molecule contains or further contains RNAi (e.g., siRNA, miRNA, shRNA, etc.). In some embodiments, the nucleic acid contains mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.).

[0074] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide, the cell-penetrating peptide is a VEPEP-3 peptide, and the cargo molecule is virus-free. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide, the cell-penetrating peptide is a VEPEP-3 peptide, and the cargo molecule is a nucleic acid. In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the nucleic acid is selected from the group consisting of RNAi (e.g., siRNA, miRNA, shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the cargo molecule comprises mRNA. In some embodiments, the cargo molecule comprises or further comprises RNAi (e.g., siRNA, miRNA, shRNA, etc.). In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the cargo comprises a DNA plasmid. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.).

[0075] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide, the cell-penetrating peptide is a VEPEP-6 peptide, and the cargo molecule does not contain a virus. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide, the cell-penetrating peptide is a VEPEP-6 peptide, and the cargo molecule contains a nucleic acid. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the nucleic acid is selected from the group consisting of RNAi (e.g., siRNA, miRNA, shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the cargo molecule comprises mRNA. In some embodiments, the cargo molecule comprises or further comprises RNAi (e.g., siRNA, miRNA, shRNA, etc.). In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.).

[0076] In some embodiments, provided is a cargo delivery complex for intracellular delivery of a cargo molecule, the cargo delivery complex comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide, the cell-penetrating peptide is a VEPEP-9 peptide, and the cargo molecule is virus-free. In some embodiments, provided is a cargo delivery complex for intracellular delivery of a cargo molecule, the cargo delivery complex comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide, the cell-penetrating peptide is a VEPEP-9 peptide, and the cargo molecule comprises a nucleic acid. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the nucleic acid is selected from the group consisting of RNAi (e.g., siRNA, miRNA, shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the cargo molecule comprises mRNA. In some embodiments, the cargo molecule comprises or further comprises RNAi (e.g., siRNA, miRNA, shRNA, etc.). In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., between about 1:1 and about 50:1, or about 20:1).

[0077] In some embodiments, a cargo delivery complex for intracellular delivery of cargo molecules, comprising a cell-penetrating peptide and a cargo molecule, wherein the cell-penetrating peptide is a retro-inverso peptide comprising the sequence of SEQ ID NO: 85 or 86, and the cargo molecule does not contain a virus, and the complex is provided. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule comprises a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of RNAi (e.g., siRNA, miRNA, shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the cargo molecule comprises mRNA. In some embodiments, the cargo molecule comprises or further comprises RNAi (e.g., siRNA, miRNA, shRNA, etc.). In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the cargo molecule (e.g., nucleic acid, etc.) is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.).

[0078] In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 - about 500 nm, about 50 - about 400 nm, about 60 - about 300 nm, about 80 - about 200 nm, or about 100 - about 160 nm, etc.).

[0079] In some embodiments, the cell-permeable peptide further comprises one or more moieties selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, cholesterol, a nuclear localization signal, a nuclear export signal, an antibody or an antibody fragment thereof, a peptide, a polysaccharide, and a targeting sequence, and the one or more moieties are covalently linked to the N-terminus of the cell-permeable peptide. In some embodiments, the one or more moieties are covalently linked to the N-terminus of the cell-permeable peptide via a linker. In some embodiments, the one or more moieties comprise an acetyl group and / or a stearyl group. In some embodiments, the one or more moieties comprise a targeting sequence. In some embodiments, the targeting sequence is covalently linked to the cell-permeable peptide via a linker. In some embodiments, the targeting sequence is covalently linked to the cell-permeable peptide without a linker.

[0080] In some embodiments, the peptide further comprises one or more moieties selected from the group consisting of cysteamide, cysteine, thiol, amide, nitrilotriacetic acid optionally substituted, carboxyl, a linear or branched C1-C6 alkyl optionally substituted, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, cholesterol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, and a targeting sequence, and the one or more moieties are covalently linked to the C-terminus of the cell-permeable peptide. In some embodiments, the one or more moieties are covalently linked to the C-terminus of the cell-permeable peptide via a linker. In some embodiments, the one or more moieties comprise an acetyl group and / or a stearyl group. In some embodiments, the one or more moieties comprise a targeting sequence. In some embodiments, the targeting sequence is covalently linked to the cell-permeable peptide via a linker. In some embodiments, the targeting sequence is covalently linked to a second cell-permeable peptide without a linker.

[0081] In some embodiments, the targeting sequence is selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, and IGSR. In some embodiments, the targeting sequence is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.

[0082] In some embodiments, the linker described herein includes a polyglycine linker. In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker includes a PEG linker moiety. In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG linker moiety is a linear PEG. In some embodiments, the PEG linker moiety is a branched PEG. In some embodiments, the linker includes beta-alanine. In some embodiments, the linker includes at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further includes serine. In some embodiments, the linker includes the GGGGS or SGGGG sequence. In some embodiments, the linker includes a glycine-beta-alanine motif. A cargo delivery complex comprising a cell-permeable peptide having a signaling sequence (i.e., a targeting moiety)

[0083] In some embodiments, provided is a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR. In some embodiments, the cell-penetrating peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm, etc.). In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide via a linker. In some embodiments, the linker comprises a polyglycine linker. In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), a PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker comprises a PEG linker moiety. In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.).In some embodiments, the PEG linker moiety is linear PEG. In some embodiments, the PEG linker moiety is branched PEG. In some embodiments, the linker comprises β-alanine. In some embodiments, the linker comprises at least about two, three, or four glycines, optionally consecutive glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises the GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-β-alanine motif. In some embodiments, the targeting sequence is covalently linked without a linker to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group.

[0084] In some embodiments, provided is a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-permeable peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, and the cargo molecule is virus-free. In some embodiments, provided is a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-permeable peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, and the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the molar ratio of the cell-permeable peptide to the cargo molecule is between about 1:1 and about 100:1 (e.g., between about 1:1 and about 50:1, or about 20:1). In some embodiments, the cell-permeable peptide is a PTD-based peptide, an amphiphilic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the cell-permeable peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-permeable peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-permeable peptide via a linker. In some embodiments, the linker comprises a polyglycine linker. In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker comprises a PEG linker moiety.In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG linker moiety is a linear PEG. In some embodiments, the PEG linker moiety is a branched PEG. In some embodiments, the linker contains β-alanine. In some embodiments, the linker contains at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further contains serine. In some embodiments, the linker contains the GGGGS or SGGGG sequence. In some embodiments, the linker contains a glycine-β-alanine motif. In some embodiments, the targeting sequence is covalently linked without a linker to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group.

[0085] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, and the cargo molecule is a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of iRNA (e.g., siRNA, miRNA, or shRNA, etc.), gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises or further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the nucleic acid is a DNA plasmid. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1). In some embodiments, the cell-penetrating peptide is a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide via a linker. In some embodiments, the linker comprises a polyglycine linker.In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker comprises a PEG linker moiety. In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG linker moiety is a linear PEG. In some embodiments, the PEG linker moiety is a branched PEG. In some embodiments, the linker comprises beta-alanine. In some embodiments, the linker comprises at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises the GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-beta-alanine motif. In some embodiments, the targeting sequence is covalently linked without a linker to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group.

[0086] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is an ADGN-100 peptide, and the cargo molecule is virus-free. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is an ADGN-100 peptide, and the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is an ADGN-100 peptide, and the cargo molecule is a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of iRNA (e.g., siRNA, miRNA, or shRNA, etc.), gRNA, mRNA, DNA, DNA, DNA plasmid, oligonucleotides, and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises or further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the nucleic acid is a DNA plasmid. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.).In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide via a linker. In some embodiments, the linker comprises a polyglycine linker. In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker comprises a PEG linker moiety. In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG linker moiety is a linear PEG. In some embodiments, the PEG linker moiety is a branched PEG. In some embodiments, the linker comprises beta-alanine. In some embodiments, the linker comprises at least about 2, 3, or 4 glycines, optionally contiguous glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises the GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-beta-alanine motif. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide without a linker. In some embodiments, the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group.

[0087] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-3 peptide, and the cargo molecule is virus-free. In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-3 peptide, and the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-3 peptide, and the cargo molecule is a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of iRNA (e.g., siRNA, miRNA, or shRNA, etc.), gRNA, mRNA, DNA, DNA, DNA plasmid, oligonucleotides, and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises or further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the nucleic acid is a DNA plasmid. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 20:1, etc.).In some embodiments, the VEPEP-3 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 75, 76, and 113-115. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide via a linker. In some embodiments, the linker comprises a polyglycine linker. In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker comprises a PEG linker moiety. In some embodiments, the PEG linker moiety consists of about 1-10 (e.g., about 1-8, 2-7, 1-5, or 6-10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is about 0.05 kDa to about 0.5 kDa (e.g., about 0.05-0.1 kDa, 0.05-0.4 kDa, 0.1-0.3 kDa, 0.05-0.25 kDa, 0.25-0.5 kDa, etc.). In some embodiments, the PEG linker moiety is a linear PEG. In some embodiments, the PEG linker moiety is a branched PEG. In some embodiments, the linker comprises beta-alanine. In some embodiments, the linker comprises at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises the GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-beta-alanine motif. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide without a linker. In some embodiments, the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group.

[0088] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-6 peptide, and the cargo molecule is virus-free. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-6 peptide, and the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-6 peptide, and the cargo molecule is a nucleic acid. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the nucleic acid is selected from the group consisting of iRNA (e.g., siRNA, miRNA, or shRNA, etc.), gRNA, mRNA, DNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises or further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition. In some embodiments, the nucleic acid is a DNA plasmid.In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., between about 1:1 and about 50:1, or between about 20:1, etc.). In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide via a linker. In some embodiments, the linker comprises a polyglycine linker. In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker comprises a PEG linker moiety. In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is from about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG linker moiety is a linear PEG. In some embodiments, the PEG linker moiety is a branched PEG. In some embodiments, the linker comprises beta-alanine. In some embodiments, the linker comprises at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises the GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-beta-alanine motif. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide without a linker. In some embodiments, the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group.

[0089] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-9 peptide, and the cargo molecule is virus-free. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-9 peptide, and the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR, the cell-penetrating peptide is the VEPEP-9 peptide, and the cargo molecule is a nucleic acid. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 78, and 116-120. In some embodiments, the nucleic acid is selected from the group consisting of iRNA (e.g., siRNA, miRNA, or shRNA, etc.), gRNA, mRNA, DNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises or further comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, and the RNAi targets an RNA, the expression of which is associated with the disease or condition. In some embodiments, the nucleic acid is a DNA plasmid.In some embodiments, the molar ratio of the cell-permeable peptide to the nucleic acid is between about 1:1 and about 100:1 (e.g., between about 1:1 and about 50:1, or between about 20:1). In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-permeable peptide. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-permeable peptide via a linker. In some embodiments, the linker comprises a polyglycine linker. In some embodiments, the linker is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker comprises a PEG linker moiety. In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10) ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa). In some embodiments, the PEG linker moiety is a linear PEG. In some embodiments, the PEG linker moiety is a branched PEG. In some embodiments, the linker comprises beta-alanine. In some embodiments, the linker comprises at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises the GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-beta-alanine motif. In some embodiments, the targeting sequence is covalently linked to the N-terminus of the cell-permeable peptide without a linker. In some embodiments, the peptide further comprises one or more moieties linked to the N-terminus of the targeting sequence, and the one or more moieties are selected from the group consisting of an acetyl group and a stearyl group.

[0090] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, comprising a) a peptide comprising a cell-permeable peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of YIGSR, and the targeting sequence is covalently linked to the N-terminus of the cell-permeable peptide without a linker. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the cell-permeable peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the cargo molecule does not include a virus. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0091] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, comprising a) a peptide comprising a cell-permeable peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of YIGSR, and the targeting sequence is covalently linked to the N-terminus of the cell-permeable peptide via a linker, and the linker comprises at least about two, three, or four glycines. In some embodiments, the linker consists of two, three, or four glycines. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the cell-permeable peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the cargo molecule does not include a virus. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0092] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising: a) a peptide comprising a cell-penetrating peptide; and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of GYVS, the targeting sequence being covalently linked via a linker to the N-terminus of the cell-penetrating peptide, the linker comprising at least about two, three, or four glycines. In some embodiments, the linker consists of two, three, or four glycines. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the cargo molecule is virus-free. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0093] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of YIGSR, the targeting sequence being covalently linked via a linker to the N-terminus of the cell-penetrating peptide, the linker comprising an Ava (5-aminopentanoic acid) moiety. In some embodiments, the Ava moiety further comprises a methylene group (i.e., CH2). In some embodiments, the Ava moiety comprises at least about 2 CH2. In some embodiments, the Ava moiety has 2 CH2. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the cargo molecule is virus-free. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotides, and analogs thereof.

[0094] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of GYVS, the targeting sequence being covalently linked via a linker to the N-terminus of the cell-penetrating peptide, the linker comprising an Ava (5-aminopentanoic acid) moiety. In some embodiments, the Ava moiety further comprises a methylene group (i.e., CH2). In some embodiments, the Ava moiety comprises at least about 2 CH2 groups. In some embodiments, the Ava moiety has 2 CH2 groups. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the cargo molecule is virus-free. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotides, and analogs thereof.

[0095] In some embodiments, a cargo delivery complex for delivering a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of YIGSR, the targeting sequence is covalently linked via a linker to the N-terminus of the cell-penetrating peptide, the linker comprises an Aun (11-amino-undecanoic acid) moiety, and a cargo delivery complex is provided. In some embodiments, the Ava moiety further comprises a methylene group (i.e., CH2). In some embodiments, the Ava moiety comprises at least 1 (e.g., at least 2, 3, 4, 5, or 6, etc.) CH2. In some embodiments, the Ava moiety comprises about 1 to 10 (e.g., about 4 to 8, about 5 to 7, or 6, etc.) CH2. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the cargo molecule does not contain a virus. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0096] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of GYVS, the targeting sequence being covalently linked via a linker to the N-terminus of the cell-penetrating peptide, the linker comprising an Aun (11-amino-undecanoic acid) moiety. In some embodiments, the Ava moiety further comprises a methylene group (i.e., CH2). In some embodiments, the Ava moiety comprises at least 1 (e.g., at least 2, 3, 4, 5, or 6, etc.) CH2. In some embodiments, the Ava moiety comprises from about 1 to 10 (e.g., about 4 to 8, about 5 to 7, or 6, etc.) CH2. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the cargo molecule is virus-free. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0097] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of GYVS or YIGSR, and the targeting sequence is covalently linked via a linker comprising a PEG linker moiety at the N-terminus of the cell-penetrating peptide. In some embodiments, the targeting sequence comprises, or consists of, YIGSR. In some embodiments, the targeting sequence comprises, or consists of, GYVS. In some embodiments, the PEG linker moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10, etc.) ethylene glycol units. In some embodiments, the PEG linker moiety consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol units. In some embodiments, the molecular weight of the PEG linker moiety is about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa, etc.). In some embodiments, the PEG linker moiety is linear PEG. In some embodiments, the PEG linker moiety is branched PEG. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the cargo molecule does not contain a virus. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0098] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, the cargo delivery complex comprising: a) a peptide comprising a cell-penetrating peptide; and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of GYVS or YIGSR, and the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 88, 89, 94-99, 101-112.

[0099] In some embodiments, provided is a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of SYTSSTM, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 128, 131, and 132. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0100] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence CKTRRVP, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 134 or 137. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0101] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of THRPPNWSPV, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 133 or 138. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0102] In some embodiments, provided is a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence TGNYKALHPDHNG, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 122 or SEQ ID NO: 123. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide and analogs thereof.

[0103] In some embodiments, provided is a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of CARPAR, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 121 or 139. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0104] In some embodiments, there is provided a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of ASSLNIA, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0105] In some embodiments, a cargo delivery complex for intracellular delivery of a cargo molecule is provided, the cargo delivery complex comprising: a) a peptide comprising a cell-penetrating peptide; and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence LSSRLDA, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, a cysteamide bridge, polyglycine (e.g., G2 or G4), a PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the cargo molecule is selected from the group consisting of a nucleic acid, a polypeptide, a protein / nucleic acid complex, a virus-like particle, and a protein complex. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmids, oligonucleotides, and analogs thereof.

[0106] In some embodiments, there is provided a cargo delivery complex for intracellular delivery of a cargo molecule, comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of KSYDTY, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 116 or 119. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide and analogs thereof.

[0107] In some embodiments, provided is a cargo delivery complex for delivering a cargo molecule into a cell, the complex comprising a) a peptide comprising a cell-penetrating peptide and b) a cargo molecule, wherein the peptide further comprises a targeting sequence comprising the sequence of CKRAV, and the targeting sequence is covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the peptide further comprises an acetyl group linked to the N-terminus of the targeting sequence. In some embodiments, the targeting sequence is linked to the N-terminus of the cell-penetrating peptide via a linker moiety. In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide or a VEPEP-6 peptide. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-70, 79, 80, 86-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-40, 77, 85, 92-100, 105, 107-109, and 129-139. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 117. In some embodiments, the cargo molecule is selected from the group consisting of nucleic acids, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. In some embodiments, the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

[0108] In some embodiments, the average diameter of the cargo delivery complex is between about 20 nm and about 1000 nm (e.g., about 20 - about 500 nm, about 50 - about 400 nm, about 60 - about 300 nm, about 80 - about 200 nm, or about 100 - about 160 nm, etc.).

[0109] In some embodiments, the cell-penetrating peptide further comprises one or more moieties selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, cholesterol, a nuclear localization signal, a nuclear export signal, an antibody or an antibody fragment thereof, a peptide, and a polysaccharide, and the one or more moieties are covalently linked to the N-terminus of the cell-penetrating peptide. In some embodiments, the one or more moieties are covalently linked to the N-terminus of the cell-penetrating peptide via a second linker. In some embodiments, the one or more moieties comprise or consist of a stearyl group.

[0110] In some embodiments, the peptide further comprises one or more moieties selected from the group consisting of cysteamide, cysteine, thiol, amide, nitrilotriacetic acid optionally substituted, carboxyl, a linear or branched C1-C6 alkyl optionally substituted, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, cholesterol, a nuclear localization signal, a nuclear export signal, an antibody, and a polysaccharide, and the one or more moieties are covalently linked to the C-terminus of the cell-penetrating peptide. In some embodiments, the one or more moieties are covalently linked to the C-terminus of the cell-penetrating peptide via a second linker. In some embodiments, the one or more moieties comprise or consist of a stearyl group.

[0111] In some embodiments, the peptide is a retro-inverso peptide. In some embodiments, the retro-inverso peptide comprises the sequence of SEQ ID NO: 85 or 86.

[0112] In some embodiments, the peptide comprises the sequence of SEQ ID NOs: 1-112.

[0113] In some embodiments, the cell-penetrating peptides described herein form a complex with one or more cargo molecules. In some embodiments, the cell-penetrating peptide forms a complex non-covalently with at least one of the one or more cargo molecules. In some embodiments, the cell-penetrating peptide forms a complex non-covalently with each of the one or more cargo molecules. In some embodiments, the cell-penetrating peptide forms a complex covalently with at least one of the one or more cargo molecules. In some embodiments, the cell-penetrating peptide forms a complex covalently with each of the one or more cargo molecules. Cell-penetrating peptide

[0114] Cell-penetrating peptides (CPPs) are one of the promising virus-independent strategies. The definition of CPPs is evolving, but they are generally described as short peptides of less than 30 amino acids derived from proteins or chimeric sequences. CPPs are usually amphiphilic and have a net positive charge (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206). CPPs can cross the cell membranes of various biomolecules to induce their movement into the cytoplasm and can permeate biological membranes to improve their intracellular routing, thereby facilitating their interaction with targets. CPPs can be subdivided into two main classes. The first class requires chemical linkage to the cargo, and the second class involves the formation of stable non-covalent complexes. CPPs of both strategies have been reported to be advantageous for the delivery of a large panel of cargos (plasmid DNA, oligonucleotides, siRNA, PNA, proteins, peptides, liposomes, nanoparticles...) to a variety of cell types and in vivo models (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206; Mickan et al. (2014) Curr Pharm Biotechnol 15, 200-209; Shukla et al. (2014) Mol Pharm 11, 3395-3408).

[0115] The concept of protein transduction domains (PTDs) was first proposed based on the observation that several proteins, mainly transcription factors, can be shuttled into cells and from one cell to another (see Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206 for reviews). The first observation was made in 1988 by Frankel and Pabo. They showed that the transcriptional transactivator (Tat) protein of HIV-1 can enter cells and translocate to the nucleus. In 1991, Prochiantz's group reached the same conclusion using the Drosophila Antennapedia homeodomain and demonstrated that this domain is internalized by neurons. These studies led to the discovery in 1994 of the first protein transduction domain: penetratin, a 16-mer peptide derived from the third helix of the Antennapedia homeodomain. In 1997, the Lebleu group identified the minimal sequence of Tat required for cellular uptake, and the Dowdy group reported the first proof of concept for the application of PTDs in vivo with respect to the delivery of small peptides and large proteins (Gump JM, and Dowdy SF (2007) Trends Mol Med 13, 443-448.). Historically, the concept of cell-penetrating peptides (CPPs) was introduced in 1998 by Langel's group, and the first chimeric peptide carrier, transportan, was designed, which was derived from the N-terminal fragment of galanin, a neuropeptide linked to mastoparan, a wasp venom peptide. Transportan was originally reported for improving the delivery of PNA (peptide nucleic acid) both in cultured cells and in vivo (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton)).In 1997, a new strategy involving CPPs for the formation of stable but non-covalent complexes of cargos was proposed by the groups of Heitz and Divita (Morris et al. (1997) Nucleic Acids Res 25, 2730-2736). This strategy was initially based on a short peptide carrier (MPG) consisting of two domains: a hydrophilic (polar) domain and a hydrophobic (apolar) domain. The MPG was designed for the delivery of nucleic acids. Subsequently, for the non-covalent delivery of proteins and peptides, the first amphiphilic peptide Pep-1 was proposed (Morris et al. (2001) Nat Biotechnol 19, 1173-1176). Then, the groups of Wender and Futaki demonstrated that a polyarginine sequence (Arg8) was sufficient to drive small and large molecules into cells and in vivo (Nakase et al. (2004) Mol Ther 10, 1011-1022; Rothbard et al. (2004) J Am Chem Soc 126, 9506-9507). Since then, many CPPs derived from natural or non-natural sequences have been identified and the list continues to grow. Peptides have been derived from the VP22 protein of herpes simplex virus, from calcitonin, from antibacterial or toxin peptides, from proteins involved in the regulation of the cell cycle, as well as from polyproline-rich peptides (Heitz et al. (2009) Br J Pharmacol 157, 195-206). More recently, new non-covalent strategies based on second-generation amphiphilic CPPs have been described. These peptides, such as the CADY and VEPEP-family, can self-organize into a helical shape using hydrophilic and hydrophobic residues on different sides of the molecule.WO2014 / 053879 discloses the VEPEP-3 peptide, WO2014 / 053881 discloses the VEPEP-4 peptide, WO2014 / 053882 discloses the VEPEP-5 peptide, WO2012 / 137150 discloses the VEPEP-6 peptide, WO2014 / 053880 discloses the VEPEP-9 peptide, WO2016 / 102687 discloses the ADGN-100 peptide, US2010 / 0099626 discloses the CADY peptide, and U.S. Patent No. 7,514,530 discloses the MPG peptide; the disclosures of which are hereby incorporated by reference in their entireties.

[0116] The cell-permeable peptides within the cargo delivery complexes or nanoparticles of the present invention are capable of forming stable complexes and nanoparticles with various cargos. Any cell-permeable peptide of any cargo delivery complex or nanoparticle described herein may comprise, or consist of, any cell-permeable peptide sequence described in this section.

[0117] In some embodiments, the cargo delivery complexes or nanoparticles described herein include cell-penetrating peptides selected from the group consisting of CADY, PEP-1, PEP-2, MPG, VEPEP-3 peptide (used interchangeably herein with ADGN-103 peptide), VEPEP-4 peptide (used interchangeably herein with ADGN-104 peptide), VEPEP-5 peptide (used interchangeably herein with ADGN-105 peptide), VEPEP-6 peptide (used interchangeably herein with ADGN-106 peptide), VEPEP-9 peptide (used interchangeably herein with ADGN-109 peptide), and ADGN-100 peptide. In some embodiments, the cell-penetrating peptide is present within the cargo delivery complex. In some embodiments, the cell-penetrating peptide is present within a cargo delivery complex that is present within the core of the nanoparticle. In some embodiments, the cell-penetrating peptide is present within the core of the nanoparticle. In some embodiments, the cell-penetrating peptide is present within the core of the nanoparticle and is associated with a cargo molecule. In some embodiments, the cell-penetrating peptide is present within an intermediate layer of the nanoparticle. In some embodiments, the cell-penetrating peptide is present within a surface layer of the nanoparticle. In some embodiments, the cell-penetrating peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by a genetic method. VEPEP-3 peptide is disclosed in WO2014 / 053879, VEPEP-4 peptide is disclosed in WO2014 / 053881, VEPEP-5 peptide is disclosed in WO2014 / 053882, VEPEP-6 peptide is disclosed in WO2012 / 137150, VEPEP-9 peptide is disclosed in WO2014 / 053880, ADGN-100 peptide is disclosed in WO2016 / 102687, CADY peptide is disclosed in US2010 / 0099626, and MPG peptide is disclosed in U.S. Patent No. 7,514,530; the disclosures of which are hereby incorporated by reference in their entireties herein. VEPEP-3 peptide

[0118] In some embodiments, the cargo delivery complexes or nanoparticles described herein have the amino acid sequence X1X2X3X4X5X2X3X4X6X7X3X8X9X 10 X 11 X 12 X 13 (SEQ ID NO: 1) and comprise a VEPEP-3 cell-penetrating peptide, where X1 is beta-A or S, X2 is K, R or L (independently of one another), X3 is F or W (independently of one another), X4 is F, W or Y (independently of one another), X5 is E, R or S, X6 is R, T or S, X7 is E, R, or S, X8 is absent, F or W, X9 is P or R, X 10 is R or L, X 11 is K, W or R, X 12 is R or F, X 13 is R or K. In some embodiments, the VEPEP-3 peptide has the amino acid sequence X1X2WX4EX2WX4X6X7X3PRX 11 RX 13 (SEQ ID NO: 2) and comprise a VEPEP-3 cell-penetrating peptide, where X1 is beta-A or S, X2 is K, R or L, X3 is F or W, X4 is F, W or Y, X5 is E, R or S, X6 is R, T or S, X7 is E, R, or S, X8 is absent, F or W, X9 is P or R, X 10 is R or L, X 11 is K, W or R, X 12 is R or F, X 13is R or K. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1KWFERWFREWPRKRR (SEQ ID NO: 3), X1KWWERWWREWPRKRR (SEQ ID NO: 4), X1KWWERWWREWPRKRK (SEQ ID NO: 5), X1RWWEKWWTRWPRKRK (SEQ ID NO: 6), or X1RWYEKWYTEFPRRRR (SEQ ID NO: 7), where X1 is beta-A or S. In some embodiments, the VEPEP-3 peptide comprises any one of the amino acid sequences of SEQ ID NOs: 1-7, where the cell-penetrating peptide is modified by substitution of the 10th amino acid with a non-natural amino acid, addition of a non-natural amino acid between the 2nd and 3rd amino acids, and addition of a hydrocarbon linkage between two non-natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1KX 14 WWERWWRX 14 WPRKRK (SEQ ID NO: 8), where X1 is beta-A or S and X 14 is a non-natural amino acid and there is a hydrocarbon linkage between two non-natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1X2X3WX5X 10 X3WX6X7WX8X9X 10 …… WX 12 R (SEQ ID NO: 9), where X1 is beta-A or S, X2 is K, R or L, X3 is F or W, X5 is R or S, X6 is R or S, X7 is R or S, X8 is F or W, X9 is R or P, X 10 is L or R, X 12is R or F. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1RWWRLWWRSWFRLWRR (SEQ ID NO: 10), X1LWWRRWWSRWWPRWRR (SEQ ID NO: 11), X1LWWSRWWRSWFRLWFR (SEQ ID NO: 12), or X1KFWSRFWRSWFRLWRR (SEQ ID NO: 13), where X1 is beta-A or S. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1 and 9-13, where the cell-penetrating peptide is modified by substitution of non-natural amino acids at the 5th and 12th amino acids, and addition of a hydrocarbon linkage between two non-natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1RWWX 14 LWWRSWX 14 RLWRR (SEQ ID NO: 14), where X1 is beta-alanine or serine, X 14is a non-natural amino acid, and there is a hydrocarbon linkage between two non-natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence set forth in SEQ ID NO: 75 or SEQ ID NO: 76. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of SEQ ID NO: 115. In some embodiments, the VEPEP-3 peptide is present within a cargo delivery complex. In some embodiments, the VEPEP-3 peptide is present within a cargo delivery complex within the core of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present within the core of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present within the core of a nanoparticle and is associated with a cargo. In some embodiments, the VEPEP-3 peptide is present within an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present within a surface layer of a nanoparticle. In some embodiments, the VEPEP-3 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by a genetic method. VEPEP-6 peptide

[0119] In some embodiments, the cargo delivery complex or nanoparticle described herein comprises a VEPEP-6 cell-penetrating peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LX2RALWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 15), X1LX2LARWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 16), and X1LX2ARLWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 17), where X1 is beta-A or S, X2 is F or W, X3 is L, W, C, or I, X4 is S, A, N, or T, X5 is L or W, X6 is W or R, X7 is K or R, X8 is A or absent, and X9 is R or S. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence X1LX2RALWRLX3RX4LWRLX5X6X7X8 (SEQ ID NO: 18), where X1 is beta-A or S, X2 is F or W, X3 is L, W, C, or I, X4 is S, A, N, or T, X5 is L or W, X6 is W or R, X7 is K or R, and X8 is A or absent. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence X1LX2RALWRLX3RX4LWRLX5X6KX7 (SEQ ID NO: 19), where X1 is beta-A or S, X2 is F or W, X3 is L or W, X4 is S, A, or N, X5 is L or W, X6 is W or R, and X7 is A or absent.In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LFRALWRLLRX2LWRLLWX3 (SEQ ID NO: 20), X1LWRALWRLWRX2LWRLLWX3A (SEQ ID NO: 21), X1LWRALWRLX4RX2LWRLWRX3A (SEQ ID NO: 22), X1LWRALWRLWRX2LWRLWRX3A (SEQ ID NO: 23), X1LWRALWRLX5RALWRLLWX3A (SEQ ID NO: 24), and X1LWRALWRLX4RNLWRLLWX3A (SEQ ID NO: 25), where X1 is beta-A or S, X2 is S or T, X3 is K or R, X4 is L, C or I, and X5 is L or I. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of Ac-X1LFRALWRLLRSLWRLLWK-cysteamide (SEQ ID NO: 26), Ac-X1LWRALWRLWRSLWRLLWKA-cysteamide (SEQ ID NO: 27), Ac-X1LWRALWRLLRSLWRLWRKA-cysteamide (SEQ ID NO: 28), Ac-X1LWRALWRLWRSLWRLWRKA-cysteamide (SEQ ID NO: 29), Ac-X1LWRALWRLLRALWRLLWKA-cysteamide (SEQ ID NO: 30), and Ac-X1LWRALWRLLRNLWRLLWKA-cysteamide (SEQ ID NO: 31), where X1 is beta-A or S. In some embodiments, the VEPEP-6 peptide comprises any one of the amino acid sequences of SEQ ID NOs: 15 to 31 and further comprises a hydrocarbon linkage between two residues at positions 8 and 12. In some embodiments, the VEPEP-6 peptide is Ac-X1LFRALWR. S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 32), Ac-X1LFLARWR S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 33), Ac-X1LFRALWS S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 34), Ac-X1LFLARWS S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 35), Ac-X1LFRALWRLLRS SLWS S LLWK-Cysteamide (SEQ ID NO: 36), Ac-X1LFLARWRLLR S SLWS S LLWK-Cysteamide (SEQ ID NO: 37), Ac-X1LFRALWRLLS S SLWS S LLWK-Cysteamide (SEQ ID NO: 38), Ac-X1LFLARWRLLS S SLWS S LLWK-Cysteamide (SEQ ID NO: 39), and Ac-X1LFAR S LWRLLRS S An amino acid sequence selected from the group consisting of LWRLLWK-Cysteamide (SEQ ID NO: 40), wherein X1 is beta-A or S, and the residue followed by a trailing "S" is a residue linked by said hydrocarbon linkage. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of SEQ ID NO: 92 or 93. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-100, 105, 107-109, and 129-139. In some embodiments, the VEPEP-6 peptide is present within a cargo delivery complex. In some embodiments, the VEPEP-6 peptide is present within a cargo delivery complex within the core of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present within the core of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present within the core of a nanoparticle and is associated with a cargo. In some embodiments, the VEPEP-6 peptide is present within an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present within a surface layer of a nanoparticle. In some embodiments, the VEPEP-6 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by genetic methods. VEPEP-9 peptide

[0120] In some embodiments, the cargo delivery complex or nanoparticle described herein comprises a VEPEP-9 cell-penetrating peptide comprising the amino acid sequence X1X2X3WWX4X5WAX6X3X7X8X9X 10 X 11 X 12 WX 13 R (SEQ ID NO: 41), wherein X1 is beta-A or S, X2 is L or absent, X3 is R or absent, X4 is L, R or G, X5 is R, W or S, X6 is S, P or T, X7 is W or P, X8 is F, A or R, X9 is S, L, P or R, X 10 is R or S, X 11 is W or absent, X 12 is A, R or absent, X 13 is W or F, and when X3 is absent, X2, X 11 and X 12 are also absent. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence X1X2RWWLRWAX6RWX8X9X 10 WX 12 WX 13 R (SEQ ID NO: 42), wherein X1 is beta-A or S, X2 is L or absent, X6 is S or P, X8 is F or A, X9 is S, L or P, X 10 is R or S, X 12 is A or R, X 13is W or F. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of X1LRWWLRWASRWFSRWAWWR (SEQ ID NO: 43), X1LRWWLRWASRWASRWAWFR (SEQ ID NO: 44), X1RWWLRWASRWALSWRWWR (SEQ ID NO: 45), X1RWWLRWASRWFLSWRWWR (SEQ ID NO: 46), X1RWWLRWAPRWFPSWRWWR (SEQ ID NO: 47), and X1RWWLRWASRWAPSWRWWR (SEQ ID NO: 48), where X1 is beta-A or S. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of X1WWX4X5WAX6X7X8RX 10 WWR (SEQ ID NO: 49), where X1 is beta-A or S, X4 is R or G, X5 is W or S, X6 is S, T or P, X7 is W or P, X8 is A or R, X 10is S or R. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of X1WWRWWASWARSWWR (SEQ ID NO: 50), X1WWGSWATPRRRWWR (SEQ ID NO: 51), and X1WWRWWAPWARSWWR (SEQ ID NO: 52), where X1 is beta-A or S. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 116-120. In some embodiments, the VEPEP-9 peptide is present within a cargo delivery complex. In some embodiments, the VEPEP-9 peptide is present within a cargo delivery complex within the core of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present within the core of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present within the core of a nanoparticle and is associated with a cargo. In some embodiments, the VEPEP-9 peptide is present within an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present within a surface layer of a nanoparticle. In some embodiments, the VEPEP-9 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by genetic methods. ADGN-100 peptide

[0121] In some embodiments, the cargo delivery complex or nanoparticle described herein comprises an ADGN-100 cell-penetrating peptide comprising the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 53), where X1 is any amino acid or absent, and X2-8 are any amino acids. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 54), where X1 is βA, S, or absent, X2 is A or V, X3 is G or L, X4 is W or Y, X5 is V or S, X6 is R, V, or A, X7 is S or L, and X8 is W or Y. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence KWRSAGWRWRLWRVRSWSR (SEQ ID NO: 55), KWRSALYRWRLWRVRSWSR (SEQ ID NO: 56), KWRSALYRWRLWRSRSWSR (SEQ ID NO: 57), or KWRSALYRWRLWRSALYSR (SEQ ID NO: 58). In some embodiments, the ADGN-100 peptide comprises two residues separated by three or six residues linked by a hydrocarbon linkage. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence KWRS S AGWR S WRLWRVRSWSR (SEQ ID NO: 59), KWR S SAGWRWR S LWRVRSWSR (SEQ ID NO: 60), KWRSAGWR S WRLWRVR S SWSR (SEQ ID NO: 61), KWRS S ALYR S WRLWRSRSWSR (SEQ ID NO: 62), KWR S SALYRWR S LWRSRSWSR (SEQ ID NO: 63), KWRSALYR S WRLWRSR S SWSR (SEQ ID NO: 64), KWRSALYRWR S LWRS S RSWSR (SEQ ID NO: 65), KWRSALYRWRLWRS S RSWSS R (Array No. 66), KWR S SALYRWR S LWRSALYSR (Array No. 67), KWRS S ALYR S WRLWRSALYSR (Array No. 68), KWRSALYRWR S LWRS S ALYSR (Array No. 69), or KWRSALYRWRLWRS S ALYS S including R (Array No. 70), wherein the residues marked with the subscript "S" are linked by a hydrocarbon linkage. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence set forth in SEQ ID NO: 79 or 80. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-91, 101-104, 106, 110-112, and 121-128. In some embodiments, the ADGN-100 peptide is present within a cargo delivery complex. In some embodiments, the ADGN-100 peptide is present within a cargo delivery complex within the core of a nanoparticle. In some embodiments, the ADGN-100 peptide is present within the core of a nanoparticle. In some embodiments, the ADGN-100 peptide is present within the core of a nanoparticle and is associated with a cargo. In some embodiments, the ADGN-100 peptide is present within the intermediate layer of a nanoparticle. In some embodiments, the ADGN-100 peptide is present within the surface layer of a nanoparticle. In some embodiments, the ADGN-100 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by a genetic method. VEPEP-4 peptide

[0122] In some embodiments, the genome editing complex or nanoparticle described herein comprises a VEPEP-4 cell-penetrating peptide comprising the amino acid sequence XWXRLXXXXXX (SEQ ID NO: 140), where X at position 1 is beta-A or S; X at positions 3, 9, and 10 are, independently of one another, W or F; X at position 6 is R if X at position 8 is S, and X at position 6 is S if X at position 8 is R; X at position 7 is L or absent; X at position 11 is R or absent, and X at position 7 is L if X at position 11 is absent. In some embodiments, the VEPEP-4 peptide comprises any one of the amino acid sequences of SEQ ID NOs: 141-144. In some embodiments, the VEPEP-4 peptide is present within a cargo delivery complex. In some embodiments, the VEPEP-4 peptide is present within a cargo delivery complex within the core of the nanoparticle. In some embodiments, the VEPEP-4 peptide is present within the core of the nanoparticle. In some embodiments, the VEPEP-4 peptide is present within the core of the nanoparticle and is associated with the cargo. In some embodiments, the VEPEP-4 peptide is present within an intermediate layer of the nanoparticle. In some embodiments, the VEPEP-4 peptide is present within the surface layer of the nanoparticle. In some embodiments, the VEPEP-4 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by genetic methods. VEPEP-5 peptide

[0123] In some embodiments, the genome editing complex or nanoparticle described herein comprises a VEPEP-5 cell-penetrating peptide comprising the amino acid sequence RXWXRLWXRLR (SEQ ID NO: 145), wherein X at position 2 is R or S, and X at positions 4 and 8 are, independently of each other, W or F. In some embodiments, the VEPEP-5 peptide comprises any one of the amino acid sequences of SEQ ID NOs: 146-151. In some embodiments, the VEPEP-5 peptide is present within a cargo delivery complex. In some embodiments, the VEPEP-5 peptide is present within a cargo delivery complex within the core of the nanoparticle. In some embodiments, the VEPEP-5 peptide is present within the core of the nanoparticle. In some embodiments, the VEPEP-5 peptide is present within the core of the nanoparticle and is associated with a cargo. In some embodiments, the VEPEP-5 peptide is present within an intermediate layer of the nanoparticle. In some embodiments, the VEPEP-5 peptide is present within a surface layer of the nanoparticle. In some embodiments, the VEPEP-5 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by a genetic method.

[0124] In some embodiments, the CPPs described herein (e.g., PEP-1, PEP-2, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) further comprise one or more moieties linked to the N-terminus of the CPP. In some embodiments, the one or more moieties are covalently linked to the N-terminus of the CPP. In some embodiments, the one or more moieties are selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, cholesterol, polyethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody or an antibody fragment thereof, a peptide, a polysaccharide, and a targeting molecule. In some embodiments, the one or more moieties are an acetyl group and / or a stearyl group. In some embodiments, the CPP comprises an acetyl group and / or a stearyl group linked to its N-terminus. In some embodiments, the CPP comprises an acetyl group linked to its N-terminus. In some embodiments, the CPP comprises a stearyl group linked to its N-terminus. In some embodiments, the CPP comprises an acetyl group and / or a stearyl group covalently linked to its N-terminus. In some embodiments, the CPP comprises an acetyl group covalently linked to its N-terminus. In some embodiments, the CPP comprises a stearyl group covalently linked to its N-terminus.

[0125] In some embodiments, the CPPs described herein (e.g., PEP-1, PEP-2, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) further comprise one or more moieties linked to the C-terminus of the CPP. In some embodiments, the one or more moieties are covalently linked to the C-terminus of the CPP. In some embodiments, the one or more moieties are selected from the group consisting of a cysteamide group, cysteine, thiol, amide, nitrilotriacetic acid, carboxyl group, linear or branched C1-C6 alkyl group, primary or secondary amine, osidic derivative, lipid, phospholipid, fatty acid, cholesterol, poly-ethylene glycol, nuclear localization signal, nuclear export signal, antibody or antibody fragment thereof, peptide, polysaccharide, and targeting molecule. In some embodiments, the one or more moieties are a cysteamide group.In some embodiments, the CPP comprises a cysteamide group linked to its C-terminus. In some embodiments, the CPP comprises a cysteamide group covalently linked to its C-terminus.

[0126] In some embodiments, the CPPs described herein (e.g., PEP-1, PEP-2, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) are stapled. As used herein, "stapled" refers to a chemical linkage between two residues within a peptide. In some embodiments, the CPP is stapled and includes a chemical linkage between two amino acids of the peptide. In some embodiments, the two amino acids linked by the chemical linkage are separated by three or six amino acids. In some embodiments, the two amino acids linked by the chemical linkage are separated by three amino acids. In some embodiments, the two amino acids linked by the chemical linkage are separated by six amino acids. In some embodiments, each of the two amino acids linked by the chemical linkage is R or S. In some embodiments, each of the two amino acids linked by the chemical linkage is R. In some embodiments, each of the two amino acids linked by the chemical linkage is S. In some embodiments, one of the two amino acids linked by the chemical linkage is R and the other is S. In some embodiments, the chemical linkage is a hydrocarbon linkage.

[0127] In some embodiments, the CPP is an L-peptide comprising L-amino acids. In some embodiments, the CPP is a retro-inverso peptide (e.g., a peptide composed of D-amino acids in a reverse sequence, and when extended, is assumed to have a side-chain topology similar to its parent molecule, but with an inverted amide peptide bond).

[0128] In some embodiments, the CPPs described herein (e.g., PEP-1, PEP-2, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) further include one or more moieties. In some embodiments, the one or more moieties are conjugated to the N-terminus or C-terminus of the CPP. In some embodiments, a first moiety is conjugated to the N-terminus of the CPP and a second moiety is conjugated to the C-terminus of the CPP.

[0129] In some embodiments, the one or more moieties include targeting molecules. In some embodiments, the targeting molecule is conjugated to the N-terminus or C-terminus of the CPP. In some embodiments, a first targeting molecule is conjugated to the N-terminus of the CPP and a second targeting molecule is conjugated to the C-terminus of the CPP. In some embodiments, the targeting molecule includes at least about 3, 4, or 5 amino acids. In some embodiments, the targeting molecule includes about 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, or 4 or fewer amino acids. In some embodiments, the targeting molecule includes about 3, 4, or 5 amino acids. In some embodiments, the targeting molecule includes a sequence selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, IGSR. In some embodiments, the sequence (e.g., the targeting sequence) is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.

[0130] In some embodiments, the sequence (e.g., the targeting sequence) is selected from the group consisting of SEQ ID NOs: 152-162.

[0131] In some embodiments, the targeting molecule is conjugated via a CPP and a linker. In some embodiments, the linker comprises a polyglycine linker. In some embodiments, the linker comprises β-alanine. In some embodiments, the linker comprises at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises a GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-β-alanine motif.

[0132] In some embodiments, one or more moieties include a polymer (e.g., PEG, polylysine, PET). In some embodiments, the polymer is conjugated to the N-terminus or C-terminus of the CPP. In some embodiments, a first polymer is conjugated to the N-terminus of the CPP and a second polymer is conjugated to the C-terminus of the CPP. In some embodiments, the polymer is PEG. In some embodiments, the PEG is linear PEG. In some embodiments, the PEG is branched PEG. In some embodiments, the molecular weight of the PEG is about 5 kDa or less, 10 kDa or less, 15 kDa or less, 20 kDa or less, 30 kDa or less, or 40 kDa or less. In some embodiments, the molecular weight of the PEG is at least about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is from about 5 kDa to about 10 kDa, from about 10 kDa to about 15 kDa, from about 15 kDa to about 20 kDa, from about 20 kDa to about 30 kDa, or from about 30 kDa to about 40 kDa. In some embodiments, the molecular weight of the PEG is about 5 kDa, 10 kDa, 20 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is selected from the group consisting of 5 kDa, 10 kDa, 20 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is about 5 kDa. In some embodiments, the molecular weight of the PEG is about 10 kDa. In some embodiments, the PEG includes at least about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG includes about 3 or fewer, 2 or fewer, or 1 or fewer ethylene glycol units. In some embodiments, the PEG includes about 1, 2, or 3 ethylene glycol units. Targeting moiety

[0133] In some embodiments, the cell-penetrating peptide includes a targeting moiety. In some embodiments, the targeting moiety is conjugated to the N-terminus of the CPP. In some embodiments, the targeting moiety is conjugated to the C-terminus of the CPP. In some embodiments, a first targeting moiety is conjugated to the N-terminus of the CPP and a second targeting moiety is conjugated to the C-terminus of the CPP.

[0134] In some embodiments, the targeting moiety comprises a targeting peptide that targets one or more organs. In some embodiments, the one or more organs are selected from the group consisting of muscle, heart, brain, spleen, lymph node, liver, lung, and kidney. In some embodiments, the targeting peptide targets the brain. In some embodiments, the targeting peptide targets muscle. In some embodiments, the targeting peptide targets the heart.

[0135] In some embodiments, the targeting moiety comprises at least about 3, 4, or 5 amino acids. In some embodiments, the targeting moiety comprises about 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, or 4 or fewer amino acids. In some embodiments, the targeting moiety comprises about 3, 4, or 5 amino acids. In some embodiments, the targeting moiety comprises a sequence selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, IGSR. In some embodiments, the sequence (e.g., the targeting sequence) is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.

[0136] In some embodiments, the targeting moiety comprises a targeting sequence selected from the group consisting of SEQ ID NOs: 152-162. In some embodiments, the targeting moiety comprises the targeting sequence SYTSSTM (SEQ ID NO: 152). In some embodiments, the targeting moiety comprises the targeting sequence CKTRRVP (SEQ ID NO: 153). In some embodiments, the targeting moiety comprises the targeting sequence THRPPNWSPV (SEQ ID NO: 154). In some embodiments, the targeting moiety comprises the targeting sequence TGNYKALHPDHNG (SEQ ID NO: 155). In some embodiments, the targeting moiety comprises the targeting sequence CARPAR (SEQ ID NO: 156). In some embodiments, the targeting moiety comprises the targeting sequence ASSLNIA (SEQ ID NO: 159). In some embodiments, the targeting moiety comprises the targeting sequence LSSRLDA (SEQ ID NO: 160). In some embodiments, the targeting moiety comprises the targeting sequence KSYDTY (SEQ ID NO: 161). In some embodiments, the targeting moiety comprises the targeting sequence CKRAV (SEQ ID NO: 162).

[0137] In some embodiments, the targeting moiety is conjugated via a linker moiety such as any one of the linker moieties described herein to a CPP. Linker moiety

[0138] In some embodiments, the cell-penetrating peptide comprises a linker moiety.

[0139] In some embodiments, the linker moiety comprises a polyglycine linker. In some embodiments, the linker comprises β-alanine. In some embodiments, the linker comprises at least about 2, 3, or 4 glycines, optionally contiguous glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises the GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-β-alanine motif.

[0140] In some embodiments, one or more moieties comprise a polymer (e.g., PEG, polylysine, PET). In some embodiments, the polymer is conjugated to the N-terminus of the CPP. In some embodiments, the polymer is conjugated to the C-terminus of the CPP. In some embodiments, a first polymer is conjugated to the N-terminus of the CPP and a second polymer is conjugated to the C-terminus of the CPP. In some embodiments, the polymer is PEG. In some embodiments, the PEG is linear PEG. In some embodiments, the PEG is branched PEG. In some embodiments, the molecular weight of the PEG is about 5 kDa or less, 10 kDa or less, 15 kDa or less, 20 kDa or less, 30 kDa or less, or 40 kDa or less. In some embodiments, the molecular weight of the PEG is at least about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is from about 5 kDa to about 10 kDa, from about 10 kDa to about 15 kDa, from about 15 kDa to about 20 kDa, from about 20 kDa to about 30 kDa, or from about 30 kDa to about 40 kDa. In some embodiments, the molecular weight of the PEG is about 5 kDa, 10 kDa, 20 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is selected from the group consisting of 5 kDa, 10 kDa, 20 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is about 5 kDa. In some embodiments, the molecular weight of the PEG is about 10 kDa. In some embodiments, the PEG comprises at least about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG consists of about 10 or fewer, 9 or fewer, 8 or fewer, or 7 or fewer ethylene glycol units. In some embodiments, the PEG consists of about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG moiety consists of from about 1 to 8, or from about 2 to 7 ethylene glycol units.

[0141] In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4, etc.), PEG linker moiety, Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker moiety comprises Aun (11-amino-undecanoic acid). In some embodiments, the linker moiety comprises Ava (5-aminopentanoic acid). In some embodiments, the linker moiety comprises Ahx (aminocaproic acid). Carbohydrate moiety

[0142] In some embodiments, the cell-penetrating peptide further comprises a carbohydrate moiety. In some embodiments, the carbohydrate moiety is GalNAc. In some embodiments, the cell-penetrating peptide is the ADGN-106 peptide. In some embodiments, the cell-penetrating peptide is the ADGN-100 peptide. In some embodiments, the alanine in the cell-penetrating peptide is modified by the carbohydrate moiety. In some embodiments, the cell-penetrating peptide is set forth in SEQ ID NO: 124 or 129. The first cell-penetrating peptide and the second cell-penetrating peptide

[0143] In some embodiments, the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, and the first peptide has no PEG moiety. In some embodiments, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is about 50 to 1 (e.g., about 25 to about 2, about 20 to about 3, about 15 to about 4, about 12 to about 4, about 12 to about 5, or about 10 to about 5, etc.).

[0144] In some embodiments, the PEG moiety is linear PEG. In some embodiments, the PEG moiety is branched PEG. In some embodiments, the molecular weight of the PEG moiety is from about 5 kDa to about 10 kDa. In some embodiments, the PEG moiety consists of about 1 to 10 ethylene glycol units. In some embodiments, the PEG moiety is conjugated to the N-terminus of a second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to the C-terminus of a second cell-penetrating peptide. Cargo molecule

[0145] In some embodiments, the cargo molecule of the above complex or nanoparticle is selected from the group consisting of nucleic acids, viruses, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes.

[0146] In some embodiments, the cargo molecule of the above complex or nanoparticle is a nucleic acid. In some embodiments, the cargo molecule is selected from the group consisting of oligonucleotides, polynucleotides, single-stranded or double-stranded oligos and polynucleotides, antisense oligonucleotides, various forms of RNAi including, for example, siRNA, shRNA, etc., microRNA (miRNA), antagomir, ribozyme, aptamer, plasmid DNA, etc., and one or more suitable combinations thereof. In some embodiments, the nucleic acid is selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide and analogs thereof. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises RNAi. In some embodiments, the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, and the RNAi targets an RNA, the expression of which is associated with the disease or condition. In some embodiments, the molar ratio of the cell-penetrating peptide to the nucleic acid is between about 1:1 and about 100:1.

[0147] In some embodiments, the cargo molecule is a protein such as, for example, an enzyme or an antibody, or a small molecule. In some embodiments, the cargo molecule comprises a plurality of cargo molecules including a combination of a nucleic acid and a protein or a small molecule. In some embodiments, the combination comprises a nucleic acid and a protein or a small molecule attached to each other by a covalent bond. In some embodiments, the combination comprises a nucleic acid and a protein or a small molecule not attached to each other by a covalent bond.

[0148] "Polynucleotide" or "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides can include modified nucleotides such as methylated nucleotides and their analogs. The term "nucleic acid" as used herein refers to a polymer in either single-stranded or double-stranded form containing at least two deoxyribonucleotides or ribonucleotides, including DNA and RNA. DNA can be, for example, in the form of antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, RNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including, for example, locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), and zip nucleic acids (ZNAs), which can be synthetic nucleic acids, naturally occurring nucleic acids, and non-naturally occurring nucleic acids, and have binding properties similar to those of reference nucleic acids. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides having binding properties similar to those of reference nucleic acids. Unless otherwise specified, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the explicitly recited sequences.Specifically, degenerate codon substitution can be achieved by generating an array in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer e al., Nucleic Acid Res., 19: 5081 (1991); Ohtsuka et a., j. Biol. Chern., 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)). "Nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked through phosphate groups. "Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, for example, modifications in which new reactive groups such as amines, alcohols, thiols, carboxylases, and alkyl halides are placed. As used herein, "oligonucleotide" generally refers to a short, generally synthetic polynucleotide, typically less than about 200 nucleotides in length, although not necessarily. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides.

[0149] In some embodiments, the nucleic acid is a single-stranded oligonucleotide. In some embodiments, the nucleic acid is a double-stranded oligonucleotide. The nucleic acids described herein can be of any of a variety of lengths, up to 200 nucleotides in the case of antisense oligonucleotides, RNAi, siRNA, shRNA, iRNA, antagomirs, although not necessarily, or up to 1000 kilobases in the case of plasmid DNA.

[0150] In some embodiments, the nucleic acid is an interfering RNA such as siRNA or shRNA. The terms "interfering RNA" or "RNAi" or "interfering RNA sequence" refer to single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (i.e., double-stranded RNA such as siRNA, aiRNA, or pre-miRNA) that can reduce or inhibit the expression of a target gene or sequence when the interfering RNA is present in the same cell as the target gene or sequence (e.g., by mediating the degradation of mRNA complementary to the interfering RNA sequence or inhibiting the translation of mRNA complementary to the interfering RNA sequence), and thus, the interfering RNA refers to a single-stranded RNA complementary to the target mRNA sequence or to a double-stranded RNA formed by two complementary strands or a single self-complementary strand. The interfering RNA can have substantial or complete identity to the target gene or sequence, or can have regions of mismatch (i.e., mismatch motifs). The sequence of the interfering RNA can correspond to the full-length target gene or a subsequence thereof. The interfering RNA includes "small interfering RNA" or "siRNA", e.g., having a length of about 15-60, 15-50, or 5-40 (double-stranded) nucleotides, more generally about 15-30, 15-25, or 19-25 (double-stranded) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (double-stranded) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA has a length of 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA has a length of about 15-60, 15-50, 15-40, 5-30, 5-25, or 19-25 base pairs in length, preferably about 8-22, 9-20, or 19-21 base pairs in length). The siRNA duplex can include 3' overhangs of about 1-about 4 nucleotides or about 2-about 3 nucleotides and 5' phosphate termini.Examples of siRNAs include, without limitation, double-stranded polynucleotide molecules assembled from two separate strand molecules, one strand being the sense strand and the other strand being the complementary antisense strand; double-stranded polynucleotide molecules assembled from single-stranded molecules in which the sense region and the antisense region are linked by a nucleic acid-based linker or a linker that is not nucleic acid-based; double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions; and circular single-stranded polynucleotide molecules having two or more loop structures and a stem with self-complementary sense and antisense regions, which can be processed in vivo or in vitro to generate active double-stranded siRNA molecules, including circular polynucleotides. The siRNA is preferably chemically synthesized. The siRNA can also be generated by cleaving long dsRNA (e.g., dsRNA longer than about 25 nucleotides in length) with E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, for example, Yang et al., Proc Natl. Acad. Sci. USA, 99: 9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99: 14236 (2002); Byrom et al., Ambion TeehNotes, 10 (1):4-6(2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science, 293: 2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243: 82 (1968)). The dsRNA is preferably at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. The dsRNA can be as long as or longer than 1000, 1500, 2000, 5000 nucleotides in length.dsRNA can encode the entire gene transcript or a partial gene transcript. In certain cases, siRNA can be encoded in a plasmid (e.g., transcribed as a sequence that automatically folds into a duplex with a hairpin loop). Small hairpin RNA (shRNA) is a sequence of RNA that forms a tight hairpin turn and can be used to silence gene expression by RNA interference. The shRNA hairpin structure is cleaved by cellular machinery into siRNA, which then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves the mRNA that matches the siRNA to which it is bound. The appropriate length of the interfering RNA is about 5 to about 200 nucleotides, or 10 to 50 nucleotides or base pairs or 15 to 30 nucleotides or base pairs. In some embodiments, the interfering RNA is substantially complementary to the corresponding target gene (e.g., at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more identical). In some embodiments, the interfering RNA is modified, for example, by incorporating non-naturally occurring nucleotides.

[0151] In some embodiments, the nucleic acid is double-stranded antisense RNA. The appropriate length of the interfering RNA is about 5 to about 200 nucleotides, or 10 to 50 nucleotides or base pairs or 15 to 30 nucleotides or base pairs. In some embodiments, the interfering RNA is substantially complementary to the corresponding target gene (e.g., at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more identical). In some embodiments, the antisense RNA is modified, for example, by incorporating non-naturally occurring nucleotides.

[0152] In some embodiments, the nucleic acid is an interfering RNA, such as siRNA that specifically targets an RNA molecule, such as mRNA encoding a protein involved in a disease such as cancer. In some embodiments, the disease is cancer such as a solid tumor or hematological malignancy, and the interfering RNA targets an mRNA encoding a protein involved in cancer, such as a protein involved in the regulation of cancer progression.

[0153] In some embodiments, the nucleic acid is an interfering RNA, such as siRNA that specifically targets an RNA molecule, such as mRNA encoding a protein involved in the negative regulation of the immune response. In some embodiments, the interfering RNA targets an mRNA encoding a negative co-stimulatory molecule. In some embodiments, examples of negative co-stimulatory molecules include, for example, PD-1, PD-L1, PD-L2, TIM-3, BTLA, VISTA, LAG-3, and CTLA-4.

[0154] In some embodiments, the nucleic acid is miRNA. MicroRNA (abbreviated miRNA) is a short ribonucleic acid (RNA) molecule found in eukaryotic cells. MicroRNA molecules have very few nucleotides (an average of 22) compared to other RNAs. miRNA is a post-transcriptional regulator that binds to complementary sequences on target messenger RNA transcripts (mRNA) and usually results in translational repression or target degradation and gene silencing. The human genome can encode more than 1000 miRNAs, which can target approximately 60% of mammalian genes and are abundant in many human cell types. The appropriate length of miRNA is about 5 to about 200 nucleotides, or 0 to 50 nucleotides or base pairs or 15 to 30 nucleotides or base pairs. In some embodiments, the miRNA is substantially complementary to the corresponding target gene (e.g., at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more identical). In some embodiments, the antisense RNA is modified, for example, by incorporating non-naturally occurring nucleotides.

[0155] In some embodiments, the nucleic acid is plasmid DNA or DNA (e.g., a DNA fragment, e.g., a DNA fragment up to about 1000 bp in length). Further, the plasmid DNA or DNA may be highly methylated or lowly methylated. In some embodiments, the plasmid DNA or DNA may encode one or more genes and contain regulatory elements necessary for the expression of the one or more genes. In some embodiments, the plasmid DNA or DNA may include one or more genes encoding a selectable marker, thereby enabling the plasmid DNA or DNA fragment to be maintained within a suitable host cell.

[0156] In some embodiments, the plasmid DNA includes a DNA sequence encoding a chimeric antigen receptor (CAR) that includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that specifically binds to a target antigen. CARs are described, for example, in U.S. Patent No. 8,822,647, U.S. Patent Application Publication No. 2015 / 0051266, WO2014 / 127261, and WO2014099671, the entire disclosures of which are specifically incorporated herein by reference. In some embodiments, the target antigen is specifically associated with cancer cells (e.g., such as expressed by cancer cells). For example, in some embodiments, the plasmid DNA includes a DNA sequence encoding a CAR that includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that specifically binds to an antigen associated with cancer. In some embodiments, the antigen associated with cancer is associated with solid tumors. In some embodiments, the antigen associated with cancer is associated with hematological malignancies such as B cell malignancies or leukemias.

[0157] In some embodiments, the cargo molecule includes a virus. In some embodiments, the virus is a recombinant virus including recombinant adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus (HSV), poxvirus, Epstein-Barr virus (EBV), vaccinia virus, and human cytomegalovirus (hCMV). In some embodiments, the recombinant virus includes a transgene for insertion into the cellular genome. In some embodiments, the transgene is a therapeutic transgene. In some embodiments, the transgene encodes a protein such as a therapeutic protein. In some embodiments, the transgene encodes an inhibitory RNA (RNAi) such as RNAi targeting an endogenous gene, for example, an endogenous gene associated with a disease. In some embodiments, the transgene encodes a CAR. In some embodiments, the virus includes a first transgene encoding RNAi. In some embodiments, the RNAi is a therapeutic RNAi targeting an endogenous gene involved in a disease or condition. In some embodiments, the therapeutic RNAi targets an endogenous gene in a disease-associated form (e.g., a gene encoding a mutant protein or a gene that results in abnormal expression of a protein). In some embodiments, the virus includes a second transgene encoding a protein. In some embodiments, the protein is a therapeutic protein useful for treating a disease or condition. In some embodiments, the second transgene is an endogenous gene in a therapeutic form (e.g., the second transgene encodes a wild-type or functional form of a mutant protein encoded by an endogenous gene, or the second transgene results in normal expression of a protein encoded by an endogenous gene). In some embodiments, a virus including the first transgene and the second transgene is provided.

[0158] In some embodiments, the cargo molecule includes both mRNA (e.g., PTEN) and siRNA (e.g., siRNA targeting an oncogene such as KRAS).

[0159] In some embodiments, the cargo molecule includes both mRNA (e.g., mRNA encoding a DNA nuclease such as Cas9) and a guide RNA (e.g., a guide RNA targeting a mutated cancer gene such as KRAS). Genome editing system

[0160] In some embodiments, the cargo includes a genome editing system molecule.

[0161] In some embodiments, the genome editing system molecule (e.g., RGEN) of the genome editing complex or nanoparticle described herein is a protein or polypeptide. For example, in some embodiments, the genome editing complex or nanoparticle described herein includes RGEN (e.g., Cas9). In some embodiments, the protein or polypeptide is between about 10 kDa and about 200 kDa (e.g., approximately, any of 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, and 200 kDa, and also includes any range between these values). In some embodiments, the genome editing complex or nanoparticle includes multiple proteins or polypeptides, and each of the multiple proteins or polypeptides is between about 10 kDa and about 200 kDa (e.g., approximately, any of 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, and 200 kDa, and also includes any range between these values).

[0162] In some embodiments, the genome editing complex or the genome editing system molecule (e.g., gRNA) of the nanoparticle described herein is a nucleic acid. In some embodiments, the nucleic acid is between about 20 nt and about 20 kb (e.g., approximately, 0.02 kb, 0.03 kb, 0.04 kb, 0.05 kb, 0.06 kb, 0.07 kb, 0.08 kb, 0.09 kb, 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, and any of 20 kb, and also includes any range between these values). For example, in some embodiments, the genome editing complex or the nanoparticle described herein includes a gRNA (e.g., Cas9 gRNA). In some embodiments, the gRNA is between about 20 nt and about 200 nt (e.g., approximately, 20 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, 110 nt, 120 nt, 130 nt, 140 nt, 150 nt, 160 nt, 170 nt, 180 nt, 190 nt, and any of 200 nt, and also includes any range between these values). In some embodiments, the nucleic acid is DNA such as a DNA plasmid encoding a genome editing system molecule. In some embodiments, the DNA plasmid includes an expression cassette for expressing the genome editing system molecule. In some embodiments, the DNA plasmid is between about 1 kb and about 20 kb (e.g., approximately, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, and any of 20 kb, and also includes any range between these values). In some embodiments, the nucleic acid is RNA such as mRNA encoding a genome editing system molecule.In some embodiments, the mRNA is between about 100 nt and about 10 kb (e.g., approximately, 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, and 10 kb, and also includes any range between these values). In some embodiments, the genome editing complex or nanoparticle includes a plurality of nucleic acids such as any of the nucleic acids described herein. For example, in some embodiments, the genome editing complex or nanoparticle includes a gRNA and a nucleic acid encoding a genome editing system molecule (e.g., a DNA plasmid or mRNA encoding a genome editing system molecule). In some embodiments, the genome editing complex or nanoparticle includes a nucleic acid encoding a plurality of genome editing system molecules (e.g., one or more DNA plasmids encoding a plurality of genome editing system molecules, or a plurality of mRNAs encoding a plurality of genome editing system molecules).

[0163] In some embodiments, the genome editing complex or nanoparticle genome editing system molecule (e.g., RGEN or gRNA) described herein is replaced with a nucleic acid encoding the genome editing system molecule. For example, in some embodiments, the genome editing complex or nanoparticle described herein comprises a nucleic acid encoding an RGEN and / or a nucleic acid encoding a gRNA. In some embodiments, the nucleic acid is DNA, such as a DNA plasmid encoding a genome editing system molecule. In some embodiments, the DNA plasmid comprises an expression cassette for expressing the genome editing system molecule. In some embodiments, the DNA plasmid is between about 1 kb and about 20 kb (e.g., approximately, any one of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, and 20 kb, and also includes any range between these values). In some embodiments, the nucleic acid is RNA, such as mRNA encoding a genome editing system molecule. In some embodiments, the mRNA is between about 100 nt and about 10 kb (e.g., approximately, any one of 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, and 10 kb, and also includes any range between these values).

[0164] In some embodiments, the cargo comprises a CRISPR-associated nuclease. In some embodiments, the CRISPR-associated nuclease is a Cas nuclease. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, such as inducible, inactivated, or split Cas proteins (see, e.g., Dominguez et al. (2015). Nature Reviews Molecular Cell Biology; Polstein, L. R., & Gersbach, C. A. (2015). Nature chemical biology, 11(3) : 198-200; Dow et al. (2015). Nature biotechnology, 33(4) : 390-394; Zetsche et al. (2015). Nature biotechnology, 33(2) : 139-142; Kleinstiver et al. (2015). Nature. 523:481-485; Bikard et al. (2013). Nucleic acids research, 41(15) : 7429-7437; Qi et al. (2013). Cell, 152(5) : 1173-1183).These enzymes are known to those skilled in the art; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found under accession number Q99ZW2 in the SwissProt database, and the amino acid sequence of the Acidaminococcus sp. Cpf1 protein can be found under accession number U2UMQ6 in the SwissProt database. In some embodiments, unmodified CRISPR enzymes, such as Cas9 for example, have DNA cleavage activity. In some embodiments, the CRISPR enzyme is Cas9 and can be Cas9 derived from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme is Cpf1 and can be Cpf1 derived from Acidaminococcus or Lachnospiraceae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the CRISPR enzyme is mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, substitution of aspartic acid to alanine (D10A) within the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves one strand). Other examples of mutations that convert Cas9 to a nickase include, without limitation, H840A, N854A, and N863A. In some embodiments, Cas9 nickase can be used in combination with guide sequences, for example, two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination allows nicks to be introduced in both strands and can be used to induce NHEJ.

[0165] As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be mutated to create a mutant Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to create a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, less than 10%, less than 5%, less than 1%, less than 0.1%, less than 0.01% of its non-mutated form, or lower. Other mutations may also be useful; when Cas9 or other CRISPR enzymes are derived from species other than S. pyogenes, mutations of the corresponding amino acids can be made to achieve similar effects.

[0166] In some embodiments, a Cas protein (e.g., Cas9, etc.) is a split Cas protein comprising an N-terminal Cas protein fragment, Cas(N), and a C-terminal Cas protein fragment, Cas(C), wherein Cas(N) is fused to a first dimerization domain and Cas(C) is fused to a second dimerization domain, and the first dimerization domain and the second dimerization domain facilitate the dimerization of Cas(N) and Cas(C) to form a complex having functional Cas nuclease activity. In some embodiments, the dimerization of the first dimerization domain and the second dimerization domain is sensitive to a dimerizing agent. For example, in some embodiments, the first dimerization domain and the second dimerization domain comprise FK506-binding protein 12 (FKBP) and the FKBP-rapamycin-binding (FRB) domain of mammalian target of rapamycin (mTOR), and the dimerizing agent is rapamycin.

[0167] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell can be from a particular organism or derived from a particular organism, including but not limited to mammals such as humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence (e.g., about 1 or more than about 1, about 2 or more than about 2, about 3 or more than about 3, about 4 or more than about 4, about 5 or more than about 5, about 10 or more than about 10, about 15 or more than about 15, about 20 or more than about 20, about 25 or more than about 25, about 50 or more than about 50, or more codons) with codons that are more frequently or most frequently used in the genes of that host cell, while maintaining the native amino acid sequence. Different species exhibit a particular bias towards certain codons for a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The prevalence of tRNAs selected in a cell generally reflects the codons that are most frequently used in peptide synthesis. Thus, genes can be adjusted based on codon optimization for optimal gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database", and these tables can be adapted in several ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).Computer algorithms are also available for codon-optimizing specific sequences for expression in specific host cells, for example, Gene Forge (Aptagen; Jacobus, Pa.) is also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in the sequence encoding the CRISPR enzyme correspond to the codons most frequently used for a particular amino acid.

[0168] In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences (NLSs), e.g., about 1 or more than about 1, about 2 or more than about 2, about 3 or more than about 3, about 4 or more than about 4, about 5 or more than about 5, about 6 or more than about 6, about 7 or more than about 7, about 8 or more than about 8, about 9 or more than about 9, about 10 or more than about 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about 1 or more than about 1, about 2 or more than about 2, about 3 or more than about 3, about 4 or more than about 4, about 5 or more than about 5, about 6 or more than about 6, about 7 or more than about 7, about 8 or more than about 8, about 9 or more than about 9, about 10 or more than about 10, or more NLSs near or at the amino terminus, or about 1 or more than about 1, about 2 or more than about 2, about 3 or more than about 3, about 4 or more than about 4, about 5 or more than about 5, about 6 or more than about 6, about 7 or more than about 7, about 8 or more than about 8, about 9 or more than about 9, about 10 or more than about 10, or more NLSs near or at the carboxy terminus, or combinations thereof (e.g., comprising one or more NLSs at the amino terminus and one or more NLSs at the carboxy terminus). If more than one NLS is present, each can be selected independently of the other NLSs, such that a single NLS may be present in more than one copy and / or may be present in combination with one or more other NLSs in one or more copies. In some embodiments, the CRISPR enzyme comprises up to 6 NLSs.In some embodiments, an NLS is considered to be near the N-terminus or C-terminus if the nearest amino acid of the NLS is within about 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, or more amino acids from the N-terminus or C-terminus along the polypeptide chain. Generally, an NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the protein surface, although other types of NLSs are also known. Non-limiting examples of NLSs include the NLS of SV40 virus large T-antigen having the amino acid sequence PKKKRKV; the NLS derived from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence of the IBB domain from importin-alpha, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; the sequences VSRKRPRP and PPKKARED of the myogenic T protein; the sequence PQPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-ab1 IV; the sequences DRLRR and PKQKKRK of influenza virus NS1; the sequence RKLKKKIKKL of hepatitis delta antigen; the sequence REKKKFLKRR of mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase; and NLS sequences derived from the sequence RKCLQAGMNLEARKTKK of the steroid hormone receptor (human) glucocorticoid.

[0169] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity to hybridize to a target polynucleotide sequence and direct the sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50% or greater than about 50%, about 60% or greater than about 60%, about 75% or greater than about 75%, about 80% or greater than about 80%, about 85% or greater than about 85%, about 90% or greater than about 90%, about 95% or greater than about 95%, about 97.5% or greater than about 97.5%, about 99% or greater than about 99%, or greater than that, when optimally aligned using an appropriate alignment algorithm. Optimal alignment can be determined using any appropriate algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).In some embodiments, the guide sequence is about 5 or more, about 10 or more, about 11 or more, about 12 or more, about 13 or more, about 14 or more, about 15 or more, about 16 or more, about 17 or more, about 18 or more, about 19 or more, about 20 or more, about 21 or more, about 22 or more, about 23 or more, about 24 or more, about 25 or more, about 26 or more, about 27 or more, about 28 or more, about 29 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 75 or more, or more nucleotides in length. In some embodiments, the guide sequence is less than about 75, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 12, or less nucleotides in length. The ability of the guide sequence to direct sequence-specific binding of the CRISPR complex to the target sequence can be evaluated by any suitable assay. For example, sufficient components of the CRISPR system to form a CRISPR complex including the guide sequence being tested are supplied to a host cell having the corresponding target sequence, and then preferential cleavage within the target sequence is evaluated, for example, by the assays or investigations described herein. Similarly, cleavage of the target polynucleotide sequence can be evaluated in vitro by providing to the target sequence the components of the CRISPR complex including the guide sequence being tested and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test guide sequence reaction and the control guide sequence reaction. Other assays are possible and will be apparent to those of skill in the art.

[0170] The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell. Exemplary target sequences include sequences unique to the target genome. In some embodiments, the guide sequence is selected such that the degree of secondary structure within the guide sequence is reduced. The secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of one such algorithm is mFold, as described in Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry of the University of Vienna using the centroid structure prediction algorithm (see, e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and P A Carr and G M Church, 2009, Nature Biotechnology 27(12): 1151-62). Yet another algorithm can be found in U.S. Patent Application No. 61 / 836,080, which is incorporated herein by reference.

[0171] Generally, to promote one or more of the following, the tracr mate array comprises any array having sufficient complementarity to the tracr array: (1) deletion of the guide array flanked by the tracr mate array in a cell containing the corresponding tracr array; and (2) formation of a CRISPR complex at the target array, where the CRISPR complex comprises a tracr mate array hybridized to the tracr array. Generally, the degree of complementarity is with reference to the optimal alignment of the tracr mate array and the tracr array and is along the length of the shorter of the two arrays. The optimal alignment can be determined by any suitable alignment algorithm, and the optimal alignment can further account for secondary structures such as self-complementarity within either the tracr array or the tracr mate array. In some embodiments, the degree of complementarity of the tracr array and the tracr mate array along the length of the shorter of the two when optimally aligned is greater than or equal to about 25%, greater than or equal to about 30%, greater than or equal to about 40%, greater than or equal to about 50%, greater than or equal to about 60%, greater than or equal to about 70%, greater than or equal to about 80%, greater than or equal to about 90%, greater than or equal to about 95%, greater than or equal to about 97.5%, greater than or equal to about 99%, or greater.In some embodiments, the tracr array is about 5 or more, about 6 or more, about 7 or more, about 8 or more, about 9 or more, about 10 or more, about 11 or more, about 12 or more, about 13 or more, about 14 or more, about 15 or more, about 16 or more, about 17 or more, about 18 or more, about 19 or more, about 20 or more, about 25 or more, about 30 or more, about 40 or more, about 50 or more, or more nucleotides in length. In some embodiments, the guide array, tracr array, and tracr mate array are contained within a single RNA (referred to herein as a "single guide RNA" or "sgRNA"), and thus hybridization of the tracr array and the tracr mate array results in a secondary structure such as a hairpin. A loop-forming sequence preferred for use in the hairpin structure is 4 nucleotides in length and most preferably has the sequence GAAA. However, longer or shorter loop sequences can also be used as alternative sequences. The sequence preferably includes a nucleotide triplet (e.g., AAA) and additional nucleotides (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In certain embodiments of the invention, the sgRNA has at least two or more hairpins. In preferred embodiments, the sgRNA has 2, 3, 4, or 5 hairpins. In further embodiments of the invention, the sgRNA has up to 5 hairpins. In some embodiments, the sgRNA further includes a transcription termination sequence, which is preferably a polyT sequence, e.g., a 6T nucleotide.

[0172] In some embodiments, a donor nucleic acid is also provided. In some embodiments, the donor nucleic acid is designed to function as a template for homologous recombination within or near a target sequence that is nicked or cleaved by a CRISPR enzyme, such as a CRISPR enzyme that is part of a CRISPR complex. The donor nucleic acid can be of any suitable length, such as a length of about 10 or more nucleotides, about 15 or more nucleotides, about 20 or more nucleotides, about 25 or more nucleotides, about 50 or more nucleotides, about 75 or more nucleotides, about 100 or more nucleotides, about 150 or more nucleotides, about 200 or more nucleotides, about 500 or more nucleotides, about 1000 or more nucleotides, or more. In some embodiments, the donor nucleic acid comprises a sequence complementary to a portion of the polynucleotide comprising the target sequence. In some embodiments, when the donor nucleic acid and the polynucleotide comprising the target sequence are optimally aligned, the donor nucleic acid overlaps with one or more nucleotides of the target sequence (e.g., about 1 or more nucleotides, about 5 or more nucleotides, about 10 or more nucleotides, about 15 or more nucleotides, about 20 or more nucleotides, about 25 or more nucleotides, about 30 or more nucleotides, about 35 or more nucleotides, about 40 or more nucleotides, about 45 or more nucleotides, about 50 or more nucleotides, about 60 or more nucleotides, about 70 or more nucleotides, about 80 or more nucleotides, about 90 or more nucleotides, about 100 or more nucleotides, or more).In some embodiments, when the polynucleotide comprising the donor nucleic acid and the target sequence is optimally aligned, the closest nucleotide of the donor nucleic acid within the region of complementarity is within about 1 nucleotide, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, 5000 nucleotides, 10000 nucleotides, or more nucleotides of the target sequence.

[0173] In some embodiments, the CRISPR enzyme is part of a fusion protein that includes one or more heterologous protein domains (e.g., in addition to the CRISPR enzyme, about 1 or more than about 1, about 2 or more than about 2, about 3 or more than about 3, about 4 or more than about 4, about 5 or more than about 5, about 6 or more than about 6, about 7 or more than about 7, about 8 or more than about 8, about 9 or more than about 9, about 10 or more than about 10, or more domains). The CRISPR enzyme fusion protein can include any additional protein sequences and, optionally, linker sequences between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and proteins having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). The CRISPR enzyme can be fused to a gene sequence encoding a protein or fragment of a protein that binds to a DNA molecule or to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.Additional domains that can form part of a fusion protein containing a CRISPR enzyme are described in US20110059502, which is incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.

[0174] In some embodiments, the cargo comprises a fusion protein comprising a nuclease that does not function as a catalyst (e.g., a Cas9 endonuclease that does not function as a catalyst, etc.) and a reverse transcriptase (e.g., a pentamutant of M-MLV reverse transcriptase, etc.). See, for example, Anzalone & Liu et al., Nature. 2019 Dec; 576 (7785): 149-157. In some embodiments, the cargo comprises a polynucleotide encoding the fusion protein.

[0175] In some embodiments, the cargo comprises a fusion protein comprising a nuclease that does not function as a catalyst (e.g., a Cas9 endonuclease that does not function as a catalyst, etc.) and a nucleobase deaminase enzyme. In some embodiments, the nucleobase deaminase enzyme is APOBEC1 cytidine deaminase. In some embodiments, the nucleobase deaminase enzyme is cytidine deaminase CDA1. In some embodiments, the fusion protein further comprises a DNA glycosylase inhibitor. In some embodiments, the DNA glycosylase inhibitor is uracil DNA glycosylase inhibitor (UGI). In some embodiments, the cargo comprises a polynucleotide encoding the fusion protein. mRNA

[0176] In some embodiments, the cargo comprises mRNA. Exemplary mRNAs encode polypeptides of interest selected from any of several target categories including, but not limited to, biological agents, antibodies, vaccines, therapeutic proteins or peptides, cell-penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, proteins bound to intracellular membranes, nuclear proteins, proteins associated with human disease, and proteins encoded by the human genome that are useful in the fields of research and discovery even though the therapeutic indication is not identified.

[0177] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein comprises a region encoding a polypeptide of interest and a region of linked nucleosides according to any of the mRNAs described in U.S. Patent Nos. 9,061,059 and 9,221,891, the entirety of each of which is incorporated herein.

[0178] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes a polypeptide variant of a reference polypeptide. In some embodiments, the polypeptide variant may have the same or similar activity as the reference polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) compared to the reference polypeptide. Generally, a variant of a particular polynucleotide or polypeptide of the invention has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide, as determined by the sequence alignment programs and parameters described herein and known to those of skill in the art.

[0179] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes a biological agent. As used herein, a "biological agent" is a polypeptide-based molecule that is made by the methods presented herein and can be used to treat, cure, alleviate, prevent, or diagnose a severe or life-threatening disease or medical condition. Biological agents according to the present invention include, but are not limited to, among others, allergen extracts (e.g., for allergy injections and tests), blood components, gene therapy products, human tissue or cell products used in transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytics, and immunomodulatory substances. In some embodiments, the biological agent is one that is currently on the market or under development.

[0180] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes an antibody or a fragment thereof (e.g., an antigen-binding fragment, etc.). In some embodiments, the antibody or fragment thereof is one that is currently on the market or under development.

[0181] The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of antibodies, i.e., a population in which the individual antibodies that make up the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in trace amounts. Monoclonal antibodies are highly specific and target a single antigenic site.

[0182] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes a vaccine. As used herein, "vaccine" is a preparation of a biological agent that improves immunity against a particular disease or infectious agent. In some embodiments, the vaccine is one that is currently on the market or in development.

[0183] In some embodiments, the vaccine encoded by the mRNA is used to treat conditions or diseases in a number of therapeutic areas including, but not limited to, cardiovascular, CNS, skin, endocrine, oncology, immune, respiratory, and infection prevention.

[0184] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes a therapeutic protein. In some embodiments, the therapeutic protein is one that is currently on the market or in development. In some embodiments, the therapeutic protein is useful for (a) replacing a deficient or abnormal protein; (b) enhancing an existing pathway; (c) providing a new function or activity; or (d) interfering with a molecule or organism. In some embodiments, therapeutic proteins include, but are not limited to, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. In some embodiments, the therapeutic protein acts by (a) binding to a target non-covalently, e.g., an mAb; (b) affecting a covalent bond, e.g., an enzyme; or (c) acting by exerting activity without specific interaction, e.g., serum albumin. In some embodiments, the therapeutic protein is a recombinant protein.

[0185] In some embodiments, therapeutic proteins encoded by mRNA are utilized to treat conditions or diseases in a number of therapeutic areas including, but not limited to, blood, cardiovascular, CNS, toxicology (including antitoxins), skin, endocrine, gene, urogenital, gastrointestinal, musculoskeletal, oncology, and immune, respiratory, sensory, and infection prevention. In some embodiments, therapeutic proteins include, but are not limited to, vascular endothelial growth factors (VEGF-A, VEGF-B, VEGF-C, VEGF-D), placental growth factor (PGF), OX40 ligand (OX40L; CD134L), interleukin 12 (IL12), interleukin 23 (IL23), interleukin 36γ (IL36γ), and CoA mutase.

[0186] In some embodiments, the therapeutic protein replaces a protein that is deficient or abnormal. In some embodiments, therapeutic proteins include, but are not limited to, alpha1-antitrypsin, frataxin, insulin, growth hormone (somatotropin), growth factors, hormones, dystrophin, insulin-like growth factor 1 (IGF1), factor VIII, factor IX, antithrombin III, protein C, β-glucocerebrosidase, alglucosidase-α, α-1-iduronidase, iduronate-2-sulfatase, galsulfase, human alpha-galactosidase A, alpha-1-proteinase inhibitor, lactase, pancreatic enzymes (including lipase, amylase, and protease), adenosine deaminase, and albumin, including recombinant forms thereof.

[0187] In some embodiments, the therapeutic protein provides a novel function or activity. In some embodiments, therapeutic proteins include, but are not limited to, botulinum toxin type A, botulinum toxin type B, collagenase, human deoxy-ribonuclease I, dornase-α, hyaluronidase, papain, L-asparaginase, rasburicase, repirudin, bivalirudin, streptokinase, and anisoylated plasminogen streptokinase activator complex (APSAC).

[0188] In some embodiments, the therapeutic protein interferes with molecules or organisms. In some embodiments, examples of therapeutic proteins include, but are not limited to, anti-VEGFA antibody, anti-EGFR antibody, anti-CD52 antibody, anti-CD20 antibody, anti-HER2 / Neu antibody, a fusion protein of the extracellular domain of human CTLA4 and a modified Fc portion of human immunoglobulin G1, interleukin 1 (IL1) receptor antagonist, anti-TNFα antibody, CD2-binding protein, anti-CD11a antibody, an antibody against the α4-subunit of α4β1 and α4β7 integrins, anti-complement protein C5 antibody, anti-thymocyte globulin, chimeric (human / mouse) IgG1, a humanized IgG1 mAb that binds to the alpha chain of CD25, anti-CD3 antibody, anti-IgE antibody, a humanized IgG1 mAb that binds to the A antigen site of the F protein of respiratory syncytial virus, an HIV envelope protein gp120 / gp41-binding peptide, a Fab fragment of chimeric (human / mouse) mAb 7E3 that binds to the glycoprotein IIb / IIIa integrin receptor, and a Fab fragment of IgG that binds to and neutralizes venom toxin.

[0189] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes a tumor suppressor protein, and the protein corresponds to a tumor suppressor gene. In some embodiments, the tumor suppressor protein is retinoblastoma protein (pRb). In some embodiments, the tumor suppressor protein is the p53 tumor suppressor protein. In some embodiments, the corresponding tumor suppressor gene is phosphatase and tensin homolog (PTEN). In some embodiments, the corresponding tumor suppressor gene is BRCA1. In some embodiments, the corresponding tumor suppressor gene is BRCA2. In some embodiments, the corresponding tumor suppressor gene is retinoblastoma RB (or RB1). In some embodiments, the corresponding tumor suppressor gene is TSC1. In some embodiments, the corresponding tumor suppressor gene is TSC2. In some embodiments, the corresponding tumor suppressor genes include, without limitation, retinoblastoma RB (or RB1), TP53, TP63, TP73, CDKN2A (INK4A), CDKN1B, CDKN1C, DLD / NP1, HEPACAM, SDHB, SDHD, SFRP1, TCF21, TIG1, MLH1, MSH2, MSH6, WT1, WT2, NF1, NF2N, VHL, KLF4, pVHL, APC, CD95, ST5, YPEL3, ST7, APC, MADR2, BRCA1, BRCA2, patched, TSC1, TSC2, PALB2, ST14, or VHL.

[0190] In some embodiments, the mRNA encodes the tumor suppressor protein PTEN. In some embodiments, the tumor suppressor protein PTEN is encoded by the human PTEN sequence. In some embodiments, the mRNA includes a sequence selected from the group consisting of the sequences of accession numbers BC005821, JF268690, U92436, CR450306, AK024986, AK313581, U96180, and U93051 and NM_000314 in NCBI GenBank.

[0191] In some embodiments, the mRNA encodes the tumor suppressor protein p53. In some embodiments, the tumor suppressor protein p53 is encoded by the human TP53 sequence. In some embodiments, the mRNA comprises a sequence selected from the group consisting of the sequences with accession numbers AF052180, NM_000546, AY429684, BT019622, AK223026, DQ186652, DQ186651, DQ186650, DQ186649, DQ186648, DQ263704, DQ286964, DQ191317, DQ401704, AF307851, AM076972, AM076971, AM076970, DQ485152, BC003596, DQ648887, DQ648886, DQ648885, DQ648884, AK225838, M14694, M14695, EF101869, EF101868, EF101867, X01405, AK312568, NM_001126117, NM_001126116, NM_001126115, NM_001126114, NM_001126113, NM_001126112, FJ207420, X60020, X60019, X60018, X60017, X60016, X60015, X60014, X60013, X60011, X60012, X60010, X02469, S66666, AB082923, NM_001126118, JN900492, NM_001276699, NM_001276698, NM_001276697, NM_001276761, NM_001276760, NM_001276696, and NM_001276695 in NCBI GenBank.

[0192] In some embodiments, the mRNA encodes the tumor suppressor protein BRCA1. In some embodiments, the tumor suppressor protein BRCA1 is encoded by the human BRCA1 sequence. In some embodiments, the mRNA comprises a sequence selected from the group consisting of the sequences with accession numbers NM_007294, NM_007297, NM_007298, NM_007304, NM_007299, NM_007300, BC046142, BC062429, BC072418, AY354539, AY751490, BC085615, BC106746, BC106745, BC114511, BC115037, U14680, AK293762, U68041, BC030969, BC012577, AK316200, DQ363751, DQ333387, DQ333386, Y08864, JN686490, AB621825, BC038947, U64805, and AF005068 in NCBI GenBank.

[0193] In some embodiments, the mRNA encodes the tumor suppressor protein BRCA2. In some embodiments, the tumor suppressor protein BRCA2 is encoded by the human BRCA2 sequence. In some embodiments, the mRNA comprises a sequence selected from the group consisting of the sequences with accession numbers BC047568, NM_000059, DQ897648, and BC026160 in NCBI GenBank.

[0194] In some embodiments, the mRNA encodes the tumor suppressor protein TSC1. In some embodiments, the tumor suppressor protein TSC1 is encoded by the human TSC1 sequence. In some embodiments, the mRNA comprises a sequence selected from the group consisting of the sequences with accession numbers BC047772, NM_000368, BC070032, AB190910, BC108668, BC121000, NM_001162427, NM_001162426, D87683, and AF013168 in NCBI GenBank.

[0195] In some embodiments, the mRNA encodes the tumor suppressor protein TSC2. In some embodiments, the tumor suppressor protein TSC2 is encoded by the human TSC2 sequence. In some embodiments, the mRNA comprises a sequence selected from the group consisting of the sequences with accession numbers BC046929, BX647816, AK125096, NM_000548, AB210000, NM_001077183, BC150300, BC025364, NM_001114382, AK094152, AK299343, AK295728, AK295672, AK294548, and X75621 in NCBI GenBank.

[0196] In some embodiments, the mRNA encodes the tumor suppressor protein retinoblastoma 1 (RB1). In some embodiments, the tumor suppressor protein RB1 is encoded by the human RB1 sequence. In some embodiments, the mRNA comprises a sequence selected from the group consisting of the sequences with accession numbers NM_000321, AY429568, AB208788, M19701, AK291258, L41870, AK307730, AK307125, AK300284, AK299179, M33647, M15400, M28419, BC039060, BC040540, and AF043224 in NCBI GenBank.

[0197] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes a protein, and the absence of the protein results in a disease or disorder. In some embodiments, the protein is frataxin. In some embodiments, the protein is alpha-1 antitrypsin. In some embodiments, the protein is factor VIII. In some embodiments, the protein is factor IX.

[0198] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes a protein, and the expression of the protein in an individual modulates the immune response to the protein in the individual. In some embodiments, the protein is an antigen. In some embodiments, the antigen is a disease-associated antigen (e.g., a tumor-associated antigen), and the expression of the antigen in an individual results in an increase in the immune response to the antigen in the individual. In some embodiments, the antigen is a self-antigen, and the expression of the antigen in an individual results in a decrease in the immune response to the antigen in the individual.

[0199] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein encodes an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a bispecific antibody such as a bispecific T cell engager (BiTE). In some embodiments, the antibody specifically binds to a disease-associated antigen such as a tumor-associated antigen.

[0200] In some embodiments, the mRNA contained in the cargo delivery complex according to any of the embodiments described herein includes reporter mRNA. In some embodiments, the mRNA includes EGFP mRNA, for example, CleanCap EGFP mRNA, CleanCap EGFP mRNA(5moU), or CleanCap Cyanine 5 EGFP mRNA(5moU). In some embodiments, the mRNA includes Luc mRNA, for example, CleanCap Fluc mRNA, CleanCap Fluc mRNA(5moU), CleanCap Cyanine 5 Fluc mRNA(5moU), CleanCap Gaussia Luc mRNA(5moU), or CleanCap Renilla Luc mRNA(5moU). In some embodiments, the mRNA includes mRNA selected from CleanCap β-gal mRNA, CleanCap β-gal mRNA(5moU), and CleanCap mCherry mRNA(5moU). RNAi

[0201] In some embodiments, the cargo molecule includes interfering RNA (RNAi). In some embodiments, examples of RNAi include, but are not limited to, siRNA, shRNA, or miRNA. In some embodiments, the RNAi is siRNA. In some embodiments, the RNAi is microRNA. In some embodiments, the RNAi targets an endogenous gene. In some embodiments, the RNAi targets an exogenous gene. In some embodiments, the RNAi targets a gene associated with a disease, for example, a gene associated with cancer such as an oncogene. In some embodiments, the RNAi targets an oncogene. In some embodiments, the oncogene is smoothened. In some embodiments, the oncogene is rasK. In some embodiments, the oncogene is KRAS.

[0202] In some embodiments, the cargo molecule comprises both mRNA (e.g., any one of the mRNAs described herein) and RNAi (e.g., siRNA).

[0203] In some embodiments, the RNAi (e.g., siRNA) targets an oncogene, and the oncogene is KRAS. In some embodiments, the individual has an abnormality of KRAS. In some embodiments, the abnormality of KRAS includes a mutation at codon 12, 13, 17, 34, or 61 of KRAS. In some embodiments, the abnormality of KRAS is selected from the group consisting of G12C, G12S, G12R, G?2F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, G13R, G13A, G13D, G13V, G13P, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A??46P, A146T, and A146V. In some embodiments, the abnormality of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, Q61K, Q61L, Q61R, and Q61H. In some embodiments, the abnormality of KRAS is selected from the group consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the abnormality of KRAS is selected from the group consisting of KRAS G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, and Q61R. In some embodiments, the abnormality of KRAS includes G12C. In some embodiments, the abnormality of KRAS includes G12D. In some embodiments, the abnormality of KRAS includes Q61K. In some embodiments, the abnormality of KRAS includes G12C and G12D. In some embodiments, the abnormality of KRAS includes G12C and Q61K. In some embodiments, the abnormality of KRAS includes G12D and Q61K. In some embodiments, the abnormality of KRAS includes G12C, G12D, and Q61K.

[0204] In some embodiments, the RNAi (e.g., siRNA) targets the mutant form of KRAS. In some embodiments, the RNAi (e.g., siRNA) specifically targets the mutant form of KRAS and does not target the wild-type form of KRAS. In some embodiments, the mutant form includes an abnormality of KRAS, and the abnormality of KRAS includes a mutation to codons 12, 13, 17, 34, or 61 of KRAS. In some embodiments, the mutant form includes an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, G13R, G13A, G13D, G13V, G13P, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the mutant form includes an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, Q61K, Q61L, Q61R, and Q61H. In some embodiments, the mutant form includes an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the mutant form includes an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of KRAS G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, and Q61R. In some embodiments, the abnormality of KRAS is selected from the group consisting of KRAS G12C, G12D, G12R, G12S, G12V, and G13D. In some embodiments, the abnormality of KRAS includes G12C. In some embodiments, the abnormality of KRAS includes G12D.In some embodiments, the KRAS abnormality includes Q61K. In some embodiments, the KRAS abnormality includes G12C and G12D. In some embodiments, the KRAS abnormality includes G12C and Q61K. In some embodiments, the KRAS abnormality includes G12D and Q61K. In some embodiments, the KRAS abnormality includes G12C, G12D and Q61K.

[0205] In some embodiments, the RNAi (e.g., siRNA) targets multiple mutant forms of KRAS. In some embodiments, the multiple mutant forms include multiple abnormalities of KRAS, and the multiple abnormalities of KRAS include at least two or more mutations to codons 12, 13, 17, 34, and / or 61 of KRAS. In some embodiments, the multiple abnormalities of KRAS include at least two or more mutations to codons 12 and 61 of KRAS. In some embodiments, the abnormality of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, G13R, G13A, G13D, G13V, G13P, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the abnormality of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, Q61K, Q,61L, Q61R, and Q61H. In some embodiments, the abnormality of KRAS is selected from the group consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the abnormality of KRAS is selected from the group consisting of KRAS G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, and Q61R. In some embodiments, the abnormality of KRAS is selected from the group consisting of KRAS G12C, G12D, G12R, G12S, G12V, and G13D. In some embodiments, the abnormality of KRAS is selected from the group consisting of KRAS G12C, G12D, and Q61K. In some embodiments, the abnormality of KRAS includes G12C and G12D. In some embodiments, the abnormality of KRAS includes G12C and Q61K.In some embodiments, the KRAS abnormality includes G12D and Q61K. In some embodiments, the KRAS abnormality includes G12C, G12D and Q61K.

[0206] In some embodiments, the RNAi (e.g., siRNA) comprises a plurality of RNAi (e.g., siRNA) including a first RNAi (e.g., a first siRNA) and a second RNAi (e.g., a second siRNA), the first RNAi targets a mutant form of the first KRAS, and the second RNAi targets a mutant form of the second KRAS. In some embodiments, the first RNAi and / or the second RNAi do not target the wild-type form of KRAS. In some embodiments, the first mutant form and / or the second mutant form comprise an abnormality of KRAS, and the abnormality of KRAS comprises a mutation to codons 12, 13, 17, 34 and / or 61 of KRAS. In some embodiments, the first mutant form and / or the second mutant form comprise an abnormality of KRAS, and the abnormality of KRAS comprises a mutation to codon 12 or 61 of KRAS. In some embodiments, the first mutant form comprises an abnormality of KRAS including a mutation to codon 12, and the second mutant form comprises an abnormality of KRAS including a mutation to codon 61. In some embodiments, the first mutant form and / or the second mutant form comprise an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, G13R, G13A, G13D, G13V, G13P, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A146P, A146T and A146V. In some embodiments, the first mutant form and / or the second mutant form comprise an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, Q61K, Q61L, Q61R, and Q61H.In some embodiments, the first mutant form and / or the second mutant form comprises an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the first mutant form and / or the second mutant form comprises an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of KRAS G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, and Q61R. In some embodiments, the first mutant form and / or the second mutant form comprises an abnormality of KRAS, and the abnormality of KRAS is selected from the group consisting of KRAS G12C, G12D, G12R, G12S, G12V, and G13D. In some embodiments, the first mutant form and / or the second mutant form comprises an abnormality of KRAS, and the abnormality of KRAS is selected from G12C, G12D, and Q61K. In some embodiments, the first mutant form comprises an abnormality of KRAS including KRAS G12C, and the second mutant form comprises an abnormality of KRAS including KRAS G12D. In some embodiments, the first mutant form comprises an abnormality of KRAS including KRAS G12C, and the second mutant form comprises an abnormality of KRAS including KRAS Q61K. In some embodiments, the first mutant form comprises an abnormality of KRAS including KRAS G12D, and the second mutant form comprises an abnormality of KRAS including KRAS Q61K.

[0207] In some embodiments, the RNAi (e.g., siRNA) comprises a plurality of RNAi (e.g., siRNA) including a first RNAi (e.g., a first siRNA), a second RNAi (e.g., a second siRNA), and a third RNAi (e.g., siRNA). In some embodiments, the first RNAi targets a first mutant form of KRAS, the second RNAi targets a second mutant form of KRAS, and the third RNAi targets a third mutant form of KRAS. In some embodiments, the first KRAS mutant form, the second KRAS mutant form, and the third KRAS mutant form each comprise an abnormality of KRAS that includes a mutation to codons 12, 13, 17, 34, and / or 61 of KRAS. In some embodiments, the first KRAS mutant form, the second KRAS mutant form, and the third KRAS mutant form each comprise an abnormality of KRAS selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, G13R, G13A, G13D, G13V, G13P, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the first KRAS mutant form, the second KRAS mutant form, and the third KRAS mutant form each comprise an abnormality of KRAS selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, Q61K, Q61L, Q61R, and Q61H. In some embodiments, the first KRAS mutant form, the second KRAS mutant form, and the third KRAS mutant form each comprise an abnormality of KRAS selected from the group consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T, and A146V.In some embodiments, the first KRAS mutant form, the second KRAS mutant form, and the third KRAS mutant form each comprise an abnormality of KRAS selected from the group consisting of KRAS G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, and Q61R. In some embodiments, the first KRAS mutant form, the second KRAS mutant form, and the third KRAS mutant form each comprise an abnormality of KRAS selected from the group consisting of KRAS G12C, G12D, G12R, G12S, G12V, G13D, and Q61K. In some embodiments, the first KRAS mutant form, the second KRAS mutant form, and the third KRAS mutant form each comprise an abnormality of KRAS selected from the group consisting of G12C, G12D, and Q61K. In some embodiments, the first mutant form comprises an abnormality of KRAS that includes KRAS G12C, the second mutant form comprises an abnormality of KRAS that includes KRAS G12D, and the third mutant form comprises an abnormality of KRAS that includes KRAS Q61K.

[0208] In some embodiments, the RNAi (e.g., siRNA) comprises an RNAi (e.g., siRNA) targeting KRAS that includes the sequences 5'-GUUGGAGCUUGUGGCGUAGTT-3' (sense), 5'-CUACGCCACCAGCUCCAACTT-3 (antisense), 5'-GAAGUGCAUACACCGAGACTT-3' (sense), 5'-GUCUCGGUGUAGCACUUCTT-3' (antisense), 5'-GUUGGAGCUGUUGGCGUAGTT-3' (sense) and / or 5'-CUACGCCAACAGCUCCAACTT-3' (antisense). In some embodiments, the RNAi (e.g., siRNA) comprises an RNAi (e.g., siRNA) targeting KRAS that includes a nucleic acid sequence selected from the sequences of SEQ ID NOs: 179, 180, 182-185. In some embodiments, the RNAi (e.g., siRNA) comprises an RNAi (e.g., siRNA) targeting KRAS that includes a sequence targeting KRAS G12S, such as, for example, the siRNA sequence disclosed in Acunzo, M. et al., Proc Natl Acad Sci USA. 2017 May 23; 114 (21): E4203-E4212. In some embodiments, the RNAi (e.g., siRNA) comprises an RNAi (e.g., siRNA) targeting KRAS that is disclosed in WO2014013995, JP2013212052, WO2014118817, WO2012129352, WO2017179660, JP2013544505, US8008474, US7745611, US7576197, US7507811, each of which is incorporated herein by reference in its entirety.

[0209] In some embodiments, RNAi includes, but is not limited to, siRNA, shRNA, and miRNA. The terms "interfering RNA", "RNAi", or "interfering RNA sequence" refer to a single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (i.e., double-stranded RNA such as siRNA, aiRNA, or pre-miRNA) that can reduce or inhibit the expression of a target gene or sequence when the interfering RNA is present in the same cell as the target gene or sequence (e.g., by mediating the degradation of mRNA complementary to the interfering RNA sequence or inhibiting the translation of mRNA complementary to the interfering RNA sequence), and thus, the interfering RNA refers to a single-stranded RNA that is complementary to the target mRNA sequence or to a double-stranded RNA formed by two complementary strands or a single self-complementary strand. The interfering RNA can have substantial or complete identity to the target gene or sequence, or can have regions of mismatch (i.e., mismatch motifs). The sequence of the interfering RNA can correspond to the full-length target gene or a partial sequence thereof. The interfering RNA includes "small interfering RNA" or "siRNA", e.g., interfering RNAs having a length of about 15-60, 15-50, or 5-40 (double-stranded) nucleotides, more generally about 15-30, 15-25, or 19-25 (double-stranded) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (double-stranded) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA has a length of 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA has a length of about 15-60, 15-50, 15-40, 5-30, 5-25, or 19-25 base pairs in length, preferably about 8-22, 9-20, or 19-21 base pairs in length). The siRNA duplex can include 3' overhangs of about 1-about 4 nucleotides or about 2-about 3 nucleotides and 5' phosphate termini.Examples of siRNA include, without limitation, double-stranded polynucleotide molecules assembled from two separate strand molecules where one strand is the sense strand and the other strand is the complementary antisense strand; double-stranded polynucleotide molecules assembled from single-stranded molecules where the sense region and the antisense region are linked by a nucleic acid-based linker or a linker that is not nucleic acid-based; double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions; and circular single-stranded polynucleotide molecules having two or more loop structures and a stem with self-complementary sense and antisense regions, which can be processed in vivo or in vitro to generate active double-stranded siRNA molecules. The siRNA is preferably chemically synthesized. The siRNA can also be generated by cleaving long dsRNA (e.g., approximately longer than 25 nucleotides in length) with E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, for example, Yang et al., Proc Natl. Acad. Set. USA, 99: 9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99: 14236 (2002); Byrom et al., Ambion TeehNotes, 10 (1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 3 1:981-987 (2003); Knight et al., Science, 293: 2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). The dsRNA is preferably at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. The dsRNA can be as long as or longer than 1000, 1500, 2000, 5000 nucleotides in length.dsRNA can encode an entire gene transcript or a partial gene transcript. In certain cases, siRNA can be encoded in a plasmid (e.g., transcribed as a sequence that automatically folds into a duplex with a hairpin loop). Small hairpin RNA (shRNA) is a sequence of RNA that forms a tight hairpin turn and can be used to silence gene expression by RNA interference. The shRNA hairpin structure is cleaved by cellular machinery into siRNA, which then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves the mRNA that matches the siRNA to which it binds. Suitable lengths for RNAi include, without limitation, about 5 to about 200 nucleotides, or 10 to 50 nucleotides or base pairs, or 15 to 30 nucleotides or base pairs. In some embodiments, the RNAi is substantially complementary to the corresponding target gene (e.g., at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more identical). In some embodiments, the RNAi is modified, for example, by incorporating non-naturally occurring nucleotides.

[0210] In some embodiments, the RNAi is double-stranded RNAi. Suitable lengths for RNAi include, without limitation, about 5 to about 200 nucleotides, or 10 to 50 nucleotides or base pairs, or 15 to 30 nucleotides or base pairs. In some embodiments, the RNAi is substantially complementary to the corresponding target gene (e.g., at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more identical). In some embodiments, the RNAi is modified, for example, by incorporating non-naturally occurring nucleotides.

[0211] In some embodiments, RNAi specifically targets RNA molecules such as mRNA encoding proteins involved in diseases such as cancer. In some embodiments, the disease is cancer such as solid tumors or hematological malignancies, and the interfering RNA targets mRNA encoding proteins involved in cancer, such as proteins involved in regulating cancer progression. In some embodiments, RNAi targets oncogenes involved in cancer.

[0212] In some embodiments, RNAi specifically targets RNA molecules such as mRNA encoding proteins involved in negative regulation of the immune response. In some embodiments, the interfering RNA targets mRNA encoding negative co-stimulatory molecules. In some embodiments, examples of negative co-stimulatory molecules include, for example, PD-1, PD-L1, PD-L2, TIM-3, BTLA, VISTA, LAG-3, and CTLA-4.

[0213] In some embodiments, RNAi is miRNA. MicroRNA (abbreviated as miRNA) is a short ribonucleic acid (RNA) molecule found in eukaryotic cells. MicroRNA molecules have very few nucleotides (an average of 22) compared to other RNAs. miRNA is a post-transcriptional regulator that binds to complementary sequences on target messenger RNA transcripts (mRNA) and usually results in translational repression or target degradation and gene silencing. The human genome can encode over 1000 miRNAs, which can target approximately 60% of mammalian genes and are abundant in many human cell types. Suitable lengths of miRNA include, witho...

Claims

**Claim 1** A composition comprising a cargo delivery complex for intracellular delivery of a cargo molecule, wherein the cargo delivery complex comprises: a) a first peptide comprising a first cell-penetrating peptide; b) a second peptide comprising a second cell-penetrating peptide; and c) a cargo molecule, wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, the first peptide has no PEG moiety, the first cell-penetrating peptide and the second cell-penetrating peptide are each independently selected from the group consisting of ADGN-100 peptide and VEPEP-6 peptide, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is from 10:1 to 4:1, the ADGN-100 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 80, 87-91, 101-104, 106, 110-112 and 124-127, and the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 92-100, 105, 107-109, 129-131 and 133-136. **Claim 2** The composition according to claim 1, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is a VEPEP-6 peptide. **Claim 3** The composition according to claim 1 or 2, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is an ADGN-100 peptide. **Claim 4** The composition according to any one of claims 1 to 3, wherein the first cell-penetrating peptide and the second cell-penetrating peptide are the same. **Claim 5** The composition according to any one of claims 1 to 4, wherein the cargo molecule is selected from the group consisting of nucleic acids, viruses, polypeptides, protein / nucleic acid complexes, virus-like particles, and protein complexes. **Claim 6** The composition according to any one of claims 1 to 5, wherein the PEG moiety is linear PEG. **Claim 7** The composition according to any one of claims 1 to 5, wherein the PEG moiety is branched PEG. **Claim 8** The composition according to any one of claims 1 to 7, wherein the molecular weight of the PEG moiety is from 0.05 kDa to 50 kDa. **Claim 9** The composition according to any one of claims 1 to 8, wherein the PEG moiety consists of 1 to 10 ethylene glycol units.

10. The composition according to any one of claims 1 to 9, wherein the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide.

11. The composition according to any one of claims 1 to 9, wherein the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

12. The first peptide and / or the second peptide further comprises one or more moieties selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, cholesterol, a nuclear localization signal, a nuclear export signal, an antibody or an antibody fragment thereof, a peptide, a polysaccharide, and a targeting sequence, and the one or more moieties are covalently linked to the N-terminus of the first cell-penetrating peptide or the second cell-penetrating peptide, or to the PEG moiety. The composition according to any one of claims 1 to 11.

13. The composition according to claim 12, wherein the one or more moieties are covalently linked to the N-terminus of the first cell-penetrating peptide, the N-terminus of the second cell-penetrating peptide, or the PEG moiety via a linker.

14. The composition according to claim 12, wherein the one or more moieties comprise a targeting sequence.

15. The composition according to claim 14, wherein the targeting sequence is selected from the group consisting of GYVSK, YIGSR, GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, and IGSR.

16. The composition according to claim 15, wherein the targeting sequence is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.

17. The composition according to any one of claims 14 to 16, wherein the targeting sequence is covalently linked to the first cell-penetrating peptide or the second cell-penetrating peptide via a linker.

18. The composition according to any one of claims 12 to 17, wherein the one or more moieties comprise an acetyl group and / or a stearyl group.

19. The first peptide and / or the second peptide is cysteamide, cysteine, thiol, amide, nitrilotriacetic acid, carboxyl, linear or branched C １ -C ６ alkyl, primary or secondary amine, oxide derivative, lipid, phospholipid, fatty acid, cholesterol, nuclear localization signal, nuclear export signal, antibody, polysaccharide and one or more moieties selected from the group consisting of targeting sequences, wherein the one or more moieties are covalently linked to the C-terminus of the first cell-permeable peptide, the C-terminus of the second cell-permeable peptide or the PEG moiety, The composition according to any one of claims 1 to 18 Substance.

20. The composition according to claim 19, wherein the one or more moieties are covalently linked via a linker to the C-terminus of the first cell-penetrating peptide, the C-terminus of the second cell-penetrating peptide, or the PEG moiety.

21. The composition according to any one of claims 1 to 20, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is a retro-inverso peptide.

22. The composition according to claim 1, wherein the first cell-penetrating peptide and / or the second cell-penetrating peptide is a retro-inverso peptide.

23. The composition according to claim 1, wherein the first peptide and / or the second peptide further comprises a targeting sequence selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.

24. The composition according to any one of claims 1 to 23, wherein the cargo molecule is a nucleic acid selected from the group consisting of siRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, oligonucleotide, and analogs thereof.

25. The composition according to claim 24, wherein the nucleic acid comprises mRNA.

26. The composition according to claim 25, wherein the nucleic acid comprises mRNA and RNAi, the mRNA encodes a therapeutic protein for treating a disease or condition, the RNAi targets an RNA, and the expression of the RNA is associated with the disease or condition.

27. The composition according to claim 25 or claim 26, wherein the mRNA encodes a therapeutic protein. **Claim 28**: The composition according to claim 27, wherein the therapeutic protein is selected from the group consisting of TP53, BRCA1, PTEN, retinoblastoma RB (or RBI), TP63, TP73, CDKN2A (INK4A), CDKN1B, CDKN1C, DLD / NP1, HEPACAM, SDHB, SDHD, SFRP1, TCF21, TIG1, MLH1, MSH2, MSH6, WT1, WT2, NF1, NF2N, VHL, KLF4, pVHL, APC, CD95, ST5, YPEL3, ST7, MADR2, BRCA2, patched, TSC1, TSC2, PALB2, ST14 or VHL, factor VIII, alpha 1-antitrypsin, frataxin, insulin, growth hormone (somatotropin), growth factor, hormone, dystrophin, insulin-like growth factor 1 (IGF1), factor IX, antithrombin III, protein C, beta-glucocerebrosidase, aglucosidase-alpha, alpha-1-iduronidase, iduronic acid-2-sulfatase, galsulfase, human alpha-galactosidase A, alpha-1-proteinase inhibitor, lactase, pancreatic enzyme, adenosine deaminase, albumin, CRISPR-related nuclease, Cas9, or a variant of a tumor suppressor gene encoding the same, or a recombinant thereof. **Claim 29**: The composition according to claim 24, wherein the cargo molecule comprises a guide RNA, and the guide RNA is KRAS gRNA. **Claim 30** A nanoparticle comprising a core comprising the cargo delivery complex according to any one of claims 1 to 29. **Claim 31** A pharmaceutical composition comprising the cargo delivery complex according to any one of claims 1 to 29 or the nanoparticle according to claim 30, and a pharmaceutically acceptable carrier. **Claim 32** **Claim 33**: A method for preparing the cargo delivery complex according to any one of claims 1 to 29, comprising: a) combining the first peptide and the second peptide to form a peptide mixture; and b) combining the peptide mixture with the cargo to form the cargo delivery complex. **Claim 34** A method for preparing the cargo delivery complex according to claim 31 or 32, comprising: a) combining the first peptide and the second peptide, thereby forming a peptide mixture; and b) combining the peptide mixture with the cargo molecule, thereby forming the cargo delivery complex.

34. An in vitro method for delivering one or more cargos into a cell, comprising contacting the cell with the cargo delivery complex according to any one of claims 1 to 29 or the nanoparticle according to claim 30, wherein the cargo delivery complex comprises one or more cargos.

35. A kit comprising the cargo delivery complex according to any one of claims 1 to 29, the nanoparticle according to claim 30, or the pharmaceutical composition according to claim 31.

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