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WO2025019713A2 - Système d'administration à base de protéines - Google Patents

Système d'administration à base de protéines Download PDF

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Publication number
WO2025019713A2
WO2025019713A2 PCT/US2024/038614 US2024038614W WO2025019713A2 WO 2025019713 A2 WO2025019713 A2 WO 2025019713A2 US 2024038614 W US2024038614 W US 2024038614W WO 2025019713 A2 WO2025019713 A2 WO 2025019713A2
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Prior art keywords
seq
ipghg
vpgag
vpggg
compound
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WO2025019713A3 (fr
Inventor
Elliot Chaikof
Feyisayo EWEJE
Jiaxuan CHEN
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Beth Israel Deaconess Medical Center Inc
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Beth Israel Deaconess Medical Center Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6435—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a connective tissue peptide, e.g. collagen, fibronectin or gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • cationic membrane disruptive peptides including cell penetrating peptides such as Tat 11 , oligoarginine 12 , and penetratin 13 or other endosomolytic peptides, as exemplified by S10 14 have been examined as vectors in the form of conjugates or as untethered peptide shuttles. 15,16 However, these peptides remain limited by low potency and cytotoxicity. Moreover, in vivo biodistribution has been difficult to control due to the cationic nature of many of these peptides and the inability to precisely control the size and morphology of the resulting macromolecular complex. Altogether, these factors have
  • the present disclosure relates in part to elastin-like polypeptides, endosomal escape peptides, and compounds comprising an elastin-like polypeptide chemically or recombinantly conjugated to one or more endosomal escape peptides.
  • the compounds provided herein may be useful in compositions comprising an agent, such as a micelle or particle, for delivery of the agent to a subject or cell.
  • the present disclosure also provides pharmaceutical compositions and kits comprising the compounds, agents, particles, or compositions provided herein.
  • the present disclosure also provides methods of treating or preventing a disease, as well as methods of delivery.
  • a compound comprising an elastin-like polypeptide chemically or recombinantly conjugated to one or more endosomal escape peptides.
  • an elastin-like polypeptide comprising a sequence of: [(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)]10–CCCCGGG– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 1) wherein: X 3 is V or A; and X 4 is E or G.
  • an endosomal escape peptide comprising a sequence selected from the group consisting of: LYKKFKKKLLKSLKRGGSGGGSLYKKFKKKLLKSLKRL; WKKWWKKWWKWWKKWWKKGGSGGGSWKKWWKKWWKWWKKWWK K; TKRVLQVWFQNARAKFRRNLLRGGSGGGSMLSFILTLKRMLKACLRAWK; MLSFLLTLKRMLRACLRAWGGSGGGSLQSTKRFIKWYNAWNEKRR; LYKKFKKKLLKSLKRLGGSGGGSYKWWQKYQKRKF; LYKKFKKKLLKSLKRGGSGGGSTKRVLQVWFQNARAKFRRNLLR; RRWVRRVRRWVRRVVRVVRRWVRRGGSGGGSFLKAIKKFGKEFKKIGAKL K; KHKHKHKHKHKHGGSGGGSKKTWWKTWTKWSQ
  • provided herein is a composition comprising a compound provided herein and an agent.
  • a kit comprising a compound or composition provided herein and instructions for using the compound or composition.
  • a method of delivering an agent to a subject, cell, or biological sample comprising administering to the subject or contacting the cell or biological sample with an effective amount of a composition provided herein.
  • a method of treating or preventing a disease in a subject comprising administering to the subject a composition provided herein.
  • FIG. 1 shows elastin-like polypeptide (ELP) nanoparticles for multimodal macromolecule delivery.
  • ELP elastin-like polypeptide
  • Micelle-forming tetrablock ELPs containing (from N- to C- terminus) a hydrophilic block, cysteine-based crosslinking block, hydrophobic block, and endosomolytic block were designed for siRNA, mRNA, and protein cargo delivery. From top to bottom, the sequences are V1: SEQ ID NO: 117, V2: SEQ ID NO: 115, V3: SEQ ID NO: 115, and V4: SEQ ID NO: 113.
  • FIGs. 2A-2M show formation of ELP-protein nanoparticles.
  • FIGs. 3A-3G show multigenerational screen to identify ELPs for effective intracellular protein delivery. a) ELP block designs of each tetrablock ELP version assessed.
  • b) Measured diameter and particle size distribution by mass of V2-ELP at varying pHs ([ELP] 10 ⁇ M).
  • FIGs. 4A-4I show discovery of enhanced endosomolytic peptides via in silico screening. a) Comparison of the observed and predicted % GFP+ of HeLa cells treated with GFP proteins and a library of membrane disruptive peptides. Data from Ref 55. b) In silico alpha-helix library screening approach.
  • V4-ELP-Gen2 Activity of V4-ELP-Gen2 and d) V4-ELP-Gen3 EEP constructs in Cre protein delivery to HEK293-RFP (average efficacies 10.1% and 27.1% respectively).
  • g) Diameter of V4-ELP- EEP13 series micelle ([ELP] 10 ⁇ M).
  • FIGs. 5A-5F show ELP-mediated protein delivery to primary cells.
  • FIGs. 6A-6H show complexation and delivery of siRNA by tetrablock ELP nanoparticles. a) Schematic of ELP-siRNA nanoparticle formation via combined electrostatic and heat-induced hydrophobic interactions.
  • FIGS. 7A-7G show complexation and delivery of mRNA-encoded Cre recombinase by tetrablock ELP nanoparticles.
  • b) Diameter and c) zeta potential of V4-ELP-EEP13 nanoparticles encapsulating Cre mRNA (n 3).
  • PDI 0.112 and 0.17, -mRNA and +mRNA.
  • % RFP+ of HEK293-RFP following treatment with Cre mRNA complexed with V4-ELP-EEP13 at varying ELP and Cre mRNA concentrations (n 3).
  • FIGs. 8A-8E show ELP-mediated delivery of therapeutic gene editor and transcription factor proteins.
  • FIGs. 10A-10D show ELP-mediated intradermal delivery of Cre protein in Ai9 mice.
  • a) Images of intradermal administration procedures. A patch of flank skin was shaved to facilitate precise injection.
  • FIGs. 10A-10D show ELP micelle size and cellular internalization.
  • PDI polydispersity index
  • FIGs. 11A-11B show protease-mediated protein cargo release from ELP micelles. a) Schematic depicting Cathepsin B cleavage sites in Cre-V1-ELP. The sequence (GGS)9 (SEQ ID NO: 91) is depicted.
  • FIG. 12 shows quantification of ELP conjugation efficiency to SpCas9.
  • FIGs. 13A-13B show CD117-targeted ELP micelle uptake via anti-CD117 mAb functionalization. a) Histograms depicting distribution of Alexa Fluor 633 MFI in CD117+ P815 cells treated with CD117-functionalized V1-ELP-AF633 compared to unmodified control. b) Z-stack confocal images of CD117-V1-ELP-AF633 treated P815 cells. [0029] FIGs.
  • FIG. 17 shows HEK293 viability following Cre-V3-ELP treatment for 48 hours.
  • FIGs. 18A-18B show optimization of protease cleavable linker sequences to improve cargo release from ELP micelles. a) Linker sequences on the C-terminal end of Cre protein variants. From top to bottom, SEQ ID NOs are 91-97.
  • FIG. 19 shows diameter and PDI of V4-ELP micelles.
  • FIG. 20 shows HEK293-RFP viability following Cre recombinase and V4-ELP-EEP co-treatment for 48 hours.
  • FIGs. 21A-21B show an assessment of Lipofectamine 3000 for Cre protein delivery to HEK293-RFP. a) Viability and b) RFP+ percentage following transfection.
  • FIGs. 22A-22B show the predicted structure and helical wheel depiction of S10. a) Predicted structure of S10 (via AlphaFold2). SEQ ID NOs: 122 (CM18 Variant), 120
  • FIG. 23 shows quantification of peptide feature importance for protein delivery efficacy.
  • FIG. 24 shows multiple linear regression model for prediction of V4-ELP-EEP construct efficiency in siGFP delivery.
  • FIG. 25 shows multi-stage ⁇ -helical database screen for the discovery of Gen3 EEPs.
  • FIGs. 26A-26B show a comparison of EEP13 and S10 in standalone format in Cre protein delivery.
  • FIGs. 28A-28F show primary cell viability following co-treatment with protein-form Cre recombinase and ELP micelles.
  • FIGs. 29A-29B show an assessment of Lipofectamine 3000 for Cre protein delivery to primary mouse fibroblasts. a) Viability and b) tdTomato expression of Ai9 fibroblasts following treated with Lipofectamine 3000 and Cre protein. [0045] FIG.
  • FIGs. 31A-31B show delivery of fluorescently labeled Cre recombinase to LS HSPCs via CD117-targeted ELP micelles.
  • n 3.
  • FIG. 35 shows Ai9-SauSpyCas9 lung fibroblast viability following treatment with mRNA-encoded Cre recombinase via V4-ELP-EEP13.
  • FIGs. 36A-36B show V4-ELP-EEP screen for a) Cre recombinase protein and b) mRNA delivery to HEK293-GFP.
  • FIG. 37 shows gating strategy for skin cell isolation.
  • FIG. 38 shows representative flow cytometry plots from isolated Ai9 skin cell populations following intradermal Cre-ELP injections.
  • FIGs. 39A-39L show ELP-mediated protein delivery to primary cells.
  • a-b) % tdTomato+ in Ai9-derived mouse lung fibroblasts, c-d) peritoneal macrophages, e-f) splenic CD4+ T cells, and g-h) Lin-Sca-1+c-kit+ (LSK) hematopoietic stem cells following Cre and ELP co-treatment (n 3).
  • c, e - [Cre] 8 ⁇ M.
  • b, d – [ELP] 8 ⁇ M.
  • FIGs. 40A-40B show complexation and delivery of siRNA by tetrablock ELP nanoparticles.
  • FIGs. 42A-42E show ELP-mediated delivery of Cre protein to the bronchial epithelium in vivo.
  • FIG. 43 shows a negative stain TEM image of V4-ELP nanoparticles.
  • [ELP] 8 ⁇ M
  • [Cre protein] 2 ⁇ M
  • n 3.
  • PDI polydispersity index
  • FIGs. 46A-46J show formation and characterization of Cre-V2-ELP and Cre-V3-ELP nanoparticles. a) PAGE depicting Cre conjugation to V2-ELP. b-d) Size distribution of V1-, V2- Cre-V1-, and Cre-V2-ELPs. e-h) Diameter of V3-ELPs after a 10 minute and 72 hour
  • FIGs. 47A-47B show optimization of protease cleavable linker sequences to improve cargo release from ELP nanoparticles. a) Linker sequences on the C-terminal end of Cre protein variants. From top to bottom, SEQ ID NOs: 91-97 are shown.
  • FIGs. 50A-50C show alpha helical peptide database screening for discovery of endosomal escape peptides. a) Quantification of the importance of peptide features for effective protein delivery for the Gen1 EEP predictive model. The five features highlighted in green were incorporated into the model parameters. b) Quantification of peptide feature importance for protein delivery efficacy for Gen2 EEP predictive model. The three features highlighted in green were incorporated into the model parameters. c) Multi-stage ⁇ -helical database screen for the discovery of Gen3 EEPs. [0066] FIGs.
  • FIGs. 52A-52B show % RFP+ HEK293-RFP cells and cell viability following treatment with protein-form Cre recombinase and V4-ELP-[EEP13]n nanoparticles.
  • FIG. 53 shows ELP-EEP-mediated endosomal escape of FITC-labeled dextran. HeLa cells were co-treated with V4-ELP-EEP13 or V4-ELP-[EEP13]2 and FITC-dextran with
  • FIG. 54A-54H show viability of primary cells following co-treatment with protein- form Cre recombinase and ELP nanoparticles.
  • FIGs. 55A-55B show assessment of Lipofectamine 3000 for Cre protein delivery to primary mouse fibroblasts. a) Cell viability and b) tdTomato expression of Ai9 fibroblasts following treated with Lipofectamine 3000 and Cre protein. [0071] FIGs. 56A-56C show cell viability and GFP expression of HeLa-d2eGFP cells after V4-ELP-EEP-mediated siGFP delivery.
  • FIGs. 57A-57E show cell viability following treatment with Cre mRNA formulated with V4-ELP-[EEP13]n nanoparticles.
  • FIG. 58 shows sequences of ELP hydrophilic blocks, as well as their charge and hydrophilicity. From top to bottom, SEQ ID NOs: 17, 18, 204, 205, and 206 are shown.
  • FIG. 59A shows PAGE analysis of V2-, V4-, and V5-ELP-EEP13 conjugates to Cre recombinase.
  • FIG. 59B shows percent conjugation of V2-, V4-, and V5-ELP-EEP13 conjugates determined by relative intensity of Cre-ELP versus ELP bands.
  • FIG. 60 shows delivery efficiency of Cre protein (FIG. 60A) and Cre mRNA (FIG. 60B) by V5-ELP particles to HEK293 Cre reporter cells.
  • FIG. 61 shows size and dispersity of V5-ELP particles with and without mRNA: V5[S]-ELP-EEP13 (FIG. 61A), V5[G] -ELP-EEP13 (FIG. 61B), V5[S]-ELP-EEP13 (FIG. 61C), V5[G]-ELP-EEP13 (FIG. 61D).
  • FIG. 62 shows PAGE depicting result of V5(S)-ELP-EEP13 reacted with LPETG- linker functionalized anti-CD117 mAb via sortase reaction.
  • FIG. 62 shows PAGE depicting result of V5(S)-ELP-EEP13 reacted with LPETG- linker functionalized anti-CD117 mAb via sortase reaction.
  • FIG. 64 shows the SEC protocol for mAb-ELP-siRNA nanoparticles.
  • FIG. 64A shows PAGE analysis of ELPs purified by SEC.
  • FIG. 64B shows SEC fractions of mAb-ELP siRNA nanoparticles, free mAb, free ELP, and free sortase.
  • FIG. 65A shows improved uptake of CD117-V5(S)-EEP13 over V5(S)-EEP13 formulated with AF647-labeled siRNA.
  • FIG. 64 shows the SEC protocol for mAb-ELP-siRNA nanoparticles.
  • FIG. 64A shows PAGE analysis of ELPs purified by SEC.
  • FIG. 64B shows SEC fractions of mAb-ELP siRNA nanoparticles, free mAb, free ELP, and free sortase.
  • FIG. 65A shows improved uptake of CD117-V5(S)-EEP13 over V5(S)-EEP13 formulated with AF6
  • FIG. 65B shows cells untreated, with V5(S)-EEP13, or with CD117-V5(S)-EEP13 at 30 minutes (top) versus 24 hours (bottom).
  • FIG. 66 shows uptake of AF647-labeled siRNA formulated ELP nanoparticles in Lineage-Sca-1+c-kit+ cells (LSK, FIG. 66A) and CD117+ cells (FIG. 66B) and overally (FIG. 66C) using CD117-V5(S)-EEP13 versus V5(S)-EEP13 nanoparticles.
  • FIGs. 67A and 67B show conjugation of an anti-CD-5 antibody to V5(S)-ELP- EEP13.
  • FIG. 67A and 67B show conjugation of an anti-CD-5 antibody to V5(S)-ELP- EEP13.
  • FIG. 68 shows a schematic for in silico discovery of EEPs.
  • FIG. 69 shows a schematic of a variational autoencoder to generate EEP sequences. From top to bottom, SEQ ID NOs: 207-210 are shown.
  • FIG. 70 shows a schematic of a transfer learning predictive model.
  • FIG. 71 shows a table of first generation ELP-E3P sequences and predicted efficacies for in vitro Cre protein delivery (normalized to V4-ELP-EEP13). From top to bottom, SEQ ID NOs: 133-164 are shown.
  • FIG. 72 shows Cre protein delivery activity of first generation ELP-E3Ps to HEK293 Cre reporter cells.
  • FIG. 73 shows a second generation predictive model to generate EEP sequences.
  • FIG. 73A shows predictive model performance on Gen0 and Gen1 ELP-EEP efficacy dataof second generation peptides.
  • FIG. 73B shows a synthetic minority oversampling with Gaussian noise technique.
  • FIG. 74 shows computational and empirical evaluation of Gen0 and Gen1EEP sequences.
  • FIG. 75 shows a table of second generation ELP-E 3 P sequences, fitness function scores, and predicted efficacies. From top to bottom, SEQ ID NOs: 165-180 are shown.
  • FIG. 76 shows Cre protein delivery activity of second generation ELP-E3Ps to HEK293 Cre reporter cells.
  • FIG. 77 shows delivery efficacy of V4-ELP-EEP13 and V4-ELP-E3P2.5 for Cre protein (FIG. 77A) and Cre mRNA (FIG. 77B) to HEK293 Cre reporter cells.
  • FIG. 77C shows the structure of E 3 P2.5. [0093] FIG.
  • FIG. 78 shows a table of third generation ELP-E3Ps. From top to bottom, SEQ ID NOs: 181-196 are shown.
  • FIG. 79A shows delivery activity of third generation ELP-E 3 Ps for Cre protein delivery to HEK293 Cre reporter cells (normalized to EEP13).
  • FIG. 79B shows a comparison of EEPs, E3P first generation, E3P second generation, and E3P third generation.
  • FIG. 80A shows strategy #1: Prevention of intramolecular crosslinking via rigid linkers.
  • (GGGGS) n corresponds to SEQ ID NO: 213
  • (EAAAK) n corresponds to SEQ ID NO: 214.
  • FIG. 80B shows strategy #2: Spacing cysteines via placement in hydrophobic block. SEQ ID NO: 132 is shown.
  • FIG. 81 shows crosslinking efficiency at 4 hours (left) and 24 hours (right) for ELP crosslinked nanoparticles.
  • FIG. 82 shows crosslinking of V4-ELP-Cys7-EEP13 and V4-ELP-[CEAAAK] 5 over time with and without siRNA.
  • FIG. 83 shows degree of crosslinking over time for V4-ELP-Cys7-EEP13 and V4- ELP-[CEAAAK]5 both with and without siRNA cargo.
  • FIG. 84 shows size analysis of Cys-containing ELP-EEP constructs with and without Cre-mRNA and over time.
  • FIG. 85 shows size analysis of Cys-containing ELPs (non-EEP13-functionalized) without mRNA.
  • FIG. 86A shows crosslinking of V4-ELP-Cys7-EEP13 particles for up to 24 hours.
  • FIG. 86B shows delivery efficacy for Cre protein and Cre mRNA after crosslinking for up to 24 hours.
  • FIG. 87A shows an image of gel band densitometry of V4-C4G3-EEP13 formulated with Cre mRNA.
  • FIG. 87B shows ELP-EEP13-mRNA nanoparticle crosslinking efficiencies.
  • FIG. 88 shows mRNA delivery efficiency of V4-ELP-EEP13 nanoparticles.
  • FIG. 89A shows V4-ELP-EEP13 +/- Cys7 at 10 uM w/500 nM siRNA, crosslinking at RT (rotating) for 24 hrs. 24% (-Cys7) and 66% (+Cys7) crosslinked per densitometry analysis.
  • FIG. 89B shows agarose gel shift assay of crosslinked and uncrosslinked ELP constructs with free and bound siRNA.
  • FIG. 90 shows biodistribution of siRNA cargo.
  • FIG. 90A shows quantification of radiant efficiency of crosslinked and uncrosslinked particles in the liver, lungs, spleen, kidneys, heart, femurs, and quads.
  • FIG. 90A shows quantification of radiant efficiency of crosslinked and uncrosslinked particles in the liver, lungs, spleen, kidneys, heart, femurs, and quads.
  • FIG. 90A shows quantification of radiant efficiency of crosslinked and uncrosslinked particles in the liver, lungs, spleen, kidneys,
  • FIG. 90B shows images of distribution of crosslinked and uncrosslinked particles to the liver, lungs, spleen, kidneys, heart, femurs, and quads.
  • FIG. 92A shows peptide placements in ELP-EEP13 variants modified with OTI peptide.
  • FIG. 92B shows ELP-EEP13 variants modified with OTI peptide.
  • FIG. 93A shows viability of untreated cells and cells treated with OTI-functionalized ELPs.
  • FIG. 93B shows delivery of ovalbumin protein using OTI-functionalized ELPs.
  • FIG. 94A shows viability of untreated cells and cells treated with varying ratios of N-terminal and middle OTI-modified ELPs.
  • FIG. 94B shows delivery of ovalbumin protein using varying ratios of N-terminal and middle OTI-modified ELPs.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Every amino acid contains an amine (-NH2) and a carboxylic acid (-COOH) functional group. Each amino acid contains a unique side chain, designated by the “R” substituent shown below.
  • Exemplary amino acids include, without limitation, alpha-amino acids such as D– and L–isomers of the 20 common naturally occurring alpha amino acids found in peptides, natural amino acids which are not the 20 common naturally occurring amino acids, and unnatural alpha-amino acids.
  • Amino acids used in the construction of peptides of the present disclosure may be prepared by organic synthesis, or obtained by other routes, such as, for example, degradation of or isolation from a natural source. Amino acids may be commercially available or may be synthesized. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • amino acid refers to a naturally occurring amino acid.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • the amino acid is an N-alkyl amino acid, where the hydrogen on any non-proline amine (N) is replaced with an alkyl (e.g., methyl (-CH 3 )) group.
  • the N-alkyl amino acid is sarcosine (Sar).
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Unnatural (or non-natural) amino acids refer to those not naturally incorporated
  • a compound provided herein comprises an amino acid side chain selected from the 20 proteinogenic amino acids (i.e., an amino acid incorporated into proteins during translation) shown in Table A.
  • amino acid may also refer to non-proteinogenic amino acids, such as, for example, selenocysteine (-CH2SeH).
  • Table A Amino acids and side chains.
  • Amino acids with hydrophobic side chains include Gly, Pro, Ala, Ile, Leu, Val, Phe, Met, Trp, and Tyr.
  • amino acids with hydrophobic side chains include Gly, Pro, Ala, Ile, Leu, Val, and Phe.
  • amino acids with hydrophobic side chains include Ala, Ile, Leu, and Val.
  • Amino acids with polar side chains include Gln, Asn, His, Ser, Thr , Tyr, Cys, Met, Trp.
  • amino acids with polar side chains include Gln, Asn, His, Ser, Thr , Tyr, Cys, Met, Trp.
  • amino acids with polar side chains include Gln, Asn, His, Ser, Thr , Tyr, Cys, Met, Trp.
  • 17/179 B0662.70120WO00 with polar side chains include Asn, Cys, Gln, Met, Ser, and Thr.
  • Amino acids with aromatic side chains include Phe, Trp, Tyr, and His.
  • Amino acids with hydrophobic aromatic side chains include Phe, Typ, and Tyr.
  • Amino acids with charged side chains include Asp, Glu, Arg, His, and Lys.
  • Negatively charged side chains include Asp and Glu.
  • Positively charged side chains include Arg, His, and Lys.
  • Neutral amino acids are selected from the group consisting of Ala, Ser, Val, Leu, Ile, Pro, Phe, Trp, Met, Gly, Thr, Cys, Tyr, Asn, and Gln.
  • a “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds.
  • the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long.
  • a protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification.
  • a protein may also be a single molecule or may be a multi-molecular complex.
  • a protein may be a fragment of a naturally occurring protein or peptide.
  • a protein may be naturally occurring, recombinant, synthetic, or any combination of these.
  • amino acid sequences one of skill will recognize that individual substitutions to a peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the term “homologous,” as used herein is an art-understood term that refers to nucleic acids or proteins that are highly related at the level of nucleotide or amino acid sequence. Nucleic acids or proteins that are homologous to each other are termed homologues.
  • Homologous may refer to the degree of sequence similarity between two sequences (i.e., nucleotide sequence or amino acid).
  • the homology percentage figures referred to herein reflect the maximal homology possible between two sequences, i.e., the percent homology when the two sequences are so aligned as to have the greatest number of matched (homologous) positions. Homology can be readily calculated by known methods
  • Methods commonly employed to determine homology between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining homology are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and PASTA Atschul, S. F. et al., J Molec. Biol., 215, 403 (1990)).
  • identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two amino acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity can be calculated by optimal alignment of the sequences using a similarity-scoring matrix such as the Blosum62 matrix described in Henikoff S. and Henikoff J.G., P.N.A.S. USA 1992, 89: 10915-10919. Calculation of the percentage identity and optimal alignment of two sequences using the
  • to “modulate” means to act as an antagonist, i.e., partially or fully inhibit, reduce, alleviate, block or prevent; or to increase or stimulate, i.e., to act as an agonist, partial agonist or inverse agonist.
  • the modulation may be direct or indirect or allosteric.
  • the term “inhibit” or “inhibition,” for example, in the context of enzymes refers to a reduction in the activity of the enzyme. In some embodiments, the term refers to a reduction of the level of enzyme activity to a level that is statistically significantly lower than an initial level, which may, for example, be a baseline level of enzyme activity.
  • the term refers to a reduction of the level of enzyme activity to a level that is less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of an initial level, which may, for example, be a baseline level of enzyme activity.
  • composition and “formulation” are used interchangeably.
  • a “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)).
  • primate e.g., cynomolgus monkey or rhesus monkey
  • commercially relevant mammal e.g., cattle, pig, horse, sheep, goat, cat, or dog
  • bird e.g., commercially relevant bird, such as
  • the non-human animal is a fish, reptile, or amphibian.
  • the non-human animal may be a male or female at any stage of development.
  • the non-human animal may be a transgenic animal or genetically engineered animal.
  • the term “patient” refers to a human subject in need of treatment of a disease.
  • tissue samples such as tissue sections and needle biopsies of a tissue
  • cell samples e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection
  • samples of whole organisms such as samples of yeasts or bacteria
  • 20/179 B0662.70120WO00 (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise).
  • Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample.
  • target tissue refers to any biological tissue of a subject (including a group of cells, a body part, or an organ) or a part thereof, including blood and/or lymph vessels, which is the object to which a compound, particle, and/or composition of the present disclosure is delivered.
  • a target tissue may be an abnormal or unhealthy tissue, which may need to be treated.
  • a target tissue may also be a normal or healthy tissue that is under a higher than normal risk of becoming abnormal or unhealthy, which may need to be prevented.
  • the target tissue is the liver.
  • the target tissue is the lung.
  • a “non-target tissue” is any biological tissue of a subject (including a group of cells, a body part, or an organ) or a part thereof, including blood and/or lymph vessels, which is not a target tissue
  • the term “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.
  • the terms “condition,” “disease,” and “disorder” are used interchangeably.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed.
  • treatment may be administered in the absence of signs or symptoms of the disease.
  • treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
  • prevent refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease.
  • the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.
  • an “effective amount” of a compound or agent described herein refers to an amount sufficient to elicit the desired biological response.
  • An effective amount of a compound or agent described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular compound, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the subject.
  • an effective amount is a therapeutically effective amount.
  • an effective amount is a prophylactic treatment.
  • an effective amount is the amount of a compound or agent described herein in a single dose.
  • an effective amount is the combined amounts of a compound or agent described herein in multiple doses.
  • the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage is delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • an effective amount of a compound or agent for administration one or more times a day to a 70 kg adult human comprises about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.
  • the compounds or agents of the present disclosure are administered orally or parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
  • dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • a “therapeutically effective amount” of a compound or agent described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a compound or agent means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.
  • a therapeutically effective amount of a compound provided herein is an amount sufficient to deliver an agent to a subject, cell, or biological sample.
  • a “prophylactically effective amount” of a compound or agent described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence.
  • a prophylactically effective amount of a compound or agent means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • a prophylactically effective amount of a compound provided herein is an amount sufficient to deliver an agent to a subject, cell, or biological sample.
  • infectious disease refers to diseases or disorders associated with infection, presence, or growth of a pathogenic agent in a host subject.
  • infectious disease is spread directly or indirectly from one host subject to another.
  • Infectious pathogenic agents include, but are not limited to, viruses, bacteria, fungi, protozoa, parasites, and aberrant proteins.
  • Viruses associated with infectious disease include but are not limited to, herpes simplex viruses, cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, herpes viruses, Vesicular stomatitis virus, Hepatitis viruses, Rhinovints, Coronavirus, Influenza viruses, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Simian Immunodeficiency viruses, Human Immunodeficiency viruses.
  • Protozoa associated with infectious disease include, but are not limited to, Entamoeba histolytica, Toxoplasma gondii, Schistosoma mansoni, Cryptosporidium sp., Leishmania donovani, Neospora caninum, and Plasmodium spp.
  • Bacteria associated with infectious disease include, but are not limited to, M. tuberculosis,
  • the term “genetic disease” refers to a disease caused by one or more abnormalities in the genome of a subject, such as a disease that is present from birth of the subject. Genetic diseases may be heritable and may be passed down from the parents’ genes. A genetic disease may also be caused by mutations or changes of the DNAs and/or RNAs of the subject. In such cases, the genetic disease will be heritable if it occurs in the germline.
  • Exemplary genetic diseases include Aarskog-Scott syndrome, Aase syndrome, achondroplasia, acrodysostosis, addiction, adreno-leukodystrophy, albinism, ablepharon-macrostomia syndrome, alagille syndrome, alkaptonuria, alpha-1 antitrypsin deficiency, Alport’s syndrome, Alzheimer’s disease, asthma, autoimmune polyglandular syndrome, androgen insensitivity syndrome, Angelman syndrome, ataxia, ataxia telangiectasia, atherosclerosis, attention deficit hyperactivity disorder (ADHD), autism, baldness, Batten disease, Beckwith- Wiedemann syndrome, Best disease, bipolar disorder, brachydactyl, breast cancer, Burkitt lymphoma, chronic myeloid leukemia, Charcot-Marie-Tooth disease, Crohn’s disease, cleft lip, Cockayne syndrome, Coffin Lowry syndrome, colon cancer, congenital adrenal hyperplasia, Cornelia de
  • a proliferative disease refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990).
  • a proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis.
  • proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases)
  • the pathological angiogenesis as in proliferative retinopathy and tumor metastasis.
  • Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.
  • angiogenesis refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue.
  • angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer.
  • Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF).
  • VEGF growth factors
  • “Pathological angiogenesis” refers to abnormal (e.g., excessive or insufficient) angiogenesis that amounts to and/or is associated with a disease.
  • the terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue.
  • a neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including
  • a “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin.
  • a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites.
  • Exemplary benign neoplasms include lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias.
  • certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor’s neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.”
  • An exemplary pre-malignant neoplasm is a teratoma.
  • a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue.
  • a malignant neoplasm generally has the capacity to metastasize to distant sites.
  • metastasis refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located.
  • a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.
  • cancer refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues.
  • the cancer may be a solid tumor.
  • the cancer may be a hematological malignancy.
  • Exemplary cancers include acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordom
  • endometrial cancer e.g., uterine cancer, uterine sarcoma
  • esophageal cancer e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma
  • Ewing’s sarcoma e.g., ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g.
  • Wilms tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic
  • HCC hepatocellular cancer
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • MLS adenocarcinoma of the lung
  • myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g.,bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’s disease
  • CML chronic myelocytic le
  • Neurodegenerative diseases refer to a type of neurological disease marked by the loss of nerve cells, including, but not limited to, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, tauopathies (including frontotemporal dementia), and Huntington’s disease.
  • neurological diseases include, but are not limited to, headache, stupor and coma, dementia, seizure, sleep disorders, trauma, infections, neoplasms, neuro-ophthalmology, movement disorders, demyelinating diseases, spinal cord disorders, and disorders of peripheral nerves, muscle and neuromuscular junctions.
  • Addiction and mental illness include, but are not limited to, bipolar disorder and schizophrenia, are also included in the definition of neurological diseases.
  • Further examples of neurological diseases include acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpers’ disease;
  • psychiatric disorder refers to a disease of the mind and includes diseases and disorders listed in the Diagnostic and Statistical Manual of Mental Disorders - Fourth Edition (DSM-IV), published by the American Psychiatric Association, Washington D. C. (1994).
  • Psychiatric disorders include, but are not limited to, anxiety disorders (e.g., acute stress disorder agoraphobia, generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, posttraumatic stress disorder, separation anxiety disorder, social phobia, and specific phobia), childhood disorders, (e.g., attention-deficit/hyperactivity disorder, conduct disorder, and oppositional defiant disorder), eating disorders (e.g., anorexia nervosa and bulimia nervosa), mood disorders (e.g., depression, bipolar disorder, cyclothymic disorder, dysthymic disorder, and major depressive disorder), personality disorders (e.g., antisocial personality disorder, avoidant personality disorder, borderline personality disorder, dependent personality disorder, histrionic personality disorder, narcissistic personality disorder, obsessive-compulsive personality disorder, paranoid personality disorder, schizoid personality disorder, and schizotypal personality disorder), psychotic disorders (e.g., brief psychotic disorder,
  • metabolic disorder refers to any disorder that involves an alteration in the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or a combination thereof.
  • a metabolic disorder is associated with either a deficiency or excess in a metabolic pathway resulting in an imbalance in metabolism of nucleic acids, proteins, lipids, and/or
  • Factors affecting metabolism include, and are not limited to, the endocrine (hormonal) control system (e.g., the insulin pathway, the enteroendocrine hormones including GLP-1, PYY or the like), the neural control system (e.g., GLP-1 in the brain), or the like.
  • Examples of metabolic disorders include, but are not limited to, diabetes (e.g., Type I diabetes, Type II diabetes, gestational diabetes), hyperglycemia, hyperinsulinemia, insulin resistance, and obesity.
  • the terms “inflammatory disease” and “inflammatory condition” are used interchangeably herein, and refer to a disease or condition caused by, resulting from, or resulting in inflammation.
  • Inflammatory diseases and conditions include those diseases, disorders or conditions that are characterized by signs of pain (dolor, from the generation of noxious substances and the stimulation of nerves), heat (calor, from vasodilatation), redness (rubor, from vasodilatation and increased blood flow), swelling (tumor, from excessive inflow or restricted outflow of fluid), and/or loss of function (functio laesa, which can be partial or complete, temporary or permanent.
  • Inflammation takes on many forms and includes, but is not limited to, acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, obliterative, parenchymatous, plastic, productive, proliferous, pseudomembranous, purulent, sclerosing, seroplastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and/or ulcerative inflammation.
  • inflammatory disease may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and/or T-lymphocytes leading to abnormal tissue damage and/or cell death.
  • An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes.
  • Inflammatory diseases include, without limitation, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis, Sjogren’s syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto’s thyroiditis, Graves’ disease, Goodpasture’s disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, per
  • An ocular inflammatory disease includes, but is not limited to, post-surgical inflammation.
  • Additional exemplary inflammatory conditions include, but are not limited to, inflammation associated with acne, anemia (e.g., aplastic anemia, haemolytic autoimmune anaemia), asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu's arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis and Reiter's arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructive pulmonary disease, cermatomyositis, diver
  • bowel disease e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis
  • IBS inflammatory bowel syndrome
  • lupus multiple sclerosis, morphea, myeasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus vulgaris, pernicious aneaemia, peptic ulcers, polymyositis, primary biliary cirrhosis, neuroinflammation associated with brain disorders (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, reperfusion injury, regional enteritis, rheumatic fever, systemic lupus erythematosus, schleroderma, scierodom
  • brain disorders e.g., Parkinson's disease, Hunt
  • the inflammatory disorder is selected from arthritis (e.g., rheumatoid arthritis), inflammatory bowel disease, inflammatory bowel syndrome, asthma, psoriasis, endometriosis, interstitial cystitis and prostatistis.
  • the inflammatory condition is an acute inflammatory condition (e.g., for example, inflammation resulting from infection).
  • the inflammatory condition is a chronic inflammatory condition (e.g., conditions resulting from asthma, arthritis and inflammatory bowel disease).
  • the compounds may also be useful in treating inflammation associated with trauma and non-inflammatory myalgia.
  • the compounds disclosed herein may also be useful in treating inflammation associated with cancer.
  • MSD muscleculoskeletal disease
  • an MSD refers to an injury and/or pain in a subject’s joints, ligaments, muscles, nerves, tendons, and structures that support limbs, neck, and back.
  • an MSD is a degenerative disease.
  • an MSD includes an inflammatory condition.
  • Body parts of a subject that may be associated with MSDs include upper and lower back, neck, shoulders, and extremities (arms, legs, feet, and hands).
  • an MSD is a bone disease, such as achondroplasia, acromegaly, bone callus, bone demineralization, bone fracture, bone marrow disease, bone marrow neoplasm, dyskeratosis congenita, leukemia (e.g., hairy cell leukemia, lymphocytic leukemia, myeloid leukemia, Philadelphia chromosome-positive leukemia, plasma cell leukemia, stem cell leukemia), systemic mastocytosis, myelodysplastic syndromes, paroxysmal nocturnal hemoglobinuria, myeloid sarcoma, myeloproliferative disorders, multiple myeloma, polycythemia vera, pearson marrow-pancreas syndrome, bone neoplasm, bone marrow neoplasm, Ewing sarcoma, osteochondroma, osteoclastoma, osteosarcoma, brachydactyly,
  • an MSD is a cartilage disease, such as cartilage neoplasm, osteochondritis, osteochondrodysplasia, Kashin-Beck disease, or Leri-Weill dyschondrosteosis.
  • an MSD is hernia, such as intervertebral disk hernia.
  • an MSD is a joint disease, such as arthralgia, arthritis (e.g., gout (e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome), Lyme disease, osteoarthritis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, Felty syndrome, synovitis, Blau syndrome, nail-patella syndrome, spondyloarthropathy, reactive arthritis, Stickler syndrome, synovial membrane disease, synovitis, or Blau syndrome.
  • arthritis e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome
  • Lyme disease e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome
  • osteoarthritis e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome
  • Lyme disease e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome
  • an MSD is a muscle disease, such as Barth syndrome, mitochondrial encephalomyopathy, MELAS syndrome, MERRF syndrome, MNGIE syndrome, mitochondrial myopathy, Kearns-Sayre syndrome, myalgia, fibromyalgia, polymyalgia rheumatica, myoma, myositis, dermatomyositis, neuromuscular disease, Kearns-Sayre syndrome, muscular dystrophy, myasthenia, congenital myasthenic syndrome, Lambert-Eaton myasthenic syndrome, myasthenia gravis, myotonia, myotonia congenita, spinal muscular atrophy, tetany, ophthalmoplegia, or rhabdomyolysis.
  • a muscle disease such as Barth syndrome, mitochondrial encephalomyopathy, MELAS syndrome, MERRF syndrome, MNGIE syndrome, mitochondrial myopathy, Kearns-Sayre syndrome, myal
  • an MSD is Proteus syndrome.
  • an MSD is a rheumatic diseases, such as arthritis (e.g., gout (e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan lyme disease)), osteoarthritis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, Felty syndrome, synovitis, Blau syndrome, gout (e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome), polymyalgia rheumatica, rheumatic fever, rheumatic heart disease, or Sjogren syndrome.
  • an MSD is Schwartz-Jampel syndrome.
  • an MSD is a skeleton disease, such as Leri-Weill dyschondrosteosis, skeleton malformations, Melnick-
  • a “hematological disease” includes a disease which affects a hematopoietic cell or tissue. Hematological diseases include diseases associated with aberrant hematological content and/or function.
  • hematological diseases include diseases resulting from bone marrow irradiation or chemotherapy treatments for cancer, diseases such as pernicious anemia, hemorrhagic anemia, hemolytic anemia, aplastic anemia, sickle cell anemia, sideroblastic anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HTV, hepatitis virus or other viruses, myelophthisic anemias caused by marrow deficiencies, renal failure resulting from anemia, anemia, polycythemia, infectious mononucleosis (EVI), acute non-lymphocytic leukemia (ANLL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute myelomonocytic leukemia (AMMoL), polycythemia vera, lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, Wilm’s tumor, Ewing’s sarcoma,
  • Immune disorders include, but are not limited to, arthritis (including rheumatoid arthritis, spondyloarthopathies, gouty arthritis, degenerative joint diseases such as osteoarthritis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, undifferentiated spondylitis, Behcet's disease, haemolytic autoimmune anaemias, multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute painful shoulder, psoriatic, and juvenile arthritis), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin condition (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itch)), enuresis, eosinophilic disease, gastrointestinal disorder (e.g., selected from p
  • Behcet's syndrome indeterminate colitis and inflammatory bowel syndrome (IBS)
  • a gastroprokinetic agent e.g., ileus, postoperative ileus and ileus during sepsis; gastroesophageal reflux disease (GORD, or its synonym GERD); eosinophilic esophagitis, gastroparesis such as diabetic gastroparesis; food intolerances and food allergies and other functional bowel disorders, such as non-ulcerative dyspepsia (NUD) and non- cardiac chest pain (NCCP, including costo-chondritis)).
  • NUD non-ulcerative dyspepsia
  • NCCP non- cardiac chest pain
  • an “autoimmune disease” or “autoimmune disorder” refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture’s disease which may affect the basement membrane in both the lung and kidney).
  • the treatment of autoimmune diseases is typically with immunosuppression, e.g., medications which decrease the immune response.
  • Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture’s syndrome, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, anti-phospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener’s granulomatosis, microscopic polyangiitis), uveitis, Sjogren’s syndrome, Crohn’s disease, Reiter’s syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barré syndrome, Hashimoto’s thyroiditis, and cardio
  • the inflammatory disorder and/or the immune disorder is a gastrointestinal disorder.
  • the gastrointestinal disorder is selected from gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)).
  • the gastrointestinal disorder is inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the skin condition is psoriasis. In certain embodiments, the skin condition is pruritis.
  • the term “cardiovascular disease” refers to diseases and disorders of the heart and circulatory system.
  • Exemplary cardiovascular diseases include, but are not limited to acute coronary syndrome, angina, arrhythmia, arteriosclerosis, atherosclerosis, atherosclerotic lesions, carotid atherosclerosis, cerebrovascular disease, cerebral infarction, congestive heart failure, congenital heart disease, coronary heart disease, coronary artery disease, coronary plaque stabilization, dyslipidemias, dyslipoproteinemias, endothelium dysfunctions, familial hypercholeasterolemia, familial combined hyperlipidemia, hypoalphalipoproteinemia, hypertriglyceridemia, hyperbetalipoproteinemia, hypercholesterolemia, hypertension, hyperlipidemia, intermittent claudication, ischemia, ischemia reperfusion injury, ischemic heart diseases, cardiac ischemia, metabolic syndrome, multi-infarct dementia, myocardial infarction, obesity, peripheral vascular disease, reperfusion injury, restenosis, renal artery atherosclerosis, rheumatic heart disease
  • lung disease refers to a disease of the lung.
  • lung diseases include, but are not limited to, bronchiectasis, bronchitis, bronchopulmonary dysplasia, interstitial lung disease, occupational lung disease, emphysema, cystic fibrosis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), asthma (e.g., intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma), chronic bronchitis, chronic obstructive pulmonary disease (COPD), emphysema, interstitial lung disease, sarcoidosis, asbestosis, aspergilloma, aspergillosis, pneumonia (e.g., lobar pneumonia, multilobar pneumonia, bronchial pneumonia, interstitial pneumonia), pulmonary fibrosis, pulmonary tuberculosis, rheumatoid lung disease, pulmonary embolism, and lung cancer (e.g., non-bronchiectasis, bronchi
  • elastin-like polypeptides 38/179 B0662.70120WO00 DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
  • compositions comprising a compound provided herein and an agent.
  • methods of delivering an agent and methods of treating or preventing a disease are also provided herein.
  • a compound comprising an elastin-like polypeptide chemically or recombinantly conjugated to one or more endosomal escape peptides.
  • the compound comprises an elastin-like polypeptide chemically conjugated to one or more endosomal escape peptides.
  • the elastin-like polypeptide is chemically conjugated to one or more endosomal escape peptides via a chemical reaction that creates a covalent bond.
  • the elastin-like polypeptide is chemically conjugated to one or more endosomal escape peptides via a chemical reaction with one or more additional reagents.
  • the elastin- like polypeptide is chemically conjugated to one or more endosomal escape peptides via reaction at a lysine residue, a cysteine residue, or a tyrosine residue. In some embodiments, the elastin-like polypeptide is chemically conjugated to one or more endosomal escape peptides via reaction with one or more of N-hydroxysuccinimide esters, isocyanates, isothiocyanates, benzoyl fluorides, maleimides, iodoacetamides, 2-thiopyridines, 3- arylpropiolonitriles, diazonium salts, or cyclic diazodicarboxyamide derivatives.
  • the elastin-like polypeptide is chemically conjugated to one or more endosomal escape peptides via acylation, disulfide bond formation, amide bond formation, electrophilic aromatic substitution, or Mannich reaction. In some embodiments, the elastin- like polypeptide is chemically conjugated to one or more endosomal escape peptides via reaction at the N-terminus. In some embodiments, the elastin-like polypeptide is chemically conjugated to one or more endosomal escape peptides via reaction at the C-terminus.
  • the elastin-like polypeptide is chemically conjugated to one or more endosomal escape peptides via functionalization of the N- or C-terminus amino acid.
  • the compound comprises an elastin-like polypeptide recombinantly conjugated to one or more endosomal escape peptides.
  • the recombinant conjugation comprises producing the compound via an oligonucleotide sequence encoding both the elastin-like polypeptide and one or more endosomal escape
  • the compound comprises an elastin-like polypeptide recombinantly conjugated to one or more endosomal escape peptides via a method provided herein.
  • the elastin-like polypeptide comprises a hydrophilic block fused to a hydrophobic block.
  • the elastin-like polypeptide comprises a hydrophilic block fused to a hydrophobic block separated by a crosslinking block.
  • the elastin-like polypeptide is conjugated via an intervening agent to one or more endosomal escape peptides.
  • the elastin-like polypeptide is conjugated at its C-terminus via an intervening agent to one or more endosomal escape peptides. In some embodiments, the elastin-like polypeptide is conjugated at its N-terminus via an intervening agent to one or more endosomal escape peptides. In some embodiments, the intervening agent is any agent provided herein. In some embodiments, the intervening agent is a peptide. In some embodiments, the peptide is ovalbumin-derived MHC class I epitope or ovalbumin-derived MHC class II epitope. In some embodiments, the peptide is ovalbumin-derived MHC class I epitope.
  • the peptide is ovalbumin-derived MHC class II epitope.
  • the hydrophilic block has the sequence: [VPGXG]n (SEQ ID NO: 114) wherein: each instance of X is independently any amino acid except proline; and n is at least 5.
  • the hydrophilic block has the sequence: [VPGXG]n (SEQ ID NO: 114) wherein: each instance of X is any amino acid except proline; and n is at least 5.
  • n is at least 5.
  • n is at least 10.
  • n is at least 20. In some embodiments, n is at least 30.
  • n is 5-200. In some embodiments, n is 5-150. In some embodiments, n is 5-100. In some embodiments, n is 5-75. In some embodiments, n is 5-50. In some embodiments, n is 10-200. In some embodiments, n is 10-150. In some embodiments, n is 10-100. In some embodiments, n is 10-75. In some embodiments, n is 10-50. In some embodiments, n is 25- 200. In some embodiments, n is 25-150. In some embodiments, n is 25-100. In some
  • n is 25-75. In some embodiments, n is 25-50. In some embodiments, n is 30- 70. [00166] In some embodiments, each instance of X is independently A, R, N, D, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, or V. In some embodiments, each instance of X is independently E, V, A, G, S, or Q. In some embodiments, each instance of X is independently G, S, or Q. In some embodiments, each instance of X is independently E, V, A, or G. In some embodiments, each instance of X is E, V, A, or G. In some embodiments, each instance of X is E, V, A, or G.
  • each instance of X is independently E or V. In some embodiments, each instance of X is E or V. In some embodiments, each instance of X is independently E or A. In some embodiments, each instance of X is independently E or G. In some embodiments, each instance of X is independently A or G. In some embodiments, each instance of X is independently A or V. In some embodiments, each instance of X is independently V or G. In some embodiments, each instance of X is independently S or G. In some embodiments, each instance of X is independently Q or G. In some embodiments, each instance of X is G.
  • each instance of X is independently X 1 or X 2 . In some embodiments, X is X 1 or X 2 .
  • each instance of X 1 is A, R, N, D, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, or V.
  • each instance of X 1 is S, Q, E, V, A, or G.
  • each instance of X 1 is S, Q, V, A, or G.
  • each instance of X 1 is E, V, A, or G.
  • each instance of X 1 is V or A.
  • each instance of X 1 is V or each instance of X 1 is A.
  • each instance of X 1 is G, S, or Q.
  • each instance of X 1 is G, each instance of X 1 is S, or each instance of X 1 is Q. In some embodiments, each instance of X 1 is A. In some embodiments, each instance of X 1 is V. In some embodiments, each instance of
  • each instance of X 1 is G.
  • each instance of X 1 is S.
  • each instance of X 1 is Q.
  • each instance of X 2 is A, R, N, D, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, or V.
  • each instance of X 2 is E, V, A, or G.
  • each instance of X 2 is E or G.
  • each instance of X 2 is E or each instance of X 2 is G.
  • each instance of X 2 is E.
  • each instance of X 2 is G.
  • each instance of X 1 is V, and each instance of X 2 is E. In some embodiments, each instance of X 1 is V, and each instance of X 2 is G. In some embodiments, each instance of X 1 is A, and each instance of X 2 is E. In some embodiments, each instance of X 1 is A, and each instance of X 2 is G. In some embodiments, each instance of X 1 is G, and each instance of X 2 is G. In some embodiments, each instance of X 1 is G, and each instance of X 2 is E. In some embodiments, each instance of X 1 is S, and each instance of X 2 is G.
  • each instance of X 1 is S, and each instance of X 2 is E. In some embodiments, each instance of X 1 is Q, and each instance of X 2 is G. In some embodiments, each instance of X 1 is Q, and each instance of X 2 is E. [00172] In some embodiments, the hydrophilic block has the sequence: [(VPGVG)(VPGEG)(VPGVG)(VPGEG)(VPGVG)]10 (SEQ ID NO: 23). [00173] In some embodiments, the hydrophilic block has the sequence: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)] 10 (SEQ ID NO: 24).
  • the hydrophilic block has the sequence: [(VPGSG)(VPGGG)(VPGSG)(VPGGG)(VPGSG)]10 (SEQ ID NO: 202). [00175] In some embodiments, the hydrophilic block has the sequence: [(VPGQG)(VPGGG)(VPGQG)(VPGGG)(VPGQG)] 10 (SEQ ID NO: 215). [00176] In some embodiments, the hydrophilic block has the sequence: [(VPGGG)(VPGGG)(VPGGG)(VPGGG)]10 (SEQ ID NO: 216). [00177] In some embodiments, the crosslinking block comprises one or more cysteine residues.
  • the crosslinking block has the sequence –CCCCGGG– (SEQ ID NO: 19). In some embodiments, the crosslinking block has the sequence –[CAP]5– (SEQ ID NO: 125). In some embodiments, the crosslinking block has the sequence –[CEAAAK] 5 – (SEQ ID NO: 126). In some embodiments, the crosslinking block has the sequence –C 5 G 3 – (SEQ ID NO: 127). [00178] In some embodiments, the crosslinking block is 4-25 amino acids in length. In some embodiments, the crosslinking block is 4-15 amino acids in length. In some embodiments, the
  • crosslinking block is 4-10 amino acids in length.
  • the crosslinking block is 6-25 amino acids in length. In some embodiments, the crosslinking block is 6-15 amino acids in length. In some embodiments, the crosslinking block is 6-10 amino acids in length. In some embodiments, the crosslinking block is 10-25 amino acids in length. In some embodiments, the crosslinking block is 10-15 amino acids in length. In some embodiments, the crosslinking block is 10-20 amino acids in length. In some embodiments, the crosslinking block is 15-25 amino acids in length. In some embodiments, the crosslinking block is 20-25 amino acids.
  • the hydrophobic block comprises one or more instances of G, H, P, A, I, L, M, F, W, Y, V, or C. In some embodiments, the hydrophobic block comprises one or more instances of G, H, P, A, I, L, M, F, W, Y, or V. In some embodiments, the hydrophobic block comprises one or more instances of G, P, H, Y, V, I, L, F, or W. In some embodiments, the hydrophobic block comprises one or more instances of G, P, H, Y, V, I, L, F, W, or C. In some embodiments, the hydrophobic block comprises one or more tyrosine residues.
  • the hydrophobic block comprises one or more cysteine residues.
  • the hydrophobic block has the sequence: (Z 1 PGZ 2 G)m (SEQ ID NO: 25) wherein: Z 1 and Z 2 are each independently a hydrophobic amino acid; and m is an integer from 5 to 400.
  • each instance of Z 1 is independently a hydrophobic amino acid.
  • each instance of Z 1 is independently P, A, I, L, M, F, W, Y, or V.
  • each instance of Z 1 is independently I, V, A, or L.
  • each instance of Z 1 is independently I, V, or L.
  • each instance of Z 1 is independently I, V, or A. In some embodiments, each instance of Z 1 is independently I, A, or L. In some embodiments, each instance of Z 1 is independently V, A, or L. In some embodiments, each instance of Z 1 is independently I or V. In some embodiments, each instance of Z 1 is independently I or L. In some embodiments, each instance of Z 1 is independently I or A. In some embodiments, each instance of Z 1 is independently V or A. In some embodiments, each instance of Z 1 is independently V or L. In some embodiments, each instance of Z 1 is independently A or L. In some embodiments, Z 1 is I. In some embodiments, Z 1 is L. In some embodiments, Z 1 is V. In some embodiments, Z 1 is A. In some embodiments, Z 1 is I. In some embodiments, Z 1 is L. In some embodiments, Z 1 is V. In some embodiments, Z 1 is A.
  • each instance of Z 2 is independently a hydrophobic amino acid. In some embodiments, each instance of Z 2 is independently C, P, A, I, L, M, F, W, Y, or V. In some embodiments, each instance of Z 2 is independently C, P, H, A, I, L, M, F, W, Y, or V. In some embodiments, each instance of Z 2 is independently C, H, Y, V, I, L, F, or W. In some embodiments, each instance of Z 2 is independently P, A, I, L, M, F, W, Y, or V.
  • each instance of Z 2 is independently P, H, A, I, L, M, F, W, Y, or V. In some embodiments, each instance of Z 2 is independently H, Y, V, I, L, F, or W. In some embodiments, each instance of Z 2 is independently C, H, or Y. In some embodiments, Z 2 is an aromatic amino acid. In some embodiments, Z 2 is H, F, Y, or W. In some embodiments, Z 2 is H, F, or Y. In some embodiments, Z 2 is H, F, or W. In some embodiments, Z 2 is H, Y, or W. In some embodiments, Z 2 is F, Y, or W. In some embodiments, Z 2 is H or F.
  • Z 2 is H or Y. In some embodiments, Z 2 is H or W. In some embodiments, Z 2 is F, or Y. In some embodiments, Z 2 is F or W. In some embodiments, Z 2 is Y or W. In some embodiments, at least one instance of Z 2 is H. In some embodiments, Z 2 is H. In some embodiments, Z 2 is F. In some embodiments, at least one instance of Z 2 is Y. In some embodiments, Z 2 is Y. In some embodiments, Z 2 is W. In some embodiments, at least one instance of Z 2 is C. [00183] In some embodiments, m is an integer from 5 to 400. In some embodiments, m is an integer from 5 to 300.
  • m is an integer from 5 to 200. In some embodiments, m is an integer from 5 to 100. In some embodiments, m is an integer from 15 to 400. In some embodiments, m is an integer from 15 to 300. In some embodiments, m is an integer from 15 to 200. In some embodiments, m is an integer from 15 to 100. In some embodiments, m is an integer from 25 to 400. In some embodiments, m is an integer from 25 to 300. In some embodiments, m is an integer from 25 to 200. In some embodiments, m is an integer from 25 to 100. In some embodiments, m is an integer from 50 to 400. In some embodiments, m is an integer from 50 to 300.
  • m is an integer from 50 to 200. In some embodiments, m is an integer from 50 to 100.
  • the hydrophobic block has the sequence: [(IPGZ 2 G)2(VPGZ 2 G)(IPGZ 2 G)2]m1 (SEQ ID NO: 26), wherein m1 is an integer from 1 to 80.
  • m1 is an integer from 1 to 70. In some embodiments, m1 is an integer from 1 to 60. In some embodiments, m1 is an integer from 1 to 50. In some embodiments, m1 is an integer from 1 to 40. In some embodiments, m1 is an integer from 1 to 30. In some embodiments, m1 is an integer from 1 to 20. In some embodiments, m1 is an
  • m1 is an integer from 5 to 30. In some embodiments, m1 is an integer from 5 to 40. In some embodiments, m1 is an integer from 5 to 50. In some embodiments, m1 is an integer from 5 to 60. In some embodiments, m1 is an integer from 5 to 70. In some embodiments, m1 is an integer from 5 to 80. In some embodiments, m1 is an integer from 1 to 20. In some embodiments, m1 is an integer from 20 to 40. In some embodiments, m1 is an integer from 40 to 60. In some embodiments, m1 is an integer from 60 to 80.
  • the hydrophobic block has the sequence: [(IPGVG)2(VPGYG)(IPGVG)2]15 (SEQ ID NO: 27). [00187] In some embodiments, the hydrophobic block has the sequence: [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 28). [00188] In some embodiments, the hydrophobic block has the sequence: [(IPGZ 2 G)2(VPGYG)(IPGZ 2 G)2]15 (SEQ ID NO: 128); wherein: each instance of Z 2 is independently C or H. [00189] In some embodiments, three instances of Z 2 are C. In some embodiments, five instances of Z 2 are C.
  • seven instances of Z 2 are C. In some embodiments, thirteen instances of Z 2 are C. In some embodiments, thirteen instances of Z 2 are C. In some embodiments, three instances of Z 2 are C and the remaining instances of Z 2 are H. In some embodiments, five instances of Z 2 are C and the remaining instances of Z 2 are H. In some embodiments, seven instances of Z 2 are C and the remaining instances of Z 2 are H. In some embodiments, thirteen instances of Z 2 are C and the remaining instances of Z 2 are H.
  • the hydrophobic block has the sequence: [IPGCGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGH GIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPG HGIPGHGIPGCGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIP GHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIP GHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIP GHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIP GHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIP
  • the elastin-like polypeptide comprises the sequence of: [VPGXG] n –CCCCGGG–(Z 1 PGZ 2 G) m (SEQ ID NO: 29) wherein: each instance of X is any amino acid except proline; n is at least 5; Z 1 and Z 2 are each independently a hydrophobic amino acid; and m is an integer from 5 to 400.
  • the elastin-like polypeptide comprises the sequence of: [(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)] n1 –CCCCGGG–(Z 1 PGZ 2 G) m (SEQ ID NO: 129) wherein: X 1 and X 2 are any amino acid except proline; each instance of X 1 is the same; each instance of X 2 is the same;
  • the elastin-like polypeptide comprises the sequence of: [VPGXG]n–CCCCGGG–[(IPGZ 2 G)2(VPGZ 2 G)(IPGZ 2 G)2]m1 (SEQ ID NO: 31) wherein: each instance of X is any amino acid except proline; n is at least 5; m1 is an integer from 1 to 80; and each instance of Z 2 is independently a hydrophobic amino acid.
  • the elastin-like polypeptide comprises the sequence of: [(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)]n1–CCCCGGG– [(IPGZ 2 G) 2 (VPGZ 2 G)(IPGZ 2 G) 2 ] m1 (SEQ ID NO: 32)
  • the elastin-like polypeptide comprises the sequence of: [(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)] 10 –CCCCGGG– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 217), wherein: each instance of X 1 is independently G, S, Q, V, or A; and each instance of X 2 is independently E or G.
  • the elastin-like polypeptide comprises the sequence of: [(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)]10–CCCCGGG– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 33), wherein: each instance of X 1 is independently V or A; and each instance of X 2 is independently E or G.
  • the elastin-like polypeptide comprises the sequence of: [(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)]10–CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 33), wherein: X 1 is V or A; and X 2 is E or G.
  • the elastin-like polypeptide comprises the sequence of: [(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)(VPGX 2 G)(VPGX 1 G)] 10 –CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 218), wherein: each instance of X 1 is independently G, S, or Q; and each instance of X 2 is G. [00201] In some embodiments, the elastin-like polypeptide comprises a sequence of:
  • the elastin-like polypeptide comprises a sequence of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)] 10 –CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 113).
  • the elastin-like polypeptide comprises a sequence of: [(VPGGG)(VPGGG)(VPGGG)(VPGGG)] 10 – CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 131). [00204] In some embodiments, the elastin-like polypeptide comprises a sequence of: [(VPGSG)(VPGGG)(VPGSG)(VPGGG)(VPGSG)] 10 – CCCCGGG– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 219).
  • the elastin-like polypeptide comprises a sequence of: [(VPGQG)(VPGGG)(VPGQG)(VPGGG)(VPGQG)]10– CCCCGGG– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 220). [00206] In some embodiments, the elastin-like polypeptide comprises a sequence of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–[CAP]5– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 199).
  • the elastin-like polypeptide comprises a sequence of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–[CEAAAK]5– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 200). [00208] In some embodiments, the elastin-like polypeptide comprises a sequence of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–C5G3– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 201).
  • the elastin-like polypeptide comprises a sequence of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–[(IPGZ 2 G)2(VPGYG)(IPG Z 2 G)2]15 (SEQ ID NO: 132); wherein: each instance of Z 2 is independently C or H.
  • the elastin-like polypeptide has the sequence of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)] 10 – [IPGCGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPG HGIPGHGVPGYGIPGHGIPGHGIPG HGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPG HGIPGHGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGCGVPGYGIPGHGIPGHGIPGHGIPGHGVPGYGIPGHGIPGHGIPGHGIPGHGVP
  • the elastin-like polypeptide is a polypeptide disclosed in Kim, W. et al. Recombinant Amphiphilic Protein Micelles for Drug Delivery. Langmuir, 2011, 27, 14329-14334, in Kim, W. et al. Targeted Antithrombotic Protein Micelles. Angew. Chem. Int. Ed., 2015, 54, 1461-1465, in Abdelghani, M. et al. Applications of elastin-like polypeptides
  • Endosomal escape peptides disrupt endosomal membranes, acting after endocytosis to trigger cargo release into the cytosol.
  • the endosomal escape peptide is a membrane active peptide.
  • the endosomal escape peptide facilitates entry of macromolecular cargo into the cytosol following endocytosis.
  • a compound provided herein e.g., an ELP-EEP compound
  • a compound provided herein e.g., an ELP-EEP compound
  • the endosomal escape peptide is not a cell penetrating peptide (CPP).
  • CPPs function to drive nanoparticle uptake into cells.
  • CPPs e.g. Tat, 11 penetratin, 41 transportan-10, 50 Aurein1.2, or oligoarginine 49 demonstrate non-specific membrane disruptive capacity that facilitates cargo internalization at the level of the plasma membrane via translocation or endocytosis, but can also induce significant cellular toxicity.
  • CPPs are generally not sufficient to enable functional cytosolic delivery.
  • the endosomal escape peptide is not one or more of S10, Penetratin, Tat, R9, Transportan-10, L17E M-Lycotoxin, or Aurein1.2.
  • the endosomal escape peptide is one or more of S10, Penetratin, Tat, R9, Transportan-10, L17E M-Lycotoxin, or Aurein1.2.
  • the endosomal escape peptide is a peptide of about 10 to about 200 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 190 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 180 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 170 amino acids in length.
  • the endosomal escape peptide is a peptide of about 10 to about 160 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 150 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 140 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 130 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10
  • the endosomal escape peptide is a peptide of about 10 to about 110 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 100 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 90 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 80 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 10 to about 70 amino acids in length.
  • the endosomal escape peptide is a peptide of about 10 to about 60 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 20 to about 60 amino acids in length. [00215] In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 200 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 190 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 180 amino acids in length.
  • the endosomal escape peptide is a peptide of about 60 to about 170 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 160 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 150 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 140 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 130 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 120 amino acids in length.
  • the endosomal escape peptide is a peptide of about 60 to about 110 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 100 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 60 to about 90 amino acids in length. [00216] In some embodiments, the endosomal escape peptide is a peptide of about 100 to about 200 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 100 to about 190 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 100 to about 180 amino acids in length.
  • the endosomal escape peptide is a peptide of about 100 to about 170 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 100 to about 160 amino acids in length. In some embodiments, the endosomal escape peptide
  • 52/179 B0662.70120WO00 peptide is a peptide of about 100 to about 150 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of about 100 to about 140 amino acids in length. [00217] In some embodiments, the endosomal escape peptide is a peptide of at least 10 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of at least 20 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of at least 30 amino acids in length. [00218] In some embodiments, the endosomal escape peptide is a peptide of at most 200 amino acids in length.
  • the endosomal escape peptide is a peptide of at most 170 amino acids in length. In some embodiments, the endosomal escape peptide is a peptide of at most 140 amino acids in length. [00219] In some embodiments, the endosomal escape peptide is a peptide comprising one or more amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising one to ten amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising two to eight amphipathic alpha helical motifs.
  • the endosomal escape peptide is a peptide comprising two to six amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising one amphipathic alpha helical motif. In some embodiments, the endosomal escape peptide is a peptide comprising one or more amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising one or two amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising one amphipathic alpha helical motifs.
  • the endosomal escape peptide is a peptide comprising two amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising two or more amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising three amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising four amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising five amphipathic alpha helical motifs.
  • the endosomal escape peptide is a peptide comprising six amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising seven amphipathic alpha helical motifs. In some embodiments, the endosomal escape peptide is a peptide comprising eight amphipathic alpha helical motifs. [00220] In some embodiments, the amphipathic alpha helical motifs comprise a positively charged hydrophilic outer face and a hydrophobic outer face. In some embodiments, the
  • an amphipathic alpha helix comprises a sequence in which a hydrophilic amino acid is present at every second or third position in the sequence. In some embodiments, an amphipathic alpha helix comprises a sequence in which a hydrophilic amino acid is present at every second position in the sequence. In some embodiments, an amphipathic alpha helix comprises a sequence in which a hydrophilic amino acid is present at every third position in the sequence. In some embodiments, an amphipathic alpha helix comprises a sequence in which a hydrophobic amino acid is present at every third or fourth position in the sequence.
  • an amphipathic alpha helix comprises a sequence in which a hydrophobic amino acid is present at every third position in the sequence. In some embodiments, an amphipathic alpha helix comprises a sequence in which a hydrophobic amino acid is present at every fourth position in the sequence.
  • the endosomal escape peptide comprises about 10% to about 85% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V (i.e., about 10% to about 85% of the amino acids of the endosomal escape peptide are selected from A, C, G, I, L, M, F, P, W, Y, R, and V).
  • the endosomal escape peptide comprises about 20% to about 75% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. In some embodiments, the endosomal escape peptide comprises about 30% to about 70% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. In some embodiments, the endosomal escape peptide comprises about 30% to about 65% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V.
  • the endosomal escape peptide comprises about 25% to about 65% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. In some embodiments, the endosomal escape peptide comprises about 40% to about 65% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. In some embodiments, the endosomal escape peptide comprises about 40% to about 55% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V.
  • the endosomal escape peptide comprises about 30% to about 55% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. In some embodiments, the endosomal escape peptide comprises about 20% to about 30% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. In some embodiments, the endosomal escape peptide comprises about 30% to about 40% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. In some embodiments, the endosomal escape peptide comprises about 40% to about 50% of any
  • the endosomal escape peptide comprises about 50% to about 60% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. In some embodiments, the endosomal escape peptide comprises about 55% to about 65% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V.
  • the endosomal escape peptide comprises about 65% to about 75% of any combination of the amino acids A, C, G, I, L, M, F, P, W, Y, R, and V. [00222] In some embodiments, the endosomal escape peptide comprises about 5% to about 65% of any combination of the amino acids K and R. In some embodiments, the endosomal escape peptide comprises about 15% to about 55% of any combination of the amino acids K and R. In some embodiments, the endosomal escape peptide comprises about 15% to about 45% of any combination of the amino acids K and R. In some embodiments, the endosomal escape peptide comprises about 25% to about 55% of any combination of the amino acids K and R.
  • the endosomal escape peptide comprises about 25% to about 45% of any combination of the amino acids K and R. In some embodiments, the endosomal escape peptide comprises about 35% to about 45% of any combination of the amino acids K and R. In some embodiments, the endosomal escape peptide comprises about 25% to about 35% of any combination of the amino acids K and R. In some embodiments, the endosomal escape peptide comprises about 15% to about 25% of any combination of the amino acids K and R. In some embodiments, the endosomal escape peptide comprises about 25% to about 35% of any combination of the amino acids K and R.
  • the endosomal escape peptide comprises about 35% to about 45% of any combination of the amino acids K and R. In some embodiments, the endosomal escape peptide comprises about 45% to about 55% of any combination of the amino acids K and R. [00223] In some embodiments, the endosomal escape peptide has a net charge between about +6 and about +20. In some embodiments, the endosomal escape peptide has a net charge between about +8 and about +18. In some embodiments, the endosomal escape peptide has a net charge between about +10 and about +16. In some embodiments, the endosomal escape peptide has a net charge between about +10 and about +18.
  • the endosomal escape peptide has a net charge between about +8 and about +16. In some embodiments, the endosomal escape peptide has a net charge between about +12 and about +16. In some embodiments, the endosomal escape peptide has a net charge between about +10 and about +14. In some embodiments, the endosomal escape peptide has a net charge between about +6 and about +8. In some embodiments, the endosomal escape peptide has a
  • the endosomal escape peptide has a net charge between about +10 and about +12. In some embodiments, the endosomal escape peptide has a net charge between about +12 and about +14. In some embodiments, the endosomal escape peptide has a net charge between about +14 and about +16. In some embodiments, the endosomal escape peptide has a net charge between about +16 and about +18. In some embodiments, the endosomal escape peptide has a net charge between about +18 and about +20. [00224] In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 2 and about 20.
  • the endosomal escape peptide has a hydrophobic moment between about 4 and about 18. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 6 and about 16. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 4 and about 16. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 6 and about 18. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 6 and about 14. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 8 and about 16. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 2 to about 6.
  • the endosomal escape peptide has a hydrophobic moment between about 6 to about 10. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 10 to about 12. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 12 to about 16. In some embodiments, the endosomal escape peptide has a hydrophobic moment between about 16 to about 20. [00225] In some embodiments, the endosomal escape peptide has a helical content between about 5% and about 80%. In some embodiments, the endosomal escape peptide has a helical content between about 10% and about 75%.
  • the endosomal escape peptide has a helical content between about 14% and about 72%. In some embodiments, the endosomal escape peptide has a helical content between about 10% and about 80%. In some embodiments, the endosomal escape peptide has a helical content between about 5% and about 75%. In some embodiments, the endosomal escape peptide has a helical content between about 10% and about 70%. In some embodiments, the endosomal escape peptide has a helical content between about 15% and about 75%. In some embodiments, the endosomal escape peptide has a helical content between about 5% to about 25%. In some embodiments, the endosomal escape peptide has a helical content between about 25% to about 45%. In
  • the endosomal escape peptide has a helical content between about 45% to about 65%. In some embodiments, the endosomal escape peptide has a helical content between about 65% to about 85%. [00226] In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 90% and about 100% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 80% and about 100% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 70% and about 100% K.
  • positively charged residues of the endosomal escape peptide comprise between about 60% and about 100% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 50% and about 100% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 40% and about 100% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 30% and about 100% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 20% and about 100% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 40% and about 90% K.
  • positively charged residues of the endosomal escape peptide comprise between about 40% and about 80% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 40% and about 70% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 40% and about 60% K. In some embodiments, wherein positively charged residues of the endosomal escape peptide comprise between about 60% and about 80% K. [00227] In some embodiments, the endosomal escape peptide is conjugated to the N-terminus of the elastin-like polypeptide.
  • the endosomal escape peptide is conjugated to the C-terminus of the elastin-like polypeptide.
  • the endosomal escape peptide is an endosomal escape peptide comprising a sequence selected from the group consisting of: LYKKFKKKLLKSLKRGGSGGGSLYKKFKKKLLKSLKRL (SEQ ID NO: 2); WKKWWKKWWKWWKKWWKKGGSGGGSWKKWWKKWWKWWKKWWK K (SEQ ID NO: 3); TKRVLQVWFQNARAKFRRNLLRGGSGGGSMLSFILTLKRMLKACLRAWK (SEQ ID NO: 4);
  • the endosomal escape peptide comprises a sequence of: LYKKFKKKLLKSLKRGGSGGGSLYKKFKKKLLKSLKRL (SEQ ID NO: 2).
  • the endosomal escape peptide comprises a sequence of: WKKWWKKWWKWWKKWWKKGGSGGGSWKKWWKKWWKWWKKWWK K (SEQ ID NO: 3).
  • the endosomal escape peptide comprises a sequence of: TKRVLQVWFQNARAKFRRNLLRGGSGGGSMLSFILTLKRMLKACLRAWK (SEQ ID NO: 4).
  • the endosomal escape peptide comprises a sequence of:
  • the endosomal escape peptide comprises a sequence of: LYKKFKKKLLKSLKRLGGSGGGSYKWWQKYQKRKF (SEQ ID NO: 6).
  • the endosomal escape peptide comprises a sequence of: LYKKFKKKLLKSLKRGGSGGGSTKRVLQVWFQNARAKFRRNLLR (SEQ ID NO: 7).
  • the endosomal escape peptide comprises a sequence of: RRWVRRVRRWVRRVVRVVRRWVRRGGSGGGSFLKAIKKFGKEFKKIGAKL K (SEQ ID NO: 8).
  • the endosomal escape peptide comprises a sequence of: KHKHKHKHKHGGSGGGSKKTWWKTWWTKWSQPKK (SEQ ID NO: 9).
  • the endosomal escape peptide comprises a sequence of: KFAKKFAKKFAKKAAKGGSGGGSFKFFKKFFKKWK (SEQ ID NO: 10).
  • the endosomal escape peptide comprises a sequence of: KLLKKLLKLWKKLLKKLKGGSGGGSFVVKKKKKVF (SEQ ID NO: 11). [00239] In some embodiments, the endosomal escape peptide comprises a sequence of: KKVVFKVKFKGGSGGGSAIWKKIIKKIIKSAKKIG (SEQ ID NO: 12). [00240] In some embodiments, the endosomal escape peptide comprises a sequence of: KKPWWKPWWPKWKKGGSGGGSGWKKFFKKAAKVGK (SEQ ID NO: 13).
  • the endosomal escape peptide comprises a sequence of: KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK (SEQ ID NO: 14). [00242] In some embodiments, the endosomal escape peptide comprises a sequence of: KWKLFKKIGKVLKVLGGSGGGSKWKKFKKIGAVLKVL (SEQ ID NO: 15). [00243] In some embodiments, the endosomal escape peptide comprises a sequence of: ALWKTLLKKVLKAAAKGGSGGGSVKFAIKKIGKKAAKKVIVKA (SEQ ID NO: 16).
  • the endosomal escape peptide comprises a sequence of: KWKLARAFARAIKKLGGSGGGSYARALRRQARTG (SEQ ID NO: 36). [00245] In some embodiments, the endosomal escape peptide comprises a sequence of: RQIKIWFQNRRMKWKK (SEQ ID NO: 37). [00246] In some embodiments, the endosomal escape peptide comprises a sequence of: CKRKKRRQRRRG (SEQ ID NO: 38).
  • the endosomal escape peptide comprises a sequence of: RRRRRRRRR (SEQ ID NO: 39). [00248] In some embodiments, the endosomal escape peptide comprises a sequence of: AGYLLGKINLKALAALAKKIL (SEQ ID NO: 40). [00249] In some embodiments, the endosomal escape peptide comprises a sequence of: IWLTALKFLGKHAAKHEAKQQLSKL(SEQ ID NO: 41).
  • the endosomal escape peptide comprises a sequence of: [00251] GLFDIIKKIAESF (SEQ ID NO: 42).
  • the endosomal escape peptide is an endosomal escape peptide comprising a sequence selected from the group consisting of: RMKFNVWAWICVVKKKKFFPHWKIRYIIGRAWLL (SEQ ID NO: 133); DVHRRIWIILLPGHRIKFNWKQWKWRFEFEFKAVRK(SEQ ID NO: 134); KLVRYISYLKLRGGVKILLAKIKPFYEVKLKLKRLSLII(SEQ ID NO: 135); YFLARARARAILIKFFGKLKPFKAKKLAKQLKWVART(SEQ ID NO: 136); ANHKFGVYFGGSGGGSGVKWQVKFGVNHANHVKF(SEQ ID NO: 137); ANHGHKFGVKLNHGGSGGGSY
  • the endosomal escape peptide is an endosomal escape peptide comprising a sequence selected from the group consisting of: KKGIGKWGKKHKKGIGGIKVVWGKVVGIWGIL(SEQ ID NO: 165); WGKLAFGKIKGIYKWGKKGWGKLAGIIGIGIKGI(SEQ ID NO: 166); GKKKLGKGIKGIIGKWGIIGIIGIKGIW(SEQ ID NO: 167); KKGIRRIGIGIGKGIGIIGKIFLSKLGWWGIW(SEQ ID NO: 168); WGIKLWGKVKGIKAIGYKAFGKIANKVINKYKGIG(SEQ ID NO: 169); KKGIGKIGKAFGKWGIKAIGIGKGALAILKVVIG(SEQ ID NO: 170); KKGIKAFIGIGKKWGGSGGGSKIWGIIGWGIAFGIAFN(SEQ ID NO: 171); WGKKSKKVW
  • the endosomal escape peptide is an endosomal escape peptide comprising a sequence selected from the group consisting of: RILSVNRIKGGSGGGSVLLKRISVLSLKGWVNW(SEQ ID NO: 181); RIKKITGLSKKVLIRISVLGKRIATGWRTGSRIRI(SEQ ID NO: 182); RIWKKVLLATGGSGGGSVNILSRIFKRISVL(SEQ ID NO: 183); RIKKFLKVNKLFSVNWAKTGQLGVLRVNRIGKLSV(SEQ ID NO: 184);
  • the endosomal escape peptide comprises a multimer. In some embodiments, the endosomal escape peptide comprises a dimer. In some embodiments, the dimer comprises two iterations of an endosomal escape peptide sequence provided herein. In some embodiments, the endosomal escape peptide comprises a trimer. In some embodiments, the trimer comprises three iterations of an endosomal escape peptide sequence provided herein.
  • the endosomal escape peptide comprises a sequence of: [KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK]–[GGSGGGS]– [KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK] (SEQ ID NO: 43).
  • the endosomal escape peptide comprises a sequence of: [KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK]–[GGSGGGS]– [KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK]–[GGSGGGS]– [KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK] (SEQ ID NO: 44).
  • the endosomal escape peptide is one or more of S10, Penetratin, Tat, R9, Transportan-10, L17E M-Lycotoxin, or Aurein1.2. In some embodiments, the endosomal escape peptide is S10, Penetratin, Tat, R9, Transportan-10, L17E M-Lycotoxin, or Aurein1.2. In some embodiments, the endosomal escape peptide is S10. In some embodiments, the endosomal escape peptide is Penetratin. In some embodiments, the endosomal escape peptide is Tat. In some embodiments, the endosomal escape peptide is R9.
  • the endosomal escape peptide is Transportan-10. In some embodiments, the endosomal escape peptide is L17E M-Lycotoxin. In some embodiments, the endosomal escape peptide is Aurein1.2.
  • the endosomal escape peptide is a peptide disclosed in Machida, S. et al. Design of a Novel Membrane-Destabilizing Peptide Selectively Acting on Acidic Liposomes. Biosci. Biotechnol. Biochem. 2000, 64, 985-994, in Krishnamurthy, S. et al. Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases. Nat. Commun. 2019, 10, 4906, or in Cerovsky, V. et al.
  • Lasioglossins Three Novel Antimicrobial Peptides from the Venom of the Eusocial Bee Lasioglossum laticeps (Hymenoptera: Halictidae). Chem. Bio. Chem. 2009, 10, 2089-2099, the contents of which are incorporated herein by reference in their entireties.
  • the compound has a sequence selected from the group consisting of: LYKKFKKKLLKSLKRGGSGGGSLYKKFKKKLLKSLKRL– [(VPGVG)(VPGEG)(VPGVG)(VPGEG)(VPGVG)]10–CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 45); WKKWWKKWWKWWKKWWKKGGSGGGSWKKWWKKWWKWWKKWWK K–[(VPGVG)(VPGEG)(VPGVG)(VPGEG)(VPGVG)]10–CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 46); TKRVLQVWFQNARAKFRRNLLRGGSGGGSMLSFILTLKRMLKACLRAWK– [(VPGVG)(VPGEG)(VPGVG)(VPGEG)(VPGEG)(VPGE
  • the compound has a sequence selected from the group consisting of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15– LYKKFKKKLLKSLKRGGSGGGSLYKKFKKKLLKSLKRL (SEQ ID NO: 67); [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15– WKKWWKKWWKWWKKWWKKGGSGGGSWKKWWKKWWKWWKKWWKK (SEQ ID NO: 68); [(VPGAG)(VPGGG)(VPGAG)(VPGAG)]10–CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)(IPGHG)(IPGHG)2]
  • the compound has a sequence selected from the group consisting of: [(VPGGG)(VPGGG)(VPGGG)(VPGGG)(VPGGG)] 10 – CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 – KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK (SEQ ID NO: 221); [(VPGSG)(VPGGG)(VPGSG)(VPGGG)(VPGSG)] 10 – CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 – KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK (SEQ ID NO: 222); and [(VPGQG)(VPGGG)(VPGQG)(VPGGG)(VPGQG)] 10 – CCCCGGG– [(VPGQG)(VPGGG
  • the compound has a sequence selected from the group consisting of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–[CAP]5– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 – KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK (SEQ ID NO: 197); [(VPGAG)(VPGGG)(VPGAG)(VPGAG)(VPGAG)]10–[CEAAAK]5– [(IPGHG)2(VPGYG)(IPGHG)2]15– KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK (SEQ ID NO: 224); [(VPGAG)(VPGGG)(VPGAG)(VPGAG)]10–C5G3– [(IPGHG)2
  • the compound has a sequence: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–[(IPG(C/H)G)2(VPGYG)(IPG (C/H)G)2]15–KNWKGIAGMAKKLLGKNWGGSGGGSVNWKKILGKIIKVVK (SEQ ID NO: 226).
  • the compound further comprises a protease cleavable linker at the C-terminus.
  • the compound further comprises a protease cleavable linker at the N-terminus.
  • the protease cleavable linker sequence is selected from the group consisting of: (GGS) 9 (SEQ ID NO: 91); GGSGGSEVRFGGSGGS (SEQ ID NO: 92); GGSGGSGGVGFLGGGS (SEQ ID NO: 93); GGSGGSGTQFFRLGGS (SEQ ID NO: 94); KKESTFEERSYWQSQV (SEQ ID NO: 95); GGSGGSRVRRGGSGGS (SEQ ID NO: 96); and NNTHDLVGDVRLAGVQSVASSRRHKRFAGV (SEQ ID NO: 97).
  • the protease cleavable linker is (GGS)9 (SEQ ID NO: 91). In some embodiments, the protease cleavable linker is GGSGGSEVRFGGSGGS (SEQ ID NO: 92). In some embodiments, the protease cleavable linker is GGSGGSGGVGFLGGGS (SEQ ID NO: 93). In some embodiments, the protease cleavable linker is GGSGGSGTQFFRLGGS (SEQ ID NO: 94). In some embodiments, the protease cleavable linker is KKESTFEERSYWQSQV (SEQ ID NO: 95).
  • the protease cleavable linker is GGSGGSRVRRGGSGGS (SEQ ID NO: 96). In some embodiments, the protease cleavable linker is NNTHDLVGDVRLAGVQSVASSRRHKRFAGV (SEQ ID NO: 97). [00267] In some embodiments, the compound comprises a protease cleavable linker conjugated to the elastin-like polypeptide.
  • compound comprises a protease cleavable linker conjugated to the elastin-like polypeptide and comprises a sequence selected from the group consisting of: (GGS) 9 –[(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)] 10 –CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 98); GGSGGSEVRFGGSGGS–[(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)] 10 – CCCCGGG–[(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 99);
  • GGSGGSGGVGFLGGGS [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)] 10 – CCCCGGG–[(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 100); GGSGGSGTQFFRLGGS–[(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10– CCCCGGG–[(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 101); KKESTFEERSYWQSQV–[(VPGAG)(VPGGG)(VPGAG)(VPGAG)]10– CCCCGGG–[(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 102); NNTHDLVGDVRLAGVQSVASSRRHKRFAGV–
  • compound comprises a protease cleavable linker conjugated to the elastin-like polypeptide and comprises a sequence selected from the group consisting of: [(VPGVG)(VPGEG)(VPGVG)(VPGEG)(VPGVG)] 10 –CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15–(GGS)9 (SEQ ID NO: 105); [(VPGVG)(VPGEG)(VPGVG)(VPGEG)(VPGVG)]10–CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15–GGSGGSEVRFGGSGGS (SEQ ID NO: 106); [(VPGVG)(VPGEG)(VPGVG)(VPGEG)(VPGVG)]10–CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15–GGSGGSGG;
  • an elastin-like polypeptide comprising a sequence of: [(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)] 10 –X * – [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 198), wherein: each instance of X 3 is independently G, S, Q, V, or A; each instance of X 4 is independently E or G; and
  • X * is -CCCCGGG- (SEQ ID NO: 19), -[CAP] 5 - (SEQ ID NO: 125), [CEAAAK] 5 (SEQ ID NO: 126), or -C5G3- (SEQ ID NO: 127). [00270] In some embodiments, X* is -CCCCGGG- (SEQ ID NO: 19) or -C5G3- (SEQ ID NO: 127). In some embodiments, X* is -[CAP] 5 - (SEQ ID NO: 125), [CEAAAK] 5 (SEQ ID NO: 126), or -C5G3- (SEQ ID NO: 127).
  • X* is -CCCCGGG- (SEQ ID NO: 19). In some embodiments, X* is -[CAP]5- (SEQ ID NO: 125). In some embodiments, X* is [CEAAAK] 5 (SEQ ID NO: 126). In some embodiments, X* is -C 5 G 3 - (SEQ ID NO: 127).
  • the elastin-like polypeptide comprises a sequence of: [(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)] 10 –CCCCGGG– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 227), wherein: X 3 is G, S, Q, V, or A; and X 4 is E or G.
  • the elastin-like polypeptide comprises a sequence of: [(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)]10–CCCCGGG– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 112) wherein: X 3 is V or A; and X 4 is E or G. [00273] In some embodiments, each instance of X 3 is V, each instance of X 3 is A, each instance of X 3 is G, each instance of X 3 is S, or each instance of X 3 is Q.
  • each instance of X 3 is V or each instance of X 3 is A. In some embodiments, each instance of X 3 is G, each instance of X 3 is S, or each instance of X 3 is Q. In some embodiments, X 3 is V. In some embodiments, X 3 is A. In some embodiments, each instance of X 3 is the same. In some embodiments, each instance of X 3 is A. In some embodiments, each instance of X 3 is V. [00274] In some embodiments, each instance of X 4 is E or each instance of X 4 is G. In some embodiments, X 4 is E. In some embodiments, X 4 is G. In some embodiments, each instance of X 4 is the same.
  • each instance of X 4 is E. In some embodiments, each instance of X 4 is G. [00275] In some embodiments, X 3 is V, and X 4 is E. In some embodiments, X 3 is V, and X 4 is G. In some embodiments, X 3 is A, and X 4 is G. In some embodiments, X 3 is A, and X 4 is E. [00276] In some embodiments, the elastin-like polypeptide comprises a sequence of:
  • the elastin-like polypeptide comprises a sequence of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)] 10 –CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 113).
  • the elastin-like polypeptide comprises a sequence of: [(VPGGG)(VPGGG)(VPGGG)(VPGGG)] 10 –CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 131); [(VPGSG)(VPGGG)(VPGSG)(VPGGG)(VPGSG)]10–CCCCGGG– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 228); or [(VPGQG)(VPGGG)(VPGQG)(VPGGG)(VPGQG)] 10 –CCCCGGG– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 229).
  • the elastin-like polypeptide comprises a sequence of: [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)] 10 –[CAP] 5 – [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 199); [(VPGAG)(VPGGG)(VPGAG)(VPGAG)]10–[CEAAAK]5– [(IPGHG) 2 (VPGYG)(IPGHG) 2 ] 15 (SEQ ID NO: 200); or [(VPGAG)(VPGGG)(VPGAG)(VPGGG)(VPGAG)]10–C5G3– [(IPGHG)2(VPGYG)(IPGHG)2]15 (SEQ ID NO: 201).
  • the elastin-like polypeptide comprises a sequence of: [(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)(VPGX 4 G)(VPGX 3 G)] 10 – [(IPGZ 4 G)2(VPGYG)(IPGZ 4 G)2]15 (SEQ ID NO: 203), wherein: each instance of X 3 is independently G, S, Q, V, or A; each instance of X 4 is independently E or G; and each instance of Z 3 is independently C or H. [00281] In some embodiments, three instances of Z 3 are C and the remaining instances of Z 3 are H.
  • an endosomal escape peptide comprising a sequence selected from the group consisting of: LYKKFKKKLLKSLKRGGSGGGSLYKKFKKKLLKSLKRL (SEQ ID NO: 2);
  • an endosomal escape peptide comprising a sequence selected from the group consisting of: RMKFNVWAWICVVKKKKFFPHWKIRYIIGRAWLL(SEQ ID NO: 133); DVHRRIWIILLPGHRIKFNWKQWKWRFEFEFKAVRK(SEQ ID NO: 134); KLVRYISYLKLRGGVKILLAKIKPFYEVKLKLKRLSLII(SEQ ID NO: 135); YFLARARARAILIKFFGKLKPFKAKKLAKQLKWVART(SEQ ID NO: 136); ANHKFGVYFGGSGGGSGVKWQVKFGVNHANHVKF(SEQ ID NO: 137); ANHGHKFGVKLNHGGSGGGSYFKFGKAPGVKW(SEQ ID NO: 138); GVGVREWKFGGSGGGSYYFGVKGHKKGVGV(SEQ ID NO: 139); GVKFG
  • an endosomal escape peptide comprising a sequence selected from the group consisting of: KKGIGKWGKKHKKGIGGIKVVWGKVVGIWGIL(SEQ ID NO: 165); WGKLAFGKIKGIYKWGKKGWGKLAGIIGIGIKGI(SEQ ID NO: 166); GKKKLGKGIKGIIGKWGIIGIIGIKGIW(SEQ ID NO: 167); KKGIRRIGIGIGKGIGIIGKIFLSKLGWWGIW(SEQ ID NO: 168); WGIKLWGKVKGIKAIGYKAFGKIANKVINKYKGIG(SEQ ID NO: 169); KKGIGKIGKAFGKWGIKAIGIGKGALAILKVVIG(SEQ ID NO: 170); KKGIKAFIGIGKKWGGSGGGSKIWGIIGWGIAFGIAFN(SEQ ID NO: 171); WGKKSKKVWGIWKVLSGWGI
  • an endosomal escape peptide comprising a sequence selected from the group consisting of: RILSVNRIKGGSGGGSVLLKRISVLSLKGWVNW(SEQ ID NO: 181); RIKKITGLSKKVLIRISVLGKRIATGWRTGSRIRI(SEQ ID NO: 182); RIWKKVLLATGGSGGGSVNILSRIFKRISVL(SEQ ID NO: 183); RIKKFLKVNKLFSVNWAKTGQLGVLRVNRIGKLSV(SEQ ID NO: 184); WKRILSKVNTGGSGGGSKNKRIIRRIKRIIRI(SEQ ID NO: 185); KRIRRIAKVGGSGGGSVNKRILSVLVNW(SEQ ID NO: 186); RRITGWARIGGSGGGSVNWKRIWVLSVL(SEQ ID NO: 187); WSVLRIRITGVLSVLGGSGGGSVRIIRIWRI(SEQ ID NO: 187); WSVLRIRITGVL
  • the term “compound provided herein” refers to a compound comprising an elastin-like polypeptide chemically or recombinantly conjugated to one or more endosomal escape peptides; to an elastin-like polypeptide; or to an endosomal escape peptide provided herein.
  • Pharmaceutical Compositions, Kits, and Administration [00287] In another aspect, provided herein is a composition comprising a compound provided herein and an agent.
  • the composition is in the form of a particle.
  • the particle is a nanoparticle.
  • the particle is a microparticle.
  • the composition is in the form of a micelle.
  • the average diameter of the particle is at least about 10 nm, at least about 50 nm, at least about 100 nm, at least about 500 nm, at least about 1 ⁇ m, at least about 5 ⁇ m, at least about 10 ⁇ m, at least about 50 ⁇ m, at least about 100 ⁇ m, at least about 500 ⁇ m, or at least about 1 mm.
  • the average diameter of the particle is less than about 1 mm, less than about 500 ⁇ m, less than about 100 ⁇ m, less than about 50 ⁇ m less than about 10 ⁇ m, less than about 5 ⁇ m, less than about 1 ⁇ m, less than about 500 nm, less than about 100 nm, less than about 50 nm, or less than about 10 nm.
  • the average diameter of the particle is about 15 nm to about 800 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 700 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 600 nm.
  • the average diameter of the particle is about 15 nm to about 500 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 400 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 300 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 250 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 200 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 150 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 100 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 90 nm.
  • the average diameter of the particle is about 15 nm to about 80 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 70 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 60 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 50 nm. In some embodiments, the average diameter of the particle is about 15 nm to about 25 nm. [00291] In some embodiments, the average diameter of the particle is about 25 nm to about 300 nm. In some embodiments, the average diameter of the particle is about 25 nm to about 250 nm.
  • the average diameter of the particle is about 25 nm to about 200 nm. In some embodiments, the average diameter of the particle is about 25 nm to about 150 nm. In some embodiments, the average diameter of the particle is about 25 nm to about 100 nm. In some embodiments, the average diameter of the particle is about 25 nm to about 90 nm. In some embodiments, the average diameter of the particle is about 25 nm to about 80
  • the average diameter of the particle is about 25 nm to about 70 nm. In some embodiments, the average diameter of the particle is about 25 nm to about 60 nm. In some embodiments, the average diameter of the particle is about 25 nm to about 50 nm. [00292] In some embodiments, the average diameter of the particle is about 50 nm to about 300 nm. In some embodiments, the average diameter of the particle is about 50 nm to about 250 nm. In some embodiments, the average diameter of the particle is about 50 nm to about 200 nm. In some embodiments, the average diameter of the particle is about 50 nm to about 150 nm.
  • the average diameter of the particle is about 50 nm to about 100 nm. [00293] In some embodiments, the average dimeter of the particle is about 20 nm to about 200 nm. In some embodiments, the average dimeter of the particle is about 30 nm to about 200 nm. In some embodiments, the average dimeter of the particle is about 40 nm to about 200 nm. In some embodiments, the average dimeter of the particle is about 60 nm to about 200 nm. In some embodiments, the average dimeter of the particle is about 75 nm to about 200 nm. [00294] In some embodiments, the average diameter of the particle is about 100 nm to about 200 nm.
  • the average diameter of the particle is about 150 nm to about 250 nm. In some embodiments, the average diameter of the particle is about 250 nm to about 350 nm. In some embodiments, the average diameter of the particle is about 350 nm to about 450 nm. In some embodiments, the average diameter of the particle is about 450 nm to about 550 nm. In some embodiments, the average diameter of the particle is about 550 nm to about 650 nm. [00295] In some embodiments, the polydispersity index is about 0.05 to about 0.8. In some embodiments, the polydispersity index is about 0.1 to about 0.6. In some embodiments, the polydispersity index is about 0.1 to about 0.5.
  • the polydispersity index is about 0.2 to about 0.4. In some embodiments, the polydispersity index is about 0.1 to about 0.3. In some embodiments, the polydispersity index is about 0.3 to about 0.5. In some embodiments, the polydispersity index is about 0.4 to about 0.6. In some embodiments, the polydispersity index is determined by light scattering. [00296] In some embodiments, the particle or micelle encapsulates the agent. [00297] In some embodiments, the compound and the agent are not covalently attached. In some embodiments, the compound and the agent are electrostatically complexed. In some embodiments, the compound and the agent are electrostatically complexed at the N-terminus
  • the compound and the agent are electrostatically complexed at the C-terminus of the compound.
  • the compound and the agent are covalently attached.
  • the compound and the agent are covalently attached via the N-terminus of the compound.
  • the compound and the agent are covalently attached via the C-terminus of the compound.
  • the compound and the agent are covalently attached via a protease cleavable linker.
  • the protease cleavable linker is selected from the group consisting of: (GGS) 9 (SEQ ID NO: 91); GGSGGSEVRFGGSGGS (SEQ ID NO: 92); GGSGGSGGVGFLGGGS (SEQ ID NO: 93); GGSGGSGTQFFRLGGS (SEQ ID NO: 94); KKESTFEERSYWQSQV (SEQ ID NO: 95); GGSGGSRVRRGGSGGS (SEQ ID NO: 96); and NNTHDLVGDVRLAGVQSVASSRRHKRFAGV (SEQ ID NO: 97).
  • the protease cleavable linker is (GGS) 9 (SEQ ID NO: 91). In some embodiments, the protease cleavable linker is GGSGGSEVRFGGSGGS (SEQ ID NO: 92). In some embodiments, the protease cleavable linker is GGSGGSGGVGFLGGGS (SEQ ID NO: 93). In some embodiments, the protease cleavable linker is GGSGGSGTQFFRLGGS (SEQ ID NO: 94). In some embodiments, the protease cleavable linker is KKESTFEERSYWQSQV (SEQ ID NO: 95).
  • the protease cleavable linker is GGSGGSRVRRGGSGGS (SEQ ID NO: 96). In some embodiments, the protease cleavable linker is NNTHDLVGDVRLAGVQSVASSRRHKRFAGV(SEQ ID NO: 97).
  • the agent is an organic molecule, protein, peptide, polypeptide, polynucleotide, an isotopically labeled chemical compound, vaccine, antibody, or an immunological agent. In some embodiments, the agent is an organic molecule, protein, polypeptide, polynucleotide, an isotopically labeled chemical compound, vaccine, or an immunological agent.
  • the agent is an organic molecule or an isotopically labeled chemical compound.
  • the agent is a protein, polypeptide, or polynucleotide.
  • the agent is a polynucleotide or polypeptide.
  • the agent is a peptide or polypeptide.
  • the agent is a vaccine or an immunological agent.
  • the agent is an organic molecule or an isotopically labeled chemical compound.
  • the agent is a protein, polypeptide, or polynucleotide.
  • the agent is a polynucleotide or polypeptide.
  • the agent is a peptide or polypeptide.
  • the agent is a vaccine or an immunological agent.
  • the agent is an organic molecule or an isotopically labeled chemical compound.
  • the agent is a protein, polypeptide, or polynucleotide.
  • the agent is a polynucleotide or polypeptide.
  • the agent is a peptide or poly
  • agent is an organic molecule.
  • the agent is a protein.
  • the agent is a peptide.
  • the agent is a polypeptide.
  • the agent is a polynucleotide.
  • the agent is an isotopically labeled chemical compound.
  • the agent is a vaccine.
  • the agent is an antibody.
  • the agent is an immunological agent. [00302] In some embodiments, the agent is an RNA.
  • the RNA is messenger RNA (mRNA), pre-messenger RNA (pre-mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Piwi- interacting RNA (piRNA), microRNA (miRNA), small interfering RNA (siRNA), antisense RNA (asRNA or aRNA), transfer-messenger RNA (tmRNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), guide RNA (gRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or lncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, polyinosinic acid
  • the RNA is messenger RNA (mRNA). In some embodiments, the RNA is pre-messenger RNA (pre-mRNA). In some embodiments, the RNA is ribosomal RNA (rRNA). In some embodiments, the RNA is transfer RNA (tRNA). In some embodiments, the RNA is small nuclear RNA (snRNA). In some embodiments, the RNA is small nucleolar RNA (snoRNA). In some embodiments, the RNA is Piwi-interacting RNA (piRNA). In some embodiments, the RNA is microRNA (miRNA). In some embodiments, the RNA is small interfering RNA (siRNA). In some embodiments, the RNA is antisense RNA (asRNA or aRNA).
  • the RNA is transfer-messenger RNA (tmRNA). In some embodiments, the RNA is single-stranded RNA (ssRNA). In some embodiments, the RNA is double-stranded RNA (dsRNA). In some embodiments, the RNA is precursor messenger RNA (pre-mRNA). In some embodiments, the RNA is small hairpin RNA or short hairpin RNA (shRNA). In some embodiments, the RNA is guide RNA (gRNA). In some embodiments, the RNA is heterogeneous nuclear RNA (hnRNA). In some embodiments, the RNA is coding RNA. In some embodiments, the RNA is non-coding RNA (ncRNA).
  • the RNA is long non-coding RNA (long ncRNA or lncRNA). In some embodiments, the RNA is satellite RNA. In some embodiments, the RNA is viral satellite RNA. In some embodiments, the RNA is signal recognition particle RNA. In some embodiments, the RNA is small cytoplasmic RNA. In some embodiments, the RNA is
  • the RNA is ribozyme. In some embodiments, the RNA is flexizyme. In some embodiments, the RNA is spliced leader RNA. In some embodiments, the RNA is viral RNA. In some embodiments, the RNA is viral satellite RNA.
  • the polynucleotide is a DNA. In some embodiments, the DNA is a plasmid DNA (pDNA).
  • the peptide is ovalbumin-derived MHC class I epitope or ovalbumin-derived MHC class II epitope.
  • the peptide is ovalbumin- derived MHC class I epitope. In some embodiments, the peptide is ovalbumin-derived MHC class II epitope.
  • the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is anti-CD117. In some embodiments, the monoclonal antibody is anti-CD5. [00306] In some embodiments, the molar ratio of the agent to the compound is from about 1:0.05 to about 1:5000. In some embodiments, the molar ratio of the agent to the compound is from about 1:1 to about 1:5000. In some embodiments, the molar ratio of the agent to the compound is from about 1:1 to about 1:3000.
  • the molar ratio of the agent to the compound is from about 1:1 to about 1:1500. In some embodiments, the molar ratio of the agent to the compound is from about 1:1 to about 1:750. In some embodiments, the molar ratio of the agent to the compound is from about 1:1 to about 1:300. In some embodiments, the molar ratio of the agent to the compound is from about 1:1 to about 1:150. In some embodiments, the molar ratio of the agent to the compound is from about 1:1 to about 1:60. In some embodiments, the molar ratio of the agent to the compound is from about 1:1 to about 1:30. In some embodiments, the molar ratio of the agent to the compound is from about 1:10 to about 1:5000.
  • the molar ratio of the agent to the compound is from about 1:10 to about 1:3000. In some embodiments, the molar ratio of the agent to the compound is from about 1:10 to about 1:1500. In some embodiments, the molar ratio of the agent to the compound is from about 1:10 to about 1:750. In some embodiments, the molar ratio of the agent to the compound is from about 1:10 to about 1:300. In some embodiments, the molar ratio of the agent to the compound is from about 1:10 to about 1:150. In some embodiments, the molar ratio of the agent to the compound is from about 1:10 to about 1:60. In some embodiments, the molar ratio of the agent to the compound is from about 1:10 to about 1:30. [00307] In some embodiments, the molar ratio of the agent to the compound is about 1:0.05 to about 1:30. In some embodiments, the molar ratio of the agent to the compound is about
  • the molar ratio of the agent to the compound is about 1:60 to about 1:150. In some embodiments, the molar ratio of the agent to the compound is about 1:150 to about 1:300. In some embodiments, the molar ratio of the agent to the compound is about 1:300 to about 1:750. In some embodiments, the molar ratio of the agent to the compound is about 1:750 to about 1:1500. In some embodiments, the molar ratio of the agent to the compound is about 1:1500 to about 1:3000. [00308] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmaceutics.
  • Such preparatory methods include bringing a compound, agent, particle, or composition described herein (e.g., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.
  • a compound, agent, particle, or composition described herein e.g., the “active ingredient”
  • Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
  • Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
  • Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross- linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
  • crospovidone cross-linked poly(vinyl-pyrrolidone)
  • sodium carboxymethyl starch sodium starch glycolate
  • Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan,
  • Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose,
  • ethylcellulose hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum ® ), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.
  • Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
  • the preservative is an antioxidant.
  • the preservative is a chelating agent.
  • antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.
  • EDTA ethylenediaminetetraacetic acid
  • salts and hydrates thereof e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like
  • citric acid and salts and hydrates thereof e.g., citric acid mono
  • antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
  • Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
  • Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
  • Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
  • Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium
  • Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic sa
  • Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
  • Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury,
  • Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.
  • Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
  • the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • the conjugates described herein are mixed with solubilizing agents such as Cremophor ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
  • solubilizing agents such as Cremophor ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that can be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, I solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as,
  • the dosage form may include a buffering agent.
  • Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • encapsulating compositions which can be used include polymeric substances and waxes.
  • Solid compositions of a similar type can be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the active ingredient can be in a micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art.
  • the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch.
  • inert diluent such as sucrose, lactose, or starch.
  • Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or
  • Dosage forms for topical and/or transdermal administration of a compound, agent, particle, or composition described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches.
  • the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required.
  • transdermal patches which often have the added advantage of providing controlled delivery of an active ingredient to the body.
  • dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium.
  • the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
  • Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration.
  • Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable.
  • Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound, agent, particle, or composition in powder form through the outer layers of the skin to the dermis are suitable.
  • Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions.
  • Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have
  • compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
  • a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
  • Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers.
  • Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
  • additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
  • Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate.
  • a flavoring agent such as saccharin sodium
  • a volatile oil such as a liquid oil
  • a buffering agent such as a liquid oil
  • a surface active agent such as methylhydroxybenzoate
  • a preservative such as methylhydroxybenzoate.
  • the droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.
  • Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder
  • Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration.
  • Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient.
  • Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein.
  • Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.
  • compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
  • Compounds, agents, particles, or compositions provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific
  • 89/179 B0662.70120WO00 therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
  • the compounds, agents, particles, and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g., oral
  • parenteral intravenous, intramuscular, intra-arterial, intramedullary
  • intrathecal subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal
  • topical as by powders, ointments, creams, and/or drops
  • Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site.
  • intravenous administration e.g., systemic intravenous injection
  • regional administration via blood and/or lymph supply e.g., via blood and/or lymph supply
  • direct administration to an affected site.
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).
  • the compound, agent, particle, or composition described herein is suitable for topical administration to the eye of a subject.
  • any two doses of the multiple doses include different or substantially the same amounts of a compound, agent, particle, or composition described herein.
  • the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks.
  • the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day.
  • the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell.
  • the duration between the first dose and last dose of the multiple doses is three months, six months, or one year.
  • the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell.
  • a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 ⁇ g and 1 ⁇ g, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound, agent, particle, or composition described herein.
  • a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound, agent, particle, or composition described herein.
  • a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound, agent, particle, or composition described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound, agent, particle, or composition described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound, agent, particle, or composition described herein. [00347] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult. [00348] A compound, agent, particle, or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents). The compounds, agents, particles, or
  • compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof, and/or in inhibiting the activity of a protein kinase in a subject or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell.
  • the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.
  • a pharmaceutical composition described herein including a compound, agent, particle, or composition described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the compound, agent, particle, or composition and the additional pharmaceutical agent, but not both.
  • the additional pharmaceutical agent achieves a desired effect for the same disorder.
  • the additional pharmaceutical agent achieves different effects.
  • the compound, agent, particle, or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies.
  • Pharmaceutical agents include therapeutically active agents.
  • Pharmaceutical agents also include prophylactically active agents.
  • Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.
  • drug compounds e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)
  • CFR Code of Federal Regulations
  • the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder).
  • a disease e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder.
  • Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent.
  • the additional pharmaceutical agents may also be administered together with each other and/or with the compound, agent, particle, or composition described herein in a single dose or composition or administered separately in different doses or compositions.
  • the particular combination to employ in a regimen will take into account compatibility of the compound,
  • 92/179 B0662.70120WO00 agent, particle, or composition described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
  • the additional pharmaceutical agents include, but are not limited to, anti- proliferative agents, anti-cancer agents, anti-angiogenesis agents, steroidal or non-steroidal anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetics, anti–coagulants, inhibitors of an enzyme, steroidal agents, steroidal or antihistamine, antigens, vaccines, antibodies, decongestant, sedatives, opioids, analgesics, anti–pyretics, hormones, and prostaglandins.
  • the additional pharmaceutical agent is an anti-proliferative agent. In certain embodiments, the additional pharmaceutical agent is an anti-cancer agent. In certain embodiments, the additional pharmaceutical agent is an anti-viral agent. In certain embodiments, the additional pharmaceutical agent is an binder or inhibitor of a protein kinase.
  • the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation.
  • epigenetic or transcriptional modulators e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors
  • antimitotic drugs e.g., taxanes and vinca
  • the compounds, agents, particles, or compositions described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.
  • Additional pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved by the US Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs,
  • CFR Code of Federal Regulations
  • kits e.g., pharmaceutical packs.
  • the kits provided may comprise a pharmaceutical composition or compound, agent, particle, or composition described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container).
  • a container e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container.
  • provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound, agent, particle, or composition described herein.
  • kits comprising a compound provided herein or a composition provided herein and instructions for using the compound or composition.
  • the kit comprises including a first container comprising a compound, agent, particle, or composition or pharmaceutical composition described herein.
  • the kits are useful for treating a disease (e.g., an infectious disease, genetic disease, or cancer) in a subject in need thereof.
  • the kits are useful for preventing a disease (e.g., an infectious disease, genetic disease, or cancer) in a subject in need thereof.
  • kits are useful for reducing the risk of developing a disease (e.g., an infectious disease, genetic disease, or cancer) in a subject in need thereof.
  • the kits are useful for delivering an agent to a cell.
  • a kit described herein further includes instructions for using the kit.
  • a kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA).
  • the information included in the kits is prescribing information.
  • the kits and instructions provide for treating a disease (e.g., an infectious disease, genetic disease, or cancer) in a subject in need thereof.
  • kits and instructions provide for preventing a disease (e.g., an infectious disease, genetic disease, or cancer) in a subject in need thereof. In certain embodiments, the kits and instructions provide for reducing the risk of developing a disease (e.g., an infectious disease, genetic disease, or cancer) in a subject in need thereof. In certain embodiments, the kits and instructions provide for delivering an agent.
  • a kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.
  • a method of treating or preventing a disease in a subject comprising administering to the subject a compound or composition provided herein.
  • the method comprises administering a compound provided herein.
  • the method comprises administering a composition provided herein.
  • the disease is a cardiovascular disease, a lung or respiratory disease, a musculoskeletal disease, a hematological disease, an immune disorder, an infectious disease, a genetic disease, cancer, a neurological disease, a psychiatric disorder, a metabolic disorder, or an inflammatory disease.
  • the disease is an infectious disease, a genetic disease, or cancer.
  • the disease is a cardiovascular disease.
  • the disease is a lung disease.
  • the disease is a respiratory disease.
  • the disease is a musculoskeletal disease.
  • the disease is a hematological disease.
  • the disease is an immune disorder.
  • the disease is an infectious disease.
  • the disease is a genetic disease.
  • the disease is cancer.
  • the disease is a neurological disease.
  • the disease is a psychiatric disorder.
  • the disease is a metabolic disorder.
  • the disease is an inflammatory disease.
  • the subject is an animal. The animal may be of either sex and may be at any stage of development.
  • the subject described herein is a human.
  • the subject is a non-human animal.
  • the subject is a mammal.
  • the subject is a non-human mammal.
  • the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat.
  • the subject is a companion animal, such as a dog or cat.
  • the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat.
  • the subject is a zoo animal.
  • the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate.
  • the animal is a genetically engineered animal.
  • the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs).
  • the subject is a fish or reptile.
  • a method of delivering an agent to a subject, cell, or biological sample comprising administering to the subject or contacting the cell or biological sample with an effective amount of a compound or composition provided herein.
  • the method comprises administering to the subject or contacting the cell or biological sample with a compound provided herein.
  • the method comprises administering to the subject or contacting the cell or biological sample with a composition provided herein.
  • the agent is any agent provided herein.
  • the agent is an organic molecule, protein, polypeptide, polynucleotide, an isotopically labeled chemical compound, vaccine, or an immunological agent.
  • the agent is a protein, polypeptide, or polynucleotide.
  • the agent is a protein.
  • the agent is a polypeptide.
  • the agent is a polynucleotide.
  • the agent is an RNA.
  • the RNA is messenger RNA (mRNA).In some embodiments, the RNA is small interfering RNA (siRNA).
  • the polynucleotide is a DNA.
  • the DNA is a plasmid DNA (pDNA).
  • the agent is delivered to a subject. In some embodiments, the agent is delivered to a target tissue of the subject. In some embodiments, the agent is delivered to a cell. In some embodiments, the cell is in vivo, e.g., in an organism. In certain embodiments, the cell is in vitro, e.g., in cell culture. In some embodiments, the cell is ex vivo, meaning the cell is removed from an organism prior to the delivery. In some embodiments, the cell is a eukaryotic cell.
  • the cell is a HeLa cell, HEK293 cell, a fibroblast, a macrophage, a T cell, or a hematopoietic stem cell.
  • the cell is a HEK293 cell.
  • the cell is a fibroblast.
  • the cell is a macrophage.
  • the cell is a T cell.
  • the cell is a hematopoietic stem cell.
  • the cell is a HeLa cell.
  • the agent is delivered from an extracellular space to the cytosol of a cell.
  • the agent is delivered from an extracellular space to the nucleus of the cell.
  • the composition is administered by any method provided herein.
  • the effective amount is sufficient to increase the transduction efficiency of the polynucleotide or polypeptide. In some embodiments, the effective amount is sufficient to increase the transduction efficiency of the polynucleotide. In some embodiments, the effective amount is sufficient to increase the transduction efficiency of the polypeptide.
  • E XAMPLES [00363] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting in their scope.
  • EXAMPLE 1 Effective intracellular delivery of biomacromolecules such as small interfering RNA (siRNA), protein-encoding messenger RNA (mRNA), and proteins can facilitate treatment of a variety of clinical disorders.
  • siRNA small interfering RNA
  • mRNA protein-encoding messenger RNA
  • ELP elastin-like polypeptide
  • EEP cationic endosomal escape peptide
  • EEP-fused ELPs demonstrated micellar self-assembly, enabling EEP shielding from the particle surface, effective nucleic acid encapsulation, as well as acidic pH-responsive micelle disassembly permitting EEP activity and intracellular cargo delivery.
  • Leveraging a machine learning-based predictive model of peptide membrane disruptive activity, in silico screening of ⁇ -helical peptide libraries led to the discovery of a various EEPs (including EEP13 (SEQ ID NO: 14)) with dramatically improved efficiency in both mRNA and protein delivery compared to previously reported EEPs. Multimerization of EEPs further improved potency.
  • ELP-EEP nanoparticles Due to the multivalent nature and micellar architecture facilitating EEP shielding from the nanoparticle surface, ELP-EEP nanoparticles also demonstrated greatly improved potency and reduced cytotoxicity as compared to standalone EEPs.
  • ELP-EEP delivery vehicles outperformed lipid-based reagents in the delivery of mRNA-encoded and Cre recombinase protein, as well as GFP-targeted siRNA to multiple cell lines and primary cells. The ability of this system to mediate in vivo delivery of Cre protein was confirmed after intradermal administration. The results establish ELP nanoparticles as a first-in-class non- viral, recombinant protein-based delivery platform for diverse macromolecular cargo of therapeutic interest.
  • ELPs Elastin-like polypeptides
  • 17–19 ELPs are composed of a short repeating peptide motif of Val-Pro- Gly-X-Gly derived from the hydrophobic domain of tropoelastin, where X represents a guest residue that can be any amino acid except proline.
  • ELPs exhibit lower critical solution temperature (LCST) phase behavior.
  • ELPs are soluble below a transition temperature (Tt), inversely correlated to guest residue (X) hydrophobicity and protein size 20,21 , but above the transition temperature reversibly phase separate to form insoluble coacervates.
  • Tt transition temperature
  • X guest residue
  • 22 ELPs can be synthesized with high precision and programmability, are biodegradable, and exhibit minimal immunogenicity.
  • 23 Previously designed amphiphilic diblock ELPs can self-assemble into micellular nanoparticles with a hydrophilic surface and hydrophobic core for targeted extracellular drug delivery, including proteins and small molecules.
  • VEGF-A vascular endothelial growth factor A
  • GLP-1 glucagon-like peptide-1
  • ELP-based delivery system Elastin-based Nanoparticles for Therapeutic delivery (ENTER)
  • ENTER Elastin-based Nanoparticles for Therapeutic delivery
  • major therapeutic macromolecule modalities including proteins, RNP, siRNA, mRNA, and plasmid DNA
  • ENTER cationic endosomal escape peptides
  • FIG. 1 An ELP-based delivery system (Elastin-based Nanoparticles for Therapeutic delivery (ENTER)) that achieves efficient intracellular delivery of major therapeutic macromolecule modalities, including proteins, RNP, siRNA, mRNA, and plasmid DNA to a variety of cell types both in vitro and in vivo was designed.
  • engineered ELP multiblock protein polymers were developed that self-assemble as crosslinked nanoparticles displaying a hydrophilic corona and a histidine-containing hydrophobic core, which includes cationic endosomal escape peptides (EEPs) (FIG. 1).
  • EEPs cationic endosomal escape peptid
  • EEP13 SEQ ID NO: 14
  • ELPs enabled efficient delivery of Cre recombinase protein and mRNA to HEK293 cells, as well as fibroblasts, macrophages, T cells, and hematopoietic stem cells (HSCs) and were easily modified for cell specific delivery.
  • EEPs as a component of ELP nanoparticles displayed dramatically enhanced potency with minimal cytotoxicity as compared to free EEP peptide shuttles.
  • this system provided an
  • 98/179 B0662.70120WO00 effective means for intracellular delivery of siRNA, mRNA, plasmid DNA, proteins, as well as Cas9 RNP and IRF5, as a prototypical transcription factor, in cell lines as well as primary fibroblasts, macrophages, T cells, and hematopoietic stem cells.
  • IRF5 interferon regulatory factor 5
  • Cre protein was confirmed in Ai9 mice.
  • Intranasal administration of ELP-EEP13 combined with Cre recombinase protein achieved effective editing of lung epithelial cells in reporter mice.
  • V1-ELP first-generation micelle-forming ELP bearing an N-terminal triglycine motif for chemical or sortase-mediated conjugation was previously reported.
  • V1-ELPs are diblock copolymers composed of a glutamic acid-containing hydrophilic block, a tyrosine- containing hydrophobic block, and a cysteine midblock domain enabling disulfide crosslinking (FIG. 1).
  • V2-ELP histidine
  • HEK293 Cre reporter cells (HEK293-RFP) were treated with Cre bearing V2-ELP nanoparticles (Cre-V2-ELP) (FIGs. 15A-15D), but RFP expression was only observed in the presence of chloroquine, a cationic amphipathic drug with known endosomolytic capacity 40 (FIG. 3D). Notably, RFP expression was greater when Cre was delivered by V2-ELPs as compared to V1-ELPs ( ⁇ 90% vs. 60%), consistent with the ability of histidine residues to enhance endosomolysis. [00370] A third generation ELP was designed to eliminate the need for an exogenous endosomolytic agent.
  • V2-ELPs were modified with endosomal escape peptides (EEPs), known to enhance intracellular delivery of macromolecular cargo (V3-ELPs).
  • EEPs endosomal escape peptides
  • 11,14,41–43 V3-ELPs were generated containing one of five EEPs positioned at the N-terminus of the protein polymer (FIGs. 16A-16E); each construct demonstrated stable size at room temperature over the course of 72 hours and was amenable to sortase-mediated functionalization with Cre recombinase.
  • HEK293-RFP cells treated with Cre-V3-ELPs for 48 hours displayed dose-dependent RFP expression, up to an order of magnitude greater than those treated with unmodified V2-ELPs (FIG. 3D).
  • Effective ELPs contained Tat 15 or or S10 14 , enabling RFP expression in up to 30% of cells at a concentration of 16 ⁇ M, though some cytotoxicity was observed at this dose (FIG. 17).
  • a linker library (SEQ ID NOs: 91-97) was screened with known susceptibility to a variety of endolysosomal proteases (FIG. 18A). 44–47 HEK293- RFP cells were treated with V3-ELPs bearing an S10 EEP (SEQ ID NO: 60) for 48 hrs.
  • a furin cleavable linker flanked by GGS repeats (GGSGGSRVRRGGSGGS)(SEQ ID NO: 96)
  • Cre delivery by V4-ELP-S10 was superior to that achieved using S10 (SEQ ID NO: 36) shuttle peptides alone (i.e., as an untethered peptide), with over 20-fold less ELP being required to achieving 50% RFP+ cells ( ⁇ 1.5 ⁇ M vs 32 ⁇ M, FIG. 3F).
  • S10 SEQ ID NO: 36
  • ELP- mediated EEP multivalency greatly improves the ability to achieve effective intracellular target protein delivery.
  • cell viability was unaffected by treatment with V4-ELPs at all doses tested, in contrast to free S10 (SEQ ID NO: 36) peptide, which was associated with substantial toxicity at its EC50 (FIG. 3G).
  • V4-ELPs also outperformed Lipofectamine 3000 in the delivery of Cre protein, with RFP expression in only 40% cells under optimal Lipofectamine conditions in contrast to 100% expression when delivered by V4-ELPs (FIGs. 21A-21B). These findings confirm that V4-ELPs are effective vectors for intracellular delivery of protein cargo. Discovery of enhanced EEPs via in silico screening [00372] To further improve the efficacy of V4-ELPs, EEPs were designed with enhanced activity. Among EEPs initially screened for Cre protein delivery in HEK293-RFP cells, the
  • S10 is comprised of amphipathic ⁇ -helical peptide variants of CM18 52 and PTD4 53 , separated by a Gly-Ser linker, in contrast to other polycationic EEPs that are either unstructured or contain a single ⁇ -helical domain (FIGs. 22A-22B).
  • the amphipathic ⁇ -helical structure is thought to facilitate intercalation within lipid bilayers, particularly negatively charged bilayers, characteristic of late endosomes. 54,55 [00373]
  • an ⁇ -helix peptide database was compiled and screened to identify next generation EEPs with enhanced functionality.
  • a multiple linear regression model was trained on a published dataset of 73 membrane disruptive peptides encoded by a set of 15 physicochemical, structural, and compositional features, labeled with efficacy values derived from an in vitro GFP protein delivery assay (Table 1).
  • 56 F-score screening of each feature with respect to peptide efficacy revealed specific physicochemical properties, including hydrophobic moment, net charge, and amphipathic ⁇ - helical structure or lack thereof, which were highly correlated with delivery efficacy (FIG. 23).
  • Predicted efficacy (%) 5.9 ⁇ hydrophobic moment + 3.2 ⁇ net charge – 3.2 ⁇ pI – 4.3 ⁇ % negative residues + 20.8 ⁇ is amphipathic helix Table 1. Physicochemical and compositional descriptors used to encode peptide library for predictive model training.
  • V4-ELPs containing each of these derived EEPs were generated and assessed for their ability to deliver free Cre protein at a dose of 100 nM to avoid effect saturation (near 100% RFP positivity) observed in prior screening studies at 2 ⁇ M.
  • the highest performing V4-ELP-EEP led to RFP expression in 25.7% of cells, but underperformed S10 (44.1%, FIG. 4C).
  • V4- ELP-EEP1 and -EEP5 were enriched in lysine over arginine among positively charged residues, with 87% and 85% of positive residues comprised of lysine, respectively.
  • V4- ELP-EEP1 and -EEP5 SEQ ID NOs: 67 and 71
  • V4-ELP-EEP13 V4-ELP-EEP14
  • EEP13 is a dimeric amphipathic ⁇ -helical peptide composed of two synthetic antimicrobial peptides (FIGs. 4E- 4F).
  • Multimerization of cell-penetrating peptides enhances membrane disruption by increasing binding affinity of multimeric cationic peptides for anionic proteoglycans in the plasma membrane.
  • BMP anionic phospholipid bis(monoacylglycero)phosphate
  • V4-ELPs containing EEP13 as a monomer, dimer, or trimer were evaluated for their ability to deliver free Cre protein.
  • V4-ELPs containing an EEP13 dimer (SEQ ID NO: 80) or trimer (SEQ ID NO: 81) outperformed those containing an EEP13 monomer (SEQ ID NO: 79) at doses between 500 nM (50.8% vs.7.02% RFP+) and 2 ⁇ M with limited cytotoxicity noted at the highest dose (FIGs. 4H-4I).
  • V4-ELP-[EEP13]2 displayed an EC50 of 500 nM in the presence of 2 ⁇ M Cre, while all three variants of V4-ELP-EEP13 led to RFP expression in 50% of cells at a Cre dose of 30 nM (FIGs. 27A-27B).
  • V4-ELP-EEPs To examine the ability of V4-ELP-EEPs to deliver cargo larger than Cre recombinase (38 kDa), FITC-labeled dextrans with molecular weight up to 2 megadaltons were tested as cargo in a co-formulation study. Both V4-ELP-EEP13 and V4-ELP-[EEP13]2 effectively facilitated cytoplasmic entry of dextrans of
  • V4-ELP-EEP13 to deliver free SpCas9 RNP formulated with a sgRNA targeting HPRT1 (hypoxanthine-guanine phosphoribosyltransferase 1) to HEK293 cells.
  • HPRT1 hyperxanthine-guanine phosphoribosyltransferase 1
  • V4-ELP-EEP13 facilitated efficient gene editing at a level ( ⁇ 65%) comparable to the positive control CRISPRMAX, a commercial lipid-based system optimized for RNP delivery (FIG. 44), establishing the potential of ENTER to serve as a delivery vehicle for gene editors.
  • ELP-mediated protein delivery to primary cells ex vivo [00378] Efficient delivery to primary cells presents another hurdle for delivery systems.
  • the ability of ELP nanoparticles to deliver Cre protein was evaluated in a variety of primary cells, including lung fibroblasts, peritoneal macrophages, splenic CD4+ T cells, and hematopoietic stem cells from reporter mice (e.g., from Ai9-SauSpyCas9 mice).
  • V4- ELP-[EEP13] 2 (SEQ ID NO: 80) significantly outperformed V4- ELP-EEP13 (SEQ ID NO: 79) particularly at low ELP concentrations, achieving nearly 90% tdTomato+ cells at 4 ⁇ M ELP with minimal cytotoxicity (FIGs. 5A-5B, 28A-28B).
  • Lipofectamine 3000 was much less effective in delivering Cre protein with tdTomato+ expression observed in fewer than 15% of cells at the highest tested dose (FIGs.29A-29B).
  • Optimal delivery efficiency to macrophages was observed following co- treatment with 4 or 8 ⁇ M V4-ELP-EEP13 (SEQ ID NO: 79) and 8 or 16 ⁇ M Cre, respectively (FIGs. 5C-5D). Viability at these effective doses was generally high ( ⁇ 80% from ⁇ 90% viability when untreated), though some ELP- and Cre-dose dependent toxicity was observed (FIGs. 28C-28D). Up to 80% of CD4+ T cells were tdTomato+ after treatment with Cre-
  • V4-ELPs 107/179 B0662.70120WO00 bearing V4-ELPs for 48 hours, with improved efficacy observed for co-delivery of V4-ELPs and free Cre protein at the lowest Cre and ELP concentrations tested (FIG. 5E).
  • treatment of CD4+ T cells result in notable dose- dependent cytotoxicity (FIG.28E).
  • monomeric V4-ELP-EEP13 was further evaluated. In fibroblasts, co-treatment with V4-ELP-EEP13 and Cre resulted in a dose-dependent increase in tdTomato+ cells of up to 70% with minimal cytotoxicity (FIGs.
  • CD4+ T cells treated with Cre and V4-ELP-EEP13 exhibited up to 50% tdTomato+ cells with both ELP and Cre dose-dependent editing efficiency (FIGs. 39E-39F). Notable cytotoxicity was observed at the highest tested concentration of ELP, while Cre dose escalation did not result in significant changes in cell viability (FIGs. 54E-54F). Finally, V4- ELP-EEP13 facilitated effective delivery of Cre into Lin-Sca-1+c-kit+ hematopoietic stem cells, enabling ⁇ 20% tdTomato+ cells at optimal dosing with minimal cytotoxicity (FIGs. 39G-39H, FIGs. 54G-54H).
  • V4-ELP-EEP13 demonstrates robust delivery efficacy across various primary cell types, including cells within hematological lineages.
  • the ability to facilitate Cre protein delivery was also examined in Sca-1+ primary bone marrow cells isolated from Ai9 mice.
  • V2-ELPs were functionalized with AF633-labeled Cre and an anti-CD117 mAb via sortase conjugation (FIG. 31A).
  • CD117 targeting enhanced Cre delivery to Lin-Sca1+ HSPCs populations (45% vs.18%) (FIGs. 30, 31B).
  • V3-ELPs bearing Cre and containing penetratin enabled expression of tdTomato+ in ⁇ 2% in Lin- Sca1+c- Kit+ (LSK) HSPCs (FIG. 5F) with some dose-dependent cytotoxicity observed (FIG. 28F).
  • ENTER The ability of ENTER to facilitate delivery of transcription factors was assessed; transcription factors represent another class of proteins of therapeutic interest due to their ability to reprogram cell state.
  • transcription factors represent another class of proteins of therapeutic interest due to their ability to reprogram cell state.
  • tumor-associated macrophages have been reprogrammed towards an antitumor phenotype by delivery of mRNA-encoded interferon regulatory factor 5 (IRF5), a proposed master transcription factor for pro-inflammatory M1 macrophages.
  • IRF5 interferon regulatory factor 5
  • V4-ELP-EEP13 was evaluated for its ability to deliver IRF5 protein to primary mouse macrophages in vitro.
  • V4-ELP-EEP13 effectively enabled the internalization of AF488-labeled IRF5 into murine peritoneal macrophages after 2 hours (FIG. 39I), supporting a synergistic uptake of IRF-5 stimulated by ELP nanoparticles. Macrophages were then treated with V4-ELP-EEP13 alone, IRF5 alone, or both for 48 hours.
  • V4-ELP-EEP13 SEQ ID NO: 79
  • V4-ELP-[EEP13] 2 SEQ ID NO: 80
  • V4- ELP-[EEP13]3 SEQ ID NO: 81
  • All three ELPs displayed effective dose-dependent complexation of siRNA via gel shift and RiboGreen RNA binding dye exclusion assays (FIG. 6B).
  • V4-ELP- EEP13 and V4-ELP-[EEP13]2 nanoparticles showed a modest ( ⁇ 10-20 nm) increase in size (FIG. 6C). This increase in size was accompanied by a decrease in zeta potential (FIG. 6D).
  • V4-ELP-EEP13 SEQ ID NO: 79
  • V4-ELP-[EEP13]2 SEQ ID NO: 80
  • V4- ELP-[EEP13]3 SEQ ID NO: 81
  • RNAiMAX lipid- based transfection reagent
  • V4-ELP-EEP13 series constructs were used to deliver an siRNA mediating GFP knockdown in HeLa cells expressing a destabilized GFP (HeLa- d2eGFP). Both the dimeric and trimeric constructs displayed high potency relative to the monomeric construct.
  • the dimeric and trimeric constructs enabled 80-85% GFP knockdown at an ELP concentration of 1 ⁇ M with 100 nM siGFP, following treatment for 24 hours with minimal cytotoxicity (FIG. 6F), and the dimeric construct displayed mediated up to 60% GFP knockdown at an ELP concentration of 5 ⁇ M with 100 nM siGFP following treatment for 24 hours, with minimal cytotoxicity (FIG. 6F-6G).
  • V4-ELP-[EEP13]2 SEQ ID NO: 80
  • V4-ELP-[EEP13]3 SEQ ID NO: 81
  • V4- ELP-EEP13 SEQ ID NO: 79
  • this effect may be related to the efficiency of siRNA release from the particles.
  • an excess of EEP-associated positive charge in V4-ELP-[EEP13]2 relative to the V4-ELP-EEP13 may be detrimental for intracellular decomplexation, reducing delivery efficiency.
  • V4-ELP-EEP13 (SEQ ID NO: 79) also effectively facilitated delivery of a GAPDH-targeted siRNA into Ai9-derived murine lung fibroblasts, inducing ⁇ 95% knockdown of mRNA expression comparable to RNAiMAX (FIG. 40A).
  • ELPs serve as an effective vehicle for in vitro siRNA delivery.
  • HEK293 cells were pretreated for 1 hour with chemical inhibitors known to primarily affect specific pathways: chlorpromazine (clathrin-mediated endocytosis, CME), nystatin (caveolin and clathrin independent carrier pathways), Dynasore (dynamin-dependent pathways, including CME), and amiloride (macropinocytosis)82, and were then treated with Alexa Fluor 647-labeled siRNA complexed with V4-ELP-EEP13 (SEQ ID NO: 79) series
  • V4-ELP- EEP13 SEQ ID NO: 79
  • V4-ELP-[EEP13] 2 SEQ ID NO: 80
  • V4- ELP-[EEP13] 3 SEQ ID NO: 81
  • Cre mRNA 1335 nt
  • ELP:mRNA molar ratios were required for effective complexation as compared to ELP- siRNA complexation, given the larger size of the mRNA cargo (150 vs 25 molar ratios for V4- ELP-EEP13 complexation of Cre mRNA and siRNA, respectively) (FIG. 7A).
  • the size and zeta potential of ELP-mRNA complexes were assessed to determine the ability of ELPs to effectively encapsulate mRNA.
  • the size of V4-ELP-EEP13 series nanoparticles increased between 5-20 nm following Cre mRNA complexation, with the largest size increase being observe with the monomeric construct (FIG.7B).
  • the zeta potential of V4-ELP-EEP13 micelles following mRNA complexation was unchanged (FIG.7C), indicative of efficient encapsulation and supporting that the majority of mRNA is shielded in the core.
  • V4-ELP-EEP13 series constructs were investigated through the delivery of mRNA-encoded Cre recombinase to HEK293-RFP cells.
  • ELPs containing EEPs generated through three generations of iterative design were assessed for their ability to deliver Cre mRNA to HEK293-RFP cells and compared to their Cre protein delivery efficiency (FIG. 36B and FIG. 7G).
  • V4-ELP-EEP13 SEQ ID NO: 79
  • -EEP14 SEQ ID NO: 82
  • V4-ELP-EEP13 SEQ ID NO: 79
  • V4-ELP-[EEP13]2 SEQ ID NO: 80
  • V4- ELP-EEP13 SEQ ID NO: 79
  • V4-ELP-EEP13 (SEQ ID NO: 79) enabled RFP+ expression at a level comparable to or greater than that achieved by MessengerMAX, a commercial lipid-based transfection agent, with significantly less observed cytotoxicity (FIGs. 34A-34B).
  • V4-ELP-EEP13 (SEQ ID NO: 79) performed comparably to Lipofectamine in delivering plasmid DNA-encoded Cre recombinase to HEK293-RFP with minimal cytotoxicity (FIG. 41A, FIG. 57C).
  • V4-ELP-EEP13 (SEQ ID NO: 79) also demonstrated effective Cre mRNA delivery to primary Ai9 lung fibroblasts in a dose-dependent manner, outperforming MessengerMAX at ELP concentrations greater than 10 ⁇ M (57.1% vs.39.9%, FIG. 7F).
  • FIG. 35 shows that increasing mRNA concentration reduces the presence of free, non- micelle associated, cell penetrating, albeit cytotoxic, ELP-EEP protein polymers.
  • V4-ELP-EEP13 SEQ ID NO: 79
  • 30 V4-ELPs containing EEPs generated through three generations of iterative design were assessed for their ability to deliver Cre mRNA and protein to HEK293- RFP cells (FIGs. 36A-36B).
  • R 0.82, FIG. 7G
  • EEP13 identified as the most effective, despite the disparate nature of the two cargos. This suggests the existence of a common mechanistic pathway by which macromolecule endosomal escape is triggered, irrespective of the cargo’s physicochemical properties.
  • V4-ELP-EEP13 SEQ ID NO: 79
  • SpCas9 mRNA or pDNA were co-complexed with a chemically modified sgRNA targeting HPRT1 at varying mRNA/pDNA:sgRNA weight ratios. In both formats, a weight ratio of 2:1
  • V4-ELP-EEP13 SEQ ID NO: 79
  • V4-ELP-S10 SEQ ID NO: 84
  • HPRT1 hyperxanthine-guanine phosphoribosyltransferase 1
  • FIG. 8B Modest editing efficiency ( ⁇ 20%) was observed at the highest doses tested (100 nM sgRNA/Cas9, 8 ⁇ M ELP, FIG. 8B) establishing the potential of ELPs to serve as a delivery vehicle for gene editors.
  • Transcription factors represent another class of proteins of therapeutic interest due to their cell polarizing activity.
  • tumor-associated macrophages have been reprogramed towards an antitumor phenotype by delivery of mRNA-encoded interferon regulatory factor 5 (IRF5), a transcription factor that regulates the pro-inflammatory M1 polarization of macrophages.
  • IRF5 interferon regulatory factor 5
  • V4-ELP-EEP13 SEQ ID NO: 79 was evaluated for its ability to deliver IRF5 to primary mouse macrophages ex vivo (FIG. 8C).
  • Peritoneal Ai9 macrophages were treated with V4-ELP-EEP13, soluble IRF5, or both for 48 hours.
  • lymphoid cells CD45+CD90.2+
  • fibroblasts CD45-CD90.2+
  • myeloid cells CD45+CD90.2- CD11b+
  • FIG. 37 tdTomato expression was observed in 10% of lymphoid cells, 2.5% of fibroblasts, and 6% of myeloid cells (FIGs. 9A-9C, 38) with comparable editing efficiency for the delivery of free or micelle bound Cre in association with S10-V3-ELP (SEQ ID NO: 60).
  • V4-ELP-EEP13- mediated delivery of Cre protein to the airway epithelium lining was assessed in mice.
  • Ai9 mice received three daily doses of V4-ELP-EEP13 (SEQ ID NO: 79) nanoparticles (100 ⁇ M, ⁇ 24 mg/kg) co-formulated with Cre recombinase protein (50 ⁇ M, ⁇ 8 mg/kg) or Cre protein alone via intranasal instillation; lungs were harvested for image-based evaluation of tdTomato expression 5 days following the final treatment (FIG. 42A).
  • mice co-treated with Cre protein and V4-ELP-EEP13 displayed significantly higher editing of the bronchial epithelium in both large airways (FIG. 42B and FIG. 42D) and small airways (FIG. 42C and FIG. 42E).
  • 25.4 ⁇ 3.7% of epithelial cells in the large airways of co-treated mice were tdTomato+ (8.9 ⁇ 1.8% in mice treated with Cre protein alone), while 9.2 ⁇ 0.8% of epithelial cells in the small airways of co-treated mice were tdTomato+ (1.8 ⁇ 0.3% in mice treated with Cre protein alone).
  • ENTER can thus be leveraged for intracellular delivery of therapeutic protein cargo in vivo.
  • a non-viral, recombinant protein nanoparticle system (Elastin-based Nanoparticles for Therapeutic delivERy (ENTER)) was developed as a versatile platform for effective cytosolic delivery of multiple types of biomacromolecules of therapeutic interest, including siRNA, mRNA, pDNA, RNPs, and protein cargo.
  • ENTER Therapeutic delivERy
  • Prior success in cytosolic delivery of mRNAs, proteins, and RNPs have largely been achieved via lipid nanoparticles and virus-like particles.
  • 98,99 114/179 B0662.70120WO00 This ELP-based platform is believed to be the first use of non-viral recombinant protein nanoparticles for intracellular mRNA and protein delivery.
  • the fourth-generation multiblock micelle copolymer design differs in multiple ways from previous ELP systems utilized for cytosolic delivery of nucleic acids, such as pDNA38,85,86, siRNA87, and CpG88.
  • nucleic acids such as pDNA38,85,86, siRNA87, and CpG88.
  • ENTER displays an organized micellar structure.
  • the hydrophilic shell ensures well-controlled surface chemistry, allowing for flexibility in modification with functional modalities like targeting moieties through sortase-mediated conjugation or genetic fusion.
  • the core-facing cationic EEP remains shielded, facilitating the encapsulation of nucleic acid cargo in the core.
  • Micelle assembly is driven by a combination of hydrophobic and electrostatic interactions, strengthened by cysteine crosslinking.
  • the presence of histidines in the ELP hydrophobic block facilitates micelle disassembly in acidic environments, enabling the exposure of EEP and cargo release only in acidic late endosomes. This feature contributes significantly to low cytotoxicity and high delivery efficiency.
  • ENTER shares similarities with other state-of-the-art delivery systems, such as lipid nanoparticles, viruses, and virus-like particles, each exhibiting organized structures with surface modules separated from core-encapsulated cargo, accompanied by a triggered release mechanism.
  • ENTER represents an advancement in engineering the structural complexity and responsiveness required for potent macromolecule delivery.
  • the effectiveness of ENTER is significantly enhanced by the identification of novel and potent EEPs.
  • ELP-S10 displayed nearly 20-fold higher potency in Cre protein delivery as compared to S10 alone (SEQ ID NO: 36) (FIG. 2F), with potency further enhanced by the incorporation of EEP dimers at the C-terminus of the ELP protein polymer (FIG. 4G).
  • a dimer [EEP13] 2 (SEQ ID NO: 80), 87 AAs) has a length that would make its manufacturing by solid phase peptide synthesis challenging. Given that the mechanism of action of such amphipathic ⁇ -helical peptides is related to their ability to intercalate, disrupt, and induce fusion of lipid membranes 54 , increased local endosomal EEP concentration afforded by ELP micelle multivalency is presumed to directly enhance this activity. [00402] Secondly, ELP-EEP fusion dramatically reduced EEP-associated cytotoxicity. Standalone EEP-mediated transfection often requires peptide removal from cell culture media after a brief incubation due to high cytotoxicity, limiting the in vivo applicability of this approach.
  • ELP-EEP ELP-EEP
  • prolonged peptide incubation was conducted in various cell types without significant cytotoxicity.
  • several factors may contribute to this phenomenon.
  • ELP nanoparticles require endocytosis and acidification- induced disassembly to expose the encapsulated EEP.
  • the kinetics of such exposure and the nature of endosomal membrane disruption likely contributes to substantially less cytotoxicity irrespective of the efficiency of cargo release into the cytosol.
  • V4-ELP-EEP13 demonstrated an EC 50 in the submicromolar to low single-digit micromolar range for delivering protein and nucleic acid cargos into various cell lines and primary cells. This represents a significant improvement in efficacy compared to previous reports, in which standalone endosomolytic peptides were functional in the 10 to 40 ⁇ M
  • V4-ELPs are comparable or even outcompete commonly used lipid-based transfection reagents like Lipofectamine 3000, RNAiMAX, and MessengerMax under a variety of conditions in the delivery of protein, siRNA and mRNA, respectively.
  • V4-ELP- EEP13 in particular exhibited comparable or superior performance in delivering proteins and nucleic acids. The enhanced efficiency of V4-ELP is especially evident when compared to Lipofectamine for Cre protein delivery.
  • V4-ELP appears to potentiate cellular uptake of both positively charged Cre and negatively charged IRF-5 as a consequence of nanoparticle stimulated collaborative uptake or macropinocytosis. 15,77 This highlights the charge-insensitive nature of the V4-ELP-EEP system and its ability to deliver positively charged cargo, which remains a challenge for lipid nanoparticles and other nonviral delivery systems.
  • V4-ELPs were also effective as a delivery system for a variety of cell lines and primary cells, including hematopoietic stem cells, macrophages, and primary T cells. This is significant as transfecting these cells has historically posed a challenge for non-viral delivery systems. These observations suggest that the mechanisms responsible for V4-ELP uptake and endosomal disruption are conserved across different cell types.
  • Multiblock micelle copolymer designs in which the cationic domain is shielded from the surface offer a number of key advantages. 66,67 Such designs reduce cytotoxicity, enable nucleic acid encapsulation, improve particle stability through combining hydrophobic and electrostatic assembly, and provide great flexibility for engineering the nanoparticle surface.
  • ELP nanoparticles were designed for efficient delivery of siRNA and mRNA (FIG. 6C and FIG. 7B). While the ability of ELPs to mediate intracellular protein delivery has not been previously reported, prior efforts directed at the delivery of nucleic acids, including pDNA 33,78,79 , siRNA 80 , CpG 81 have been in the form of amorphous polyion complexes (PICs). Such complexes do not display organized structure or the stability of the V4-ELP nanoparticles. [00407] The uniqueness of ELPs as a recombinant protein offers several advantages over other nanoparticle systems, such as LNPs or synthetic polymeric nanoparticles. The genetically defined composition of ELPs enables greater manufacturing precision when
  • ELPs and ENTER represent a promising in vivo and ex vivo delivery platform for various cargos across numerous therapeutic indications.
  • Methods Plasmid Cloning [00410] DNA sequences coding ELP were codon optimized for E. Coli expression, synthesized, and cloned into previously reported pQE-80L backbone using commercial services from GenScript. 34 Cre and Cas9 with LPETG tag and GGS linker were initially subcloned in house using previously reported genome editor coding plasmids. 35 Cre with variable cleavable linkers were cloned using GenScript.
  • a constitutively active mutant of human IRF5 splice variant 5 carrying five serine to aspartic acid mutation (IRF-5/4D) with furin cleavable linker and LPETG tag was codon optimized for E. Coli expression, synthesized, and cloned into pET backbone by GenScript.
  • 92 Protein expression and purification [00411] All plasmids were transformed into E. coli strain BL21(DE3) (Invitrogen, Thermo Fisher Scientific; C601003) for expression. E. coli were grown in Luria-Bertani medium supplemented with 100 ug/ml of ampicillin at 37 °C with continuous shaking at 225 rpm till
  • ELPs were precipitated in hot cycle by incubating with 3M NaCl at 30 °C for 50 minutes. Following centrifugation, ELP pellets were resolubilized in PBS with 10mM DTT at 4C during in the cold cycle. Un-resoluble impurities were removed by centrifugation at 20,000g at 4 °C for 30 minutes.
  • cationic exchange chromatography HiTrap SP HP, Cytiva was utilized as the second step of purification following manufacturer’s protocol under denaturing condition. ELP-EEP13 and ELP-(EEP13)2 were eluted at about 200mM and 300 mM NaCl concentrations respectively.
  • Unconjugated Cas9 was removed by glycerol gradient ultracentrifugation. Briefly, solution was layered on top of a glycerol gradience from 5 to 55% and centrifuged at 39,000RPM in a Beckman XPN-80 ultracentrifuge for 4 hours at room temperature (about 22 °C). Unconjugated Cas9 presented in 5 to 15% glycerol fractions whereas formed Cas9-ELP nanoparticles resided in 35 to 45% fractions.
  • Anti-CD117 antibody was azide-modified using the SiteClick Antibody Azido Modification kit (Invitrogen, Thermo Fisher Scientific; S10900) according to the manufacturer’s protocol. Azide modified antibody was successfully conjugated to ELP via both sortase reaction and chemical conjugation.
  • KLPETGG SEQ ID NO: 118
  • peptide with N-terminus amine acetylated was commercially synthesized (GenScript) and the only primary amine on the side chain of lysine was coupled with DBCO-NHCO-PEG13-NHS ester linker (BroadPharm).
  • DBCO- NHCO-PEG13-NHS ester linker (BroadPharm) was incubated with V1-ELP-A633, which only carries one free amine at N-terminus for bio-orthogonal conjugation. Unreacted linkers were removed via 10KD centrifugal filter. Purified DBCO-Diblcok-cysteine-A633 was then incubated with azide modified CD117(2B8) antibody at room temperature overnight with constant agitation. Unreacted Ab was removed via 100KD centrifugal filter at 37 °C.
  • ELP-RNA nanoparticle formation [00418] To form ELP-RNA NPs, siRNA or mRNAs were added to ELPs at varying molar ratios. An ELP stock concentration of 200 ⁇ M was generally used. For siRNA particle formation, the NPs were incubated at room temperature with continuous shaking at 500 rpm for 30 minutes.
  • siRNAs used in the described studies include a GFP-targeted siRNA (green fluorescent protein) (Thermo Fisher Scientific), AF647-labeled siRNA (AllStars), and a scrambled siRNA (Applied Biosystems, Thermo Fisher Scientific; 4390843).
  • GFP-targeted siRNA green fluorescent protein
  • AllStars AF647-labeled siRNA
  • scrambled siRNA Applied Biosystems, Thermo Fisher Scientific; 4390843
  • the NPs were first incubated on ice with continuous shaking at 500 RPM for 30 minutes, followed by a 30- minute room temperature (about 22 °C) incubation at 500 RPM.5-methoxyuridine-modified Cre mRNA (TriLink) was used.
  • mRNA encapsulation efficiency of each LNP formulation was calculated using the Quant-iT RiboGreen (Thermo Fisher Scientific) or electrophoretic mobility shift assays (gel shift assays).
  • Gel shift assays ELP-RNA nanoparticles were formed as described above then run in a 1% agarose gel at 100 V for 30 minutes.
  • RiboGreen assays ELP-RNA nanoparticles were formed in 1x PBS at varying molar ratios then diluted 1:1 in 1x TE buffer containing the fluorescent RiboGreen reagent. A final RNA concentration of 133 ng/mL was
  • HeLa cells for AF647-labeled siRNA uptake assessment
  • HeLa-d2eGFP cells for GFP knockdown assessment
  • siRNA-containing ELPs prepared as described above were used to treat cells for up to 24 hours.
  • GFP expression and siRNA uptake were evaluated using flow cytometry.
  • Cell viability was monitored by Zombie Aqua live/dead cell staining (Thermo Fisher Scientific) prior to flow analysis.
  • RNAiMAX (Thermo Fisher Scientific) was used as a positive control.
  • HEK293-RFP cells were seeded in 96-well plates at a density of 20,000 cells/well and left overnight.
  • Cre pDNA pCAG-Cre, Addgene Plasmid #13775
  • Cre mRNA Trilink
  • Cre protein-ELP particles prepared as described above were used to treat cells for 48 hours (mRNA) or 72 hours (pDNA).
  • RFP expression was evaluated using flow cytometry. Cell viability was monitored by Zombie Aqua live/dead cell staining (Thermo Fisher Scientific) prior to flow analysis. For mRNA transfection studies, MessengerMAX (Thermo Fisher Scientific) was used as a positive control.
  • HEK293-RFP were treated with ELP nanoparticles either 1) containing pDNA/mRNA- encoded SpCas9 alongside a chemically modified sgRNA against HPRT1 (Synthego), or 2) co- formulated with True SpCas9 (Thermo Fisher Scientific) RNP complexing the same HPRT1 sgRNA.
  • Genomic DNA was isolated from treated cells after 72 hours, then the target locus was amplified via PCR and sequenced via Sanger sequencing. Editing efficiency was quantified using TIDE analysis 101 .
  • the Alpha Helix Database (AHDB) screened to identify enhanced EEPs was constructed by combining peptides from three sources: 1) the Antimicrobial Peptide Database (APD) 57 , from which peptides with a known helical structure were selected, 2) the Database of Antimicrobial Activity and Structure of Peptides (DBAASP) 58 , from which all monomeric peptides without chemical modifications were selected, and 3) the Therapeutic Peptide Design database (TP- DB) 59 , a database that contains all ⁇ -helical peptides extracted from the PDB. The TP-DB was filtered to select peptides likely retain an ⁇ -helical secondary structure outside of their native protein.
  • API Antimicrobial Peptide Database
  • DBAASP Database of Antimicrobial Activity and Structure of Peptides
  • TP- DB Therapeutic Peptide Design database
  • AHDB contained a total of 11,084 peptides.
  • the predicted efficacy of each peptide was calculated.
  • a library of dimers was formed by combining peptide monomers with predicted efficacies > 10%. From these dimers, sequences to be recombinantly incorporated into ELP constructs for in vitro selection were manually selected. The selected dimeric peptides had a predicted efficacy greater than the most effective peptide in the training dataset, were composed of sequences of non-microbial origin, and were less than 50 residues in length (Gen1 EEPs).
  • Gen2 EEPs enriched in lysine were generated through a second in silico screen. Using a dataset derived from a screen of V4-ELPs for siRNA delivery, a second linear regression model was trained (FIG. 24) which was used to rescreen the compiled ⁇ -helical
  • Macrophages were cultured in complete medium: Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (Hyclone, GE Healthcare, IL) and treatments were conducted within 1 week.
  • DMEM Modified Eagle Media
  • fetal bovine serum Hyclone, GE Healthcare, IL
  • CD4+ T cells were isolated as previously described. 95 Spleens were harvested and red blood cells were lysed by osmotic shock, and single-cell suspensions were collected using a 70- um cell strainer.
  • CD4+ T cells were isolated by negative selection using a T cell isolation kit (Miltenyi Biotec) and stimulated with plate-bound CD3 (10 ⁇ g/ml) and soluble CD28 antibody (5 ⁇ g/ml) in RPMI medium supplemented 10% FBS, 1%P.S., 50 ⁇ M 2-mercaptoethanol and 50U/ml IL-2 for 4 days before treatment.
  • plate-bound CD3 10 ⁇ g/ml
  • soluble CD28 antibody 5 ⁇ g/ml
  • RPMI medium supplemented 10% FBS, 1%P.S., 50 ⁇ M 2-mercaptoethanol and 50U/ml IL-2 for 4 days before treatment.
  • Bone marrow Sca1+ progenitor cells were isolated and cultured as previously described. 96 Briefly, tibias, femur and iliac crests were dissected from reporter mice and crushed by pestle in mortar to release bone marrow cells.
  • Sca1+ cells were isolated using Sca1 enrichment kit according to the manufacture’s protocol (Miltenyi Biotech). Sca1 enriched cells were cultured in serum free StemSpanTM SFEM II medium (Stemcell technologies) supplied with mouse SCF (50 ng/ml), mouse TPO (50 ng/ml), mouse Flt3L (50 ng/ml) and human IL- 11 (50 ng/ml) (Peprotech) for 3 days before treatment.
  • serum free StemSpanTM SFEM II medium Stemtechnologies supplied with mouse SCF (50 ng/ml), mouse TPO (50 ng/ml), mouse Flt3L (50 ng/ml) and human IL- 11 (50 ng/ml) (Peprotech) for 3 days before treatment.
  • V4-ELP-EEP13 100 ⁇ M, ⁇ 24 mg/kg was formulated with Cre protein (50 ⁇ M, ⁇ 8 mg/kg) in HBSS and administered to 8-week-old male Ai9 via intranasal instillation of a 50 ⁇ L volume. Mice were anesthetized with ketamine via intraperitoneal injection. Mice were
  • tdTomato+ epithelial cells in mouse airways [00432] Lung tissues were fixed with 4% paraformaldehyde by tracheal instillation and cryopreserved. The tissue was placed ventral side down for sectioning. After reaching a depth of sectioning at which all lung lobes were visible, a total of 16 sections were collected at 4 levels, spaced ⁇ 112 ⁇ m apart (4 sections per level, 2 sections per slide). One slide at each level was stained by H&E for histopathologic assessment.
  • the other slide was systemically imaged for quantification of tdTomato expression in the bronchial epithelium.
  • Airways were defined as large or small airways based on their size and anatomical position.
  • the left and right main bronchi were categorized as large airways.
  • Small and large airway views to be imaged were identified through evaluation of the DAPI-stained nuclei pattern alone to prevent biased selections.
  • ELP-mediated Cell Targeted Delivery [00434] The functionalization of cargo-loaded ELP nanoparticles with moieties to facilitate cell receptor-targeted internalization can facilitate functional delivery in specific cell types.
  • an enzyme- and click chemistry-based conjugation reaction scheme was designed and implemented to functionalize ELP nanoparticles with monoclonal antibodies (mAbs).
  • mAbs monoclonal antibodies
  • an anti-CD117 mAb was functionalized with azide groups, followed by conjugation of LPETG-containing linker to the antibody via click chemistry (FIG. 2I).
  • the mAb-linker was conjugated to an V1-ELP micelle by sortase-mediated transpeptidation.
  • ELP sequences were identified to improve accessibility of surface- exposed N-terminus and enable protein-protein conjugation, for example, increasing the hydrophilicity of ELP backbone to improve terminal end exposure and protein ligand presentation.
  • V4-ELP which has a neutral hydrophilic shell in comparison to the anionic shell in V1-V3, displayed lower sortase conjugation efficiency than V1-V3 designs.
  • G V5(G)
  • S V5(S)
  • Q V5(QQ) guest residues instead of A would increase stability and improve ligand presentation.
  • V5-ELP-EEP13 designs i.e., V5(G), V5(S), V5(Q)
  • sortase and Cre-LPETG FIG. 58
  • Conjugation efficiency of the V5-ELP- EEP13 designs were quantified by SDS-PAGE by comparing the relative intensity of Cre- ELP bands versus the ELP bands and demonstrated improved sortase conjugation efficiency in comparison to V4-ELP-EEP13 (e.g., V4) (FIGs. 59A and 59B).
  • HSC hematopoietic stem cell
  • the present example also describes a size exclusion-based purification method to isolate excess antibody from mAb-ELP nanoparticles.
  • Nanoparticles were formulated by adding 50 uM of ELP-EEP13 with varying amounts (e.g., ELP:siRNA molar ratio of 1:0, 1:100, 1:50, 1: 25, or 1:10) of AF647-labeled siRNA (e.g., ELP:siRNA molar ratio of 1:0, 1:100, 1:50, 1: 25, or 1:10), which was formulated in water, followed by addition of PBS to 1x concentration.
  • the nanoparticles were purified by size exclusion chromatography using qEVoriginal column (Izon).
  • particle solution was loaded onto the column and washed with buffer multiple times (28 total), and 400 uL fractions were collected. Analysis of the fractions revealed that the particle formulated with higher ELP:siRNA molar ratios (with more siRNA) enabled more effective ELP nanoparticle purification via SEC (FIG. 63). Without being bound by theory, it is thought that the ionic bonds between EEP13 and siRNA create additional crosslinks, stabilizing particles against dilution that occurs during SEC. Purification of mAb-ELP-siRNA nanoparticles was determined by subsequent analysis of SEC fractions (FIG. 64) .
  • EXAMPLE 3 Endosomal Escape Peptide (EEP) Development for ELP-EEP Fusion
  • E3Ps enhanced endosomal escape peptides
  • the pipeline combined (1) models trained to predict EEP efficacy with (2) a generative model for creation of EEP-like sequences and (3) an optimizer algorithm to modify starting sequences towards improving their predicted efficacy (FIG. 68).
  • the predictive model was iteratively refined using data from each generation of characterized EEPs.
  • the present disclosure characterized 68 de novo sequences and identified 1 (E3P2.5) with comparable activity to EEP13.
  • the EEP generator model used in step (1) was a variation autoencoder (VAE) trained for EEP sequence generation (FIG. 69).
  • VAE variation autoencoder
  • the encoder and decoder were both Long short-term memory (LSTMs), which is a type of recurrent neural network).
  • LSTMs Long short-term memory
  • the first generation of the predictive model was constructed using a transfer learning approach (FIG. 70).
  • the training data comprised a library of 397 peptide sequences assessed for GFP protein delivery to HeLa cells (shuttle peptide dataset, via Feldan Tx).
  • a multilayer perceptron (MLP) regressor was used to compare/distinguish data of true GFP+ shuttle peptides and predicted GFP+ shuttled peptides.
  • the first generation of the predictive model produced an ELP-EEP dataset of 32 sequences (also referred to as the “Gen1 ELP-E 3 Ps”) (FIG. 71).
  • the second generation of the predictive model leveraged synthetic examples of positive peptides and did not utilize transfer-learning.
  • the model utilized (1) direct encoding with physicochemical feature set (propy), (2) fully connected neural network architecture, and (3) augmentation of dataset using synthetic data points (SMOGN), which is a technique for addressing class imbalances in training datasets, creates synthetic examples by interpolation between existing minority class samples (FIG. 73).
  • SMOGN synthetic data points
  • a function combining computational and empirical Assessment was used to quantify peptide fitness during Gen2 in silico directed evolution (FIG. 74).
  • Fifty ELP-E3P Gen2 peptides were designed, and sixteen were selected to be synthesized for in vitro assessment (FIG. 75).
  • Each of the selected sixteen peptides had a predicted efficacy that exceeded that of EEP13.
  • the sixteen sequences were selected as follows: top 8 Efficacy x Score designs (where the score is a fitness function that assess peptides with physicochemical similarities to previously identified effective peptides as more
  • ELP Nanoparticle Crosslinking Stabilizing self-assembled ELP micelles improves their integrity in circulation following intravenous administration. To accomplish this, reversible disulfide-based ELP crosslinking was evaluated. The reversible disulfide-based cross-linking was enabled by cysteine residues incorporated into ELP constructs. Multiple cysteine-modified ELP designs have been designed, synthesized, and characterized. Cysteine-modified ELP constructs demonstrated efficient crosslinking. In ELP constructs with a C4G3 crosslinking block, crosslinking was reversible intracellularly, did not impede cargo delivery, and also sufficiently stabilized particles to enable distribution into peripheral organs.
  • a library of cysteine-containing ELP constructs was synthesized and assessed for disulfide-based crosslinking efficiency and nanoparticle formation (FIGs. 80A-80B, D12). In one strategy, cysteine residues were incorporated into a crosslinking block (FIG. 80A). In a second strategy, cysteine residues were incorporated into the hydrophobic block (FIG. 80B). Both cysteine-based design approaches enabled effective ELP nanoparticle crosslinking (FIG. 81). [00449] Cys-containing ELPs were conjugated to EEP13 to form Cys-containing ELP-EEP constructs.
  • Crosslinked Cys-containing ELP-EEP particles displayed varied sizes and
  • V4-ELP-C4G3-EEP13 was formulated with varying concentrations of Cre recombinase mRNA, crosslinked overnight via air oxidation.
  • HEK293-loxP-RFP Re reporter cell line
  • Crosslinked particles performed comparably in mRNA delivery efficiency to uncrosslinked particles (FIG. 88).
  • the inventors posited that lower crosslink density improved disassembly relative to the Cys7 design.
  • Pharmacokinetics and biodistribution of Cys-crosslinked ELP nanoparticles were assessed.
  • Cys-crosslinked ELP nanoparticles were formulated with 0.5 mg/kg siRNA + 50 mg/kg ELP (5 uM siRNA + 100 uM ELP in 200 uL injection volume) complexed in HBG buffer via 30 min shaking at RT, then rotation overnight. 15 uL blood was collected at 0, 5, 15, 30, 60, 120, 240 minutes, added to 30 ⁇ L 2% Triton X, sonicated for 10 min, then 75 ⁇ L DMSO was added. The mixture was centrifuged at 21,000 g for 5 min to pellet solid material. 65 ⁇ L of supernatant was used for assessing siRNA concentration in plasma via plate reader
  • ELP-Mediated Cancer Vaccination [00456] This example reports ELP nanoparticle-based cancer vaccines designed to facilitate intracellular delivery of tumor-associated peptide antigens to antigen-presenting cells.
  • ELP- EEP13 constructs modified with peptide epitopes derived from an antigen were designed, synthesized, and tested.
  • ELP-EEP13 variants were modified with OTI peptide (ovalbumin-derived MHC class I epitope) and assessed for intracellular peptide antigen delivery to antigen presenting cells (APCs).
  • ELP-EEP13 variants with different peptide placements FIG. 92 and 93
  • ratios FIG. 94
  • Modified ELPs were evaluated for effective, non-toxic intracellular peptide delivery to dendritic cells in vitro (FIG. 93).
  • DC2.4 cells murine dendritic cell line
  • ELPs V4-ELP-EEP13, OTI-V4-EEP13, V4-OTI-EEP13, or V4- EEP13-OTI
  • OTI peptide alone as a control.
  • 50 ⁇ M ovalbumin protein was used with 10 ⁇ M V4-ELP-EEP13.
  • Cells were stained with anti MHC- I/OTI complex mAb, and peptide presentation was assessed via flow cytometry after 24 hours.
  • ELP nanoparticles facilitated intracellular ovalbumin protein delivery in vitro ( ⁇ 10% OT-I+) and also enabled effective OTI-MHC Class I presentation ( ⁇ 60% in optimal conditions) (FIG. 2CB). Aggregation was observed with N-terminus OTI-modified ELP (OTI-V4-EEP13) and middle OTI-modified ELP (V4-OTIEEP13), resulting in low cell viability at the 10 ⁇ M concentration (FIG. 93A). [00458] Modifying the formulation ratios of OTI-ELP variants enabled mitigated aggregation while retaining effective delivery.
  • Biniossek ML Nägler DK, Becker-Pauly C, Schilling O. Proteomic Identification of Protease Cleavage Sites Characterizes Prime and Non-prime Specificity of Cysteine Cathepsins B, L, and S. J Proteome Res 2011;10:5363–73. https://doi.org/10.1021/pr200621z. 46. Abboud-Jarrous G, Atzmon R, Peretz T, Palermo C, Gadea BB, Joyce JA, et al. Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *.
  • Islam MdZ Ariyama H, Alam JMd, Yamazaki M.
  • APD3 the antimicrobial peptide database as a tool for research and education.
  • DBAASP v3 database of antimicrobial/cytotoxic activity and structure of peptides as a resource for development of new therapeutics.
  • Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells.
  • the present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

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Abstract

La présente invention concerne des polypeptides de type élastine, des peptides d'échappement endosomal et des composés comprenant un polypeptide de type élastine conjugué de manière chimique ou par recombinaison à un ou plusieurs peptides d'échappement endosomal. La présente invention concerne également des compositions comprenant un composé selon la présente invention et un agent, des méthodes d'administration d'un agent et des méthodes de traitement ou de prévention d'une maladie (par exemple, une maladie cardiovasculaire, une maladie pulmonaire ou respiratoire, une maladie musculo-squelettique, une maladie hématologique, un trouble immunitaire, une maladie infectieuse, une maladie génétique, un cancer, une maladie neurologique, un trouble psychiatrique, un trouble métabolique ou une maladie inflammatoire).
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Non-Patent Citations (117)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
ABBOUD-JARROUS GATZMON RPERETZ TPALERMO CGADEA BBJOYCE JACATHEPSIN L ET AL.: "Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 283, 2008, pages 18167 - 76, Retrieved from the Internet <URL:https://doi.org/10.1074/jbc.M801327200>
ABDELGHANI, M ET AL.: "Applications of elastin-like polypeptides in nanomedicine", BIOMEDICAL ENGINEERING
ADAMS DGONZALEZ-DUARTE AO'RIORDAN WDYANG C-CUEDA MKRISTEN AV ET AL.: "Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis", NEW ENGLAND, vol. 379, 2018, pages 11 - 21, XP055841361, Retrieved from the Internet <URL:https://doi.org/10.1056/NEJMoa1716153.> DOI: 10.1056/NEJMoa1716153
AKISHIBA MTAKEUCHI TKAWAGUCHI YSAKAMOTO KYU H-HNAKASE I ET AL.: "Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide", NAT CHEM, vol. 9, 2017, pages 751 - 61, XP055636443, Retrieved from the Internet <URL:https://doi.org/10.1038/nchem.2779> DOI: 10.1038/nchem.2779
ANGSANA JCHEN JSMITH SXIAO JWEN JLIU L ET AL.: "Syndecan-1 modulates the motility and resolution responses of macrophages", ARTERIOSCLER THROMB VASC BIOL, vol. 35, 2015, pages 332 - 40, Retrieved from the Internet <URL:https://doi.org/10.1161/ATVBAHA.114.304720>
ARAFILES JVVHIROSE HAKISHIBA MTSUJI SIMANISHI MFUTAKI S: "Stimulating Macropinocytosis for Intracellular Nucleic Acid and Protein Delivery: A Combined Strategy with Membrane-Lytic Peptides To Facilitate Endosomal Escape", BIOCONJUGATE CHEM, vol. 31, 2020, pages 547 - 53, XP055788002, Retrieved from the Internet <URL:https://doi.org/10.1021/acs.bioconjchem.0c00064> DOI: 10.1021/acs.bioconjchem.0c00064
ASAADI YJOUNEGHANI FFJANANI SRAHBARIZADEH F: "A comprehensive comparison between camelid nanobodies and single chain variable fragments", BIOMARKER RESEARCH, vol. 9, 2021, pages 87, Retrieved from the Internet <URL:https://doi.org/10.1186/s40364-021-00332-6>
BADEN LREL SAHLY HMESSINK BKOTLOFF KFREY SNOVAK R ET AL.: "Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine", N ENGL J MED, vol. 384, 2021, pages 403 - 16, XP093093433, DOI: 10.1056/NEJMoa2035389
BANSKOTA SRAGURAM ASUH SDU SWDAVIS JRCHOI EH ET AL.: "Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins", CELL, vol. 185, 2022, pages 250 - 265, Retrieved from the Internet <URL:https://doi.org/10.1016/j.cell.2021.12.021.>
BINIOSSEK MLNÄGLER DKBECKER-PAULY CSCHILLING O: "Proteomic Identification of Protease Cleavage Sites Characterizes Prime and Non-prime Specificity of Cysteine Cathepsins B, L, and S", J PROTEOME RES, vol. 10, 2011, pages 5363 - 73, Retrieved from the Internet <URL:https://doi.org/10.1021/pr200621z>
BISWAS AJOO K-ILIU JZHAO MFAN GWANG P ET AL.: "Endoprotease-Mediated Intracellular Protein Delivery Using Nanocapsules", ACS NANO, vol. 5, 2011, pages 1385 - 94, XP055144462, DOI: 10.1021/nn1031005
BLASTPBLASTNPASTA ATSCHUL, S. F ET AL., J MOLEC. BIOL., vol. 215, 1990, pages 403
BOISGUERIN PKONATE KJOSSE EVIVES EDESHAYES S: "Peptide-Based Nanoparticles for Therapeutic Nucleic Acid Delivery", BIOMEDICINES, vol. 9, 2021, pages 583
BOYKEN SEBENHAIM MABUSCH FJIA MBICK MJCHOI H ET AL.: "De novo design of tunable, pH-driven conformational changes", SCIENCE, vol. 364, 2019, pages 658 - 64, XP093185999, Retrieved from the Internet <URL:https://doi.org/10.1126/science.aav7897> DOI: 10.1126/science.aav7897
BRAVO-ANAYA LM, GARBAY B, NANDO-RODRIGUEZ JLE, CARVAJAL RAMOS F, IBARBOURE E, BATHANY K: "Nucleic acids complexation with cationic elastin-like polypeptides:Stoichiometry and stability of nano-assemblies", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 557, 2019, pages 777 - 92, XP085876629, DOI: 10.1016/j.jcis.2019.09.054
BRINKMAN EKCHEN TAMENDOLA MVAN STEENSEL B: "Easy quantitative assessment of genome editing by sequence trace decomposition", NUCLEIC ACIDS RES, vol. 42, 2014, pages e168, XP055788071, Retrieved from the Internet <URL:https://doi.org/10.1093/nar/gku936> DOI: 10.1093/nar/gku936
BROCK DJKONDOW-MCCONAGHY HALLEN JBRKLJAČA ZKUSTIGIAN LJIANG M ET AL.: "Mechanism of Cell Penetration by Permeabilization of Late Endosomes: Interplay between a Multivalent TAT Peptide and Bis(monoacylglycero)phosphate", CELL CHEMICAL BIOLOGY, vol. 27, 2020, pages 1296 - 1307, Retrieved from the Internet <URL:https://doi.org/10.1016/j.chembiol.2020.07.015>
BUTTERFIELD GLLAJOIE MJGUSTAFSON HHSELLERS DLNATTERMANN UELLIS D ET AL.: "Evolution of a designed protein assembly encapsulating its own RNA genome", NATURE, vol. 552, 2017, pages 415 - 20, Retrieved from the Internet <URL:https://doi.org/10.1038/nature25157>
CARILLO, H.LIPMAN, D., SIAM J APPLIED MATH., vol. 48, 1988, pages 1073
CEROVSKY, V ET AL.: "Lasioglossins: Three Novel Antimicrobial Peptides from the Venom of the Eusocial Bee Lasioglossum laticeps (Hymenoptera: Halictidae", CHEM. BIO. CHEM, vol. 10, 2009, pages 2089 - 2099, XP072143947, DOI: 10.1002/cbic.200900133
CHADE ARTULLOS NAHARVEY TWMAHDI FBIDWELL GLI: "Renal Therapeutic Angiogenesis Using a Bioengineered Polymer-Stabilized Vascular Endothelial Growth Factor Construct", JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, vol. 27, 2016, pages 1741, XP055485293, Retrieved from the Internet <URL:https://doi.org/10.1681/ASN.2015040346> DOI: 10.1681/ASN.2015040346
CHEN JHALLER CAJERNIGAN FEKOERNER SKWONG DJWANG Y ET AL.: "Modulation of lymphocyte-mediated tissue repair by rational design of heterocyclic aryl hydrocarbon receptor agonists", SCI ADV, vol. 6, 2020, pages eaay8230, Retrieved from the Internet <URL:https://doi.org/10.1126/sciadv.aay8230>
CHENG T-FBRZOSTEK SANDO OVAN SCOY SKUMAR KPREICH NC: "Differential Activation of IFN Regulatory Factor (IRF)-3 and IRF-5 Transcription Factors during Viral Infection1", THE JOURNAL OF IMMUNOLOGY, vol. 176, 2006, pages 7462 - 70, Retrieved from the Internet <URL:https://doi.org/10.4049/jimmunol.176.12.7462>
CHO SDONG SPARENT KNCHEN M: "Immune-tolerant elastin-like polypeptides (iTEPs) and their application as CTL vaccine carriers", JOURNAL OF DRUG TARGETING, vol. 24, 2016, pages 328 - 39, XP055334119, DOI: 10.3109/1061186X.2015.1077847
CHONG S-EOH JHMIN KPARK SCHOI SAHN JH ET AL.: "Intracellular delivery of immunoglobulin G at nanomolar concentrations with domain Z-fused multimeric α-helical cell penetrating peptides", JOURNAL OF CONTROLLED RELEASE, vol. 330, 2021, pages 161 - 72, Retrieved from the Internet <URL:https://doi.org/10.1016/j.jconrel.2020.12.020.>
COSTA SAMOZHDEHI DDZURICKY MJISAACS FJBRUSTAD EMCHILKOTI A: "Active Targeting of Cancer Cells by Nanobody Decorated Polypeptide Micelle with Bio-orthogonally Conjugated Drug", NANO LET, vol. 19, 2019, pages 247 - 54, XP093154429, DOI: 10.1021/acs.nanolett.8b03837
CZECHOWICZ APALCHAUDHURI RSCHECK AHU YHOGGATT JSAEZ B ET AL.: "Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation", NAT COMMUN, vol. 10, 2019, pages 617, XP055617538, Retrieved from the Internet <URL:https://doi.org/10.1038/s41467-018-08201-x> DOI: 10.1038/s41467-018-08201-x
DEL'GUIDICE TLEPETIT-STOFFAES J-PBORDELEAU L-JROBERGE JTHÉBERGE VLAUVAUX C ET AL.: "Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells", PLOS ONE, vol. 13, 2018, pages e0195558, Retrieved from the Internet <URL:https://doi.org/10.1371/journal.pone.0195558>
DEROSSI DJOLIOT AHCHASSAING GPROCHIANTZ A: "The third helix of the Antennapedia homeodomain translocates through biological membranes", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, 1994, pages 10444 - 50, Retrieved from the Internet <URL:https://doi.org/10.1016/S0021-9258(17)34080-2>
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. 1, 1984, pages 387
DHANDHUKIA JPSHI PPEDDI SLI ZALURI SJU Y ET AL.: "Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo", BIOCONJUGATE CHEM, vol. 28, 2017, pages 2715 - 28, XP093002756, DOI: 10.1021/acs.bioconjchem.7b00469
DORR BMHAM HOAN CCHAIKOF ELLIU DR: "Reprogramming the specificity of sortase enzymes", PNAS, vol. 111, 2014, pages 13343 - 8, XP055340492, Retrieved from the Internet <URL:https://doi.org/10.1073/pnas.1411179111> DOI: 10.1073/pnas.1411179111
DOUDNA JA: "The promise and challenge of therapeutic genome editing", NATURE 2020, vol. 578, pages 229 - 36, XP037019025, Retrieved from the Internet <URL:https://doi.org/10.1038/s41586-020-1978-5> DOI: 10.1038/s41586-020-1978-5
DREHER MRSIMNICK AJFISCHER KSMITH RJPATEL ASCHMIDT M ET AL.: "Temperature Triggered Self-Assembly of Polypeptides into Multivalent Spherical Micelles", J AM, no. 130, 2008, pages 687 - 94, Retrieved from the Internet <URL:https://doi.org/10.1021/ja0764862.>
E. PUGSLEY CELWYN ISAAC RJ. WARREN NJ. CAYRE O: "Linear ABC amphiphilic triblock copolymers for complexation and protection of dsRNA", POLYMER CHEMISTRY, vol. 13, 2022, pages 5707 - 17, Retrieved from the Internet <URL:https://doi.org/10.1039/D2PY00914E.>
EL-SAYED A, MASUDA T, KHALIL I, AKITA H, HARASHIMA H.: " Enhanced gene expression by a novel stearylated INF7 peptide derivative through fusion independent endosomal escape.", J CONTROL RELEASE, vol. 138, 2009, pages 160 - 7, XP026394868, Retrieved from the Internet <URL:https://doi.org/10.1016/j.jconrel.2009.05.018>
ENGEL JEWILLIAMS EWILLIAMS MLBIDWELL GLCHADE AR: "Targeted VEGF (Vascular Endothelial Growth Factor) Therapy Induces Long-Term Renal Recovery in Chronic Kidney Disease via Macrophage Polarization", HYPERTENSION, vol. 74, 2019, pages 1113 - 23, Retrieved from the Internet <URL:https://doi.org/10.1161/HYPERTENSIONAHA.119.13469>
FEBS LETTERS, vol. 584, 2010, pages 1806 - 13, Retrieved from the Internet <URL:https://doi.org/10.1016/j.febslet.2009.11.046>
FOSS DVMULDOON JJNGUYEN DNCARR DSAHU SUHUNSINGER JM ET AL.: "Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes", NAT BIOMED ENG, vol. 2023, pages 1 - 14, Retrieved from the Internet <URL:https://doi.org/10.1038/s41551-023-01032-2>
FRANGOUL HALTSHULER DCAPPELLINI MDCHEN Y-SDOMM JEUSTACE BK ET AL.: "CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia", N ENGL J MED, vol. 384, 2021, pages 252 - 60, XP093005338, Retrieved from the Internet <URL:https://doi.org/10.1056/NEJMoa2031054> DOI: 10.1056/NEJMoa2031054
FREEMAN EC, WEILAND LM, MENG WS.: "Examining Proton Sponge Effectiveness for Enhancing Intracellular Gene Delivery through Multiscale Modeling", J BIOMATER SCI POLYM ED, vol. 24, 2013, pages 398 - 416
GAGNER JEKIM WCHAIKOF EL: "Designing protein-based biomaterials for medical applications", ACTA BIOMATER, vol. 10, 2014, pages 1542 - 57, XP055429789, Retrieved from the Internet <URL:https://doi.org/10.1016/j.actbio.2013.10.001> DOI: 10.1016/j.actbio.2013.10.001
GILLMORE JDGANE ETAUBEL JKAO JFONTANA MMAITLAND ML ET AL.: "CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis", NEW ENGLAND JOURNAL OF MEDICINE, vol. 385, 2021, pages 493 - 502, XP055978811, Retrieved from the Internet <URL:https://doi.org/10.1056/NEJMoa2107454> DOI: 10.1056/NEJMoa2107454
GUAY DDEL'GUIDICE TLEPETIT-STOFFAES J-P, POLYPEPTIDE-BASED SHUTTLE AGENTS FOR IMPROVING THE TRANSDUCTION EFFICIENCY OF POLYPEPTIDE CARGOS TO THE CYTOSOL OF TARGET EUKARYOTIC CELLS, USES THEREOF, METHODS AND KITS RELATING TO SAME
GUTERSTAM PMADANI FHIROSE HTAKEUCHI T, FUTAKI S, EL ANDALOUSSI S ET AL., BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - BIOMEMBRANES, vol. 1788, 2009, pages 2509 - 17
HAJIMOLAALI M, MOHAMMADIAN H, TORABI A, SHIRINI A, KHALIFE SHAL M, BARAZANDEH NEZHAD H: "Application of chloroquine as an endosomal escape enhancing agent: new frontiers for an old drug", EXPERT OPINION ON DRUG DELIVERY, vol. 18, 2021, pages 877 - 89
HENIKOFF SHENIKOFF J.G., P.N.A.S. USA, vol. 89, 1992, pages 10915 - 10919
HO ASCHWARZE SRMERMELSTEIN SJ: "Synthetic Protein Transduction Domains: Enhanced Transduction Potential in Vitro and in Vivo", CANCER RES., vol. 61, 2001, pages 474 - 7
ISLAM MDZARIYAMA HALAM JMDYAMAZAKI M: "Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating Across Lipid Membrane and Its Induced Pores", BIOCHEMISTRY, vol. 53, 2014, pages 386 - 96, XP055225478, Retrieved from the Internet <URL:https://doi.org/10.1021/bi401406p> DOI: 10.1021/bi401406p
JARAK IPEREIRA-SILVA MSANTOS ACVEIGA FCABRAL HFIGUEIRAS A: "Multifunctional polymeric micelle-based nucleic acid delivery: Current advances and future perspectives", APPLIED MATERIALS TODAY, vol. 25, 2021, pages 101217, Retrieved from the Internet <URL:https://doi.org/10.1016/j.apmt.2021.101217>
JENKINS ICMILLIGAN JJCHILKOTI A: "Genetically Encoded Elastin-Like Polypeptides for Drug Delivery", ADVANCED HEALTHCARE MATERIALS, vol. 10, 2021, pages 2100209, Retrieved from the Internet <URL:https://doi.org/10.1002/adhm.202100209>
KABELKA IVÁCHA R: "Advances in Molecular Understanding of a-Helical Membrane-Active Peptides", ACC CHEM RES, vol. 54, 2021, pages 2196 - 204, XP055877006, DOI: 10.1021/acs.accounts.1c00047
KELLY GMILLIGAN JJMASTRIA EMKIM SZELENETZ SRDOBBINS J ET AL.: "Intratumoral delivery of brachytherapy and immunotherapy by a thermally triggered polypeptide depot", JOURNAL OF CONTROLLED RELEASE, vol. 343, 2022, pages 267 - 76, XP087005418, Retrieved from the Internet <URL:https://doi.org/10.1016/j.jconrel.2022.01.024.> DOI: 10.1016/j.jconrel.2022.01.024
KIM BSKIM HJOSAWA SHAYASHI KTOH KNAITO M ET AL.: "Dually Stabilized Triblock Copolymer Micelles with Hydrophilic Shell and Hydrophobic Interlayer for Systemic Antisense Oligonucleotide Delivery to Solid Tumor", ACS BIOMATER SCI ENG, vol. 5, 2019, pages 5770 - 80
KIM W, BRADY C, CHAIKOF EL.: " Amphiphilic protein micelles for targeted in vivo imaging.", ACTA BIOMATER, vol. 8, 2012, pages 2476 - 82, XP028511594, Retrieved from the Internet <URL:https://doi.org/10.1016/j.actbio.2012.04.011> DOI: 10.1016/j.actbio.2012.04.011
KIM W, XIAO J, CHAIKOF EL: "Recombinant Amphiphilic Protein Micelles for Drug", LANGMUIR, vol. 27, 2011, pages 14329 - 34, Retrieved from the Internet <URL:https://doi.org/10.1021/1a202864x>
KIM WCHAIKOF EL: "Recombinant elastin-mimetic biomaterials: Emerging applications in medicine", ADV DRUG DELIV REV, vol. 62, 2010, pages 1468 - 78, XP027544541, Retrieved from the Internet <URL:https://doi.org/10.1016/j.addr.2010.04.007>
KIM WHALLER CDAI EWANG XHAGEMEYER CELIU DR ET AL.: "Targeted antithrombotic protein micelles", ANGEW CHEM INT ED ENGL, vol. 54, 2015, pages 1461 - 5, XP072068740, Retrieved from the Internet <URL:https://doi.org/10.1002/anie.201408529> DOI: 10.1002/anie.201408529
KIM WTHÉVENOT JIBARBOURE ELECOMMANDOUX SCHAIKOF EL: "Self-assembly of thermally responsive amphiphilic diblock copolypeptides into spherical micellar nanoparticles", ANGEW CHEM INT ED ENGL, vol. 49, 2010, pages 4257 - 60, XP055312668, Retrieved from the Internet <URL:https://doi.org/10.1002/anie.201001356> DOI: 10.1002/anie.201001356
KIM, W ET AL.: "Recombinant Amphiphilic Protein Micelles for Drug Delivery", LANGMUIR, vol. 27, 2011, pages 14329 - 14334
KIM, W ET AL.: "Targeted Antithrombotic Protein Micelles", ANGEW. CHEM. INT, vol. 54, 2015, pages 1461 - 1465, XP072068740, DOI: 10.1002/anie.201408529
KOBAYASHI TBEUCHAT M-HCHEVALLIER JMAKINO AMAYRAN NESCOLA J-M ET AL.: "Separation and Characterization of Late Endosomal Membrane Domains", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, 2002, pages 32157 - 64, Retrieved from the Internet <URL:https://doi.org/10.1074/jbc.M202838200>
KOREN EAPTE ASAWANT RRGRUNWALD JTORCHILIN VP: "Cell-penetrating TAT peptide in drug delivery systems: Proteolytic stability requirements", DRUG DELIV, vol. 18, 2011, pages 377 - 84, Retrieved from the Internet <URL:https://doi.org/10.3109/10717544.2011.567310>
KRAUSGRUBER TBLAZEK KSMALLIE TALZABIN SLOCKSTONE HSAHGAL N ET AL.: "IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses", NAT IMMUNOL, vol. 12, 2011, pages 231 - 8, XP055025301, Retrieved from the Internet <URL:https://doi.org/10.1038/ni.1990> DOI: 10.1038/ni.1990
KRISHNAMURTHY SWOHLFORD-LENANE CKANDIMALLA SSARTRE GMEYERHOLZ DKTHÉBERGE V ET AL.: "Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia", NAT COMMUN, vol. 10, 2019, pages 4906, Retrieved from the Internet <URL:https://doi.org/10.1038/s41467-019-12922-y>
KRISHNAMURTHY, S ET AL.: "Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases", NAT. COMMUN, vol. 10, 2019, pages 4906
KWON H-SLOGAN ACCHHABRA APANG WWCZECHOWICZ ATATE K ET AL.: "Anti-human CD 117 antibody-mediated bone marrow niche clearance in nonhuman primates and humanized NSG mice", BLOOD, vol. 133, 2019, pages 2104 - 8, Retrieved from the Internet <URL:https://doi.org/10.1182/blood-2018-06-853879>
LI NKGARCIA QUIROZ FHALL CKCHILKOTI AYINGLING YG: "Molecular description of the LCST behavior of an elastin-like polypeptide", BIOMACROMOLECULES, vol. 15, 2014, pages 3522 - 30, Retrieved from the Internet <URL:https://doi.org/10.1021/bm500658w>
LI YYE ZYANG HXU Q: "Tailoring combinatorial lipid nanoparticles for intracellular delivery of nucleic acids, proteins, and drugs", ACTA PHARMACEUTICA SINICA B, vol. 12, 2022, pages 2624 - 39, Retrieved from the Internet <URL:https://doi.org/10.1016/j.apsb.2022.04.013>
LUGINBUHL KMSCHAAL JLUMSTEAD BMASTRIA EMLI XBANSKOTA S ET AL.: "One-week glucose control via zero-order release kinetics from an injectable depot of glucagon-like peptide-1 fused to a thermosensitive biopolymer", NAT BIOMED ENG, vol. 1, 2017, pages 1 - 14, Retrieved from the Internet <URL:https://doi.org/10.1038/s41551-017-0078>
MACHIDA SNIIMI SSHI XANDO YYU Y: "Design of a Novel Membrane-Destabilizing Peptide Selectively Acting on Acidic Liposomes", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 64, 2000, pages 985 - 94, XP001053323, Retrieved from the Internet <URL:https://doi.org/10.1271/bbb.64.985> DOI: 10.1271/bbb.64.985
MACHIDA, S ET AL.: "Design of a Novel Membrane-Destabilizing Peptide Selectively Acting on Acidic Liposomes", BIOSCI. BIOTECHNOL. BIOCHEM., vol. 64, 2000, pages 985 - 994, XP001053323, DOI: 10.1271/bbb.64.985
MARCANDALLI JFIALA BOLS SPEROTTI MDE VAN DER SCHUEREN WSNIJDER J ET AL.: "Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus", CELL, vol. 176, 2019, pages 1420 - 1431, Retrieved from the Internet <URL:https://doi.org/10.1016/j.cell.2019.01.046>
MENDES BBCONNIOT JAVITAL AYAO DJIANG XZHOU X ET AL.: "Nanodelivery of nucleic acids", NAT REV METHODS PRIMERS, vol. 2, 2022, pages 1 - 21, XP093106995, Retrieved from the Internet <URL:https://doi.org/10.1038/s43586-022-00104-Y>
MÓNICA BRAVO-ANAYA LROSSELGONG JGRICELDA FERNANDEZ-SOLIS KXIAO YVAX AIBARBOURE E ET AL.: "Coupling of RAFT polymerization and chemoselective post-modifications of elastin-like polypeptides for the synthesis of gene delivery hybrid vectors", POLYMER CHEMISTRY, vol. 12, 2021, pages 226 - 41, Retrieved from the Internet <URL:https://doi.org/10.1039/DOPY01293A>
NEEDLEMANWUNSCH, J. MOL. BIOL, vol. 48, 1970, pages 443 - 453
NIELSEN EJBYOSHIDA SKAMEI NIWAMAE RKHAFAGY E-SOLSEN J ET AL.: "In vivo proof of concept of oral insulin delivery based on a co-administration strategy with the cell-penetrating peptide penetratin", JOURNAL OF CONTROLLED RELEASE, vol. 189, 2014, pages 19 - 24, Retrieved from the Internet <URL:https://doi.org/10.1016/j.jconrel.2014.06.022>
NUMATA KHAMASAKI JSUBRAMANIAN BKAPLAN DL: "Gene delivery mediated by recombinant silk proteins containing cationic and cell binding motifs", JOURNAL OF CONTROLLED RELEASE, vol. 146, 2010, pages 136 - 43, XP027181138, Retrieved from the Internet <URL:https://doi.org/10.1016/j.jconrel.2010.05.006>
OH JHCHONG S-ENAM SHYUN SCHOI SGYE H ET AL.: "Multimeric Amphipathic α-Helical Sequences for Rapid and Efficient Intracellular Protein Transport at Nanomolar Concentrations", ADVANCED SCIENCE, vol. 5, 2018, pages 1800240, XP055765910, DOI: 10.1002/advs.201800240
OLSHEFSKY ARICHARDSON CPUN SHKING NP: "Engineering Self-Assembling Protein Nanoparticles for Therapeutic Delivery", BIOCONJUGATE CHEM, vol. 33, 2022, pages 2018 - 34, Retrieved from the Internet <URL:https://doi.org/10.1021/acs.bioconjchem.2c00030>
OSAWA S, OSADA K, HIKI S, DIRISALA A, ISHII T, KATAOKA K.: "Polyplex Micelles with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection.", BIOMACROMOLECULES, vol. 17, 2016, pages 354 - 61, XP055563342, Retrieved from the Internet <URL:https://doi.org/10.1021/acs.biomac.5b01456> DOI: 10.1021/acs.biomac.5b01456
PARAYATH NNSTEPHAN SBKOEHNE ALNELSON PSSTEPHAN MT: "In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo", NAT COMMUN, vol. 11, 2020, pages 6080, Retrieved from the Internet <URL:https://doi.org/10.1038/s41467-020-19486-2>
PETERSON JJ, MEARES CF.: "Cathepsin Substrates as Cleavable Peptide Linkers in Bioconjugates, Selected from a Fluorescence Quench Combinatorial Library.", BIOCONJUGATE CHEM, vol. 9, 1998, pages 618 - 26, XP002591052, Retrieved from the Internet <URL:https://doi.org/10.1021/bc980059>
PIRTSKHALAVA MAMSTRONG AAGRIGOLAVA MCHUBINIDZE MALIMBARASHVILI EVISHNEPOLSKY B ET AL.: "DBAASP v3: database of antimicrobial/cytotoxic activity and structure of peptides as a resource for development of new therapeutics", NUCLEIC ACIDS RESEARCH, vol. 49, 2021, pages D288 - 97, Retrieved from the Internet <URL:https://doi.org/10.1093/nar/gkaa991>
POLACK FPTHOMAS SJKITCHIN NABSALON JGURTMAN ALOCKHART S ET AL.: "Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine", N ENGL J MED, vol. 383, 2020, pages 2603 - 15, XP055820495, Retrieved from the Internet <URL:https://doi.org/10.1056/NEJMoa2034577> DOI: 10.1056/NEJMoa2034577
RAI DKQIAN S: "Interaction of the Antimicrobial Peptide Aurein 1.2 and Charged Lipid Bilayer", SCI REP, vol. 7, 2017, pages 3719, Retrieved from the Internet <URL:https://doi.org/10.1038/s41598-017-03795-6>
RAMASWAMY STONNU NTACHIKAWA KLIMPHONG PVEGA JBKARMALI PP ET AL.: "Systemic delivery of factor IX messenger RNA for protein replacement therapy", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 114, 2017, pages E1941 - 50, XP055756678, DOI: 10.1073/pnas.1619653114
RENNICK JJJOHNSTON APRPARTON RG: "Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics", NAT NANOTECHNOL, vol. 16, 2021, pages 266 - 76, XP037409683, Retrieved from the Internet <URL:https://doi.org/10.1038/s41565-021-00858-8> DOI: 10.1038/s41565-021-00858-8
RILO-ALVAREZ HLEDO AMVIDAL AGARCIA-FUENTES M: "Delivery of transcription factors as modulators of cell differentiation", DRUG DELIV AND TRANSL RES, vol. 11, 2021, pages 426 - 44, XP037393818, DOI: 10.1007/s13346-021-00931-8
RITT NAYAOU AZENTEL R: "RAFT Synthesis of Reactive Multifunctional Triblock-Copolymers for Polyplex Formation", MACROMOLECULAR CHEMISTRY AND PHYSICS, vol. 222, 2021, pages 2100122, Retrieved from the Internet <URL:https://doi.org/10.1002/macp.202100122>
RURIK JGTOMBÁCZ IYADEGARI AMENDEZ FERNÁNDEZ POSHEWALE SVLI L ET AL.: "CAR T cells produced in vivo to treat cardiac injury", SCIENCE, vol. 375, 2022, pages 91 - 6
SÁNCHEZ-NAVARRO M: "Advances in peptide-mediated cytosolic delivery of proteins", ADVANCED DRUG DELIVERY REVIEWS, vol. 171, 2021, pages 187 - 98
SATO HFEIX JB: "Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic a-helical antimicrobial peptides", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - BIOMEMBRANES, vol. 1758, 2006, pages 1245 - 56, XP025062927, DOI: 10.1016/j.bbamem.2006.02.021
SELUANOV AVAIDYA AGORBUNOVA V: "Establishing primary adult fibroblast cultures from rodents", J VIS EXP, vol. 2010, pages 2033, Retrieved from the Internet <URL:https://doi.org/10.3791/2033>
SHA FSALZMAN GGUPTA AKOIDE S: "Monobodies and other synthetic binding proteins for expanding protein science", PROTEIN SCI, vol. 26, 2017, pages 910 - 24, XP055893730, Retrieved from the Internet <URL:https://doi.org/10.1002/pro.3148> DOI: 10.1002/pro.3148
SHILOVA ONDEYEV SM: "DARPins: Promising Scaffolds for Theranostics", ACTA NATURAE, vol. 11, 2019, pages 42 - 53, XP055730993, Retrieved from the Internet <URL:https://doi.org/10.32607/20758251-2019-11-4-42-53> DOI: 10.32607/20758251-2019-11-4-42-53
SLANINOVÁ JMLSOVÁ VKROUPOVÁ HALÁN LTUMOVÁ TMONINCOVÁ L ET AL.: "Toxicity study of antimicrobial peptides from wild bee venom and their analogs toward mammalian normal and cancer cells", PEPTIDES, vol. 33, 2012, pages 18 - 26, XP009160673, Retrieved from the Internet <URL:https://doi.org/10.1016/j.peptides.2011.11.002> DOI: 10.1016/j.peptides.2011.11.002
TECHNISCHE UNIVERSITEIT EINDHOVEN, 2 September 2021 (2021-09-02)
TIAN Y, TIRRELL MV, LABELLE JL.: " Harnessing the Therapeutic Potential of Biomacromolecules through Intracellular Delivery of Nucleic Acids, Peptides, and Proteins", ADV HEALTHCARE MATERIALS, vol. 11, 2022, pages 2102600
TRAN NT, SOMMERMANN T, GRAF R, TROMBKE J, PEMPE J, PETSCH K: "Efficient CRISPR/Cas9- Mediated Gene Knockin in Mouse Hematopoietic Stem and Progenitor", CELL REPORTS, vol. 28, 2019, pages 3510 - 3522, Retrieved from the Internet <URL:https://doi.org/10.1016/j.celrep.2019.08.065>
TSAI C-YSALAWU EOLI HLIN G-YKUO T-YVOON L ET AL.: "Helical structure motifs made searchable for functional peptide design", NAT COMMUN, vol. 13, 2022, pages 102, Retrieved from the Internet <URL:https://doi.org/10.1038/s41467-021-27655-0>
UEDA GANTANASIJEVIC AFALLAS JASHEFFLER WCOPPS JELLIS D ET AL.: "Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens", ELIFE, vol. 9, 2020, pages e57659, Retrieved from the Internet <URL:https://doi.org/10.7554/eLife.57659>
URRY DW: "Physical Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers", J PHYS CHEM B, vol. 101, 1997, pages 11007 - 28, XP001021863
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
WALKER: "Cambridge Dictionary of Biology;", 1990, CAMBRIDGE UNIVERSITY PRESS
WANG GLI XWANG Z: "APD3: the antimicrobial peptide database as a tool for research and education", NUCLEIC ACIDS RESEARCH, vol. 44, 2016, pages 1087 - 93
WEI TCHENG QMIN Y-LOLSON ENSIEGWART DJ: "Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing", NAT COMMUN, vol. 11, 2020, pages 3232, XP093095942, Retrieved from the Internet <URL:https://doi.org/10.1038/s41467-020-17029-3>
WINKELJANN BKEUL DCMERKEL OM: "Engineering poly- and micelleplexes for nucleic acid delivery - A reflection on their endosomal escape", JOURNAL OF CONTROLLED RELEASE, vol. 353, 2023, pages 518 - 34, Retrieved from the Internet <URL:https://doi.org/10.1016/j.jconrel.2022.12.008>
XIE JBI YZHANG HDONG STENG LLEE RJ ET AL.: "Cell-Penetrating Peptides in Diagnosis and Treatment of Human Diseases: From Preclinical Research to Clinical Application", FRONTIERS IN PHARMACOLOGY, vol. 11, 2020, XP055965167, DOI: 10.3389/fphar.2020.00697
YI ASIM DLEE S-BSARANGTHEM VPARK R-W: "Application of bioengineered elastin-like polypeptide-based system for targeted gene delivery in tumor cells", BIOMATERIALS AND, vol. 6, 2022, pages 10050
YI ASIM DLEE Y-JSARANGTHEM VPARK R-W: "Development of elastin-like polypeptide for targeted specific gene delivery in vivo", JOURNAL OF NANOBIOTECHNOLOGY, vol. 18, 2020, pages 15, Retrieved from the Internet <URL:https://doi.org/10.1186/s12951-020-0574-z>
YI, A: "Development of elastin-like polypeptide for targeted specific gene delivery in vivo", J. NANOBIOTECHNOL.,, vol. 18, 2020, pages 15
ZHANG FPARAYATH NNENE CISTEPHAN SBKOEHNE ALCOON ME ET AL.: "Genetic programming of macrophages to perform anti-tumor functions using targeted mRNA nanocarriers", NAT COMMUN, vol. 10, 2019, pages 3974, Retrieved from the Internet <URL:https://doi.org/10.1038/s41467-019-11911-5>
ZHANG SSHEN JLI DCHENG Y: "Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing", THERANOSTICS, vol. 11, 2021, pages 614 - 48, XP055926382, Retrieved from the Internet <URL:https://doi.org/10.7150/thno.47007> DOI: 10.7150/thno.47007
ZURIS JATHOMPSON DBSHU YGUILINGER JPBESSEN JLHU JH ET AL.: "Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo", NAT BIOTECHNOL, vol. 33, 2015, pages 73 - 80, XP055562063, Retrieved from the Internet <URL:https://doi.org/10.1038/nbt.3081> DOI: 10.1038/nbt.3081

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