WO2017175072A1 - Peptide shuttle based gene disruption - Google Patents

Peptide shuttle based gene disruption Download PDF

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Publication number
WO2017175072A1
WO2017175072A1 PCT/IB2017/000512 IB2017000512W WO2017175072A1 WO 2017175072 A1 WO2017175072 A1 WO 2017175072A1 IB 2017000512 W IB2017000512 W IB 2017000512W WO 2017175072 A1 WO2017175072 A1 WO 2017175072A1
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cells
gfp
polypeptide
protein
cell
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PCT/IB2017/000512
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French (fr)
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David Guay
Thomas DEL'GUIDICE
Jean-Pascal LEPETIT-STOFFAES
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Feldan Bio Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present description relates to synthetic peptides useful for increasing the transduction efficiency of polypeptide cargos to the cytosol of target eukaryotic cells. More specifically, the present description relates to synthetic peptides and polypeptide-based shuttle agents comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD.
  • ELD endosome leakage domain
  • CPD cell penetrating domain
  • CPD histidine-rich domain
  • ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 7, 2017, is named 49446-702_601_SL.txt and is 81 ,784 bytes in size.
  • polypeptide-based transduction agents may be useful for introducing purified recombinant proteins directly into target cells, for example, to help bypass safety concerns regarding the introduction of foreign DNA.
  • Lipid- or cationic polymer-based transduction agents exist, but introduce safety concerns regarding chemical toxicity and efficiency, which hamper their use in human therapy.
  • Protein transduction approaches involving fusing a recombinant protein cargo directly to a cell- penetrating peptide e.g., HIV transactivating protein TAT
  • endosomal membrane disrupting peptides have been developed to try and facilitate the escape of endosomally-trapped cargos to the cytosol.
  • many of these endosomolytic peptides are intended to alleviate endosomal entrapment of cargos that have already been delivered intracellular ⁇ , and do not by themselves aid in the initial step of shuttling the cargos intracellular ⁇ across the plasma membrane (Salomone et al., 2012; Salomone et al., 2013; Erazo-Oliveras et al., 2014; Fasoli et al., 2014).
  • shuttle agents capable of increasing the transduction efficiency of polypeptide cargos, and delivering the cargos to the cytosol of target eukaryotic cells.
  • the present description stems from the surprising discovery that synthetic peptides comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD) and optionally a histidine-rich domain, have the ability to increase the proportion of cells that can be transduced with a polypeptide cargo of interest, without the synthetic peptide being covalently bound to the polypeptide cargo.
  • the synthetic peptides may facilitate the ability of endosomally-trapped polypeptide cargos to gain access to the cytosol, and optionally be targeted to various subcellular comparts (e.g., the nucleus).
  • a method of editing a genome of one or more eukaryotic cells comprising contacting a eukaryotic cell with a polypeptide-based shuttle and a DNA cleavage protein, such that the DNA cleavage protein is delivered to the nucleus and binds to at least one target DNA sequence, thereby editing the genome, wherein the polypeptide-based shuttle comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or functional fragment thereof having endosomolytic activity, and wherein the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones.
  • CPD cell penetrating domain
  • ELD endosome leakage domain
  • methods of editing a genome of one or more eukaryotic cells comprising contacting a population of eukaryotic cells with a polypeptide-based shuttle and a DNA cleavage protein, such that the DNA cleavage protein is delivered to the nucleus and binds to at least one target DNA sequence, thereby editing the genome, wherein the polypeptide-based shuttle comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or functional fragment thereof having endosomolytic activity, and wherein the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones.
  • CPD cell penetrating domain
  • ELD endosome leakage domain
  • the polypeptide-based shuttle is present at a concentration sufficient to increase the percentage or proportion of the population eukaryotic cells into which the DNA cleavage protein is delivered across the plasma membrane, as compared to in the absence of said polypeptide-based shuttle.
  • the polypeptide-based shuttle is complexed with the DNA cleavage protein.
  • the polypeptide-based shuttle is not complexed with the DNA cleavage protein.
  • the DNA cleavage protein is a variant or functional derivative of a DNA cleavage protein. Further provided herein are methods wherein the polypeptide-based shuttle and the DNA cleavage protein are not covalently linked.
  • the polypeptide-based shuttle further comprises a histidine rich domain.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity.
  • the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the DNA-cleavage protein comprises a subcellular targeting domain.
  • the subcellular targeting domain comprises a nuclear localization signal (NLS).
  • the NLS comprises at least one of SEQ ID NO. 28-50.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity
  • the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof.
  • the DNA cleavage protein comprises an RNA-guided nuclease.
  • the RNA-guided nuclease comprises a Cas protein.
  • the Cas protein comprises a Type I, Type II, Type III, Type IV, Type V, or a Type VI Cas protein or protein complex. Further provided herein are methods wherein the Cas protein comprises Cas9, Cpf1 , or at least one functional fragment or derivative thereof. Further provided herein are methods wherein the population of eukaryotic cells is further contacted with at least one guiding RNA. Further provided herein are methods wherein the eukaryotic cell is further contacted with at least one guiding RNA. Further provided herein are methods wherein the polypeptide- based shuttle and the RNA-guided nuclease are additionally complexed with at least one guiding RNA.
  • RNA-guided nuclease comprises Cas9 and wherein the at least one guiding RNA comprises a crRNA and a trRNA. Further provided herein are methods wherein the crRNA and trRNA have independent phosphodiester backbones. Further provided herein are methods wherein the crRNA and trRNA share a common phosphodiester backbone.
  • the crRNA is engineered to hybridize with the at least one target DNA sequence
  • the trRNA is engineered to hybridize with the crRNA
  • the crRNA and the trRNA form a complex with Cas9, thereby targeting Cas9 to the at least one target DNA sequence such that the at least one target DNA sequence is cleaved by Cas9.
  • the RNA-guided nuclease comprises Cpf1 and wherein the at least one guiding RNA comprises a crRNA.
  • the crRNA is engineered to hybridize with the at least one target DNA sequence, and wherein the crRNA forms a complex with Cpf1 , thereby targeting Cpf1 to the at least one target DNA sequence such that the at least one target DNA sequence is cleaved by Cpf1.
  • binding to at least one target further comprises cleavage of the at least one target DNA sequence, such that cleavage creates a double strand break which is repaired by a non-homologous end joining (NHEJ) repair mechanism, thereby editing the at least one target DNA molecule.
  • NHEJ non-homologous end joining
  • binding to at least one target further comprises cleavage of the at least one target DNA sequence, such that cleavage creates a double strand break which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the at least one target DNA sequence, thereby editing the target DNA molecule.
  • the Cas9 protein is a nickase variant of Cas9 which comprises at least one mutation in at least one of a RuvC domain and a HNH domain such binding to at least one target further comprises cleavage of only one strand of the at least one target DNA sequence.
  • the method comprises editing the at least one target DNA sequence by insertion of a sequence for a donor polynucleotide into the cleaved strand of the at least one target DNA sequence. Further provided herein are methods wherein the method comprises editing the at least one target DNA sequence by a homology directed repair mechanism which incorporates a sequence of a donor polynucleotide into the at least one target DNA sequence, thereby editing the at least one target DNA molecule. Further provided herein are methods wherein incorporation of a sequence of a donor polynucleotide results in insertion, deletion, or substitution of one or more nucleotides.
  • RNA-guided nuclease is multiplexed with at least two guiding RNAs, such that at least two target DNA sequences are cleaved.
  • the Cas protein is catalytically dead such that the Cas protein binds but does not cleave the at least one target DNA sequence.
  • binding of the catalytically dead Cas protein blocks functional transcription the at least one target DNA sequence.
  • the DNA-cleavage protein is a zinc finger nuclease, TALEN, or a meganuclease.
  • the eukaryotic cell is a mammalian cell.
  • the cell is a human cell. Further provided herein are methods wherein the cell is a T cell. Further provided herein are methods wherein the cell is a megakaryocyte. Further provided herein are methods wherein the cell is a NK cell. Further provided herein are methods wherein the cell is a stem cell. Further provided herein are methods wherein the cell is a hematopoietic stem cell. Further provided herein are methods wherein the population of eukaryotic cells is a mammalian cell population, a human cell population, a T cell population, a megakaryocyte population, a NK cell population, a stem cell population, or a hematopoietic stem cell population.
  • the target DNA sequence is implicated in a genetic disease, such that the genome editing treats said genetic disease.
  • the genetic disease is a blood-related disease.
  • the target DNA sequence is implicated in an infection, such that the genome editing treats said infection.
  • the infection is a viral infection.
  • the target DNA sequence is implicated in immunogenicity, such that the genome editing deletes or ameliorates the immunogenicity.
  • the genome editing results in non-immunogenic cells.
  • the target DNA sequence comprises a human leukocyte antigen gene complex sequence.
  • the target sequence comprises a major histocompatibility complex gene sequence.
  • the non-immunogenic cells comprise universal stem cells.
  • the non-immunogenic cells comprise CAR-T cells.
  • incorporation of a sequence of a donor polynucleotide results in insertion of one or more nucleotides, wherein the one or more nucleotides comprises a heterologous gene.
  • the heterologous gene is GALC.
  • the heterologous gene is HEXA.
  • the heterologous gene is IDUA.
  • the eukaryotic cell is a mammalian cell. Further provided herein are methods wherein the cell is a stem cell. Further provided herein are methods wherein the cell is a hematopoietic stem cell. Further provided herein are methods wherein the cell is a central nervous system cell. Further provided herein are methods wherein the cell is a microgilia cell. Further provided herein are methods wherein the cell is a neuron. Further provided herein are methods wherein the cell is a liver cell. Further provided herein are methods wherein the cell is a liver endothelial cell. Further provided herein are methods wherein the cell is a hepatocyte. Further provided herein are methods wherein the target DNA sequence is in a gene locus.
  • the gene locus is an abnormal GALC gene. Further provided herein are methods wherein the gene locus is an abnormal HEXA gene. Further provided herein are methods wherein the gene locus is an abnormal IDUA gene. Further provided herein are methods wherein the gene locus is an albumin gene. Further provided herein are methods wherein incorporation of the heterologous gene treats or ameliorates the symptoms of a genetic disease. Further provided herein are methods wherein the genetic disease is Krabbe Disease. Further provided herein are methods wherein the genetic disease is Tay-Sachs Disease. Further provided herein are methods wherein the genetic disease is Hurler Syndrome.
  • a patient with a condition by administering to the patient in need thereof non-immunogenic cells obtained by any of the methods disclosed herein.
  • the condition is a genetic disease.
  • the condition is a blood disorder.
  • the condition is a malignant condition.
  • the condition is a non-malignant condition.
  • the condition is thrombocytopenia.
  • the condition is a genetic disease.
  • condition is a blood disorder. Further provided herein are uses wherein the condition is a malignant condition. Further provided herein are uses wherein the condition is a non-malignant condition. Further provided herein are uses wherein the condition is thrombocytopenia.
  • methods of editing a genome of one or more megakaryocyte cells comprising contacting a megakaryocyte with a polypeptide-based shuttle complexed with Cas9 and at least one guiding RNA such that Cas9 is delivered to the nucleus and cleaves at least one target DNA sequence, thereby editing the genome
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, and wherein the polypeptide- based shuttle and Cas9 have independent protein backbones and are not covalently linked
  • the at least one guiding RNA comprises a crRNA and a trRNA with independent phosphodiester backbones
  • the crRNA is engineered to hybridize with the at least one target DNA sequence, wherein the target DNA sequence comprises a major histocompatibility complex gene sequence, and wherein the genome editing results in non-immunogenic megakaryocytes.
  • non- immunogenic megakaryocytes obtained by any of the methods disclosed herein.
  • kits for treating a patient with a condition by administering to the patient in need thereof cells obtained by any of the methods disclosed herein.
  • the condition is a genetic disease.
  • the genetic disease is Krabbe Disease.
  • the genetic disease is Tay-Sachs Disease.
  • the genetic disease is Hurler Syndrome.
  • non-immunogenic cells obtained by any of the methods disclosed herein for treatment of a patient in need thereof due to a condition.
  • the condition is a genetic disease.
  • the genetic disease is Krabbe Disease.
  • the genetic disease is Tay-Sachs Disease.
  • the genetic disease is Hurler Syndrome.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.
  • protein or “polypeptide” means any peptide-linked chain of amino acids, which may or may not comprise any type of modification (e.g., post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc).
  • post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc.
  • the expression “is or is from” or “is from” comprises functional variants of a given protein domain (CPD or ELD), such as conservative amino acid substitutions, deletions, modifications, as well as variants or function derivatives, which do not abrogate the activity of the protein domain.
  • CPD or ELD functional variants of a given protein domain
  • Figures 1A-1 B show a typical result of a calcein endosomal escape assay in which HEK293A cells were loaded with the fluorescent dye calcein ("100 ⁇ calcein”), and were then treated (or not) with a shuttle agent that facilitates endosomal escape of the calcein ("100 ⁇ calcein + CM18-TAT 5 ⁇ ").
  • Figure 1A shows the results of a fluorescence microscopy experiment
  • Figure 1 B shows the results of a flow cytometry experiment.
  • Figure 2 shows the results of a calcein endosomal escape flow cytometry assay in which HeLa cells were loaded with calcein ("calcein 100 ⁇ "), and were then treated with increasing concentrations of the shuttle agent CM18-TAT-Cys (labeled "CM18-TAT").
  • Figures 3 and 4 show the results of calcein endosomal escape flow cytometry assays in which HeLa cells ( Figure 3) or primary myoblasts ( Figure 4) were loaded with calcein ("calcein 100 ⁇ "), and were then treated with 5 ⁇ or 8 ⁇ of the shuttle agents CM18-TAT-Cys or CM18-Penetratin-Cys (labeled "CM18-TAT” and "CM18-Penetratin", respectively).
  • FIG 5 shows the results of a GFP transduction experiment visualized by fluorescence microscopy in which a GFP cargo protein was co-incubated with 0, 3 or 5 ⁇ of CM18-TAT-Cys (labeled "CM18-TAT”), and then exposed to HeLa cells. The cells were observed by bright field (upper pictures in Figure 5) and fluorescence microscopy (lower pictures in Figure 5).
  • CM18-TAT CM18-TAT-Cys
  • FIGS 6A-6B show the results of a GFP transduction efficiency experiment in which GFP cargo protein (10 ⁇ ) was co-incubated with different concentrations of CM18-TAT-Cys (labeled "CM18-TAT”), prior to being exposed to HeLa cells.
  • CM18-TAT CM18-TAT-Cys
  • Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cell is shown in Figure 6A, and corresponding cell toxicity data is shown in Figure 6B.
  • Figures 7A-7B show the results of a GFP transduction efficiency experiment in which different concentrations of GFP cargo protein (10, 5 or 1 ⁇ ) were co-incubated with either 5 ⁇ of CM18-TAT- Cys ( Figure 7A, labeled "CM18TAT”), or 2.5 ⁇ of dCM18-TAT-Cys ( Figure 7B, labeled "dCM18TAT”), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentages of fluorescent (GFP-positive) cells are shown.
  • FIGS 8 and 9 show the results of GFP transduction efficiency experiments in which GFP cargo protein (10 ⁇ ) was co-incubated with different concentrations and combinations of CM18-TAT- Cys (labeled "CM18TAT”), CM18-Penetratin-Cys (labeled "CM18penetratin”), and dimers of each (dCM18-TAT-Cys (labeled "dCM18TAT”), dCM18-Penetratin-Cys (labeled "dCM18penetratin”), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentages of fluorescent (GFP-positive) cells are shown.
  • FIG 10 shows typical results of a TAT-GFP transduction experiment in which TAT-GFP cargo protein (5 ⁇ ) was co-incubated with 3 ⁇ of CM18-TAT-Cys (labeled "CM18-TAT”), prior to being exposed to HeLa cells.
  • CM18-TAT CM18-TAT-Cys
  • Cells and GFP fluorescence were visualized by bright field and fluorescence microscopy at 10x and 40x magnifications. Arrows indicate the endosome delivery of TAT- GFP in the absence of CM18-TAT-Cys, as well as its nuclear delivery in the presence of CM18-TAT- Cys.
  • FIGS 11A-11 B show the results of a TAT-GFP transduction efficiency experiment in which
  • TAT-GFP cargo protein (5 ⁇ ) was co-incubated with different concentrations of CM18-TAT-Cys (labeled "CM18TAT"), prior to being exposed to HeLa cells.
  • CM18TAT CM18-TAT-Cys
  • Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cell is shown in Figure 11 A, and corresponding cell toxicity data is shown in Figure 11 B.
  • Figure 12 shows typical results of a GFP-NLS transduction experiment in which GFP-NLS cargo protein (5 ⁇ ) was co-incubated with 5 ⁇ of CM18-TAT-Cys (labeled "CM18-TAT”), prior to being exposed to HeLa cells for 5 minutes.
  • CM18-TAT CM18-TAT-Cys
  • FIGS 13A-13B show the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 ⁇ ) was co-incubated with different concentrations of CM18-TAT-Cys (labeled "CM18TAT”), prior to being exposed to HeLa cells.
  • CM18TAT CM18-TAT-Cys
  • Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cell is shown in Figure 13A, and corresponding cell toxicity data is shown in Figure 13B.
  • FIGS 14 and 15 show the results of GFP-NLS transduction efficiency experiments in which GFP-NLS cargo protein (5 ⁇ ) was co-incubated with different concentrations and combinations of CM18-TAT (labeled "CM18TAT”), CM18-Penetratin (labeled "CM18penetratin”), and dinners of each (dCM18-TAT-Cys, dCM18-Penetratin-Cys; labeled “dCM18TAT” and "dCM18penetratin”, respectively), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentages of fluorescent (GFP-positive) cells are shown.
  • CM18TAT CM18-TAT
  • CM18penetratin CM18penetratin
  • Figure 16 shows the results of a GFP-NLS transduction efficiency experiment in which GFP-
  • NLS cargo protein (5 ⁇ ) was co-incubated with either CM18-TAT-Cys (3.5 ⁇ , labeled "CM18TAT”) alone or with dCM18-Penetratin-Cys (1 ⁇ , labeled "dCM18pen”) for 5 minutes or 1 hour in plain DMEM media (“DMEM”) or DMEM media containing 10% FBS (“FBS”), before being subjected to flow cytometry analysis. The percentages of fluorescent (GFP-positive) cells are shown. Cells that were not treated with shuttle agent or GFP-NLS (“ctrl”), and cells that were treated with GFP-NLS without shuttle agent (“GFP-NLS 5 ⁇ ”) were used as controls.
  • FIGs 17A-17B show the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 ⁇ ) was co-incubated with or without 1 ⁇ CM18-TAT-Cys (labeled "CM18TAT”), prior to being exposed to THP-1 cells.
  • CM18TAT 1 ⁇ CM18-TAT-Cys
  • Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cells is shown in Figure17A, and corresponding cell toxicity data is shown in Figure17B.
  • Figures 18A-18C show the results of a transduction efficiency experiment in which the cargo protein, FITC-labeled anti-tubulin antibody (0.5 ⁇ ), was co-incubated with 5 ⁇ of CM18-TAT-Cys (labeled "CM18-TAT”), prior to being exposed to HeLa cells.
  • Functional antibody delivery was visualized by bright field (20x- Figure 18A) and fluorescence microscopy (20x- Figure 18B and 40x- Figure 18C), in which fluorescent tubulin fibers in the cytoplasm were visualized.
  • FIGS 19A-19B show the results of an FITC-labeled anti-tubulin antibody transduction efficiency experiment in which the antibody cargo protein (0.5 ⁇ ) was co-incubated with 3.5 ⁇ of CM18-TAT-Cys (labeled "CM18TAT”), CM18-Penetratin-Cys (labeled "CM18pen”)or dCM18-Penetratin- Cys (labeled “dCM18pen”), or a combination of 3.5 ⁇ of CM18-TAT-Cys and 0.5 ⁇ of dCM18- Penetratin-Cys, prior to being exposed to HeLa cells.
  • Cells were evaluated by flow cytometry and the percentage of fluorescent (FITC-positive) cell is shown in Figure19A, and corresponding cell toxicity data is shown in Figure 19B.
  • Figure 20 shows the results of DNA transfection efficiency experiment in which plasmid DNA (pEGFP) was labeled with a Cy5TM dye was co-incubated with 0, 0.05, 0.5, or 5 ⁇ of CM18-TAT-Cys (labeled "CM18-TAT”), prior to being exposed to HEK293A cells.
  • CM18-TAT CM18-TAT-Cys
  • Figures 21A-21 B show the results of a GFP-NLS transduction efficiency experiment in which the GFP-NLS cargo protein (5 ⁇ ) was co-incubated with 1 , 3, or 5 ⁇ of CM18-TAT-Cys (labeled "CM18TAT”), of His-CM18-TAT (labeled "His-CM18TAT”), prior to being exposed to HeLa cells.
  • CM18TAT CM18-TAT-Cys
  • His-CM18-TAT labeled "His-CM18TAT”
  • Figures 22A-22B show the results of a transduction efficiency experiment in which GFP-NLS cargo protein was intracellularly delivered using the shuttle His-CM18-PTD4 in HeLa cells. GFP-NLS transduction efficiency was evaluated by flow cytometry and the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data (“viability (%)”) are shown.
  • Figure 22A shows a comparison of GFP-NLS transduction efficiencies using different transduction protocols (Protocol A vs. B).
  • Figure 22B shows the effect of using different concentrations of the shuttle His- CM18-PTD4 when using Protocol B.
  • Figures 23A-23D, Figures 24A-24B, Figures 25A-25B and Figures 26A-26C are microscopy images showing the results of transduction experiments in which GFP-NLS ( Figures 23A-23D, 24A, 24B, 25A-B and 26A-26C) cargo protein was intracellularly delivered with the shuttle His-CM18-PTD4 in HeLa cells.
  • Figures 23D, 24A, 26A, and Figures 23A to 23C, 24B, 25A-B, 26B-C show the bright field and fluorescence images, respectively, of living cells.
  • the cells were fixed, permeabilized and subjected to immuno-labelling with an anti-GFP antibody and a fluorescent secondary antibody.
  • White triangle windows indicate examples of areas of co-labelling between nuclei (DAPI) and GFP-NLS signals.
  • Figure 26A-26C shows images captured by confocal microscopy.
  • Figures 27A-27D show microscopy images of a kinetic (time-course) transduction experiment in HeLa cells, where the fluorescence of GFP-NLS cargo protein was tracked after 45, 75, 100, and 120 seconds following intracellular delivery with the shuttle His-CM18-PTD4.
  • the diffuse cytoplasmic fluorescence pattern observed after 45 seconds gradually becomes a more concentrated nuclear pattern at 120 seconds ( Figure 27D).
  • Figures 28A-28D show microscopy images of co-delivery transduction experiment in which two cargo proteins (GFP-NLS and mCherryTM-NLS) are simultaneously delivered intracellularly by the shuttle His-CM18-PTD4 in HeLa cells.
  • Cells and fluorescent signals were visualized by ( Figure 28A) bright field and ( Figures 28B-28D) fluorescence microscopy.
  • White triangle windows indicate examples of areas of co-labelling between nuclei (DAPI) and GFP-NLS or mCherryTM.
  • Figures 29A-29I show the results of GFP-NLS transduction efficiency experiments in HeLa cells using different shuttle agents or single-domain/control peptides.
  • GFP-NLS transduction efficiency was evaluated by flow cytometry and the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data (“viability (%)”) are shown in Figures 29A, 29B, 29D-29G, and 29I.
  • Pos cells (%) percentage of GFP fluorescent cells
  • viability (%) percentage of GFP fluorescent cells
  • Figure 29A and Figure29D-29F cells were exposed to the cargo/shuttle agent for 10 seconds.
  • Figure 29I cells were exposed to the cargo/shuttle agent for 1 minute.
  • Figures 29B, 29C, 29G and 29H cells were exposed to the cargo/shuttle agent for 1 , 2, or 5 min.
  • Relative fluorescence intensity (FL1-A)" or “Signal intensity” corresponds to the mean of all fluorescence intensities from each cell with a GFP fluorescent signal after GFP-NLS fluorescent protein delivery with the shuttle agent.
  • Figure 29D shows the results of a control experiment in which only single-domain peptides (ELD or CDP) or the peptide His-PTD4 (His-CPD) were used for the GFP-NLS transduction, instead of the multi-domain shuttle agents.
  • Figure 30A-30F shows microscopy images of HeLa cells transduced with GFP-NLS using the shuttle agent (Figure 30A) TAT-KALA, ( Figure 30B) His-CM18-PTD4, (Figure 30C) His-C(LLKK) 3 C- PTD4, (Figure 30D) PTD4-KALA, ( Figure 30E) EB1-PTD4, and ( Figure 30F) His-CM18-PTD4-His.
  • the insets in the row of the lower pictures in Figures 30A-30F show the results of corresponding flow cytometry analyses, indicating the percentage of cells exhibiting GFP fluorescence.
  • Figure 31 shows the results of a transduction efficiency experiment in which GFP-NLS cargo protein was intracellularly delivered using the shuttle His-CM18-PTD4 in THP-1 cells using different Protocols (Protocol A vs C).
  • GFP-NLS transduction efficiency was evaluated by flow cytometry and the percentage of GFP fluorescent cells ("Pos cells (%)”), as well as corresponding cell viability data (“viability (%)”) are shown.
  • Pos cells (%) the percentage of GFP fluorescent cells
  • viability (%) cell viability
  • “Ctrl” corresponds to THP-1 cells exposed to GFP-NLS cargo protein in the absence of a shuttle agent.
  • Figures 32A-32D show microscopy images of THP-1 cells transduced with GFP-NLS cargo protein using the shuttle His-CM18-PTD4. Images captured under at 4x, 10x and 40x magnifications are shown in Figures 32A-32C, respectively.
  • White triangle windows in Figure 32C indicate examples of areas of co-labelling between cells (bright field) and GFP-NLS fluorescence.
  • Figure 32D shows the results of corresponding flow cytometry analyses, indicating the percentage of cells exhibiting GFP fluorescence.
  • Figures 33A-33D show microscopy images of THP-1 cells transduced with GFP-NLS cargo protein using the shuttle His-CM18-PTD4.
  • White triangle windows indicate examples of areas of co- labelling between cells (bright field; Figure33A-33B), and GFP-NLS fluorescence (Figure 33C-33D).
  • Figure 33E shows FACS analysis of GFP-positive cells.
  • Figures 34A-34B show the results of GFP-NLS transduction efficiency experiments in THP-1 cells using the shuttle TAT-KALA, His-CM18-PTD4, or His-C(LLKK) 3 C-PTD4.
  • the cargo protein/shuttle agents were exposed to the THP-1 cells for 15, 30, 60 or 120 seconds.
  • GFP-NLS transduction efficiency was evaluated by flow cytometry and the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data (“viability (%)”) are shown in Figure 34A.
  • “Relative fluorescence intensity (FL1 -A)” corresponds to the mean of all fluorescence intensities from each cell with a GFP fluorescent signal after GFP-NLS fluorescent protein delivery with the shuttle agent.
  • Figures 35A-35F show the results of transduction efficiency experiments in which THP-1 cells were exposed daily to GFP-NLS cargo in the presence of a shuttle agent for 2.5 hours. His-CM18- PTD4 was used in Figures 35A-35E, and His-C(LLKK) 3 C-PTD4 was used in Figure 35F.
  • GFP-NLS transduction efficiency was determined by flow cytometry at Day 1 or Day 3, and the results are expressed as the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data ("viability (%)") in Figures 35A-35C and in Figure 35F.
  • Figure 35D shows the metabolic activity index of the THP-1 cells after 1 , 2, 4, and 24h
  • Figure 35E shows the metabolic activity index of the THP-1 cells after 1 to 4 days, for cells exposed to the His-CM18-PTD4 shuttle.
  • Figure 36 shows a comparison of the GFP-NLS transduction efficiencies in a plurality of different types of cells (e.g., adherent and suspension, as well as cell lines and primary cells) using the shuttle His-CM18-PTD4, as measured by flow cytometry.
  • the results are expressed as the percentage of GFP fluorescent cells ("Pos cells (%)”), as well as corresponding cell viability data (“viability (%)”).
  • Figures 37A-37H show fluorescence microscopy images of different types of cells transduced with GFP-NLS cargo using the shuttle His-CM18-PTD4. GFP fluorescence was visualized by fluorescence microscopy at a 10x magnification. The results of parallel flow cytometry experiments are also provided in the insets (viability and percentage of GFP-fluorescing cells).
  • Figures 38A-38B show fluorescence microscopy images of primary human myoblasts transduced with GFP-NLS using the shuttle His-CM18-PTD4. Cells were fixed and permeabilized prior to immuno-labelling GFP-NLS with an anti-GFP antibody and a fluorescent secondary antibody. Immuno-labelled GFP is shown in Figure 38A, and this image is overlaid with nuclei (DAPI) labelling in Figure 38B.
  • DAPI nuclei
  • Figures 39A-39E show a schematic layout ( Figures 39A, 39B and 39C) and sample fluorescence images (D and E) of a transfection plasmid surrogate assay used to evaluate the activity of intracellular ⁇ delivered CRISPR/Cas9-NLS complex.
  • Figure 39A At Day 1, cells are transfected with an expression plasmid encoding the fluorescent proteins mCherryTM and GFP, with a STOP codon separating their two open reading frames. Transfection of the cells with the expression plasmid results in only mCherryTM expression as shown in Figure 39D.
  • a CRISPR/Cas9-NLS complex which has been designed/programmed to cleave the plasmid DNA at the STOP codon, is then delivered intracellular ⁇ to the transfected cells expressing mCherryTM, resulting double-stranded cleavage of the plasmid DNA at the STOP codon as shown in Figure 39B
  • random non-homologous DNA repair of the cleaved plasmid occurs and results in removal of the STOP codon ( Figure 39C), and thus GFP expression and fluorescence (Figure 39E).
  • White triangle windows indicate examples of areas of co-labelling of mCherryTM and GFP fluorescence.
  • Figures 40A-40H show fluorescence microscopy images of HeLa cells expressing mCherryTM and GFP, indicating CRISPR/Cas9-NLS-mediated cleavage of plasmid surrogate DNA.
  • HeLa cells were co-transfected with three plasmids: the plasmid surrogate as described in the brief description of Figures 39A-39E, and two other expression plasmids encoding the Cas9-NLS protein and crRNA/tracrRNAs, respectively.
  • Figure 41A shows the products of a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR/Cas9-mediated cleavage of cellular genomic DNA.
  • T7E1 assay DNA cleavage assay
  • HeLa cells were transduced with a CRISPR-Cas9-NLS complex programmed to cleave the PPIB gene.
  • Figure 41 B shows the products of a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR/Cas9-mediated cleavage of cellular genomic DNA (PPIB DNA sequences).
  • T7E1 assay DNA cleavage assay
  • PPIB DNA sequences CRISPR/Cas9-mediated cleavage of cellular genomic DNA
  • the left picture of the Figure 41 B shows the cleavage product of the amplified PPIB DNA sequence by the CRIPR/Cas9 complex after the delivery of the complex with the shuttle agent His-CM18-PTD4 in HeLa cells.
  • the right picture of the Figure 41 B shows amplified DNA sequence before the T7E1 digestion procedure as a negative control.
  • Figure 41 C shows the products of a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR/Cas9-mediated cleavage of cellular genomic DNA (PPIB DNA sequences).
  • T7E1 assay DNA cleavage assay
  • PPIB DNA sequences The left picture of the Figure 41 C shows the amplified PPIB DNA sequence after incubation of the HeLa cells with the Cas9/RNAs complex in presence of a lipidic transfection agent (DharmaFectTM transfection reagent # T-20XX-01) (positive control).
  • the right picture of the Figure 41C shows amplified DNA sequence before the T7E1 digestion procedure as a negative control.
  • Figures 42-44 show the transcriptional activity of THP-1 cells that have been transduced with the transcription factor HOXB4 using different concentrations of the shuttle His-CM18-PTD4 and different cargo/shuttle exposure times.
  • Successful intra-nuclear delivery of HOXB4 was determined by monitoring mRNA levels of a target gene by real-time PCR, and the results are normalized against those in the negative control (HOXB4 without shuttle agent) and expressed as "Fold over control” (left bars).
  • Total cellular RNA (ng/ ⁇ ) was quantified and used a marker for cell viability (right bars).
  • "0" or “Ctrl” means "no treatment”;
  • TF means “Transcription Factor alone”;
  • FS means "shuttle alone”.
  • Figures 45A-45D show fluorescence microscopy images of HeLa cells transduced with wild- type HOXB4 cargo using the shuttle His-CM18-PTD4. After a 30-minute incubation to allow transduced HOXB4-WT to accumulate in the nucleus, the cells were fixed, permeabilized and HOXB4-WT was labelled using a primary anti-HOXB4 monoclonal antibody and a fluorescent secondary antibody ( Figures 45B and 45D). Nuclei were labelled with DAPI ( Figures 45A and 45C). White triangle windows indicate examples of areas of co-labelling between nuclei and HOXB4 - compare Figures 45A vs 45B (x20 magnification), and Figures 45C vs 45D (x40 magnification).
  • Figures 46A-46B show the products of a DNA cleavage assay separated by agarose gel electrophoresis, which is used to measure CRISPR/Cas9-mediated cleavage of cellular genomic DNA (HPTR sequence) after intracellular delivery of the complex with different shuttle agents.
  • Figure 46A shows the results with the shuttle agents: His-CM18-PTD4, His-CM18-PTD4-His, and His-C(LLKK)3C- PTD4 in HeLa cells.
  • Figure 46B shows the results with His-CM18-PTD4-His and His-CM18-L2-PTD4 in Jurkat cells.
  • Negative controls show amplified HPTR DNA sequence after incubation of the cells with the CRISPR/Cas9 complex without the presence of the shuttle agent.
  • Positive controls show the amplified HPTR DNA sequence after incubation of the cells with the Cas9/RNAs complex in presence of a commercial lipidic transfection agent.
  • Figure 47 shows the transcriptional activity of THP-1 cells that have been transduced with the transcription factor HOXB4 using the shuttle agents His-CM18-PTD4, TAT-KALA, EB1-PTD4, His- C(LLKK)3C-PTD4 and His-CM18-PTD4-His.
  • Successful intra-nuclear delivery of HOXB4 was determined by monitoring mRNA levels of a target gene by real-time PCR, and the results were normalized against those in the negative control (HOXB4 without shuttle agent) and expressed as "Fold over control" (left bars).
  • Total cellular RNA (ng/ ⁇ .) was quantified and used a marker for cell viability (right bars).
  • "0" or "Ctrl” means "no treatment”
  • 'TF means "Transcription Factor alone”
  • FS means "shuttle alone”.
  • Figures 48A-48D show in vivo GFP-NLS delivery in rat parietal cortex by His-CM18-PTD4. Briefly, GFP-NLS (20 ⁇ ) was injected in the parietal cortex of rat in presence of the shuttle agent His- CM18-PTD4 (20 ⁇ ) for 10 min. Dorso-ventral rat brain slices were collected and analysed by fluorescence microscopy at (Figure 48A) 4x, ( Figure 48C) 10x and ( Figure 48D) 20x magnifications. The injection site is located in the deepest layers of the parietal cortex (PCx).
  • PCx parietal cortex
  • Figure 48B shows the stereotaxic coordinates of the injection site (black arrows) from the rat brain atlas of Franklin and Paxinos.
  • the injection of GFP-NLS in presence of His-CM18-PTD4 was performed on the left part of the brain, and the negative control (injection of GFP-NLS alone), was done on the contralateral site.
  • the black circle and connected black lines in Figure 48B show the areas observed in the fluorescent pictures ( Figure 48A, 48C and 48D).
  • Figures 49A-49B depict an example of homologous-directed recombination with 6His-CM18-
  • Figure 50 shows an example of 6His-CM18-PTD4-mediated delivery of CRISPR/Cas9 RNP system in NK cells. Genomic cleavage analysis on agarose gel electrophoresis in NK cells. Cas9-NLS and specific crRNA were used for the cleavage of the HPRT in NK cells.
  • Figure 51 shows an example of genomic cleavage analysis of multiple exons on the B2M gene with T7E1 assay in HeLa cells after separation by agarose gel electrophoresis.
  • Three CRISPR/Cpfl RNP complexes targeting the exons 1 and 2 of the B2M gene were co-delivered in presence of 6His- CM18-PTD4.
  • Electrophoresis gels show that crRNA-2 (black thick arrows), crRNA-3 (red thick arrows) and crRNA-4 (blue thick arrows) were cleaved by respective CRISPR/Cpfl -crRNA complexes.
  • the present description stems from the surprising discovery that multi-domain synthetic peptides comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD) can significantly increase the transduction efficiency of an independent polypeptide cargo to the cytosol of eukaryotic target cells.
  • ELD endosome leakage domain
  • CPD cell penetrating domain
  • the present description relates to a polypeptide-based shuttle agent comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD, for use in increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell.
  • ELD endosome leakage domain
  • CPD cell penetrating domain
  • CPD histidine-rich domain and a CPD
  • synthetic polypeptide is intended to refer to non-naturally occurring molecules that can be produced in vitro (e.g., synthesized chemically and/or produced using recombinant DNA technology). The purities of various synthetic preparations may be assessed by for example high-performance liquid chromatography analysis and mass spectroscopy. Chemical synthesis approaches may be advantageous over cellular expression systems (e.g., yeast or bacteria protein expression systems), as they may preclude the need for extensive recombinant protein purification steps (e.g., required for clinical use). In contrast, longer synthetic polypeptides may be more complicated and/or costly to produce via chemical synthesis approaches and such polypeptides may be more advantageously produced using cellular expression systems.
  • cellular expression systems e.g., yeast or bacteria protein expression systems
  • the peptides or shuttle agent of the present description may be chemically synthesized (e.g., solid- or liquid phase peptide synthesis), as opposed to expressed from a recombinant host cell.
  • the peptides or shuttle agent of the present description may lack an N-terminal methionine residue.
  • a person of skill in the art may adapt a synthetic peptide or shuttle agent of the present description by using one or more modified amino acids (e.g., non-naturally-occurring amino acids), or by chemically modifying the synthetic peptide or shuttle agent of the present description, to suit particular needs of stability or other needs.
  • polypeptide-based when used here in the context of a shuttle agent of the present description, is intended to distinguish the presently described shuttle agents from non- polypeptide or non-protein-based shuttle agents such as lipid- or cationic polymer-based transduction agents, which are often associated with increased cellular toxicity and may not be suitable for use in human therapy.
  • the expression “increasing transduction efficiency” refers to the ability of a shuttle agent (e.g., a polypeptide-based shuttle agent of the present description) to improve the percentage or proportion of a population of target cells into which a cargo of interest (e.g., a polypeptide cargo) is delivered intracellularly across the plasma membrane.
  • a shuttle agent e.g., a polypeptide-based shuttle agent of the present description
  • a cargo of interest e.g., a polypeptide cargo
  • Immunofluorescence microscopy, flow cytometry, and other suitable methods may be used to assess cargo transduction efficiency.
  • a shuttle agent of the present description may enable a transduction efficiency of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, for example as measure by immunofluorescence microscopy, flow cytometry, FACS, and other suitable methods.
  • a shuttle agent of the present description may enable one of the aforementioned transduction efficiencies together wish a cell viability of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, for example as measure by the assay described in Example 3.3a, or by another suitable assay known in the art.
  • independent is generally intended refer to molecules or agents which are not covalently bound to one another.
  • independent polypeptide cargo is intended to refer to a polypeptide cargo to be delivered intracellularly that is not covalently bound (e.g., not fused) to a shuttle agent of the present description.
  • having shuttle agents that are independent of (not fused to) a polypeptide cargo may be advantageous by providing increased shuttle agent versatility - e.g., not being required to re-engineer a new fusion protein for different polypeptide cargoes, and/or being able to readily vary the ratio of shuttle agent to cargo (as opposed to being limited to a 1 : 1 ratio in the case of a fusion protein).
  • shuttle agents of the present description may facilitate the delivery of a cargo of interest (e.g., a polypeptide cargo) to the cytosol of target cells.
  • a cargo of interest e.g., a polypeptide cargo
  • efficiently delivering an extracellular cargo to the cytosol of a target cell using approaches based on cell penetrating peptides can be challenging, as the cargo often becomes trapped in intracellular endosomes after crossing the plasma membrane, which may limit its intracellular availability and may result in its eventual metabolic degradation.
  • use of the protein transduction domain from the HIV-1 Tat protein has been reported to result in massive sequestration of the cargo into intracellular vesicles.
  • shuttle agents of the present description may facilitate the ability of endosomally-trapped cargo to escape from the endosome and gain access to the cytoplasmic compartment.
  • the expression "to the cytosol" in the phrase “increasing the transduction efficiency of an independent polypeptide cargo to the cytosol,” is intended to refer to the ability of shuttle agents of the present description to allow an intracellular ⁇ delivered cargo of interest to escape endosomal entrapment and gain access to the cytoplasmic compartment. After a cargo of interest has gained access to the cytosol, it may be subsequently targeted to various subcellular compartments (e.g., nucleus, nucleolus, mitochondria, peroxisome).
  • the expression "to the cytosol” is thus intended to encompass not only cytosolic delivery, but also delivery to other subcellular compartments that first require the cargo to gain access to the cytoplasmic compartment.
  • a “domain” or “protein domain” generally refers to a part of a protein having a particular functionality or function. Some domains conserve their function when separated from the rest of the protein, and thus can be used in a modular fashion. By combining such domains from different proteins of viral, bacterial, or eukaryotic origin, it becomes possible in accordance with the present description to not only design multi-domain polypeptide-based shuttle agents that are able to deliver a cargo intracellular ⁇ , but also enable the cargo to escape endosomes and reach the cytoplasmic compartment.
  • the modular characteristic of many protein domains can provide flexibility in terms of their placement within the shuttle agents of the present description. However, some domains may perform better when engineered at certain positions of the shuttle agent (e.g., at the N- or C-terminal region, or therebetween). The position of the domain within its endogenous protein is sometimes an indicator of where the domain should be engineered within the shuttle agent, and of what type/length of linker should be used. Standard recombinant DNA techniques can be used by the skilled person to manipulate the placement and/or number of the domains within the shuttle agents of the present description in view of the present disclosure.
  • assays disclosed herein can be used to assess the functionality of each of the domains within the context of the shuttle agents (e.g., their ability to facilitate cell penetration across the plasma membrane, endosome escape, and/or access to the cytosol). Standard methods can also be used to assess whether the domains of the shuttle agent affect the activity of the cargo to be delivered intracellularly.
  • the expression “operably linked” as used herein refers to the ability of the domains to carry out their intended function(s) (e.g., cell penetration, endosome escape, and/or subcellular targeting) within the context of the shuttle agents of the present description.
  • the expression “operably linked” is meant to define a functional connection between two or more domains without being limited to a particular order or distance between same.
  • synthetic peptide or polypeptide-based shuttle agent of the present description may comprise a minimum length of 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues and a maximum length of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues.
  • shorter synthetic peptide or polypeptide-based shuttle agents are particularly advantageous because they may be more easily synthesized and purified by chemical synthesis approaches, which may be more suitable for clinical use (as opposed to recombinant proteins that must be purified from cellular expression systems).
  • synthetic peptide or polypeptide-based shuttle agent of the present description may comprise a predicted net charge of at least +5, +6, +7, at least +8, at least +9, at least +10, at least +11 , at least +12, at least +13, at least +14, or at least +15 at physiological pH.
  • positive charges are generally conferred by the greater presence of positively-charged lysine and/or arginine residues, as opposed to negatively charged aspartate and/or glutamate residues.
  • synthetic peptide or polypeptide-based shuttle agent of the present description may be soluble in aqueous solution (e.g., at physiological pH), which facilitates their use in for example cell culture media to delivery cargoes intracellularly to live cells.
  • synthetic peptide or polypeptide-based shuttle agent of the present description may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 57-59, 66- 72, or 82-102, or a functional variant thereof having at least 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 % 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to any one of SEQ ID NOs: 57-59, 66-72, or 82-102.
  • synthetic peptide or polypeptide-based shuttle agents of the present description may comprise oligomers (e.g., dimers, trimers, etc.) of a synthetic peptide or polypeptide- based shuttle agent as defined herein.
  • oligomers e.g., dimers, trimers, etc.
  • Such oligomers may be constructed by covalently binding the same or different types of shuttle agent monomers (e.g., using disulfide bridges to link cysteine residues introduced into the monomer sequences).
  • the synthetic peptide or polypeptide-based shuttle agent of the present description may comprise an N-terminal and/or a C-terminal cysteine residue.
  • ELDs Endosome leakage domains
  • synthetic peptide or polypeptide-based shuttle agents of the present description may comprise an endosome leakage domain (ELD) for facilitating endosome escape and access to the cytoplasmic compartment.
  • ELD endosome leakage domain
  • endosome leakage domain refers to a sequence of amino acids which confers the ability of endosomally-trapped macromolecules to gain access to the cytoplasmic compartment.
  • endosome leakage domains are short sequences (often derived from viral or bacterial peptides), which are believed to induce destabilization of the endosomal membrane and liberation of the endosome contents into the cytoplasm.
  • endosomolytic peptide is intended to refer to this general class of peptides having endosomal membrane-destabilizing properties. Accordingly, in some embodiments, synthetic peptide or polypeptide-based shuttle agents of the present description may comprise an ELD which is an endosomolytic peptide. The activity of such peptides may be assessed for example using the calcein endosome escape assays described in Example 2.
  • the ELD may be a peptide that disrupts membranes at acidic pH, such as pH-dependent membrane active peptide (PMAP) or a pH-dependent lytic peptide.
  • PMAP pH-dependent membrane active peptide
  • the peptides GALA and INF-7 are amphiphilic peptides that form alpha helixes when a drop in pH modifies the charge of the amino acids which they contain. More particularly, without being bound by theory, it is suggested that ELDs such as GALA induce endosomal leakage by forming pores and flip-flop of membrane lipids following conformational change due to a decrease in pH (Kakudo, Chaki et al., 2004, Li, Nicol et al., 2004).
  • ELDs such as INF-7 induce endosomal leakage by accumulating in and destabilizing the endosomal membrane (El-Sayed, Futaki et al., 2009). Accordingly in the course of endosome maturation, the concomitant decline in pH causes a change in the conformation of the peptide and this destabilizes the endosome membrane leading to the liberation of the endosome contents.
  • the same principle is thought to apply to the toxin A of Pseudomonas (Varkouhi, Scholte et al., 2011).
  • the ELD may be an antimicrobial peptide (AMP) such as a linear cationic alpha-helical antimicrobial peptide (AMP).
  • AMP antimicrobial peptide
  • these peptides play a key role in the innate immune response due to their ability to strongly interact with bacterial membranes. Without being bound by theory, these peptides are thought to assume a disordered state in aqueous solution, but adopt an alpha-helical secondary structure in hydrophobic environments. The latter conformation thought to contribute to their typical concentration-dependent membrane-disrupting properties. When accumulated in endosomes at a certain concentrations, some antimicrobial peptides may induce endosomal leakage.
  • the ELD may be an antimicrobial peptide (AMP) such as Cecropin-
  • AMP antimicrobial peptide
  • CM series A/Melittin hybrid (CM series) peptide. Such peptides are thought to be among the smallest and most effective AMP-derived peptides with membrane-disrupting ability.
  • Cecropins are a family of antimicrobial peptides with membrane-perturbing abilities against both Gram-positive and Gram- negative bacteria.
  • Cecropin A (CA) the first identified antibacterial peptide, is composed of 37 amino acids with a linear structure.
  • Melittin (M) a peptide of 26 amino acids, is a cell membrane lytic factor found in bee venom.
  • Cecropin-melittin hybrid peptides have been shown to produce short efficient antibiotic peptides without cytotoxicity for eukaryotic cells (i.e., non-hemolytic), a desirable property in any antibacterial agent.
  • These chimeric peptides were constructed from various combinations of the hydrophilic N-terminal domain of Cecropin A with the hydrophobic N-terminal domain of Melittin, and have been tested on bacterial model systems.
  • Two 26-mers, CA(1-13)M(1-13) and CA(1-8) M(1 -18) (Boman et al., 1989), have been shown to demonstrate a wider spectrum and improved potency of natural Cecropin A without the cytotoxic effects of melittin.
  • synthetic peptide or polypeptide-based shuttle agents of the present description may comprise an ELD which is or is from CM series peptide variants, such as those described above.
  • the ELD may be the CM series peptide CM18 composed of residues 1- 7 of Cecropin-A (KWKLFKKIGAVLKVLTTG) (SEQ ID NO: 104) fused to residues 2-12 of Melittin (YGRKKRRQRRR) (SEQ ID NO: 105), [C(1-7)M(2-12)].
  • CM18 was shown to independently cross the plasma membrane and destabilize the endosomal membrane, allowing some endosomally-trapped cargos to be released to the cytosol (Salomone et al., 2012).
  • the ELD may be CM18 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 1 and having endosomolytic activity.
  • the ELD may be a peptide derived from the N terminus of the HA2 subunit of influenza hemagglutinin (HA), which may also cause endosomal membrane destabilization when accumulated in the endosome.
  • HA hemagglutinin
  • synthetic peptide or polypeptide-based shuttle agents of the present description may comprise an ELD which is or is from an ELD set forth in Table A, or a variant thereof having endosome escape activity and/or pH-dependent membrane disrupting activity.
  • ALEHPELSELKTVTGTNPVFAGANYAAWA (Uherek, Fominaya et uipntneria toxin VNVA Q V
  • shuttle agents of the present description may comprise one or more ELD or type of ELD. More particularly, they can comprise at least 2, at least 3, at least 4, at least 5, or more ELDs. In some embodiments, the shuttle agents can comprise between 1 and 10 ELDs, between 1 and 9 ELDs, between 1 and 8 ELDs, between 1 and 7 ELDs, between 1 and 6 ELDs, between 1 and 5 ELDs, between 1 and 4 ELDs, between 1 and 3 ELDs, etc.
  • the order or placement of the ELD relative to the other domains (CPD, histidine-rich domains) within the shuttle agents of the present description may be varied provided the shuttling ability of the shuttle agent is retained.
  • the ELD may be a variant or fragment of any one those listed in Table A, and having endosomolytic activity.
  • the ELD may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 1 -15, 63, or 64, or a sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91 %, 92%, 93%, 94%, or 95% identical to any one of SEQ ID NOs: 1-15, 63, or 64, and having endosomolytic activity.
  • Cell penetration domains CPDs
  • the shuttle agents of the present description may comprise a cell penetration domain (CPD).
  • CPD cell penetration domain
  • the expression "cell penetration domain” refers to a sequence of amino acids which confers the ability of a macromolecule (e.g., peptide or protein) containing the CPD to be transduced into a cell.
  • the CPD may be (or may be from) a cell-penetrating peptide or the protein transduction domain of a cell-penetrating peptide.
  • Cell-penetrating peptides can serve as carriers to successfully deliver a variety of cargos intracellular ⁇ (e.g., polynucleotides, polypeptides, small molecule compounds or other macromolecules/compounds that are otherwise membrane- impermeable).
  • cargos intracellular ⁇ e.g., polynucleotides, polypeptides, small molecule compounds or other macromolecules/compounds that are otherwise membrane- impermeable.
  • Cell-penetrating peptides often include short peptides rich in basic amino acids that, once fused (or otherwise operably linked) to a macromolecule, mediate its internalization inside cells (Shaw, Catchpole et al., 2008).
  • the first cell-penetrating peptide was identified by analyzing the cell penetration ability of the HIV-1 trans-activator of transcription (Tat) protein (Green and Loewenstein 1988, Vives, Brodin et al., 1997).
  • This protein contains a short hydrophilic amino acid sequence, named 'TAT", which promotes its insertion within the plasma membrane and the formation of pores. Since this discovery, many other cell-penetrating peptides have been described.
  • the CPD can be a cell-penetrating peptide as listed in Table B, or a variant thereof having cell-penetrating activity.
  • SynB1 RGGRLSYSRRRFSTSTGR 26 (Drin, Cottin et al., 2003)
  • SynB3 RRLSYSRRRF 27 (Drin, Cottin et al., 2003)
  • PTD4 YARAAARQARA 65 Ho et al, 2001
  • cell-penetrating peptides are thought to interact with the cell plasma membrane before crossing by pinocytosis or endocytosis.
  • TAT peptide its hydrophilic nature and charge are thought to promote its insertion within the plasma membrane and the formation of a pore (Herce and Garcia 2007).
  • Alpha helix motifs within hydrophobic peptides (such as SP) are also thought to form pores within plasma membranes (Veach, Liu et al., 2004).
  • shuttle agents of the present description may comprise one or more CPD or type of CPD. More particularly, they may comprise at least 2, at least 3, at least 4, or at least 5 or more CPDs. In some embodiments, the shuttle agents can comprise between 1 and 10 CPDs, between 1 and 6 CPDs, between 1 and 5 CPDs, between 1 and 4 CPDs, between 1 and 3 CPDs, etc.
  • the CPD may be TAT having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 % 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 17 and having cell penetrating activity; or Penetratin having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 % 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 17
  • the CPD may be PTD4 having the amino acid sequence of SEQ ID NO: 65, or a variant thereof having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 % 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 65.
  • the order or placement of the CPD relative to the other domains (ELD, histidine-rich domains) within the shuttle agents of the present description may be varied provided the shuttling ability of the shuttle agent is retained.
  • the CPD may be a variant or fragment of any one those listed in Table
  • the CPD may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65, or a sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91 %, 92%, 93%, 94%, or 95% identical to any one of SEQ ID NOs: 16-27 or 65, and having cell penetrating activity. Histidine-rich domains
  • the shuttle agents of the present description may comprise a histidine-rich domain.
  • the shuttle agents of the present description may be combined/used together with a further independent synthetic peptide comprising or consisting essentially of a histidine- rich domain and a CPD (e.g., but lacking an ELD).
  • a further independent synthetic peptide comprising or consisting essentially of a histidine- rich domain and a CPD (e.g., but lacking an ELD).
  • This latter approach may provide the added advantage of allowing the concentration of the histidine-rich domain to be varied or controlled independently from the concentration of the ELD and the CPD contained in the shuttle agent.
  • the histidine-rich domain may act as a proton sponge in the endosome, providing another mechanism of endosomal membrane destabilization.
  • the histidine-rich domain may be a stretch of at least 2, at least 3, at least 4, at least 5, or at least 6 amino acids comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues.
  • the histidine-rich domain may comprise at least 2, at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, or at least 9 consecutive histidine residues.
  • the histidine-rich domain in the shuttle agent may act as a proton sponge in the endosome through protonation of their imidazole groups under acidic conditions of the endosomes, providing another mechanism of endosomal membrane destabilization and thus further facilitating the ability of endosomally-trapped cargos to gain access to the cytosol.
  • the histidine-rich domain may be located at the N or C terminus of the synthetic peptide or shuttle agent.
  • the histidine-rich domain may be located N-terminal or C terminal to the CPD and/or ELD.
  • the order or placement of the histidine-rich domain relative to the other domains (CPD, ELD) within the shuttle agents of the present description may be varied provided the shuttling ability of the shuttle agent is retained.
  • the shuttle agents of the present description may comprise more than one histidine-rich domain (e.g., histidine-rich domains at the amino and carboxyl termini).
  • suitable linkers can be used to operably connect the domains (CPDs, ELDs, or histidine-rich domains) to one another within the context of synthetic peptides and shuttle agents of the present description.
  • linkers may be formed by adding sequences of small hydrophobic amino acids without rotatory potential (such as glycine) and polar serine residues that confer stability and flexibility. Linkers may be soft and allow the domains of the shuttle agents to move. In some embodiments, prolines may be avoided since they can add significant conformational rigidity.
  • the linkers may be serine/glycine-rich linkers (e.g., GGS, GGSGGGS (SEQ ID NO: 106), GGSGGGSGGGS (SEQ ID NO: 107), or the like).
  • the use shuttle agents comprising a suitable linker may be advantageous for delivering an independent polypeptide cargo to suspension cells, rather than to adherent cells.
  • the synthetic peptide or polypeptide-based shuttle agent of the present description may be useful for delivering an independent cargo (e.g., a polypeptide cargo) to the cytosol of a target eukaryotic cell.
  • the polypeptide cargo may be fused to one or more CPDs to further facilitate intracellular delivery.
  • the CPD fused to the polypeptide cargo may be the same or different from the CPD of the shuttle agent of the present description.
  • Such fusion proteins may be constructed using standard recombinant technology.
  • the independent polypeptide cargo may be fused, complexed with, or covalently bound to a second biologically active cargo (e.g., a biologically active polypeptide or compound).
  • the polypeptide cargo may comprise a subcellular targeting domain.
  • the polypeptide cargo must be delivered to the nucleus for it to carry out its intended biological effect.
  • the cargo is a polypeptide intended for nuclear delivery (e.g., a transcription factor).
  • nuclear localization signals e.g., nuclear localization signals
  • the NLS sequences are recognized by proteins (importins a and ⁇ ), which act as transporters and mediators of translocation across the nuclear envelope.
  • NLSs are generally enriched in charged amino acids such as arginine, histidine, and lysine, conferring a positive charge which is partially responsible for their recognition by importins.
  • the polypeptide cargo may comprise an NLS for facilitating nuclear delivery, such as one or more of the NLSs as listed in Table C, or a variant thereof having nuclear targeting activity.
  • the polypeptide cargo may comprise its natural NLS.
  • Pho4p PYLNKRKGKP 44 (Welch, Franke et al., 1999)
  • recombinant proteins are exposed to protein trafficking system of eukaryotic cells. Indeed, all proteins are synthetized in the cell's cytoplasm and are then redistributed to their final subcellular localization by a system of transport based on small amino acid sequences recognized by shuttle proteins (Karniely and Pines 2005, Stojanovski, Bohnert et al., 2012). In addition to NLSs, other localization sequences can mediate subcellular targeting to various organelles following intracellular delivery of the polypeptide cargos of the present description.
  • polypeptide cargos of the present description may comprise a subcellular localization signal for facilitating delivery of the shuttle agent and cargo to specific organelles, such as one or more of the sequences as listed in Table D, or a variant thereof having corresponding subcellular targeting activity.
  • the cargo can be a biologically active compound such as a biologically active (recombinant) polypeptide (e.g., a transcription factor, a cytokine, or a nuclease) intended for intracellular delivery.
  • a biologically active polypeptide e.g., a transcription factor, a cytokine, or a nuclease
  • biologically active refers to the ability of a compound to mediate a structural, regulatory, and/or biochemical function when introduced in a target cell.
  • the cargo may be a recombinant polypeptide intended for nuclear delivery, such as a transcription factor.
  • the transcription factor can be HOXB4 (Lu, Feng et al., 2007), NUP98-HOXA9 (Takeda, Goolsby et al., 2006), Oct3/4, Sox2, Sox9, Klf4, c-Myc (Takahashi and Yamanaka 2006), MyoD (Sung, Mun et al., 2013), Pdx1 , Ngn3 and MafA (Akinci, Banga et al., 2012), Blimp-1 (Lin, Chou et al., 2013), Eomes, T-bet (Gordon, Chaix et al., 2012), FOX03A (Warr, Binnewies et al., 2013), NF-YA (Dolfini, Minuzzo et al., 2012), SALL4 (Aguila, Liao et
  • the cargo may be a recombinant polypeptide intended for nuclear delivery, such as a nuclease useful for genome editing technologies.
  • the nuclease may be an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1 (Zetsche et al., 2015), a zinc-finger nuclease (ZFN), a Transcription activator-like effector nuclease (TALEN) (Cox et al., 2015), a homing endonucleas
  • the nuclease may be fused to a nuclear localization signal (e.g., Cas9-NLS; Cpfl-NLS; ZFN-NLS; TALEN-NLS).
  • the nuclease may be complexed with a nucleic acid (e.g., one or more guide RNAs, a crRNA, a tracrRNAs, or both a crRNA and a tracrRNA).
  • the nuclease may possess DNA or RNA-binding activity, but may lack the ability to cleave DNA.
  • the shuttle agents of the present description may be used for intracellular delivery (e.g., nuclear delivery) of one or more CRISPR endonucleases, for example one or more of the CRISPR endonucleases described below.
  • Type I and its subtypes A, B, C, D, E, F and I including their respective Cas1 , Cas2, Cas3, Cas4, Cas6, Cas7 and Cas8 proteins, and the signature homologs and subunits of these Cas proteins including Cse1 , Cse2, Cas7, Cas5, and Cas6e subunits in E. coli (type l-E) and Csy1 , Csy2, Csy3, and Cas6f in Pseudomonas aeruginosa (type l-F) (Wiedenheft et al., 2011 ; Makarova et al, 2011).
  • Type II and its subtypes A, B, C including their respective Cas1 , Cas2 and Cas9 proteins, and the signature homologs and subunits of these Cas proteins including Csn complexes (Makarova et al, 2011).
  • Type III and its subtypes A, B and MTH326-like module including their respective Cas1 , Cas2, Cas6 and Cas10 proteins, and the signature homologs and subunits of these Cas proteins including Csm and CMR complexes (Makarova et al, 2011).
  • Type IV represents the Csf3 family of Cas proteins. Members of this family show up near CRISPR repeats in Acidithiobacillus ferrooxidans ATCC 23270, Azoarcus sp.
  • Type V includes the enzyme C2c2, which reported shares little homology to known sequences.
  • the shuttle agents of the present description may be used in conjunction with one or more of the nucleases, endonucleases, RNA-guided endonuclease, CRISPR endonuclease described above, for a variety of applications, such as those described herein.
  • CRISPR systems interact with their respective nucleic acids, such as DNA binding, RNA binding, helicase, and nuclease motifs (Marakova et al, 2011 ; Barrangou & Marraffini, 2014).
  • CRISPR systems may be used for different genome editing applications including:
  • NHEJ non-homologous end-joining
  • HDR Homologous-directed recombination
  • dCas catalytically dead Cas
  • a catalytically dead Cas that can also be fused to different functional proteins domains as a method to bring enzymatic activities at specific sites of the genome including transcription repression, transcription activation, chromatin remodeling, fluorescent reporter, histone modification, recombinase system acetylation, methylation, ubiquitylation, phosphorylation, sumoylation, ribosylation and citrullination (Gilbert et al, 2013).
  • the present shuttle agents although exemplified with Cas9 in the present examples, may be used with other nucleases as described herein.
  • nucleases such as Cpf1 , Cas9, and variants of such nucleases or others, are encompassed by the present description. It should be understood that, in one aspect, the present description may broadly cover any cargo having nuclease activity, such an RNA-guided endonuclease, or variants thereof (e.g., those that can bind to DNA or RNA, but have lost their nuclease activity; or those that have been fused to a transcription factor).
  • cargo having nuclease activity such an RNA-guided endonuclease, or variants thereof (e.g., those that can bind to DNA or RNA, but have lost their nuclease activity; or those that have been fused to a transcription factor).
  • the polypeptide cargo may be a cytokine such as a chemokine, an interferon, an interleukin, a lymphokine, or a tumour necrosis factor.
  • the polypeptide cargo may be a hormone or growth factor.
  • the cargo may be an antibody (e.g., a labelled antibody).
  • the cargo can be a detectable label (fluorescent polypeptide or reporter enzyme) that is intended for intracellular delivery, for example, for research and/or diagnostic purposes.
  • the cargo may be a globular protein or a fibrous protein. In some embodiments, the cargo may have a molecule weight of any one of about 5, 10, 15, 20, 25, 30, 35, 40, 45, to 50 to about 150, 200, 250, 300, 350, 400, 450, 500 kDa or more. In some embodiments, the cargo may have a molecule weight of between about 20 to 200 kDa.
  • synthetic peptides and shuttle agents of the present description may be non-toxic to the intended target eukaryotic cells at concentrations up to 50 ⁇ , 45 ⁇ , 40 ⁇ , 35 ⁇ , 30 ⁇ , 25 ⁇ , 20 ⁇ , 15 ⁇ , 10 ⁇ , 9 ⁇ , 8 ⁇ , 7 ⁇ , 6 ⁇ , 5 ⁇ , 4 ⁇ , 3 ⁇ , 2 ⁇ , 1 ⁇ , 0.5 ⁇ 0.1 ⁇ , or 0.05 ⁇ .
  • Cellular toxicity of shuttle agents of the present description may be measured using any suitable method.
  • transduction protocols may be adapted (e.g., concentrations of shuttle and/or cargo used, shuttle/cargo exposure times, exposure in the presence or absence of serum), to reduce or minimize toxicity of the shuttle agents, and/or to improve/maximize transfection efficiency.
  • synthetic peptides and shuttle agents of the present description may be readily metabolizable by intended target eukaryotic cells.
  • the synthetic peptides and shuttle agents may consist entirely or essentially of peptides or polypeptides, for which the target eukaryotic cells possess the cellular machinery to metabolize/degrade.
  • the intracellular half-life of the synthetic peptides and polypeptide-based shuttle agents of the present description is expected to be much lower than the half-life of foreign organic compounds such as fluorophores.
  • fluorophores can be toxic and must be investigated before they can be safely used clinically (Alford et al., 2009).
  • synthetic peptides and shuttle agents of the present description may be suitable for clinical use. In some embodiments, the synthetic peptides and shuttle agents of the present description may avoid the use of domains or compounds for which toxicity is uncertain or has not been ruled out.
  • the present description relates to a composition
  • a composition comprising a cocktail of at least 2, at least 3, at least 4, or at least 5 different types of the synthetic peptides or polypeptide- based shuttle agents as defined herein.
  • combining different types of synthetic peptides or polypeptide-based shuttle agents may provide increased versatility for delivering different polypeptide cargos intracellular ⁇ .
  • combining lower concentrations of different types of shuttle agents may help reduce cellular toxicity associated with using a single type of shuttle agent (e.g., at higher concentrations).
  • the present description relates to a method for increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell.
  • the method may comprise contacting the target eukaryotic cell with the synthetic peptide, polypeptide-based shuttle agent, or composition as defined herein, and the polypeptide cargo.
  • the synthetic peptide, polypeptide-based shuttle agent, or composition may be pre-incubated with the polypeptide cargo to form a mixture, prior to exposing the target eukaryotic cell to that mixture.
  • the type of CPD may be selected based on the amino acid sequence of the polypeptide cargo to be delivered intracellular ⁇ .
  • the type of CPD and ELD may be selected to take into account the amino acid sequence of the polypeptide cargo to be delivered intracellularly, the type of cell, the type of tissue, etc.
  • the method may comprise multiple treatments of the target cells with the synthetic peptide, polypeptide-based shuttle agent, or composition (e.g., 1 , 2, 3, 4 or more times per day, and/or on a pre-determined schedule). In such cases, lower concentrations of the synthetic peptide, polypeptide-based shuttle agent, or composition may be advisable (e.g., for reduced toxicity).
  • the cells may be suspension cells or adherent cells.
  • the person of skill in the art will be able to adapt the teachings of the present description using different combinations of shuttles, domains, uses and methods to suit particular needs of delivering a polypeptide cargo to particular cells with a desired viability.
  • the methods of the present description may apply to methods of delivering a polypeptide cargo intracellularly to a cell in vivo. Such methods may be accomplished by parenteral administration or direct injection into a tissue, organ, or system.
  • the synthetic peptide, polypeptide-based shuttle agent, or composition, and the polypeptide cargo may be exposed to the target cell in the presence or absence of serum.
  • the method may be suitable for clinical or therapeutic use.
  • the present description relates to a kit for increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell.
  • the kit may comprise the synthetic peptide, polypeptide-based shuttle agent, or composition as defined herein, and a suitable container.
  • the target eukaryotic cells may be an animal cell, a mammalian cell, or a human cell.
  • the target eukaryotic cells may be a stem cell (e.g., embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, peripheral blood stem cells), primary cells (e.g., myoblasts, fibroblasts), or an immune cell (e.g., T cells, NK cells, dendritic cells, antigen presenting cells).
  • the present description relates to an isolated cell comprising a synthetic peptide or polypeptide-based shuttle agent as defined herein.
  • the cell may be a protein- induced pluripotent stem cell. It will be understood that cells that are often resistant or not amenable to protein transduction may be interesting candidates for the synthetic peptides or polypeptide-based shuttle agents of the present description.
  • a polypeptide-based shuttle as disclosed herein.
  • delivery of cargo is performed for therapeutic applications.
  • the polypeptide-based shuttles disclosed herein increase the efficiency of 1) translocation across the cell member, 2) escape from the endosome, 3) allowing delivery to the targeted subcellular location.
  • the increased efficiency of these steps leads to a desired outcome, such as a genetic engineering event by the delivery of a genetic engineering protein such as Cas9, or expression modification by the delivery of proteins such as transcription factors.
  • polypeptide-based shuttles disclosed herein are non-toxic to the host cells and are able to be degraded by the host cell after the delivery function is complete.
  • methods for delivering cargo to a cell using a polypeptide-based shuttle as described herein are methods for expression modification of target genes.
  • Cargo is any combination of polypeptide, nucleic acid, and/or other molecule.
  • the cargo is a polypeptide or protein.
  • the cargo includes DNA or RNA. Sometime the cargo is a transcription factor or functional fragment thereof.
  • the transcription factor is HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Klf4, c-Myc, MyoD, Pdx1 , Ngn3 and MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1 , FoxA1 , Nanog, Esrrb, Lin28, HI F1 -alpha, Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5, Bcl-6, Sox9, or Yamanaka factors.
  • the transcription factor is HoxB4.
  • the cargo In cases where the cargo is a protein or polypeptide, the cargo often comprises a subcellular localization domain.
  • the subcellular localization domain can be a nuclear localization signal, nucleolar localization signal, mitochondrial localization signal, peroxisome localization signal, or cytosol localization signal.
  • Cargo is delivered into a cell using any polypeptide-based shuttle disclosed herein.
  • the cell is a eukaryotic cell. Often the eukaryotic cell is a human cell. Alternatively, the cell is a mammalian, animal, plant, archaea, or bacterial cell.
  • the cell is a stem cell or stem-cell derived cell. For example, the cell is a hematopoietic stem cell or a megakaryocyte. It will be readily recognize by one of skill in the art that many cell types would be useful in these methods.
  • the cells are isolated from patients or human donors. Often the cells are isolated or maintained ex vivo. In other instances, the cells are in vivo, or alternatively, in vitro.
  • the cargo protein complexed with the polypeptide-based shuttle is intended for delivery to the nucleus.
  • the transcription factor HoxB4 is functional in the nucleus and the transcription factor's inherent NLS and/or an additional NLS ensure deliver of said transcription factor to the nucleus.
  • the polypeptide-based shuttle has a cell penetrating domain (CPD), for transport of HoxB4 across the cell membrane, and an endosome leakage domain (ELD), to reduce sequestration of HoxB4 in the endosome.
  • the cargo protein, such as HoxB4 comprises a nuclear localization signal (NLS), to target HoxB4 to the nucleus.
  • NLS nuclear localization signal
  • the cargo comprises a subcellular targeting domain for delivery of the cargo to a desired subcellular localization.
  • a mitochondrial or peroxisome localization domain is used to target cargo to the mitochondria or peroxisome respectively.
  • Other known subcellular targeting domains, including those disclosed herein, are envisioned for use and would be readily recognized by one of skill in the art as being sufficient for use in the methods disclosed herein.
  • the cargo protein is a transcription factor which is selected in order to initiate a specific function within the cell.
  • HoxB4 is used for the expansion of hematopoietic stem cells.
  • HoxB4 is complexed with a polypeptide-based shuttle as disclosed herein, which is subsequently delivered to the nucleus where HoxB4 functions to expand the HSC population such that the expanded population is greater in number than the starting population.
  • the cargo protein is a transcription factor that modifies the expression of target proteins.
  • the expression modification leads to cell expansion. In other examples, expression modification leads to cell differentiation. Other outcomes of transcription factor activity are envisioned as well and are well known in the art.
  • the condition is a genetic condition.
  • the condition is a genetic disorder or blood malignancy.
  • the condition is ischemic heart failure or an indication requiring a hematopoietic stem cell transplant.
  • the condition is thrombocytopenia.
  • the cells used in the methods provided herein are autologous cells isolated from the patient.
  • the cells are isolated from a different person.
  • the cells will be further engineered in order to make them immunogenic so they will not be rejected by the patient or cause another sort of adverse effect associated with immunogenicity.
  • the cells obtained from methods described herein are subsequently treated further before being used to treat a patient.
  • an expanded hematopoietic stem cell population can be treated with differentiation factors to generate megakaryocytes which are subsequently used to treat a patient in need thereof as described herein.
  • an expanded stem cell population can be differentiated into a desired downstream cell type using methods known in the art.
  • isolated cells can be treated with transcription factors, such as Yamanaka factors in order to generate induced pluripotent stem cells.
  • transcription factors such as Yamanaka factors
  • cells are treated with the transcription factor Sox9 in order to generate chondrocytes.
  • the generated cells are sometimes further treated.
  • the cells are sometimes further engineered to generate non-immunogenic cells.
  • expanded hematopoietic stem cell-derived megakaryocytes can be further treated to generate non-immunogenic megakaryocytes.
  • the non-immunogenic megakaryocytes are generated by disrupting a major histocompatibility complex gene sequences.
  • target gene disruption is achieved, for example, by a DNA nuclease.
  • the DNA nuclease is an RNA- guided nuclease such as Cas9 or Cpfl
  • the RNA-guided nuclease is a Cas protein.
  • the gene disruption is achieve through the activity of TALENs or ZFNs.
  • Method for gene disruption is achieved, in some examples, by the delivery of a DNA-disrupting agent complexed with a polypeptide-based shuttle as disclosed herein.
  • DNA-disrupting agents include proteins or other molecules capable of disrupting a nucleic acid sequence, such as DNA-binding agents, DNA-degrading agents, or DNA-cleaving agents.
  • DNA-cleaving agents include RNA-guided nucleases such as Cas proteins.
  • Cas proteins include Cas9 and Cpfl
  • Other DNA-cleaving agents include TALENs or ZFNs. Many other DNA-disrupting agents are known in the art and would be readily recognized as sufficient for methods disclosed herein.
  • the gene-disrupting agent is delivered to the nucleus by a polypeptide- based shuttle and NLS as disclosed herein.
  • Cas9 or Cpfl are delivered to the cell and escape the endosome through a complexed interaction with a polypeptide-based shuttle, and are subsequently delivered to the nucleus due to the nuclear localization signal fused to Cas9 or Cpfl as disclosed herein.
  • the gene-disrupting agent is small enough or otherwise suited to diffuse into the nucleus without need for an NLS.
  • the gene- disrupting agent comprises an inherent NLS, while in other cases the gene-disrupting agent is engineered to comprise an NLS or an additional NLS. Many appropriate nuclear localization signals are known in the art.
  • Gene-disrupting agents delivered with a polypeptide-based shuttle as disclosed herein are used to target a nucleic acid sequence of interest.
  • Cas9 is guided to a target DNA sequence by an engineered crRNA and corresponding trRNA.
  • Cpfl is guided to a target DNA sequence by an engineered crRNA.
  • Cas9 or Cpfl cleave the target DNA sequence leading to disruption of the gene product. Cleavage occurs through the generation of a double strand break, or alternatively a single strand break when using a modified Cas9 or Cpfl protein.
  • NJEJ non-homologous end joining
  • HDR homology driven recombination
  • the deleted sequence comprises the target sequence of interest, which often comprises a target gene of target gene fragment of interest.
  • the donor sequence comprises a selective marker, reporter marker, or other exogenous gene of interest.
  • the DNA when using a catalytically dead RNA-guided nuclease, the DNA is not cleaved; instead the dead nuclease binds to the target DNA sequence and blocks transcription from occurring, thereby disrupting production of the gene product.
  • Other methods for targeted gene disruption are well-known in the art and are sufficient for incorporation into the methods provided herein.
  • a gene disrupting agent-peptide shuttle complex is used to delete or disrupt a human leukocyte antigen (HLA) sequence or a major histocompatibility complex (MHC) gene sequence.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • other immunogenicity target sequences are deleted or disrupted.
  • immunogenicity targets are disrupted in megakaryocytes.
  • immunogenicity targets are disrupted in stem cells to generate, as an example, universal stem cells.
  • immunogenicity targets are deleted or disrupted in T cells or chimeric antigen receptor t cells.
  • Non-immunogenic cells are used for treatment of a variety of diseases. Such diseases include cancer, thrombocytopenia, other blood malignancies, and other genetic diseases.
  • disruption or deletion of a target sequence through methods disclosed herein are used for treatment of infection.
  • viral sequences or other infective nucleotide sequences are targeted for disruption or deletion using a DNA-disrupting agent, such as Cas9, complexed with a polypeptide-based shuttle as disclosed herein.
  • the viral sequence can be HIV.
  • a sequence is targeted using methods disclosed herein in order to treat a genetic disease. In some of these cases, the genetic disease is a blood disease.
  • cells are eukaryotic cells.
  • the cells are human.
  • the cells are mammalian, animal, plant, or any other eukaryotic cell.
  • the cells are human, they are often isolated from a patient. In many of these cases, the cells are an autologous population.
  • human cells are isolated from a donor. In many of these cases, the isolated cells are an allogenic population.
  • the isolated human cells are stem cells. Stem cells are isolated from a variety of sources, including peripheral blood, bone marrow, and umbilical cord blood. Therapeutic applications- hematopoietic stem cell expansion
  • HSC hematopoietic stem cell
  • the HSCs to be transplanted can be isolated from the patient (autologous sample) or from a donor (allogenic sample). Autologous samples are often preferred since the patient will not have an adverse immunogenic response or rejection of the sample since it is derived from the patient's own peripheral blood, bone marrow, or umbilical cord blood.
  • the number of HSCs to be transplanted into the patient will affect the prognosis of the transplant, and a threshold population size is often required before a transplant can proceed. Unfortunately, for many patients in need of a HSC transplant, the number of cells isolated from the patient is below this threshold.
  • the expanded HSC population is subsequently transplanted into a patient in need thereof.
  • the HSC population is an autologous population from the patient to be treated.
  • the expanded HSC population is above the threshold needed for transplantation.
  • HSC expansion is achieved by delivery of a transcription factor involved in HSC expansion.
  • the transcription factor in some embodiments is a Hox family transcription factor. I n specific examples, the transcription factor is HoxB4.
  • HoxB4 is delivered to a HSC nucleus by being complexed with a polypeptide-based shuttle comprising a cell penetrating domain (CPD) and endosome leakage domain (ELD).
  • the transcription factor comprises a nuclear localization signal (NLS) as disclosed herein.
  • the transcription factor is engineered to comprise an NLS or an additional NLS.
  • the polypeptide-based shuttle is any polypeptide-based shuttle or combination of CPD and ELD disclosed herein.
  • Cargo proteins such as transcription factors destined for the nucleus comprise any NLS sequences disclosed herein or native NLS sequences.
  • the patient to be treated with the expanded HSC population is in need of a HSC transplantation due to a condition.
  • the condition is a malignant condition.
  • Malignant conditions include acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma, neuroblastoma, Ewing sarcoma, myelodysplasia syndrome, glioma, solid tumors, and some genetic diseases, among others.
  • the condition is a non-malignant condition.
  • Non-malignant conditions include thalassemia, sickle cell amenia, aplastic anemia, Fanconi anemia, immune deficiency syndromes, inborn errors of metabolism, and some genetic diseases, among others.
  • expanded stem cell populations are subsequently differentiated into other cells of interest. Differentiation is achieved by contacting the cell population with differentiation factors. Differentiation factors vary depending on the desired cell type and combinations of differentiation factors are well known in the art.
  • an expanded HSC population generated using the methods disclosed herein are subsequently differentiated into megakaryocytes. Differentiated cells are subsequently used to treat patients in need thereof. For example, an expanded megakaryocyte population is used in some examples to treat a patient with thrombocytopenia. Therapeutic applications- non-immunogenic megakaryocytes
  • a common treatment for patients with certain condition is transplantation with specific cells.
  • thrombocytopenia can be treated with transplantation of megakaryocytes.
  • autologous samples are not able to be acquired, and therefore allogenic samples from other donors are used in the transplantation.
  • immune rejection is a major health risk to the patient.
  • Non-immunogenic cells are often subsequently transplanted into patients in need.
  • non- immunogenic megakaryocytes are obtained from methods disclosed herein.
  • Non-immunogenic cells are generated by disruption of immunogenicity targets or genes, for example HLA or MHC genes.
  • the starting megakaryocytes are be isolated from a patient or donor.
  • HSCs are isolated from a donor or patient and subsequently expanded and differentiated into megakaryocytes using methods disclosed herein.
  • a disorder such as a genetic disease
  • a nucleic acid sequence, or a gene results in treatment or amelioration of symptoms of a disease or disorder.
  • the genetic disorder is Krabbe Disease, Tay-Sachs Disease, or Hurler Syndrome.
  • the gene being inserted is GALC, HEXA, or IDUA.
  • the gene being inserted is a normal copy of a gene, and in some of these cases, the normal gene can be inserted into a corresponding abnormal gene locus.
  • the insertion site is an abnormal GALC, HEXA, or IDUA gene. In some examples, the insertion site is an albumin gene. In some examples the cell is a eukaryotic cell. In some examples, the cell is a stem cell, hematopoietic cell, central nervous system cell, microgilia cell, neuron, liver cell, hepatocyte, or liver endothelia cell. In some examples, the peptide shuttle and donor gene or donor nucleic acid is delivered ex vivo, or by direct injection. In some examples, direct injection comprises direct CNS injection, or liver intra-arterial injection.
  • hematopoietic stem cell comprising contacting the HSC with a polypeptide-based shuttle complexed with the cargo protein, wherein the polypeptide-based shuttle comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, and wherein the cargo protein and the polypeptide-based shuttle have independent protein backbones.
  • CPD cell penetrating domain
  • ELD endosome leakage domain
  • the polypeptide-based shuttle further comprises a histidine rich domain.
  • the cargo protein comprises a subcellular targeting domain
  • the subcellular targeting domain is an organelle localization domain.
  • the organelle localization domain is a nuclear localization signal (NLS).
  • the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity.
  • the cargo protein and the polypeptide-based shuttle are not covalently linked.
  • the cargo protein comprises a transcription factor, or a functional fragment thereof.
  • the transcription factor comprises a Hox family transcription factor, or functional fragment thereof.
  • the transcription factor comprises a mammalian Hox family transcription factor, or a functional fragment thereof.
  • the transcription factor comprises a human Hox family transcription factor, or functional fragment thereof.
  • the transcription factor comprises HoxB4, or a functional fragment thereof.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity
  • the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof.
  • the polypeptide-based shuttle is non-toxic and/or metabolizable.
  • the HSC is a mammalian HSC.
  • the mammalian HSC is a human HSC.
  • HSC hematopoietic stem cell
  • the polypeptide-based shuttle comprises comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity
  • the cargo protein comprises a subcellular targeting domain
  • the polypeptide-based shuttle and the cargo protein have independent protein backbones.
  • polypeptide-based shuttle further comprises a histidine rich domain. Further provided herein are methods wherein the polypeptide-based shuttle and the cargo protein are not covalently linked. Further provided herein are methods wherein the subcellular targeting domain comprises a nuclear localization signal (NLS). Further provided herein are methods wherein the protein cargo comprises a transcription factor, or a functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a mammalian Hox family transcription factor, or a functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a human Hox family transcription factor, or functional fragment thereof.
  • NLS nuclear localization signal
  • the transcription factor comprises HoxB4, or a functional fragment thereof.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity.
  • the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity
  • the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof.
  • the HSC population is a mammalian HSC population.
  • the HSC population is a human HSC population.
  • the HSC population is an autologous HSC population. Further provided herein are methods wherein the HSC population is an allogenic HSC population. Further provided herein are methods wherein the HSC population is a CD34+ population. Further provided herein are methods wherein the HSC population is isolated from cord blood. Further provided herein are methods wherein the HSC population is isolated from bone marrow. Further provided herein are methods wherein the HSC population is isolated from peripheral blood. Further provided herein are methods wherein the expanded HSC population is differentiated to produce a differentiated population. Further provided herein are methods further comprising transplantation of the expanded population into a patient in need thereof due to a condition. Further provided herein are methods wherein the condition is a genetic disease.
  • condition is a blood malignancy. Further provided herein are methods wherein the condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are methods wherein the condition is an indication requiring a hematopoietic stem cell transplantation. Further provided herein are methods wherein the differentiated population is an autologous differentiated population. Further provided herein are methods wherein the differentiated population is an allogenic differentiated population. Further provided herein are methods wherein the differentiated population is a megakaryocyte population. Further provided herein are methods of treating a patient with a condition by administering to the patient in need thereof an expanded HSC population obtained by any of the methods disclosed herein.
  • condition is a genetic disease. Further provided herein are methods wherein the condition is a blood disorder. Further provided herein are methods wherein the condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are uses of an expanded HSC population obtained by aby of the methods disclosed herein for treatment of a patient in need thereof due to a condition. Further provided herein are uses wherein the condition is a genetic disease. Further provided herein are uses wherein the condition is a blood disorder. Further provided herein are uses wherein the condition is a malignant condition. Further provided herein are uses wherein the condition is a non-malignant condition.
  • a population of megakaryocytes comprising, contacting a hematopoietic stem cell (HSC) population having a starting population size with a polypeptide-based shuttle complexed with a transcription factor, such that the HSC population expands beyond the starting population size, and contacting the expanded HSC population with differentiating factors such that at least a portion of the HSC population differentiates into megakaryocytes, thereby generating a megakaryocyte population
  • the polypeptide-based shuttle comprises comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity
  • the transcription factor comprises a nuclear localization signal (NLS)
  • NLS nuclear localization signal
  • the polypeptide-based shuttle further comprises a histidine rich domain. Further provided herein are methods wherein the polypeptide-based shuttle and the transcription factor are not covalently linked. Further provided herein are methods wherein the transcription factor comprises a Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a mammalian Hox family transcription factor, or a functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a human Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises HoxB4, or a functional fragment thereof.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity.
  • the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity
  • the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof.
  • the HSC population is human HSC population.
  • the human HSC population is a CD34+ population.
  • the HSC population is isolated from cord blood.
  • the HSC population is isolated from bone marrow.
  • the HSC population is isolated from peripheral blood.
  • the condition is a genetic disease.
  • condition is a blood malignancy. Further provided herein are methods wherein the condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are methods wherein the condition is an indication requiring a hematopoietic stem cell transplant. Further provided herein are methods wherein the condition is thrombocytopenia. Further provided herein are methods wherein the megakaryocyte population is an autologous population. Further provided herein are methods wherein the megakaryocyte population is an allogenic population. Further provided herein are methods wherein the megakaryocyte population is non-immunogenic.
  • a non-immunogenic megakaryocyte comprising contacting a megakaryocyte with a polypeptide-based shuttle complexed with a DNA cleavage protein, such that the DNA cleavage protein cleaves at least one immunogenic target DNA sequence within the megakaryocyte, thereby rendering the at least one immunogenic target DNA sequence non-functional
  • the polypeptide-based shuttle comprises comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity
  • the DNA cleavage protein comprises a nuclear localization signal (NLS)
  • the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones.
  • the polypeptide-based shuttle further comprises a histidine rich domain.
  • the CPD comprises at least one of SEQ ID NOs: 16- 27 or 65, or a variant or functional fragment thereof having cell penetrating activity.
  • the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity, and wherein the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof.
  • the DNA cleavage protein is an RNA-guided nuclease.
  • the RNA-guided nuclease comprises a Cas protein.
  • the Cas protein comprises Cas9, Cpf1 , or a functional fragment thereof.
  • the polypeptide-based shuttle and the RNA-guided nuclease are further complexed with at least one guiding RNA.
  • RNA-guided nuclease comprises Cas9 and is further complexed with a crRNA and a trRNA. Further provided herein are methods wherein the RNA-guided nuclease comprises Cpf1 and is further complexed with a guiding RNA. Further provided herein are methods wherein the at least one guiding RNA is engineered to target the at least one immunogenic target DNA. Further provided herein are methods wherein the RNA-guided nuclease and at least one guiding RNA contact and cleave the at least one immunogenic target DNA.
  • cleavage of the at least one immunogenic target DNA results in a disrupted immunogenic target gene following DNA repair, such that the immunogenic target gene or gene product is non-functional.
  • the at least one immunogenic target DNA comprises an MHC gene.
  • the at least one immunogenic target DNA comprises a sequence sharing at least 90% identity to a nucleic acid sequence comprised within an MHC gene.
  • the condition is a genetic disease.
  • the condition is a blood malignancy.
  • condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are methods wherein the condition is an indication requiring a hematopoietic stem cell transplant. Further provided herein are methods wherein the condition is thrombocytopenia. Further provided herein are methods of treating a patient with a condition by administering to the patient in need thereof non-immunogenic cells obtained by any of the methods disclosed herein. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood disorder. Further provided herein are methods wherein the condition is thrombocytopenia.
  • non- immunogenic cells obtained by any of the methods disclosed herein for treatment of a patient in need thereof due to a condition.
  • the condition is a genetic disease.
  • the condition is a blood disorder.
  • the condition is thrombocytopenia.
  • a polypeptide-based shuttle complexed with a transcription factor
  • the polypeptide-based shuttle comprises comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity
  • the transcription factor comprises a subcellular targeting domain and wherein the transcription factor and the polypeptide-based shuttle have independent protein backbones.
  • the polypeptide-based shuttle and the transcription factor are not covalently linked.
  • the polypeptide-based shuttle further comprises a histidine rich domain.
  • the subcellular targeting domain comprises a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity.
  • the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity
  • the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof.
  • the transcription factor is a mammalian transcription factor.
  • the transcription factor is a human transcription factor.
  • the cell is within a population.
  • modified gene expression leads to expansion of the cell population. Further provided herein are methods wherein the expanded cell population is transplanted into a patient in need thereof due to a condition. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood malignancy. Further provided herein are methods wherein the condition is an indication requiring a hematopoietic stem cell transplant. Further provided herein are methods wherein the condition is thrombocytopenia. Further provided herein are methods wherein the cell is a stem cell. Further provided herein are methods wherein the cell is a hematopoietic stem cell. Further provided herein are methods wherein the transcription factor comprises HoxB4, or a functional fragment thereof. Further provided herein are methods wherein the modified gene expression leads to differentiation of the cell population. Further provided herein are methods wherein the cell is a stem cell. Further provided herein are methods wherein the cell is a hematopoietic stem cell.
  • hematopoietic stem cell comprising contacting the HSC with a polypeptide-based shuttle complexed with the cargo protein, wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs. 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the cargo protein and the polypeptide-based shuttle have independent protein backbones, wherein the cargo protein and the polypeptide-based shuttle are not covalently linked, wherein the cargo protein comprises a HoxB4 transcription factor, or a functional fragment thereof, and wherein the HSC is a human HSC.
  • HSC hematopoietic stem cell
  • HSC hematopoietic stem cell
  • methods for expanding a hematopoietic stem cell (HSC) population comprising contacting the HSC population having a starting population size with a polypeptide-based shuttle complexed with a cargo protein such that the HSC population expands beyond the starting population size, wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the polypeptide-based shuttle and the cargo protein have independent protein backbones, wherein the polypeptide-based shuttle and the cargo protein are not covalently linked, wherein the protein cargo comprises a HoxB4 transcription factor, or a functional fragment thereof, wherein the HSC population is a human HSC population.
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the polypeptide-based shuttle and the cargo protein have independent protein backbones
  • a population of megakaryocytes comprising, contacting a hematopoietic stem cell (HSC) population having a starting population size with a polypeptide-based shuttle complexed with a transcription factor, such that the HSC population expands beyond the starting population size, and contacting the expanded HSC population with differentiating factors such that at least a portion of the HSC population differentiates into megakaryocytes, thereby generating a megakaryocyte population
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the polypeptide-based shuttle and the cargo protein have independent protein backbones, wherein the polypeptide-based shuttle and the transcription factor are not covalently linked, wherein the transcription factor comprises HoxB4, or a functional fragment thereof, wherein the HSC population is human HSC population.
  • a non-immunogenic megakaryocyte comprising contacting a megakaryocyte with a polypeptide-based shuttle complexed with a DNA cleavage protein, such that the DNA cleavage protein cleaves at least one immunogenic target DNA sequence within the megakaryocyte, thereby rendering the at least one immunogenic target DNA sequence non-functional
  • the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones, wherein the polypeptide-based shuttle and the DNA cleavage protein are not covalently linked
  • the DNA cleavage protein comprises Cas9 and is further complexed with a crRNA and a trRNA, wherein the crRNA is engineered to target an MHC gene sequence, such that Cas9 contacts and cleaves the MHC gene sequence, and wherein clea
  • non-immunogenic megakaryocytes obtained by any of the methods disclosed herein.
  • the present description may additionally or alternatively relate to the following aspects:
  • a synthetic peptide comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD.
  • a polypeptide-based shuttle agent comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD, for use in increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell.
  • amino acid residues 24, 25, 26, 27, 28, 29, or 30 amino acid residues and a maximum length of 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues;
  • (b) has a predicted net charge of at least +6, +7, +8, +9, +10, +11 , +12, +13, +14, or +15 at physiological pH;
  • (c) is soluble in aqueous solution; or (d) any combination of (a) to (c).
  • the ELD is or is from: an endosomolytic peptide; an antimicrobial peptide (AMP); a linear cationic alpha-helical antimicrobial peptide; a Cecropin-A/Melittin hybrid (CM series) peptide; pH- dependent membrane active peptide (PAMP); a peptide amphiphile; a peptide derived from the N terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; Diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1 ; VSVG; Pseudomonas toxin; melittin; KALA; JST-1 ; C(LLKK) 3 C (SEQ ID NO: 63); G(LLKK) 3 G (SEQ ID NO:
  • a CPD which is TAT or PTD4 having the amino acid sequence of SEQ ID NO: 17 or 65, or a variant thereof having at least 85%, 90%, or 95% identity to SEQ ID NO: 17 or 65, and having cell penetrating activity; or Penetratin having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least 85%, 90%, or 95% identity to SEQ ID NO: 18 and having cell penetrating activity;
  • a histidine-rich domain comprising at least 6 consecutive histidine residues; or (d) any combination of (a) to (c).
  • a composition comprising: (a) the synthetic peptide or polypeptide-based shuttle agent as defined in any one of (1) to (10), and a further independent synthetic peptide comprising a histidine-rich domain and a CPD; and/or (b) a cocktail of at least 2, at least 3, at least 4, or at least 5 different types of the synthetic peptides or polypeptide-based shuttle agents as defined in any one of (1) to (10).
  • a method for increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell comprising contacting the target eukaryotic cell with the synthetic peptide, polypeptide-based shuttle agent, or composition as defined in any one of (1) to
  • kits for increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell comprising the synthetic peptide, polypeptide-based shuttle agent, or composition as defined in any one of (1) to (11), and a suitable container.
  • polypeptide cargo (a) comprises or lacks a CPD or a CPD as defined in (4)(b); (b) is a recombinant protein; (c) comprises a subcellular targeting domain; (d) is complexed with a DNA and/or RNA molecule; or (e) any combination of (a) to (d).
  • NLS nuclear localization signal
  • the NLS is from: E1 a, T-Ag, c-myc, T-Ag, op-T-NL
  • polypeptide cargo is a transcription factor, a nuclease, a cytokine, a hormone, a growth factor, or an antibody.
  • a eukaryotic cell comprising the synthetic peptide or polypeptide-based shuttle agent as defined in any one of (1 ) to (10), or the composition of (11 ).
  • a method for delivering an independent polypeptide cargo to the cytosol of a target eukaryotic cell comprising contacting said target eukaryotic cell with the synthetic peptide or polypeptide-based shuttle agent as defined in any one of (1) to (10), or the composition of (11); and an independent polypeptide cargo to be delivered intracellular ⁇ by said synthetic peptide or polypeptide-based shuttle agent.
  • a method for increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell comprising contacting said target eukaryotic cell with a synthetic peptide and said independent polypeptide cargo, wherein said synthetic peptide:
  • (a) comprises an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, operably linked to a cell penetrating domain (CPD), wherein said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64;
  • (c) has an overall length of between 20 and 100 amino acid residues
  • said CPD enables intracellular delivery of said synthetic peptide
  • said ELD enables escape of endosomally trapped independent polypeptide cargo to the cytosol of the target eukaryotic cell.
  • a method for increasing the transduction efficiency and cytosolic delivery of an independent polypeptide cargo in a population of target eukaryotic cells comprising contacting said target eukaryotic cells with said independent polypeptide cargo and a concentration of a synthetic peptide sufficient to increase the percentage or proportion of the population of target eukaryotic cells into which the independent polypeptide is delivered intracellular ⁇ across the plasma membrane, as compared to in the absence of said synthetic peptide, wherein said synthetic peptide:
  • (a) comprises an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, operably linked to a cell penetrating domain (CPD), wherein said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1 -15, 63, or 64;
  • (c) has an overall length of between 20 and 100 amino acid residues
  • said CPD enables intracellular delivery of said synthetic peptide
  • said ELD enables escape of endosomally trapped independent polypeptide cargo to the cytosol of the target eukaryotic cells, thereby increasing the transduction efficiency and cytosolic delivery of the independent polypeptide cargo in the population of target eukaryotic cells.
  • said synthetic peptide further comprises a histidine- rich domain consisting of a stretch of at least 6 amino acids comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues; and/or comprises at least 2, at least 3, at least 4, at least 5, or at least 6 consecutive histidine residues.
  • said transcription factor is: HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Sox9, Klf4, c-Myc, MyoD, Pdx1 , Ngn3, MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1 , FoxA1, Nanog, Esrrb, Lin28, HIF1 -alpha, Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5, Bcl-6, or any combination thereof; or
  • said nuclease is an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1 , a zinc-finger nuclease (ZFNs), a Transcription activator-like effector nucleases (TALENs), a homing endonuclease, or a meganuclease.
  • RNA-guided endonuclease RNA-guided endonuclease
  • CRISPR endonuclease a type I CRISPR endonuclease
  • nuclease further comprises a guide RNA, a crRNA, a tracrRNA, or both a crRNA and a tracrRNA.
  • said transcription factor is: HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Sox9, Klf4, c-Myc, MyoD, Pdx1 , Ngn3, MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1 , FoxA1, Nanog, Esrrb, Lin28, HIF1 -alpha, Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5, Bcl-6, or any combination thereof; or
  • said nuclease is an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1 , a zinc -finger nuclease (ZFNs), a Transcription activator-like effector nucleases (TALENs), a homing endonuclease, or a meganuclease.
  • RNA-guided endonuclease RNA-guided endonuclease
  • CRISPR endonuclease a
  • a method for increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell comprising contacting said target eukaryotic cell with a synthetic peptide and said independent polypeptide cargo, wherein said synthetic peptide: (a) comprises an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), wherein said ELD is an endosomolytic peptide which is, or is derived from: a linear cationic alpha-helical antimicrobial peptide; a Cecropin-A/Melittin hybrid (CM series) peptide; pH-dependent membrane active peptide (PAMP); a peptide amphiphile; a peptide derived from the N terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; Diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5W
  • (c) has an overall length of between 20 and 100 amino acid residues
  • said CPD enables intracellular delivery of said synthetic peptide
  • said ELD enables escape of endosomally trapped independent polypeptide cargo to the cytosol of the target eukaryotic cell.
  • a method for increasing the transduction efficiency and cytosolic delivery of an independent polypeptide cargo in a population of target eukaryotic cells comprising contacting said target eukaryotic cells with said independent polypeptide cargo and a concentration of a synthetic peptide sufficient to increase the percentage or proportion of the population of target eukaryotic cells into which the independent polypeptide is delivered intracellular ⁇ across the plasma membrane, as compared to in the absence of said synthetic peptide, wherein said synthetic peptide:
  • (a) comprises an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), wherein said ELD is an endosomolytic peptide which is, or is derived from: a linear cationic alpha-helical antimicrobial peptide; a Cecropin-A/Melittin hybrid (CM series) peptide; pH-dependent membrane active peptide (PAMP); a peptide amphiphile; a peptide derived from the N terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; Diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1; VSVG; Pseudomonas toxin; melittin; KALA; JST-1 ; C(LLKK)3C; or G(LLKK)3G;
  • ELD endosome leakage domain
  • (c) has an overall length of between 20 and 100 amino acid residues
  • (e) is soluble in aqueous solution at physiological pH, wherein said CPD enables intracellular delivery of said synthetic peptide, and said ELD enables escape of endosomally trapped independent polypeptide cargo to the cytosol of the target eukaryotic cells,
  • said synthetic peptide further comprises a histidine- rich domain consisting of a stretch of at least 3 amino acids comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues; and/or comprises at least 2, at least 3, at least 4, at least 5, or at least 6 consecutive histidine residues.
  • transcription factor is: HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Klf4, c-Myc, MyoD, Pdx1 , Ngn3, MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1,
  • nuclease is an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1, a zinc-finger nuclease (ZFNs), a Transcription activator-like effector nucleases (TALENs), a homing endonuclease, or a meganuclease.
  • a method for increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell comprising contacting said target eukaryotic cell with a synthetic peptide and said independent polypeptide cargo which is not covalently bound to said synthetic peptide, wherein said synthetic peptide comprises an endosome leakage domain (ELD) operably linked to a cell penetrating domain, or an ELD operably linked to a CPD and a histidine-rich domain, wherein:
  • ELD endosome leakage domain
  • said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64;
  • said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65;
  • said histidine-rich domain comprises at least two consecutive histidine residues.
  • a method for increasing the transduction efficiency and cytosolic delivery of an independent polypeptide cargo in a population of target eukaryotic cells comprising contacting said target eukaryotic cell with said independent polypeptide cargo and a concentration of a synthetic peptide sufficient to increase the percentage or proportion of the population of target eukaryotic cells into which the independent polypeptide is delivered intracellular ⁇ across the plasma membrane, as compared to in the absence of said synthetic peptide, which is not covalently bound to said synthetic peptide, wherein said synthetic peptide comprises an endosome leakage domain (ELD) operably linked to a cell penetrating domain, or an ELD operably linked to a CPD and a histidine-rich domain, wherein:
  • ELD endosome leakage domain
  • said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1 -15, 63, or 64;
  • said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65;
  • said histidine-rich domain comprises at least two consecutive histidine residues, thereby increasing the transduction efficiency and cytosolic delivery of the independent polypeptide cargo in the population of target eukaryotic cells.
  • a method for delivering a CRISPR associated protein 9 (Cas9) to the nucleus of a target eukaryotic cell comprising contacting said eukaryotic cell with a Cas9 recombinant protein comprising a nuclear localization signal, and a separate synthetic peptide shuttle agent less than 100 residues in length and comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain, or an ELD operably linked to a CPD and a histidine-rich domain, wherein:
  • said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64;
  • said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65;
  • said histidine-rich domain comprises at least two consecutive histidine residues.
  • a method for delivering a CRISPR associated protein 9 (Cas9) to the nucleus of a population of eukaryotic cells comprising contacting said population of eukaryotic cells with Cas9 recombinant protein comprising a nuclear localization signal, and a separate synthetic peptide shuttle agent less than 100 residues in length and comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain, or an ELD operably linked to a CPD and a histidine-rich domain, wherein:
  • said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64;
  • said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65;
  • said histidine-rich domain comprises at least two consecutive histidine residues, wherein said synthetic peptide is at a concentration sufficient to increase the percentage or proportion of the population of eukaryotic cells into which the independent polypeptide is delivered across the plasma membrane, as compared to in the absence of said synthetic peptide
  • HeLa, HEK293A, HEK293T, THP-1 , CHO, NIH3T3, CA46, Balb3T3 and HT2 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured following the manufacturer's instructions.
  • Myoblasts are primary human cells kindly provided by Professor J.P. Tremblay (Universite Laval, Quebec, Canada).
  • Myoblasts Human (13 kindly provided MB1 15% FBS ITS 1x, FGF 2 10 ng/mL, (primary months) by Professor JP Dexamethasone adherent myoblasts Tremblay 0.39Mg/mL, cells) BSA 0.5mg/mL,
  • HT2 T lymphocytes ATCCTM CRL- RPM1 1640 10% FBS 200 lU/mL IL-2
  • FBS Fetal bovine serum
  • Fusion proteins were expressed in bacteria (£. coli BL21 DE3) under standard conditions using an isopropyl ⁇ -D- -thiogalactopyranoside (IPTG) inducible vector containing a T5 promoter.
  • Culture media contained 24 g yeast extract, 12 g tryptone, 4 mL glycerol, 2.3 g KH2PO4, and 12.5 g K2HPO4 per liter.
  • Bacterial broth was incubated at 37°C under agitation with appropriate antibiotic (e.g., ampicillin). Expression was induced at optical density (600 nm) between 0.5 and 0.6 with a final concentration of 1 mM IPTG for 3 hours at 30°C.
  • Bacteria were recuperated following centrifugation at 5000 RPM and bacterial pellets were stored at -20°C.
  • Bacterial pellets were resuspended in Tris buffer (Tris 25 mM pH 7.5, NaCI l OOmM, imidazole 5 mM) with phenylmethylsulfonyl fluoride (PMSF) 1 mM, and lysed by passing 3 times through the homogenizer Panda 2KTM at 1000 bar. The solution was centrifuged at 15000 RPM, 4°C for 30 minutes. Supernatants were collected and filtered with a 0.22 ⁇ filtration device.
  • Tris buffer Tris 25 mM pH 7.5, NaCI l OOmM, imidazole 5 mM
  • PMSF phenylmethylsulfonyl fluoride
  • Solubilized proteins were loaded, using a FPLC (AKTA Explorer 100R), on HisTrapTM FF column previously equilibrated with 5 column volumes (CV) of Tris buffer. The column was washed with 30 column volumes (CV) of Tris buffer supplemented with 0.1 % TritonTM X-114 followed with 30 CV of Tris buffer with imidazole 40 mM. Proteins were eluted with 5 CV of Tris buffer with 350 mM Imidazole and collected. Collected fractions corresponding to specific proteins were determined by standard denaturing SDS-PAGE.
  • Purified proteins were diluted in Tris 20 mM at the desired pH according to the protein's pi and loaded on an appropriate ion exchange column (Q SepharoseTM or SP SepharoseTM) previously equilibrated with 5 CV of Tris 20 mM, NaCI 30 mM. The column was washed with 10 CV of Tris 20 mM, NaCI 30 mM and proteins were eluted with a NaCI gradient until 1 M on 15 CV. Collected fractions corresponding to specific proteins were determined by standard denaturing SDS-PAGE. Purified proteins were then washed and concentrated in PBS 1X on Amicon UltraTM centrifugal filters 10,000 MWCO. Protein concentration was evaluated using a standard Bradford assay.
  • Peptide shuttle agents facilitate escape of endosomally-trapped calcein
  • Microscopy-based and flow cytometry-based fluorescence assays were developed to study endosome leakage and to determine whether the addition of the shuttle agents facilitates endosome leakage of the polypeptide cargo.
  • Calcein is a membrane-impermeable fluorescent molecule that is readily internalized by cells when administered to the extracellular medium. Its fluorescence is pH-dependent and calcein self- quenches at higher concentrations. Once internalized, calcein becomes sequestered at high concentrations in cell endosomes and can be visualized by fluorescence microscopy as a punctate pattern. Following endosomal leakage, calcein is released to the cell cytoplasm and this release can be visualized by fluorescence microscopy as a diffuse pattern.
  • mammalian cells e.g., HeLa, HEK293A, or myoblasts
  • the cells were allowed to attach by incubating overnight in appropriate growth media, as described in Example 1.
  • the media was removed and replaced with 300 ⁇ of fresh media without FBS containing 62.5 pg/mL (100 ⁇ ) of calcein, except for HEK293A (250 pg/mL, 400 ⁇ ).
  • the shuttle agent(s) to be tested was added at a predetermined concentration. The plate was incubated at 37°C for 30 minutes.
  • the cells were washed with 1x PBS (37°C) and fresh media containing FBS was added. The plate was incubated at 37°C for 2.5 hours. The cells were washed three times and were visualized by phase contrast and fluorescence microscopy (1X81TM, Olympus).
  • Calcein fluorescence is optimal at physiological pH (e.g., in the cytosol), as compared to the acidic environment of the endosome.
  • mammalian cells e.g., HeLa, HEK293, or myoblasts
  • mammalian cells e.g., HeLa, HEK293, or myoblasts
  • the cells were allowed to attach by incubating overnight in appropriate growth media, as described in Example 1.
  • the media in wells was removed and replaced with 50 ⁇ of fresh media without serum containing 62.5 pg/mL (100 ⁇ ) of calcein, except for HEK293A (250 g/mL, 400 ⁇ ).
  • the shuttle agent(s) to be tested was added at a predetermined concentration. The plate was incubated at 37°C for 30 minutes.
  • the cells were washed with 1x PBS (37°C) and fresh media containing 5-10% serum was added. The plate was incubated at 37°C for 2.5 hours. The cells were washed with 1x PBS and detached using trypsinization. Trypsinization was stopped by addition of appropriate growth media, and calcein fluorescence was quantified using flow cytometry (Accuri C6, Becton, Dickinson and Company (BD)).
  • Untreated calcein-loaded cells were used as a control to distinguish cells having a baseline of fluorescence due to endosomally-trapped calcein from cells having increased fluorescence due to release of calcein from endosomes.
  • Fluorescence signal means (“mean counts") were analyzed for endosomal escape quantification. In some cases, the "Mean Factor" was calculated, which corresponds to the fold-increase of the mean counts relative to control (untreated calcein-loaded cells).
  • the events scanned by flow cytometry corresponding to cells (size and granularity) were analyzed. The cellular mortality was monitored with the percentage of cells in the total events scanned. When it became lower than the control, it was considered that the number of cellular debris was increasing due to toxicity and the assay was discarded.
  • FIG. 1 B A typical result is shown in Figure 1 B, in which an increase in fluorescence intensity (right-shift) is observed for calcein-loaded HeLa cells treated with a shuttle agent that facilitates endosomal escape ("Calcein 100 ⁇ + CM18-TAT 5 ⁇ ", right panel in Figure 1 B), as compared to untreated calcein- loaded HeLa cells ("Calcein 100 ⁇ ", left panel in Figure 1 B).
  • the increase in calcein fluorescence is caused by the increase in pH associated with the release of calcein from the endosome (acidic) to the cytoplasm (physiological).
  • HeLa cells were cultured and tested in the endosomal escape assays as described in Example 2.1.
  • the results of flow cytometry analyses are summarized below. In each case, the flow cytometry results were also confirmed by fluorescence microscopy (data not shown).
  • Tables 2.1 and 2.2 show that treating calcein-loaded HeLa cells with the shuttle agents CM18-Penetratin-Cys and CM18-TAT-Cys (having the domain structure ELD-CPD) results in increased mean cellular calcein fluorescence intensity, as compared to untreated control cells or cells treated with single-domain peptides used alone (CM18, TAT-Cys, Penetratin-Cys) or together (CM18 + TAT-Cys, CM18 + Penetratin-Cys).
  • HEK293A cells were cultured and tested in the endosomal escape assays as described in Example 2.1.
  • the results of flow cytometry analyses are summarized below in Table 2.8 and in Figure 1 B.
  • TAT-Cys facilitates escape of endosomally-trapped calcein in a dose-dependent manner in primary myoblasts.
  • Concentrations of CM18-TAT-Cys above 10 ⁇ were associated with an increase in cell toxicity in myoblast cells, as for HeLa cells.
  • Table 2.11 Monomers v. dimers CM18-TAT-Cys and CM18-Penetratin-Cys in primary myoblasts
  • Peptide shuttle agents increase GFP transduction efficiency
  • mammalian cells e.g., HEK293, CHO, HeLa, THP-1 , and myoblasts
  • mammalian cells e.g., HEK293, CHO, HeLa, THP-1 , and myoblasts
  • the cells were incubated overnight in appropriate growth media containing FBS (see Example 1).
  • cargo protein at 0.5 to 10 ⁇ (GFP, TAT-GFP, GFP-NLS, or FITC-labeled anti-tubulin antibody) was pre-mixed (pre-incu bated) for 10 min at 37°C with shuttle agents (0.5 to 5 ⁇ ) in 50 L of fresh medium without serum (unless otherwise specified).
  • GFP, GFP-NLS and TAT-GFP are recombinant proteins developed and produced by Feldan (see Example 3.4 below).
  • FITC-labeled anti-tubulin antibody was purchased from Abeam (ab64503). The media in wells was removed and the cells were washed three times with freshly prepared phosphate buffered saline (PBS) previously warmed at 37°C. The cells were incubated with the cargo protein/shuttle agent mixture at 37°C for 5 or 60 min. After the incubation, the cells were quickly washed three times with freshly prepared PBS and/or heparin (0.5 mg/mL) previously warmed at 37°C.
  • PBS phosphate buffered saline
  • the washes with heparin were required for human THP-1 blood cells to avoid undesired cell membrane-bound protein background in subsequent analyses (microscopy and flow cytometry).
  • the cells were finally incubated in 50 ⁇ of fresh medium with serum at 37°C before analysis.
  • the delivery of fluorescent protein cargo in cytosolic and nuclear cell compartments was observed with an Olympus IX70TM microscope (Japan) equipped with a fluorescence lamp (Model U- LH100HGAPO) and different filters.
  • the Olympus filter U-MF2TM (C54942-Exc495/Em510) was used to observe GFP and FITC-labeled antibody fluorescent signals.
  • the Olympus filter HQ-TRTM V-N41004- Exc555-60/Em645-75) was used to observe mCherryTM and GFP antibody fluorescent signals.
  • the Olympus filter U-MWU2TM (Exc330/Em385) was used to observe DAPI or Blue Hoechst fluorescent signals.
  • the cells incubated in 50 L of fresh medium were directly observed by microscopy (Bright- field and fluorescence) at different power fields (4x to 40x).
  • the cells were observed using a CoolSNAP-PROTM camera (Series A02D874021) and images were acquired using the Image-ProplusTM software.
  • Adherent cells were plated on a sterile glass strip at 1.5x10 5 cells per well in a 24-plate well and incubated overnight at 37°C.
  • For fixation cells were incubated in 500 L per well of formaldehyde (3.7% v/v) for 15 minutes at room temperature, and washed 3 times for 5 minutes with PBS.
  • For permeabilization cells were incubated in 500 L per well of TritonTM X-100 (0.2%) for 10 minutes at room temperature, and washed 3 times for 5 minutes with PBS.
  • For blocking cells were incubated in 500 [il per well of PBS containing 1 % BSA (PBS/BSA) for 60 minutes at room temperature.
  • Primary mouse monoclonal antibody was diluted PBS/BSA (1 %).
  • the fluorescence of GFP was quantified using flow cytometry (Accuri C6, Becton, Dickinson and Company (BD)). Untreated cells were used to establish a baseline in order to quantify the increased fluorescence due to the internalization of the fluorescent protein in treated cells. The percentage of cells with a fluorescence signal above the maximum fluorescence of untreated cells, "mean %” or “Pos cells (%)", is used to identify positive fluorescent cells. "Relative fluorescence intensity (FL1 -A)” corresponds to the mean of all fluorescence intensities from each cell with a fluorescent signal after fluorescent protein delivery with the shuttle agent. Also, the events scanned by flow cytometry corresponding to cells (size and granularity) were analyzed. The cellular toxicity (% cell viability) was monitored comparing the percentage of cells in the total events scanned of treated cells comparatively to untreated cells.
  • the viability of cells was assessed with a rezazurine test.
  • Rezazurine is a sodium salt colorant that is converted from blue to pink by mitochondrial enzymes in metabolically active cells. This colorimetric conversion, which only occurs in viable cells, can be measured by spectroscopy analysis in order to quantify the percentage of viable cells.
  • the stock solution of rezazurine was prepared in water at 1 mg/100 mL and stored at 4°C. 25 L of the stock solution was added to each well of a 96-well plate, and cells were incubated at 37°C for one hour before spectrometry analysis. The incubation time used for the rezazurine enzymatic reaction depended on the quantity of cells and the volume of medium used in the wells.
  • the GFP-encoding gene was cloned in a T5 bacterial expression vector to express a GFP protein containing a 6x histidine tag (SEQ ID NO: 113) and a serine/glycine rich linker in the N-terminal end, and a serine/glycine rich linker and a stop codon (-) at the C-terminal end.
  • Recombinant GFP protein was purified as described in Example 1.4.
  • the sequence of the GFP construct was:
  • GFP sequence is underlined 3.5 GFP transduction by CM18-TAT-Cys in HeLa cells: Fluorescence microscopy
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein was co-incubated with 0, 3 or 5 ⁇ of CM18-TAT, and then exposed to HeLa cells for 1 hour. The cells were observed by bright field and fluorescence microscopy as described in Example 3.2. The results presented in Figure 5 show that GFP was delivered intracellular ⁇ to HeLa cells in the presence of the shuttle agent CM18-TAT.
  • Table 3.1 and Figure 6A show the results of flow cytometry analysis of the fluorescence intensity of HeLa cells transduced with GFP (5 ⁇ ) without or with 5, 3, 1 , and 0.5 ⁇ of CM18-TAT- Cys. Corresponding cellular toxicity data are presented in Table 3.1 and in Figure 6B. These results suggest that the shuttle agent CM18-TAT-Cys increases the transduction efficiency of GFP in a dose- dependent manner.
  • Table 3.2 and Figures 7A-7B show the results of flow cytometry analysis of the fluorescence intensity of HeLa cells transduced with different concentrations of GFP (1 to 10 ⁇ ) without or with 5 ⁇ of CM18-TAT-Cys ( Figure 7A) or 2.5 ⁇ dCM18-TAT-Cys ( Figure 7B).
  • 3.7 GFP transduction in HeLa cells Dose responses of CM18-TAT-Cys and CM18- Penetratin-Cys, and dimers thereof
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein (5 ⁇ ) was co-incubated with different concentrations and combinations of CM18-TAT-Cys, CM18-Penetratin-Cys, and dimers of each (dCM18-TAT-Cys, dCM18- Penetratin-Cys), and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 3.3 and Figure 8, as well as in Table 3.4 and Figure 9.
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein (5 ⁇ ) was co-incubated with 5 ⁇ of each of the following peptide(s): TAT-Cys; CM18; Penetratin-Cys; TAT-Cys + CM18; Penetratin-Cys + CM18; and CM18- TAT-Cys, and then exposed to HeLa cells for 1 hour. GFP fluorescence was visualized by bright field and fluorescence microscopy. The microscopy results (data not shown) showed that GFP was successfully delivered intracellularly using CM18-TAT-Cys.
  • Peptide shuttle agents increase TAT-GFP transduction efficiency
  • Example 3 showed the ability of shuttle agents to deliver GFP intracellularly.
  • the experiments presented in this example show that the shuttle agents can also increase the intracellular delivery of a GFP cargo protein that is fused to a CPD (TAT-GFP).
  • TAT-GFP CPD
  • TAT-GFP protein was purified as described in Example 1.4.
  • the sequence of the TAT- GFP construct was: MHHHHHHGGGGSGGGGSGGASTGTGRKKRRQRRRPPQGGGGSGGGGSGGGTG
  • HeLa cells were cultured and tested in the protein transduction assay described in Example
  • TAT-GFP recombinant protein (5 ⁇ ) was co-incubated with 3 ⁇ of CM18-TAT-Cys and then exposed to HeLa cells for 1 hour. Cells and GFP fluorescence were visualized by bright field and fluorescence microscopy (as described in Example 3.2) at 10x and 40x magnifications, and sample results are shown in Figure 10. The microscopy results revealed that in the absence of CM18-TAT- Cys, TAT-GFP shows a low intensity, endosomal distribution as reported in the literature. In contrast, TAT-GFP is delivered to the cytoplasm and to the nucleus in the presence of the shuttle agent CM18- TAT-Cys.
  • TAT peptide itself may act as a nuclear localization signal (NLS), explaining the nuclear localization of TAT-GFP.
  • NLS nuclear localization signal
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, TAT-GFP-Cys recombinant protein (5 ⁇ ) was co-incubated with different concentrations of CM18-TAT-Cys (0, 0.5, 1 , 3, or 5 ⁇ ) and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 4.3 and Figure 11 A. Corresponding cellular toxicity data are presented in Figure 11 B. Table 4.3: Data from Figure 11A and 11 B
  • Peptide shuttle agents increase GFP-NLS transduction efficiency and nuclear localization
  • the experiments in Examples 3 and 4 showed the ability of shuttle agents to deliver GFP and TAT-GFP intracellularly.
  • the experiments presented in this example show that the shuttle agents can facilitate nuclear delivery of a GFP protein cargo fused to a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • Serine/glycine rich linkers are in bold 5.2 Nuclear delivery of GFP-NLS by CM18-TAT-Cys in HeLa cells in 5 minutes: Visualisation by fluorescence microscopy
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ) was co-incubated with 5 ⁇ of CM18-TAT-Cys, and then exposed to HeLa cells. GFP fluorescence was visualized by bright field and fluorescence microscopy after 5 minutes (as described in Example 3.2) at 10x, 20x and 40x magnifications, and sample results are shown in Figure 12. The microscopy results revealed that GFP-NLS is efficiently delivered to the nucleus in the presence of the shuttle agent CM18-TAT-Cys, after only 5 minutes of incubation.
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1.
  • GFP-NLS recombinant protein (5 ⁇ ) was co-incubated with 0, 0.5, 1 , 3, or 5 ⁇ of CM18-TAT- Cys, and then exposed to HeLa cells for 1 hour.
  • the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 5.1 and Figure 13A. Corresponding cellular toxicity data are presented in Figure 13B.
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1.
  • GFP-NLS recombinant protein (5 ⁇ ) was co-incubated with different concentrations and combinations of CM18-TAT-Cys, CM18-Penetratin-Cys, and dimers of each (dCM18-TAT-Cys, dCM18- Penetratin-Cys), and then exposed to HeLa cells for 1 hour.
  • the cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Tables 5.2 and 5.3, and in Figures 14 and 15.
  • HeLa cells were cultured and tested in the protein transduction assay described in Example
  • GFP-NLS recombinant protein (5 ⁇ ) was co-incubated with either CM18-TAT-Cys (3.5 ⁇ ) alone or with dCM18-Penetratin-Cys (1 ⁇ ). Cells were incubated for 5 minutes or 1 hour in plain DMEM media ("DMEM") or DMEM media containing 10% FBS ("FBS”), before being subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 5.4, and in Figure 16. Cells that were not treated with shuttle agent or GFP-NLS (“ctrl”), and cells that were treated with GFP- NLS without shuttle agent (“GFP-NLS 5 ⁇ ”) were used as controls.
  • DMEM plain DMEM media
  • FBS DMEM media containing 10% FBS
  • THP-1 cells which is an acute monocytic leukemia cell line that grows in suspension.
  • THP-1 cells were cultured (see Example 1) and tested in the protein transduction assay described in Example 3.1.
  • GFP-NLS recombinant protein (5 ⁇ ) was co-incubated with or without 1 ⁇ CM18-TAT-Cys, and exposed to the THP-1 cells for 5 minutes, before being subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 5.5 and in Figure 17A. Corresponding cellular toxicity data are presented in Figure 17B.
  • the experiments in Examples 3-5 showed the ability of shuttle agents to increase the transduction efficiency of GFP, TAT-GFP, and GFP-NLS.
  • the experiments presented in this example show that the shuttle agents can also deliver a larger protein cargo: an FITC-labeled anti-tubulin antibody.
  • the FITC-labeled anti-tubulin antibody was purchased from (Abeam, ab64503) and has an estimated molecular weight of 150 KDa.
  • the delivery and microscopy protocols are described in Example 3. 6.1 Transduction of a functional antibody by CM18-TAT-Cys in HeLa cells: Visualization by microscopy
  • FITC-labeled anti-tubulin antibody (0.5 ⁇ ) was co-incubated with 5 ⁇ of CM18-TAT-Cys and exposed to HeLa cells for 1 hour. Antibody delivery was visualized by bright field (20x) and fluorescence microscopy (20x and 40x). As shown in Figure 18, fluorescent tubulin fibers in the cytoplasm were visualized, demonstrating the functionality of the antibody inside the cell.
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1.
  • FITC-labeled anti-tubulin antibody (0.5 ⁇ ) was co-incubated with 3.5 ⁇ of CM18-TAT-Cys, CM18-Penetratin-Cys or dCM18-Penetratin-Cys, or a combination of 3.5 ⁇ of CM18-TAT-Cys and 0.5 ⁇ of dCM18-Penetratin-Cys, and exposed to HeLa cells for 1 hour.
  • the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 6.1 and Figure 19A. Corresponding cellular toxicity data are presented in Figure 19B.
  • CM18-TAT-Cys and dCM18-Penetratin-Cys allowed for higher intracellular delivery as compared with CM18-TAT-Cys alone, and with less cell toxicity as compared to CM18-Penetratin-Cys and dCM18-Penetratin-Cys (see Figure 19A and 19B).
  • CM18-TAT-Cys enables intracellular plasmid DNA delivery but poor plasmid expression
  • CM 18-TAT-Cys shuttle agent The ability of the CM 18-TAT-Cys shuttle agent to deliver plasmid DNA intracellularly was tested in this example on HEK293A cells using a plasmid encoding GFP.
  • mammalian cells HEK293A
  • HEK293A mammalian cells
  • the cells were incubated overnight in appropriate growth media containing FBS.
  • pEGFP labeled with a Cy5TM fluorochrome was mixed for 10 min at 37°C with CM18- TAT-Cys (0.05, 0.5, or 5 ⁇ ) in fresh PBS at a final 100 ⁇ volume.
  • the media in wells was removed and the cells were quickly washed three times with PBS and 500 L of warm media without FBS was added.
  • the pEGFP and CM 18-TAT-Cys solution was added to the cells and incubated at 37°C for 4 hours. After the incubation, cells were washed with PBS and fresh media containing FBS was added. Cells were incubated at 37°C before being subjected to flow cytometry analysis as described in Example 3.
  • Plasmid DNA (pEGFP) was labeled with a Cy5TM dye following the manufacturer's instructions (Mirus Bio LLC). Cy5TM Moiety did not influence transfection efficiency when compared to unlabelled plasmid using standard transfection protocol (data not shown).
  • Flow cytometry analysis allowed quantification of Cy5TM emission, corresponding to DNA intracellular delivery, and GFP emission, corresponding to successful nuclear delivery, DNA transcription and protein expression. The results are shown in Table 7.1 and in Figure 20.
  • Table 7.1 Data from Figure 20
  • GFP-NLS (5 ⁇ ; see Example 5) was co-incubated with 5 ⁇ of CM18-TAT-Cys or His- CM18-TAT and exposed to HeLa cells for 1 hour. Nuclear fluorescence of intracellular ⁇ delivered GFP- NLS was confirmed by fluorescence microscopy (data not shown), indicating successful delivery of GFP-NLS to the nucleus.
  • HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1.
  • GFP-NLS (5 ⁇ ) was co-incubated with 0, 1 , 3, or 5 ⁇ of CM18-TAT-Cys or His-CM18-TAT, and exposed to HeLa cells for 1 hour.
  • the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 8.1 and Figure 21 A. Corresponding cellular toxicity data are presented in Figure 21 B.
  • combining the shuttle agents with a further independent synthetic peptide containing a histidine-rich domain fused to a CPD may provide a similar advantage for protein transduction, with the added advantage of allowing the concentration of the histidine-rich domain to be varied or controlled independently from the concentration of the shuttle agent.
  • the histidine-rich domain may act as a proton sponge in the endosome, providing another mechanism of endosomal membrane destabilization.
  • His-CM18-PTD4 increases transduction efficiency and nuclear delivery of GFP-NLS, mCherryTM-
  • Protocol A Protein transduction assay for delivery in cell culture medium
  • Protocol B Protein transduction assay for adherent cells in PBS
  • shuttle agents were diluted in sterile distilled water at room temperature (if the cargo is or comprised a nucleic acid, nuclease-free water was used). Cargo protein(s) were then added to the shuttle agents and, if necessary, sterile PBS was added to obtain the desired concentrations of shuttle agent and cargo in a sufficient final volume to cover the cells (e.g., 10 to 100 L per well for a 96-well plate). The shuttle agent/cargo mixture was then immediately used for experiments.
  • At least three controls were included for each experiment, including: (1) shuttle agent alone (e.g., at highest concentration tested); (2) cargo alone; and (3) without any cargo or shuttle agent.
  • the media in wells was removed, cells were washed once with PBS previously warmed at 37°C, and the shuttle agent/cargo mixture was then added to cover all cells for the desired length of time.
  • the shuttle agent/cargo mixture in wells was removed, the cells were washed once with PBS, and fresh complete medium was added. Before analysis, the cells were washed once with PBS and fresh complete medium was added.
  • Protocol C Protein transduction assay for suspension cells in PBS
  • suspension cells in exponential growth phase were harvested and plated in a 96-well plate (20,000 cells per well). The cells were incubated overnight in appropriate growth media containing serum (see Example 1). The next day, in separate sterile 1.5-mL tubes, shuttle agents were diluted in sterile distilled water at room temperature (if the cargo is or comprised a nucleic acid, nuclease-free water was used).
  • Cargo protein(s) were then added to the shuttle agents and, if necessary, sterile PBS or cell culture medium (serum-free) was added to obtain the desired concentrations of shuttle agent and cargo in a sufficient final volume to resuspend the cells (e.g., 10 to 100 ⁇ per well in a 96-well plate).
  • the shuttle agent/cargo mixture was then immediately used for experiments. At least three controls were included for each experiment, including: (1) shuttle agent alone (e.g., at highest concentration tested); (2) cargo alone; and (3) without any cargo or shuttle agent.
  • the cells were centrifuged for 2 minutes at 400g, the medium was then removed and the cells were resuspended in PBS previously warmed at 37°C.
  • the cells were centrifuged again 2 minutes at 400g, the PBS removed, and the cells were resuspended in the shuttle agent/cargo mixture. After the desired incubation time, 100 ⁇ of complete medium was added directly on the cells. Cells were centrifuged for 2 minutes at 400g and the medium was removed. The pellet was resuspended and washed in 200 ⁇ of PBS previously warmed at 37°C. After another centrifugation, the PBS was removed and the cells were resuspended in 100 ⁇ of complete medium. The last two steps were repeated one time before analysis.
  • Protocol B As compared to Protocol A.
  • GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co-incubated with 35 ⁇ of His- CM18-PTD4, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. The cells were then subjected to fluorescence microscopy analysis as described in Examples 3.2 and 3.2a.
  • the upper panels in Figures 23A, 23B and 23C show nuclei labelling (DAPI) at 4x, 20x and 40x magnifications, respectively, while the lower respective panels show corresponding GFP-NLS fluorescence.
  • white triangle windows indicate examples of areas of co-labelling between nuclei (DAPI) and GFP-NLS signals.
  • the upper and bottom panels show sample bright field images of the HeLa cells, and the middle panel shows the results of a corresponding FACS analysis (performed as described in Example 3.3), which indicates the percentage of cells in a 96-plate with a GFP signal. No significant GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
  • Figures 24A-24B shows bright field (Figure 24A) and fluorescent images (Figure 24B).
  • the inset in Figure 24B shows the results of a corresponding FACS analysis (performed as described in Example 3.3), which indicates the percentage of cells in a 96-plate well with a GFP signal. No significant GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
  • Figure 26A-26C shows sample images captured with confocal microscopy at 63x magnification of living cells.
  • Figure 26A shows a bright field image
  • Figure 26B shows the corresponding fluorescent GFP-NLS.
  • Figure 26C is an overlay between the images in Figures 26A and 26B. No significant GFP-NLS fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
  • FITC-labeled anti-tubulin antibody (0.5 ⁇ ; Abeam, ab64503) was co-incubated with 50 ⁇ of His-CM18-PTD4, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. The cells were then subjected to fluorescence microscopy analysis as described in Examples 3.2 and 3.2a, wherein the FITC fluorescence of the anti-tubulin antibody in the HeLa cells was immediately visualized by bright field and fluorescence microscopy at 20x magnification after the final washing step. No significant FITC fluorescence was observed in negative control samples (i.e., cells exposed to the FITC-labeled anti-tubulin antibody without any shuttle agent; data not shown).
  • Examples 9.4 and 9.4a show that GFP-NLS and FITC-labeled anti- tubulin antibody cargos are successfully transduced and delivered to the nucleus and/or the cytosol of HeLa cells in the presence of the shuttle agent His-CM18-PTD4.
  • GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co-incubated with 50 ⁇ of His- CM18-PTD4, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. After a washing step, the GFP fluorescence of the HeLa cells was immediately visualized by fluorescence microscopy (Example 3.2) at 20x magnification after different intervals of time. Typical results are shown in Figures 27A to 27D, in which fluorescence microscopy images were captured after 45, 75, 100, and 120 seconds (see Figures 27A, 27B, 27C and 27D, respectively).
  • FIG. 27A diffuse cellular GFP fluorescence was generally observed after 45 seconds, with areas of lower GFP fluorescence in the nucleus in many cells. These results suggest predominantly cytoplasmic and low nuclear distribution of the GPF-NLS delivered intracellular ⁇ via the shuttle agent after 45 seconds.
  • Figures 27B to 27D show the gradual redistribution of GFP fluorescence to the cell nuclei at 75 seconds (Figure 27B), 100 seconds (Figure 27C), and 120 seconds (Figure 27D) following exposure to the His-CM18-PTD4 shuttle agent and GFP-NLS cargo. No significant cellular GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
  • Example 9.5 show that GFP-NLS is successfully delivered to the nucleus of
  • mCherryTM-NLS recombinant protein was constructed, expressed and purified from a bacterial expression system as described in Example 1.4.
  • the sequence of the mCherryTM-NLS recombinant protein was:
  • White triangle windows indicate examples of areas of co-labelling between GFP-NLS and mCherryTM fluorescence signals in cell nuclei. No significant cellular GFP or mCherryTM fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS or mCherryTM without any shuttle agent; data not shown).
  • THP-1 cells were cultured and tested in the protein transduction assays using Protocols A and C as described in Example 9.1.
  • GFP-NLS (5 ⁇ ; see Example 5.1) was co-incubated with 1 ⁇ of His-CM18-PTD4 and exposed to THP-1 cells for 1 hour (Protocol A), or was co-incubated with 5 ⁇ of His-CM18-PTD4 and exposed to THP-1 cells for 15 seconds (Protocol C).
  • the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 9.3 and in Figure 31. Table 9.3: Data from Figure 31
  • GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co-incubated with 5 ⁇ of His- CM18-PTD4, and then exposed to THP-1 cells for 15 seconds using Protocol C as described in Example 9.1. The cells were subjected to microscopy visualization as described in Example 3.2.
  • Figure 33A- 33D Additional results are shown in Figure 33A- 33D, in which Figures 33A and 33B show bright field images, and Figures 33C and 33D show corresponding fluorescence images.
  • White triangle windows indicate examples of areas of co-labelling between Figures 33A and 33C, as well as Figures 33B and 33D.
  • the right-most panel shows typical results of a corresponding FACS analysis (performed as described in Example 3.3), which indicates the percentage of cells in a 96-plate well with a GFP signal.
  • HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co- incubated with 50 ⁇ of different shuttle agents and exposed to the HeLa cells for 10 seconds. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.1 and Figure 29A. "Pos cells (%)” is the mean percentages of all cells that emanate a GFP signal. The negative control (“Ctrl”) corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any shuttle agent.
  • HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co- incubated with 10 ⁇ of TAT-KALA, His-CM18-PTD4, or His-C(LLKK) 3 C-PTD4 for 1 , 2, or 5 minutes. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.2 and Figure 29B. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control (“Ctrl”) corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any shuttle agent. Table 10.2: Data from Figure 29B
  • HeLa cells were cultured and tested in the protein transduction assays using Protocol C as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co- incubated with 5 ⁇ of TAT-KALA, His-CM18-PTD4, or His-C(LLKK) 3 C-PTD4 for 1 , 2, or 5 minutes. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.3 and Figure 29C. The negative control (“Ctrl”) corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any shuttle agent.
  • HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co- incubated with 50 ⁇ of different shuttle agents (see Table 1.3 for amino acid sequences and properties) and exposed to the HeLa cells for 10 seconds. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Tables 10.3a & 10.3b and Figures 29E & 29F. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control (“Ctrl”) corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any shuttle agent.
  • HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co- incubated with 10 ⁇ of TAT-KALA, His-CM18-PTD4, or His-C(LLKK) 3 C-PTD4 for 1 , 2, or 5 minutes. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Tables 10.3c & 10.3b and Figures 29G and 29H. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control (“Ctrl”) corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any shuttle agent.
  • the shuttle agent CM18-PTD4 was used as a model to demonstrate the modular nature of the individual protein domains, as well as their ability to be modified. More particularly, the presence or absence of: an N-terminal cysteine residue ("Cys"); different flexible linkers between the ELD and CPD domains (11": GGS; 12": GGSGGGS (SEQ ID NO: 106); and 13": GGSGGGSGGGS (SEQ ID NO: 107)) and different lengths, positions, and variants to histidine-rich domains; were studied.
  • Cys N-terminal cysteine residue
  • HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co- incubated with 20 ⁇ of different shuttle peptide variants (see Table 1.3 for amino acid sequences and properties) of the shuttle agent His-CM18-PTD4 for 1 minute. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.3e and Figure 29I. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control (“Ctrl”) corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any shuttle agent.
  • CM18-PTD4 variations in a given shuttle
  • CM18-PTD4 an N-terminal cysteine residue
  • GFP- NLS transduction efficiency decreased GFP- NLS transduction efficiency by 11 % (from 47.6% to 36.6%), but increased cell viability from 33.9% to 78.7%.
  • histidine-rich domain(s) did not result in a complete loss of transduction efficiency and cell viability of His-CM18-PTD4 (see 3His-CM18-PTD4, 12His-CM18-PTD4, HA-CM18-PTD4, 3HA-CM18-PTD4, CM18-His-PTD4, and His-CM18-PTD4-His).
  • adding a second histidine-rich domain at the C terminus of His-CM18-PTD4 i.e., His-CM18-PTD4-His
  • HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co- incubated with 50 ⁇ of different single-domain peptides (TAT; PTD4; Penetratin; CM18; C(LLKK) 3 C (SEQ ID NO: 63); KALA) or the two-domain peptide His-PTD4 (lacking an ELD), and exposed to the HeLa cells for 10 seconds. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.4 and Figure 29D.
  • Pos cells (%) is the mean percentages of all cells that emanate a GFP signal.
  • the negative control (“Ctrl”) corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any single- domain peptide or shuttle agent.
  • GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co-incubated with 50 ⁇ of shuttle agent, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. The cells were visualized by microscopy as described in Example 3.2, after an incubation time of 2 minutes.
  • THP-1 cells were cultured and tested in the protein transduction assays using Protocol C as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co- incubated with 1 ⁇ of TAT-KALA, His-CM18-PTD4, or His-C(LLKK) 3 C-PTD4 for 15, 30, 60, or 120 seconds. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. The mean percentages of cells emanating a GFP signal ("Pos cells (%)") are shown in Table 10.4 and in Figure 34A. The mean fluorescence intensity is shown in Table 10.5 and Figure
  • the negative control corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any shuttle agent.
  • THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1, but with the following modifications.
  • GFP-NLS recombinant protein (5, 2.5, or 1 ⁇ ; see Example 5.1) was co-incubated with 0.5 or 0.8 ⁇ of His-CM18-PTD4, or with 0.8 ⁇ of His-C(LLKK)3C-PTD4, and then exposed to THP-1 cells each day for 150 min in the presence of cell culture medium containing serum. Cells were washed and subjected to flow cytometry analysis as described in Example 3.3 after 1 or 3 days of repeated exposure to the shuttle agent/cargo. The results are shown in Table 11.1 and in Figures 35A, 35B, 35C and 35F.
  • the negative control (“Ctrl”) corresponds to cells that were incubated with GFP-NLS recombinant protein (5 ⁇ ) without any shuttle agent.
  • THP-1 cells The viability of THP-1 cells repeatedly exposed to His-CM18-PTD4 and GFP-NLS was determined as described in Example 3.3a. The results are shown in Tables 11.2 and 11.3 and in Figures 35D and 35E. The results in Table 11.2 and Figure 35D show the metabolic activity index of the THP-1 cells after 1 , 2, 4, and 24h, and the results in Table 11.3 and Figure 35E show the metabolic activity index of the THP-1 cells after 1 to 4 days.
  • Example 11 shows that repeated daily (or chronic) treatments with relatively low concentrations of His-CM18-PTD4 or His-C(LLKK) 3 C-PTD4 in the presence of serum result in intracellular delivery of GFP-NLS in THP-1 cells.
  • the results also suggest that the dosages of the shuttle agents and the cargo can be independently adjusted to improve cargo transduction efficiency and/or cell viability.
  • Example 12 shows that the dosages of the shuttle agents and the cargo can be independently adjusted to improve cargo transduction efficiency and/or cell viability.
  • His-CM18-PTD4 increases transduction efficiency and nuclear delivery of GFP-NLS in a plurality of cell lines
  • the ability of the shuttle agent His-CM18-PTD4 to deliver GFP-NLS to the nuclei of different adherent and suspension cells using Protocols B (adherent cells) or C (suspension cells) as described in Example 9.1 was examined.
  • GFP-NLS (5 ⁇ ; see Example 5.1) was co-incubated with 35 ⁇ of His-CM18-PTD4 and exposed to adherent cells for 10 seconds (Protocol B), or was co-incubated with 5 ⁇ of His-CM18-PTD4 and exposed to suspension cells for 15 seconds (Protocol C). Cells were washed and subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 12.1 and Figure 36. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal.
  • GFP-NLS recombinant protein (5 ⁇ ; see Example 5.1) was co-incubated with 35 ⁇ of His- CM18-PTD4 and exposed to adherent cells for 10 seconds using Protocol A, or was co-incubated with 5 ⁇ of His-CM18-PTD4 and exposed to suspension cells for 15 seconds using Protocol B, as described in Example 9.1. After washing the cells, GFP fluorescence was visualized by bright field and fluorescence microscopy.
  • FIG. 37A-37H Sample images captured at 10x magnifications showing GFP fluorescence are shown in Figures 37A-37H for (Figure 37A) 293T, ( Figure 37B) Balb3T3, ( Figure 37C) CHO, (Figure 37D) Myoblasts, (Figure 37E) Jurkat, ( Figure 37F) CA46, ( Figure 37G) HT2, and ( Figure 37H) NIH3T3 cells.
  • the insets show corresponding flow cytometry results performed as described in Example 3.3, indicating the percentage of GFP-NLS-positive cells. No significant cellular GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
  • GFP-NLS nuclear localization of the GFP-NLS was further confirmed in fixed and permeabilized myoblasts using cell immuno-labelling as described in Example 3.2a.
  • GFP-NLS was labeled using a primary mouse monoclonal anti-GFP antibody (Feldan, #A017) and a secondary goat anti-mouse AlexaTM-594 antibody (Abeam #150116). Nuclei were labelled with DAPI.
  • Sample results for primary human myoblast cells are shown in Figures 38A-38B, in which GFP immuno-labelling is shown in Figure 38A, and an overlay of the GFP immuno-labelling and DAPI labelling is shown in Figure 38B. No significant cellular GFP labelling was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
  • His-CM18-PTD4 enables transduction of a CRISPR/Cas9-NLS system and genome editing
  • Cas9-NLS recombinant protein was constructed, expressed and purified from a bacterial expression system as described in Example 1.4.
  • the sequence of the Cas9-NLS recombinant protein produced was:
  • Serine/glycine rich linkers are in bold 13.2 Transfection plasmid surrogate assay
  • This assay enables one to visually identify cells that have been successfully delivered an active CRISPR/Cas9 complex.
  • the assay involves transfecting cells with an expression plasmid DNA encoding the fluorescent proteins mCherryTM and GFP, with a STOP codon separating their two open reading frames. Transfection of the cells with the expression plasmid results in mCherryTM expression, but no GFP expression ( Figure 39B).
  • a CRISPR/Cas9 complex which has been designed/programmed to cleave the plasmid DNA at the STOP codon, is then delivered intracellular ⁇ to the transfected cells expressing mCherryTM ( Figure 39D).
  • DNA plasmids for different experimental conditions are diluted in DMEM (50 L) in separate sterile 1.5-mL tubes, vortexed and briefly centrifuged.
  • DMEM fetal calf serum
  • FastfectTM transfection reagent was diluted in DMEM (50 [il) with no serum and no antibiotics at a ratio of 3:1 (3 ⁇ of FastfectTM transfection reagent for 1 g of DNA) and then quickly vortexed and briefly centrifuged. The FastfectTM/DMEM mixture was then added to the DNA mix and quickly vortexed and briefly centrifuged.
  • the FastfectTM/DMEM/DNA mixture is then incubated for 15-20 min at room temperature, before being added to the cells (100 L per well).
  • the cells are then incubated at 37°C and 5% CO2 for 5h.
  • the media is then changed for complete medium (with serum) and further incubated at 37°C and 5% CO2 for 24-48h.
  • the cells are then visualized under fluorescent microscopy to view the mCherryTM signal.
  • RNAs (crRNA & tracrRNA) were designed to target a nucleotide sequence of the EMX1 gene, containing a STOP codon between the mCherryTM and GFP coding sequences in the plasmid of Example 13.2.
  • the sequences of the crRNA and tracrRNA used were as follows:
  • HeLa cells were cultured and subjected to the transfection plasmid surrogate assay as described in Example 13.2).
  • the HeLa cells were transfected with a plasmid surrogate encoding the mCherryTM protein as shown in Figure 39A.
  • a mix of Cas9-NLS recombinant protein (2 ⁇ ; see Example 13.1) and RNAs (crRNA & tracrRNA; 2 ⁇ ; see above) were co-incubated with 50 ⁇ of His-CM18-PTD4, and the mixture (CRISPR/Cas9 complex) was exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1.
  • Double-stranded plasmid DNA cleavage by the CRISPR/Cas9 complex at the STOP codon between the mCherryTM and GFP coding sequences ( Figure 39B), and subsequent non-homologous repair by the cell in some cases results in removal of the STOP codon ( Figure 39C), thereby allowing expression of both the mCherryTM and GFP fluorescent proteins in the same cell on Day 3 ( Figure 39D-39E).
  • White triangle windows in Figures 39D and 39E indicate examples of areas of co-labelling between mCherryTM and GFP.
  • HeLa cells were cultured and co- transfected with three plasmids: the plasmid surrogate (as described in Example 13.2) and other expression plasmids encoding the Cas9-NLS protein (Example 13.1) and the crRNMracrRNAs (Example 13.3).
  • Typical fluorescence microscopy results are shown in Figure 40A to 40D.
  • Figures 40A and 40B show cells 24 hours post-transfection
  • Figures 40C and 40D show cells 72 hours post-transfection.
  • Figure 40E-40H shows the results of a parallel transfection plasmid surrogate assay performed using 35 ⁇ of the shuttle His-CM18-PTD4, as described for Figure 39A-39E.
  • Figures 40E and 40F show cells 24 hours post-transduction, while Figures 40G and 40H show cells 48 hours post- transduction.
  • Figures 40E and 40G show mCherryTM fluorescence, and Figures 40F and 40H show GFP fluorescence, the latter resulting from removal of the STOP codon by the transduced CRISPR/Cas9-NLS complex and subsequent non-homologous repair by the cell. No significant cellular GFP fluorescence was observed in negative control samples (i.e., cells exposed to CRISPR/Cas9-NLS complex without any shuttle agent; data not shown).
  • the T7E1 assay was performed with the Edit-RTM Synthetic crRNA Positive Controls (Dharmacon #U-007000-05) and the T7 Endonuclease I (NEB, Cat #M0302S). After the delivery of the CRISPR/Cas9 complex, cells were lysed in 100 ⁇ _ of PhusionTM High-Fidelity DNA polymerase (NEB #M0530S) laboratory with additives. The cells were incubated for 15-30 minutes at 56°C, followed by deactivation for 5 minutes at 96°C. The plate was briefly centrifuged to collect the liquid at bottom of the wells. 50- ⁇ . PCR samples were set up for each sample to be analyzed.
  • PCR samples were heated to 95°C for 10 minutes and then slowly (>15 minutes) cooled to room temperature. PCR product ( ⁇ 5 ⁇ .) was then separated on an agarose gel (2%) to confirm amplification. 15 of each reaction was incubated with T7E1 nuclease for 25 minutes at 37 °C. Immediately, the entire reaction volume was run with the appropriate gel loading buffer on an agarose gel (2%).
  • a mix composed of a Cas9-NLS recombinant protein (25 nM; Example 13.1) and crRNA/tracrRNA (50 nM; see below) targeting a nucleotide sequence of the PPIB gene were co- incubated with 10 ⁇ of His-CM18-PTD4 or His-C(LLKK) 3 C-PTD4, and incubated with HeLa cells for 16h in medium without serum using Protocol A as described in Example 9.1.
  • Figure 41A shows an agarose gel with the PPIB DNA sequences after PCR amplification.
  • Lane A shows the amplified PPIB DNA sequence in HeLa cells without any treatment (i.e., no shuttle or Cas9/RNAs complex).
  • Lanes B The two bands framed in white box #1 are the cleavage product of the PPIB DNA sequence by the CRIPR/Cas9 complex after the delivery of the complex with the shuttle His- C(LLKK) 3 C-PTD4.
  • Lane C These bands show the amplified PPIB DNA sequence after incubation of the HeLa cells with the Cas9/RNAs complex without shuttle (negative control).
  • Lane D The bands framed in white box #2 show the amplified PPIB DNA sequence after incubation of the HeLa cells with the Cas9/RNAs complex in presence of a lipidic transfection agent (DharmaFectTM tranfection reagent # T-20XX-01) (positive control). Similar results were obtained using the shuttle His-CM18-PTD4 (data not shown).
  • a lipidic transfection agent DharmaFectTM tranfection reagent # T-20XX-01
  • Figure 41 B shows an agarose gel with the PPIB DNA sequences after PCR amplification.
  • the left panel in Figure 41 B shows the cleavage product of the amplified PPIB DNA sequence by the CRIPR/Cas9 complex after the delivery of the complex with the shuttle agent His-CM18-PTD4 in HeLa cells.
  • the right panel Figure 41 B shows amplified DNA sequence before the T7E1 digestion procedure as a negative control.
  • Figure 41 C shows an agarose gel with the PPIB DNA sequences after PCR amplification.
  • the left panel Figure 41 C shows the amplified PPIB DNA sequence after incubation of the HeLa cells with the Cas9/RNAs complex in presence of a lipidic transfection agent (DharmaFectTM transfection reagent # T-20XX-01) (positive control).
  • the right panel Figure 41 C shows amplified DNA sequence before the T7E1 digestion procedure as a negative control.
  • a mix composed of a Cas9-NLS recombinant protein (2.5 ⁇ ; Example 13.1) and crRNA/tracrRNA (2 ⁇ ; see below) targeting a nucleotide sequence of the HPTR gene were co- incubated with 35 ⁇ of His-CM18-PTD4, His-CM18-PTD4-His, His-C(LLKK)3C-PTD4, or EB1-PTD4, and incubated with HeLa or Jurkat cells for 2 minutes in PBS using Protocol B as described in Example 9.1.
  • the sequences of the crRNA and tracrRNAs constructed and their targets were:
  • Figures 46A-46B shows an agarose gel with the HPTR DNA sequences after PCR amplification and the cleavage product of the amplified HPTR DNA sequence by the CRISPR/Cas9 complex after the delivery of the complex with the different shuttle agents.
  • Figure 46A shows the results with the shuttle agents: His-CM18-PTD4, His-CM18-PTD4-His, and His-C(LLKK)3C-PTD4 in HeLa cells.
  • Figure 46B shows the results with His-CM18-PTD4 and His-CM18-L2-PTD4 in Jurkat cells.
  • Negative controls show amplified HPTR DNA sequence after incubation of the cells with the CRISPR/Cas9 complex without the presence of the shuttle agent.
  • Positive controls (lane 5 in Figures 46A and 46B) show the amplified HPTR DNA sequence after incubation of the cells with the Cas9/RNAs complex in presence of a lipidic transfection agent (Lipofectamine® RNAiMAXTM Transfection Reagent ThermoFisher Product No. 13778100).
  • a mix composed of a Cpf1 -NLS recombinant protein (1.33 ⁇ ) and crRNA (2 ⁇ ; see below) targeting a nucleotide sequence of the DNMT1 gene was co-incubated with different concentrations of His-CM18-PTD4 and incubated with HeLa cells for 2 min in PBS using Protocol A as described in Example 9.1.
  • crRNA guide RNAs
  • the sequences of the guide RNAs and the DNA template constructed and their targets were: - Feldan tracrR A [SEQ ID NO: 77] :
  • Figures 49A-49B show agarose gels with PCR amplification of the HPTR or the DNMT1 DNA sequences and the cleavage products of the amplified HPTR and DNMT1 DNA sequences after His-CM18-PTD4-mediated delivery of (upper panel of Figure 49A) the CRISPR/Cas9 complex and (upper panel of Figure 49B) the CRISPR/Cpfl complex in HeLa cells, (bottom panel of Figure 49A) PCR amplification of the DNA template delivered in HeLa cells in the HPRT gene, (bottom panel of Figure 49B) PCR amplification of the DNMT1 gene from HeLa cells extracts after the genomic insertion of the DNA template in this gene, and exposure of the DNMT1 sequence to the restriction enzyme Ecorl Negative controls show amplified HPTR or DNMT1 DNA sequences after incubation of the cells with the CRISPR/Cas9-NLS or CRISPR/Cpfl -NLS complex and respective DNA template without the presence of a His-CM18-
  • Positive controls show the amplified HPTR or DNMT1 DNA sequence after incubation of the cells with the Cas9/RNAs or Cpf1/RNAs complex in presence of the lipid transfection agent Lipofectamine CRISPRMax (product #B25642). Dotted arrows indicate the bands corresponding to the target gene, and thick black arrows indicate the bands corresponding to the cleavage products of this target gene, which indicate the successful delivery of functional CRISPR RNP systems for genome editing. An imaging software was used to quantify the relative signal intensities of each of the different bands directly on gels.
  • a mix composed of a Cas9-NLS recombinant protein (2.5 ⁇ ; Example 13.1) and guide RNAs (crRNA & tracrRNA) (2 ⁇ ; see below) targeting a nucleotide sequence of the HPTR gene were co- incubated with 4 ⁇ of His-CM18-PTD4 and incubated with NK cells for 2 minutes in PBS using Protocol C as described in Example 9.1.
  • Figure 50 shows agarose gels with the HPTR DNA sequences after PCR amplification and the cleavage product of the amplified HPTR DNA sequence by the CRISPR/Cas9 complex after the delivery of the complex with His-CM18-PTD4 in NK cells.
  • Negative controls show amplified HPTR or DNMT1 DNA sequences after incubation of the cells with the CRISPR/Cas9-NLS or CRISPR/Cpfl -NLS complex and respective DNA template without the presence of a His-CM18-PTD4.
  • Positive control shows the amplified HPTR sequence after incubation of the cells with the Cas9/RNAs complex in presence of the lipidic transfection agent Lipofectamine CRISPRMax (product # B25642).
  • a mix composed of a Cpf1 -NLS recombinant protein (1 ⁇ ; Example 13.7) and three guide RNAs (crRNA2, crRNA3 and crRNA4) (1.2 ⁇ ; see below) targeting exon 1 (crRNA2) and exon2 (crRNAs 3 & 4) of the B2M gene were co-incubated with 20 ⁇ of His-CM18-PTD4 and incubated with HeLa cells for 2 minutes in PBS using Protocol B as described in Example 9.1. After 2 minutes, HeLa cells were washed with PBS and incubated in medium with serum for 48h. HeLa cells were harvested to proceed with the T7E1 protocol assay as described in Example 13.4.
  • Figure 51 shows agarose gels with the B2M sequence after PCR amplification and the cleavage products of the amplified (upper panel) B2M exons 1 and 2 sequences by the three CRISPR/Cpf1 complexes after their delivery in presence of His-CM18-PTD4 in HeLa cells (upper panels).
  • Figure 51 also shows agarose gels with the B2M sequence after PCR amplification of the B2M exons 1 and 2 sequences before enzymatic T7E1 assay (bottom panels). Exons 1 and 2 have been cleaved in presence of respective CRISPR/Cpf1 -NLS complexes or in presence of the three CRISPR/Cpf1 -NLS complexes.
  • Indels (%) indicated successful genome editing although the presence of non-specific complexes reduced gene cutting. It indicated that His-CM18-PTD4 can deliver a limited amount of each CRISPR complex in cells and/or each complex competes for cell entrance. Negative controls show amplified B2M exonl sequence after incubation of the cells without any CRISPR/Cpf1 -NLS complex. The sequences of the crRNA used was as follows:
  • His-CM18-PTD4 enables transduction of the transcription factor H0XB4 in THP-1 cells
  • Human H0XB4 recombinant protein was constructed, expressed and purified from a bacterial expression system as described in Example 1.4.
  • the sequence of the H0XB4-WT recombinant protein produced was:
  • the initiator methionine and the 6x Histidine tag are shown in bold. 14.2 Real-Time Polymerase Chain Reaction (rt-PCR)
  • Control and treated cells are transferred to separate sterile 1.5-mL tubes and centrifuged for 5 minutes at 300g.
  • the cell pellets are resuspended in appropriate buffer to lyse the cells.
  • RNAase-free 70% ethanol is then added followed by mixing by pipetting.
  • the lysates are transferred to an RNeasyTM Mini spin column and centrifuged 30 seconds at 13000 RPM. After several washes with appropriate buffers and centrifugation steps, the eluates are collected in sterile 1.5-mL tubes on ice, and the RNA quantity in each tube is then quantified with a spectrophotometer.
  • DNase treatment 2 g of RNA is diluted in 15 L of RNase-free water.
  • Newly synthesized cDNA is transferred in sterile 1.5-mL tubes and diluted in 2 ⁇ of nuclease-free water. 18 L per well of a qPCR machine (CFX-96TM) mix is then added in a PCR plate for analysis.
  • CFX-96TM qPCR machine
  • THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were plated at 30 000 cells/well one day before transduction. HOXB4-WT recombinant protein (0.3, 0.9, or 1.5 ⁇ ; Example 14.1) was co-incubated with different concentrations of His-CM18-PTD4 (0, 0.5, 7.5, 0.8 or 1 ⁇ ) and then exposed to THP-1 cells for 2.5 hours in the presence of serum.
  • the cells were subjected to real time-PCR analysis as described in Example 14.2 to measure the mRNA levels of a target gene as a marker for HOXB4 activity, which was then normalized to the target gene mRNA levels detected in the negative control cells (no treatment), to obtain a "Fold over control" value. Total RNA levels (ng/ ⁇ .) were also measured as a marker for cell viability. Results are shown in Table 14.1 and Figure 42.
  • THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were plated at 30 000 cells/well one day before the first time course experiment. HOXB4-WT recombinant protein (1.5 ⁇ ; Example 14.1) was co-incubated with His-CM18-PTD4 (0.8 ⁇ ) and then exposed to THP-1 cells for 0, 2.5, 4, 24 or 48 hours in presence of serum. The cells were subjected to real time-PCR analysis as described in Example 14.2 to measure mRNA levels of a target gene as a marker for HOXB4 activity, which was then normalized to the target gene mRNA levels detected in the negative control cells (no treatment), to obtain a "Fold over control" value. Total RNA levels (ng/ ⁇ .) were also measured as a marker for cell viability. Results are shown in Table 14.2 and Figure 43. Table 14.2: Data from Figure 43
  • THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were plated at 30 000 cells/well one day before the first time course experiment. HOXB4-WT recombinant protein (0.3 ⁇ ; Example 14.1) was co-incubated with His-CM18-PTD4 (0.8 ⁇ ) and then exposed to THP-1 cells for 0, 0.5, 1 , 2, 2.5, 3 or 4 hours in presence of serum.
  • the cells were subjected to real time-PCR analysis as described in Example 14.2 to measure mRNA levels of a target gene as a marker for HOXB4 activity, which was then normalized to target gene mRNA levels detected in the negative control cells (no treatment), to obtain a "Fold over control" value. Total RNA levels (ng/ ⁇ .) were also measured as a marker for cell viability. Results are shown in Table 14.3 and Figure 44.
  • HOXB4-WT transcription factor (25 ⁇ ; Example 14.1) was co-incubated with 35 ⁇ of His-CM18-PTD4 and exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. After a 30-minute incubation to allow transduced HOXB4-WT to accumulate in the nucleus, the cells were fixed, permeabilized and immuno-labelled as described in Example 3.2a.
  • HOXB4-WT was labelled using a primary mouse anti-HOXB4 monoclonal antibody (Novus Bio #NBP2- 37257) diluted 1/500, and a secondary anti-mouse antibody AlexaTM-594 (Abeam #150116) diluted 1/1000.
  • Nuclei were labelled with DAPI.
  • the cells were visualized by bright field and fluorescence microscopy at 20x and 40x magnifications as described in Example 3.2, and sample results are shown in Figures 45A-45D.
  • Co-localization was observed between nuclei labelling ( Figures 45A and 45C) and HOXB4-WT labelling ( Figures 45B and 45D), indicating that HOXB4-WT was successfully delivered to the nucleus after 30 min in the presence of the shuttle agent His-CM18-PTD4.
  • White triangle windows show examples of areas of co-localization between the nuclei (DAPI) and HOXB4-WT immuno-labels.
  • THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were plated at 30 000 cells/well one day before the first time course experiment. HOXB4-WT recombinant protein (1.5 ⁇ ; Example 14.1) co-incubated with the shuttle agents His-CM18-PTD4, TAT-KALA, EB1-PTD4, His-C(LLKK)3C-PTD4 and His-CM18- PTD4-His at 0.8 ⁇ , and then exposed to THP-1 cells for 2.5 hours in presence of serum.
  • HOXB4-WT recombinant protein 1.5 ⁇ ; Example 14.1 co-incubated with the shuttle agents His-CM18-PTD4, TAT-KALA, EB1-PTD4, His-C(LLKK)3C-PTD4 and His-CM18- PTD4-His at 0.8 ⁇ , and then exposed to THP-1 cells for 2.5 hours in presence of serum.
  • the cells were subjected to real time-PCR analysis as described in Example 14.2 to measure mRNA levels of a target gene as a marker for HOXB4 activity, which was then normalized to target gene mRNA levels detected in the negative control cells (no treatment), to obtain a "Fold over control" value. Total RNA levels (ng/ ⁇ ) were also measured as a marker for cell viability. Results are shown in Table 14.4 and Figure 47.
  • shuttle agent His-CM18-PTD4 was diluted in sterile distilled water at room temperature.
  • GFP-NLS used as cargo protein, was then added to the shuttle agent and, if necessary, sterile PBS was added to obtain the desired concentrations of shuttle agent and cargo in a sufficient final volume for injection in rat brain (e.g., 5 ⁇ per each injection brain site).
  • the shuttle agent/cargo mixture was then immediately used for experiments.
  • One negative control was included for the experiment, which corresponds to the injection of the GFP-NLS alone.
  • Antero-posterior (AP), lateral (L), and dorso-ventral (DV) coordinates were taken relative to the bregma: (a) AP 0.48 mm, L ⁇ 3 mm, V - 5 mm; (b) AP - 2 mm, L ⁇ 1.3 mm, V - 1.5 mm; (c) AP - 2.6 mm, L ⁇ 1.5 mm, V - 1.5 mm.
  • the infused volume of the shuttle/cargo mix or cargo alone was 5 ⁇ l per injection site and the injection was performed for 10 minutes. After that, experimenter waited 1 min before removing the needle from the brain. All measures were taken before, during, and after surgery to minimize animal pain and discomfort. Animals were sacrificed by perfusion with paraformaldehyde (4%) 2 h after surgery, and brain were collected and prepared for microcopy analysis. Experimental procedures were approved by the Animal Care Committee in line with guidelines from the Canadian Council on Animal Care.
  • This experiment demonstrated the cell delivery of the cargo GFP-NLS after its stereotaxic injection in the rat parietal cortex in the presence of the shuttle agent His-CM18-PTD4.
  • Results show the delivery of the GFP-NLS in the nucleus of cells from the deeper layers of the parietal cortex (injection site) to the corpus callus and the dorsal level of the striatum (putamen).
  • the negative control in which GFP-NLS is only detectable locally around the injection site.
  • This experiment shows that shuttle agent induced nuclear delivery of the cargo in the injection site (parietal cortex) and its diffusion through both neighboring brain areas (corpus callus and striatum rat brain).
  • the following examples are prophetic examples.
  • HSCs hematopoietic stem cells
  • HoxB4 is involved in HSC expansion. Therefore, HoxB4 is selected for delivery to the HSC nuclease with the goal of HSC population expansion in order to generate an adequate population size for treatment of a patient in need of a HSC transplantation.
  • HoxB4 comprising a fused cell permeable domain (TAT) is cloned, expressed, and purified.
  • TAT fused cell permeable domain
  • the TAT-HoxB4 fusion protein is added to a population of ex vivo HSCs isolated from the patient. Following translocation across the cell membrane, the HoxB4 protein is sequestered in the endosome and the majority is eventually degraded. Therefore, very little HoxB4 protein reaches the nucleus and cell expansion occurs at a low frequency due to the inefficient delivery of HoxB4. As a result, the patient undergoes a transplantation with a sub-optimal HSC population size.
  • HSCs are isolated from the patient described in Example 16 and maintained ex vivo.
  • HoxB4 is complexed with a peptide shuttle as disclosed herein and is added to the ex vivo population of HSCs.
  • the peptide shuttle has a cell penetrating domain (CPD), which aids in transport of HoxB4 across the cell membrane, and an endosome leakage domain (ELD), which ensures HoxB4 does not become sequestered in the endosome.
  • HoxB4 comprises a nuclear localization signal (NLS), which targets HoxB4 to the nucleus, where HoxB4 functions to initiate expansion of the population.
  • NLS nuclear localization signal
  • the HoxB4-peptide shuttle complex successfully escapes the endosome and is targeted to the nucleus.
  • the HSC population expands and surpasses the threshold needed for transplantation.
  • the expanded HSC population is transplanted into the patient.
  • megakaryocytes which are platelet-producing cells derived from hematopoietic stem cells.
  • the success of megakaryocyte transplantation is effected by the population size and immunogenicity of the cells to be transplanted.
  • HSCs are isolated from the patient and maintained ex vivo.
  • HoxB4 is complexed with a peptide shuttle as disclosed herein and is added to the ex vivo population of HSCs.
  • the peptide shuttle has a cell penetrating domain (CPD), which aids in transport of HoxB4 across the cell membrane, and an endosome leakage domain (ELD), which ensures HoxB4 does not become sequestered in the endosome.
  • HoxB4 comprises a nuclear localization signal (NLS), which targets HoxB4 to the nucleus, where HoxB4 functions to initiate differentiation of the HSC to a megakaryocyte. Following translocation across the HSC cell membrane, the HoxB4-peptide shuttle complex successfully escapes the endosome and is targeted to the nucleus. As a result, the HSC population expands.
  • NLS nuclear localization signal
  • the cells are treated with differentiation factors which initiate differentiation of the HSCs into megakaryocytes. Because of the expanded starting HSC population, the number of generated megakaryocytes surpasses the threshold needed for transplantation. The expanded HSC population-derived megakaryocytes are transplanted into the patient.
  • Example 18 A patient similar to the one described in Example 18 has thrombocytopenia and is need of a megakaryocyte transplantation. An attempt to isolate HSCs from the patient is unsuccessful, therefore donor HSCs from a different person are obtained. These non-autologous HSCs are expanded with a HoxB4-peptide shuttle complex as described in Examples 16-17.
  • the cells are treated with differentiation factors which initiate differentiation of the HSCs into megakaryocytes. Because of the expanded starting HSC population, the number of generated megakaryocytes surpasses the threshold needed for transplantation. However, the megakaryocytes are immunogenic to the patient and will be rejected during transplantation.
  • a Cas9-peptide shuttle complex is used to delete a major histocompatibility complex (MHC) gene from the cells.
  • the peptide shuttle is the same as that described in Examples 16-17 and contains a cell penetrating domain and an endosome leakage domain.
  • Cas9 is engineered to comprise a nuclear localization signal.
  • the Cas9-peptide shuttle complex also contains a crRNA engineered to target the MHC DNA sequence and a corresponding trRNA.
  • the Cas9-peptide shuttle complex successfully escapes the endosome and is targeted to the nucleus.
  • the crRNA and trRNA guide Cas9 to the MHC DNA target sequence, followed by Cas9-based cleavage of the target sequence. Cleavage of the target sequence leads to disruption of the MHC gene product and results in non-immunogenic megakaryocytes.
  • the non-immunogenic megakaryocytes are transplanted into the patient.

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Abstract

The present description relates to synthetic peptides useful for increasing the transduction efficiency of polypeptide cargos to the cytosol of target eukaryotic cells. More specifically, the present description relates to synthetic peptides and polypeptide-based shuttle agents comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD. Compositions, kits, methods and uses relating to same are also described. Methods for using the disclosed polypeptide-based shuttles for delivering a transcription factor and/or nuclease to eukaryotic cell nucleus and uses of the derived cells are also described.

Description

PEPTIDE SHUTTLE BASED GENE DISRUPTION
The present description relates to synthetic peptides useful for increasing the transduction efficiency of polypeptide cargos to the cytosol of target eukaryotic cells. More specifically, the present description relates to synthetic peptides and polypeptide-based shuttle agents comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD.
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Provisional Application Ser. No. 62/320,043, filed April 8,
2016 and Provisional Application Ser. No. 62/320,065 filed April 8, 2016, the disclosures of each of which are incorporated herein by reference in their entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in
ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 7, 2017, is named 49446-702_601_SL.txt and is 81 ,784 bytes in size.
BACKGROUND
Cell delivery technologies to transport large molecules inside eukaryotic cells have a wide range of applications, particularly in the biopharmaceutical industry. While some soluble chemical substances (e.g., small molecule drugs) may passively diffuse through the eukaryotic cell membrane, larger cargos (e.g., biologies, polynucleotides, and polypeptides) require the help of shuttle agents to reach their intracellular targets.
An area that would greatly benefit from advances in cell delivery technologies is the field of cell therapy, which has made enormous leaps over the last two decades. Deciphering the different growth factors and molecular cues that govern cell expansion, differentiation and reprogramming open the door to many therapeutic possibilities for the treatment of unmet medical needs. For example, induction of pluripotent stem cells directly from adult cells, direct cell conversion (trans-differentiation), and genome editing (Zinc finger nuclease, TALEN, and CRISPR/Cas9 technologies) are examples of methods that have been developed to maximize the therapeutic value of cells for clinical applications. Presently, the production of cells with high therapeutic activity usually requires ex vivo manipulations, mainly achieved by viral transduction, raising important safety and economical concerns for human applications. The ability to directly deliver active proteins such as transcription factors or artificial nucleases, inside these cells, may advantageously circumvent the safety concerns and regulatory hurdles associated with more risky gene transfer methods.
In this regard, polypeptide-based transduction agents may be useful for introducing purified recombinant proteins directly into target cells, for example, to help bypass safety concerns regarding the introduction of foreign DNA. Lipid- or cationic polymer-based transduction agents exist, but introduce safety concerns regarding chemical toxicity and efficiency, which hamper their use in human therapy. Protein transduction approaches involving fusing a recombinant protein cargo directly to a cell- penetrating peptide (e.g., HIV transactivating protein TAT) require large amounts of the recombinant protein and often fail to deliver the cargo to the proper subcellular location, leading to massive endosomal trapping and eventual degradation. Several endosomal membrane disrupting peptides have been developed to try and facilitate the escape of endosomally-trapped cargos to the cytosol. However, many of these endosomolytic peptides are intended to alleviate endosomal entrapment of cargos that have already been delivered intracellular^, and do not by themselves aid in the initial step of shuttling the cargos intracellular^ across the plasma membrane (Salomone et al., 2012; Salomone et al., 2013; Erazo-Oliveras et al., 2014; Fasoli et al., 2014). Thus, there is a need for improved shuttle agents capable of increasing the transduction efficiency of polypeptide cargos, and delivering the cargos to the cytosol of target eukaryotic cells.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY
The present description stems from the surprising discovery that synthetic peptides comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD) and optionally a histidine-rich domain, have the ability to increase the proportion of cells that can be transduced with a polypeptide cargo of interest, without the synthetic peptide being covalently bound to the polypeptide cargo. Following successful transduction, the synthetic peptides may facilitate the ability of endosomally-trapped polypeptide cargos to gain access to the cytosol, and optionally be targeted to various subcellular comparts (e.g., the nucleus).
Provided herein are methods of editing a genome of one or more eukaryotic cells comprising contacting a eukaryotic cell with a polypeptide-based shuttle and a DNA cleavage protein, such that the DNA cleavage protein is delivered to the nucleus and binds to at least one target DNA sequence, thereby editing the genome, wherein the polypeptide-based shuttle comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or functional fragment thereof having endosomolytic activity, and wherein the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones. Additionally provided herein are methods of editing a genome of one or more eukaryotic cells comprising contacting a population of eukaryotic cells with a polypeptide-based shuttle and a DNA cleavage protein, such that the DNA cleavage protein is delivered to the nucleus and binds to at least one target DNA sequence, thereby editing the genome, wherein the polypeptide-based shuttle comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or functional fragment thereof having endosomolytic activity, and wherein the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones. In some cases, the polypeptide-based shuttle is present at a concentration sufficient to increase the percentage or proportion of the population eukaryotic cells into which the DNA cleavage protein is delivered across the plasma membrane, as compared to in the absence of said polypeptide-based shuttle. In some cases, the polypeptide-based shuttle is complexed with the DNA cleavage protein. In some cases, the polypeptide-based shuttle is not complexed with the DNA cleavage protein. In some cases, the DNA cleavage protein is a variant or functional derivative of a DNA cleavage protein. Further provided herein are methods wherein the polypeptide-based shuttle and the DNA cleavage protein are not covalently linked. Further provided herein are methods wherein the polypeptide-based shuttle further comprises a histidine rich domain. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity. Further provided herein are methods wherein the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the DNA-cleavage protein comprises a subcellular targeting domain. Further provided herein are methods wherein the subcellular targeting domain comprises a nuclear localization signal (NLS). Further provided herein are methods wherein the NLS comprises at least one of SEQ ID NO. 28-50. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity, and the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof. Further provided herein are methods wherein the DNA cleavage protein comprises an RNA-guided nuclease. Further provided herein are methods wherein the RNA-guided nuclease comprises a Cas protein. Further provided herein are methods wherein the Cas protein comprises a Type I, Type II, Type III, Type IV, Type V, or a Type VI Cas protein or protein complex. Further provided herein are methods wherein the Cas protein comprises Cas9, Cpf1 , or at least one functional fragment or derivative thereof. Further provided herein are methods wherein the population of eukaryotic cells is further contacted with at least one guiding RNA. Further provided herein are methods wherein the eukaryotic cell is further contacted with at least one guiding RNA. Further provided herein are methods wherein the polypeptide- based shuttle and the RNA-guided nuclease are additionally complexed with at least one guiding RNA. Further provided herein are methods wherein the RNA-guided nuclease comprises Cas9 and wherein the at least one guiding RNA comprises a crRNA and a trRNA. Further provided herein are methods wherein the crRNA and trRNA have independent phosphodiester backbones. Further provided herein are methods wherein the crRNA and trRNA share a common phosphodiester backbone. Further provided herein are methods wherein the crRNA is engineered to hybridize with the at least one target DNA sequence, wherein the trRNA is engineered to hybridize with the crRNA, and wherein the crRNA and the trRNA form a complex with Cas9, thereby targeting Cas9 to the at least one target DNA sequence such that the at least one target DNA sequence is cleaved by Cas9. Further provided herein are methods wherein the RNA-guided nuclease comprises Cpf1 and wherein the at least one guiding RNA comprises a crRNA. Further provided herein are methods wherein the crRNA is engineered to hybridize with the at least one target DNA sequence, and wherein the crRNA forms a complex with Cpf1 , thereby targeting Cpf1 to the at least one target DNA sequence such that the at least one target DNA sequence is cleaved by Cpf1. Further provided herein are methods wherein binding to at least one target further comprises cleavage of the at least one target DNA sequence, such that cleavage creates a double strand break which is repaired by a non-homologous end joining (NHEJ) repair mechanism, thereby editing the at least one target DNA molecule. Further provided herein are methods wherein binding to at least one target further comprises cleavage of the at least one target DNA sequence, such that cleavage creates a double strand break which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the at least one target DNA sequence, thereby editing the target DNA molecule. Further provided herein are methods wherein the Cas9 protein is a nickase variant of Cas9 which comprises at least one mutation in at least one of a RuvC domain and a HNH domain such binding to at least one target further comprises cleavage of only one strand of the at least one target DNA sequence. Further provided herein are methods wherein the method comprises editing the at least one target DNA sequence by insertion of a sequence for a donor polynucleotide into the cleaved strand of the at least one target DNA sequence. Further provided herein are methods wherein the method comprises editing the at least one target DNA sequence by a homology directed repair mechanism which incorporates a sequence of a donor polynucleotide into the at least one target DNA sequence, thereby editing the at least one target DNA molecule. Further provided herein are methods wherein incorporation of a sequence of a donor polynucleotide results in insertion, deletion, or substitution of one or more nucleotides. Further provided herein are methods wherein the RNA-guided nuclease is multiplexed with at least two guiding RNAs, such that at least two target DNA sequences are cleaved. Further provided herein are methods wherein the Cas protein is catalytically dead such that the Cas protein binds but does not cleave the at least one target DNA sequence. Further provided herein are methods wherein binding of the catalytically dead Cas protein blocks functional transcription the at least one target DNA sequence. Further provided herein are methods wherein the DNA-cleavage protein is a zinc finger nuclease, TALEN, or a meganuclease. Further provided herein are methods wherein the eukaryotic cell is a mammalian cell. Further provided herein are methods wherein the cell is a human cell. Further provided herein are methods wherein the cell is a T cell. Further provided herein are methods wherein the cell is a megakaryocyte. Further provided herein are methods wherein the cell is a NK cell. Further provided herein are methods wherein the cell is a stem cell. Further provided herein are methods wherein the cell is a hematopoietic stem cell. Further provided herein are methods wherein the population of eukaryotic cells is a mammalian cell population, a human cell population, a T cell population, a megakaryocyte population, a NK cell population, a stem cell population, or a hematopoietic stem cell population. Further provided herein are methods wherein the target DNA sequence is implicated in a genetic disease, such that the genome editing treats said genetic disease. Further provided herein are methods wherein the genetic disease is a blood-related disease. Further provided herein are methods wherein the target DNA sequence is implicated in an infection, such that the genome editing treats said infection. Further provided herein are methods wherein the infection is a viral infection. Further provided herein are methods wherein the target DNA sequence is implicated in immunogenicity, such that the genome editing deletes or ameliorates the immunogenicity. Further provided herein are methods wherein the genome editing results in non-immunogenic cells. Further provided herein are methods wherein the target DNA sequence comprises a human leukocyte antigen gene complex sequence. Further provided herein are methods wherein the target sequence comprises a major histocompatibility complex gene sequence. Further provided herein are methods wherein the non-immunogenic cells comprise universal stem cells. Further provided herein are methods wherein the non-immunogenic cells comprise CAR-T cells. Further provided herein are methods wherein incorporation of a sequence of a donor polynucleotide results in insertion of one or more nucleotides, wherein the one or more nucleotides comprises a heterologous gene. Further provided herein are methods wherein the heterologous gene is GALC. Further provided herein are methods wherein the heterologous gene is HEXA. Further provided herein are methods wherein the heterologous gene is IDUA. Further provided herein are methods wherein the eukaryotic cell is a mammalian cell. Further provided herein are methods wherein the cell is a stem cell. Further provided herein are methods wherein the cell is a hematopoietic stem cell. Further provided herein are methods wherein the cell is a central nervous system cell. Further provided herein are methods wherein the cell is a microgilia cell. Further provided herein are methods wherein the cell is a neuron. Further provided herein are methods wherein the cell is a liver cell. Further provided herein are methods wherein the cell is a liver endothelial cell. Further provided herein are methods wherein the cell is a hepatocyte. Further provided herein are methods wherein the target DNA sequence is in a gene locus. Further provided herein are methods wherein the gene locus is an abnormal GALC gene. Further provided herein are methods wherein the gene locus is an abnormal HEXA gene. Further provided herein are methods wherein the gene locus is an abnormal IDUA gene. Further provided herein are methods wherein the gene locus is an albumin gene. Further provided herein are methods wherein incorporation of the heterologous gene treats or ameliorates the symptoms of a genetic disease. Further provided herein are methods wherein the genetic disease is Krabbe Disease. Further provided herein are methods wherein the genetic disease is Tay-Sachs Disease. Further provided herein are methods wherein the genetic disease is Hurler Syndrome.
Provided herein are methods of treating a patient with a condition by administering to the patient in need thereof non-immunogenic cells obtained by any of the methods disclosed herein. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood disorder. Further provided herein are methods wherein the condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are methods wherein the condition is thrombocytopenia. Further provided herein are uses of non-immunogenic cells obtained by any of the methods disclosed herein for treatment of a patient in need thereof due to a condition. Further provided herein are uses wherein the condition is a genetic disease. Further provided herein are uses wherein the condition is a blood disorder. Further provided herein are uses wherein the condition is a malignant condition. Further provided herein are uses wherein the condition is a non-malignant condition. Further provided herein are uses wherein the condition is thrombocytopenia.
Provided herein are methods of editing a genome of one or more megakaryocyte cells comprising contacting a megakaryocyte with a polypeptide-based shuttle complexed with Cas9 and at least one guiding RNA such that Cas9 is delivered to the nucleus and cleaves at least one target DNA sequence, thereby editing the genome, wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, and wherein the polypeptide- based shuttle and Cas9 have independent protein backbones and are not covalently linked, wherein the at least one guiding RNA comprises a crRNA and a trRNA with independent phosphodiester backbones, wherein the crRNA is engineered to hybridize with the at least one target DNA sequence, wherein the target DNA sequence comprises a major histocompatibility complex gene sequence, and wherein the genome editing results in non-immunogenic megakaryocytes. Further provided herein are methods of treating a patient with thrombocytopenia by administering to the patient in need thereof non- immunogenic megakaryocytes obtained by any of the methods disclosed herein. Further provided herein are uses of non-immunogenic megakaryocytes obtained by any of the methods disclosed herein for treatment of a thrombocytopenia patient in need thereof.
Provided herein are methods of treating a patient with a condition by administering to the patient in need thereof cells obtained by any of the methods disclosed herein. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the genetic disease is Krabbe Disease. Further provided herein are methods wherein the genetic disease is Tay-Sachs Disease. Further provided herein are methods wherein the genetic disease is Hurler Syndrome.
Provided herein are uses of non-immunogenic cells obtained by any of the methods disclosed herein for treatment of a patient in need thereof due to a condition. Further provided herein are uses wherein the condition is a genetic disease. Further provided herein are uses wherein the genetic disease is Krabbe Disease. Further provided herein are uses wherein the genetic disease is Tay-Sachs Disease. Further provided herein are uses wherein the genetic disease is Hurler Syndrome.
General Definitions
Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.
The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".
The term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology "about" is meant to designate a possible variation of up to 10%. Therefore, a variation of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term "about". Unless indicated otherwise, use of the term "about" before a range applies to both ends of the range.
As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. As used herein, "protein" or "polypeptide" means any peptide-linked chain of amino acids, which may or may not comprise any type of modification (e.g., post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc).
As used herein, the expression "is or is from" or "is from" comprises functional variants of a given protein domain (CPD or ELD), such as conservative amino acid substitutions, deletions, modifications, as well as variants or function derivatives, which do not abrogate the activity of the protein domain. Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
Figures 1A-1 B show a typical result of a calcein endosomal escape assay in which HEK293A cells were loaded with the fluorescent dye calcein ("100 μΜ calcein"), and were then treated (or not) with a shuttle agent that facilitates endosomal escape of the calcein ("100 μΜ calcein + CM18-TAT 5 μΜ"). Figure 1A shows the results of a fluorescence microscopy experiment, while Figure 1 B shows the results of a flow cytometry experiment.
Figure 2 shows the results of a calcein endosomal escape flow cytometry assay in which HeLa cells were loaded with calcein ("calcein 100 μΜ"), and were then treated with increasing concentrations of the shuttle agent CM18-TAT-Cys (labeled "CM18-TAT").
Figures 3 and 4 show the results of calcein endosomal escape flow cytometry assays in which HeLa cells (Figure 3) or primary myoblasts (Figure 4) were loaded with calcein ("calcein 100 μΜ"), and were then treated with 5 μΜ or 8 μΜ of the shuttle agents CM18-TAT-Cys or CM18-Penetratin-Cys (labeled "CM18-TAT" and "CM18-Penetratin", respectively).
Figure 5 shows the results of a GFP transduction experiment visualized by fluorescence microscopy in which a GFP cargo protein was co-incubated with 0, 3 or 5 μΜ of CM18-TAT-Cys (labeled "CM18-TAT"), and then exposed to HeLa cells. The cells were observed by bright field (upper pictures in Figure 5) and fluorescence microscopy (lower pictures in Figure 5).
Figures 6A-6B show the results of a GFP transduction efficiency experiment in which GFP cargo protein (10 μΜ) was co-incubated with different concentrations of CM18-TAT-Cys (labeled "CM18-TAT"), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cell is shown in Figure 6A, and corresponding cell toxicity data is shown in Figure 6B. Figures 7A-7B show the results of a GFP transduction efficiency experiment in which different concentrations of GFP cargo protein (10, 5 or 1 μΜ) were co-incubated with either 5 μΜ of CM18-TAT- Cys (Figure 7A, labeled "CM18TAT"), or 2.5 μΜ of dCM18-TAT-Cys (Figure 7B, labeled "dCM18TAT"), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentages of fluorescent (GFP-positive) cells are shown.
Figures 8 and 9 show the results of GFP transduction efficiency experiments in which GFP cargo protein (10 μΜ) was co-incubated with different concentrations and combinations of CM18-TAT- Cys (labeled "CM18TAT"), CM18-Penetratin-Cys (labeled "CM18penetratin"), and dimers of each (dCM18-TAT-Cys (labeled "dCM18TAT"), dCM18-Penetratin-Cys (labeled "dCM18penetratin"), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentages of fluorescent (GFP-positive) cells are shown.
Figure 10 shows typical results of a TAT-GFP transduction experiment in which TAT-GFP cargo protein (5 μΜ) was co-incubated with 3 μΜ of CM18-TAT-Cys (labeled "CM18-TAT"), prior to being exposed to HeLa cells. Cells and GFP fluorescence were visualized by bright field and fluorescence microscopy at 10x and 40x magnifications. Arrows indicate the endosome delivery of TAT- GFP in the absence of CM18-TAT-Cys, as well as its nuclear delivery in the presence of CM18-TAT- Cys.
Figures 11A-11 B show the results of a TAT-GFP transduction efficiency experiment in which
TAT-GFP cargo protein (5 μΜ) was co-incubated with different concentrations of CM18-TAT-Cys (labeled "CM18TAT"), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cell is shown in Figure 11 A, and corresponding cell toxicity data is shown in Figure 11 B.
Figure 12 shows typical results of a GFP-NLS transduction experiment in which GFP-NLS cargo protein (5 μΜ) was co-incubated with 5 μΜ of CM18-TAT-Cys (labeled "CM18-TAT"), prior to being exposed to HeLa cells for 5 minutes. Cells and GFP fluorescence were visualized by bright field and fluorescence microscopy at 10x, 20x, and 40x magnifications. Arrows indicate areas of nuclear delivery of GFP-NLS.
Figures 13A-13B show the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 μΜ) was co-incubated with different concentrations of CM18-TAT-Cys (labeled "CM18TAT"), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cell is shown in Figure 13A, and corresponding cell toxicity data is shown in Figure 13B.
Figures 14 and 15 show the results of GFP-NLS transduction efficiency experiments in which GFP-NLS cargo protein (5 μΜ) was co-incubated with different concentrations and combinations of CM18-TAT (labeled "CM18TAT"), CM18-Penetratin (labeled "CM18penetratin"), and dinners of each (dCM18-TAT-Cys, dCM18-Penetratin-Cys; labeled "dCM18TAT" and "dCM18penetratin", respectively), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentages of fluorescent (GFP-positive) cells are shown.
Figure 16 shows the results of a GFP-NLS transduction efficiency experiment in which GFP-
NLS cargo protein (5 μΜ) was co-incubated with either CM18-TAT-Cys (3.5 μΜ, labeled "CM18TAT") alone or with dCM18-Penetratin-Cys (1 μΜ, labeled "dCM18pen") for 5 minutes or 1 hour in plain DMEM media ("DMEM") or DMEM media containing 10% FBS ("FBS"), before being subjected to flow cytometry analysis. The percentages of fluorescent (GFP-positive) cells are shown. Cells that were not treated with shuttle agent or GFP-NLS ("ctrl"), and cells that were treated with GFP-NLS without shuttle agent ("GFP-NLS 5 μΜ") were used as controls.
Figures 17A-17B show the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 μΜ) was co-incubated with or without 1 μΜ CM18-TAT-Cys (labeled "CM18TAT"), prior to being exposed to THP-1 cells. Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cells is shown in Figure17A, and corresponding cell toxicity data is shown in Figure17B.
Figures 18A-18C show the results of a transduction efficiency experiment in which the cargo protein, FITC-labeled anti-tubulin antibody (0.5 μΜ), was co-incubated with 5 μΜ of CM18-TAT-Cys (labeled "CM18-TAT"), prior to being exposed to HeLa cells. Functional antibody delivery was visualized by bright field (20x- Figure 18A) and fluorescence microscopy (20x- Figure 18B and 40x- Figure 18C), in which fluorescent tubulin fibers in the cytoplasm were visualized.
Figures 19A-19B show the results of an FITC-labeled anti-tubulin antibody transduction efficiency experiment in which the antibody cargo protein (0.5 μΜ) was co-incubated with 3.5 μΜ of CM18-TAT-Cys (labeled "CM18TAT"), CM18-Penetratin-Cys (labeled "CM18pen")or dCM18-Penetratin- Cys (labeled "dCM18pen"), or a combination of 3.5 μΜ of CM18-TAT-Cys and 0.5 μΜ of dCM18- Penetratin-Cys, prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentage of fluorescent (FITC-positive) cell is shown in Figure19A, and corresponding cell toxicity data is shown in Figure 19B.
Figure 20 shows the results of DNA transfection efficiency experiment in which plasmid DNA (pEGFP) was labeled with a Cy5™ dye was co-incubated with 0, 0.05, 0.5, or 5 μΜ of CM18-TAT-Cys (labeled "CM18-TAT"), prior to being exposed to HEK293A cells. Flow cytometry analysis allowed quantification of Cy5™ emission (corresponding to DNA intracellular delivery; y-axis) and GFP emission (corresponding to successful nuclear delivery of DNA; percentage indicated above each bar). Figures 21A-21 B show the results of a GFP-NLS transduction efficiency experiment in which the GFP-NLS cargo protein (5 μΜ) was co-incubated with 1 , 3, or 5 μΜ of CM18-TAT-Cys (labeled "CM18TAT"), of His-CM18-TAT (labeled "His-CM18TAT"), prior to being exposed to HeLa cells. Cells were evaluated by flow cytometry and the percentage of fluorescent (GFP-positive) cell is shown in Figure 21 A, and corresponding cell toxicity data is shown in Figure21 B.
Figures 22A-22B show the results of a transduction efficiency experiment in which GFP-NLS cargo protein was intracellularly delivered using the shuttle His-CM18-PTD4 in HeLa cells. GFP-NLS transduction efficiency was evaluated by flow cytometry and the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data ("viability (%)") are shown. Figure 22A shows a comparison of GFP-NLS transduction efficiencies using different transduction protocols (Protocol A vs. B). Figure 22B shows the effect of using different concentrations of the shuttle His- CM18-PTD4 when using Protocol B.
Figures 23A-23D, Figures 24A-24B, Figures 25A-25B and Figures 26A-26C are microscopy images showing the results of transduction experiments in which GFP-NLS (Figures 23A-23D, 24A, 24B, 25A-B and 26A-26C) cargo protein was intracellularly delivered with the shuttle His-CM18-PTD4 in HeLa cells. Figures 23D, 24A, 26A, and Figures 23A to 23C, 24B, 25A-B, 26B-C show the bright field and fluorescence images, respectively, of living cells. In Figure 25A-25B, the cells were fixed, permeabilized and subjected to immuno-labelling with an anti-GFP antibody and a fluorescent secondary antibody. White triangle windows indicate examples of areas of co-labelling between nuclei (DAPI) and GFP-NLS signals. Figure 26A-26C shows images captured by confocal microscopy.
Figures 27A-27D show microscopy images of a kinetic (time-course) transduction experiment in HeLa cells, where the fluorescence of GFP-NLS cargo protein was tracked after 45, 75, 100, and 120 seconds following intracellular delivery with the shuttle His-CM18-PTD4. The diffuse cytoplasmic fluorescence pattern observed after 45 seconds (Figure27A) gradually becomes a more concentrated nuclear pattern at 120 seconds (Figure 27D).
Figures 28A-28D show microscopy images of co-delivery transduction experiment in which two cargo proteins (GFP-NLS and mCherry™-NLS) are simultaneously delivered intracellularly by the shuttle His-CM18-PTD4 in HeLa cells. Cells and fluorescent signals were visualized by (Figure 28A) bright field and (Figures 28B-28D) fluorescence microscopy. White triangle windows indicate examples of areas of co-labelling between nuclei (DAPI) and GFP-NLS or mCherry™.
Figures 29A-29I show the results of GFP-NLS transduction efficiency experiments in HeLa cells using different shuttle agents or single-domain/control peptides. GFP-NLS transduction efficiency was evaluated by flow cytometry and the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data ("viability (%)") are shown in Figures 29A, 29B, 29D-29G, and 29I. In Figure 29A and Figure29D-29F, cells were exposed to the cargo/shuttle agent for 10 seconds. In Figure 29I, cells were exposed to the cargo/shuttle agent for 1 minute. In Figures 29B, 29C, 29G and 29H, cells were exposed to the cargo/shuttle agent for 1 , 2, or 5 min. "Relative fluorescence intensity (FL1-A)" or "Signal intensity" corresponds to the mean of all fluorescence intensities from each cell with a GFP fluorescent signal after GFP-NLS fluorescent protein delivery with the shuttle agent. Figure 29D shows the results of a control experiment in which only single-domain peptides (ELD or CDP) or the peptide His-PTD4 (His-CPD) were used for the GFP-NLS transduction, instead of the multi-domain shuttle agents.
Figure 30A-30F shows microscopy images of HeLa cells transduced with GFP-NLS using the shuttle agent (Figure 30A) TAT-KALA, (Figure 30B) His-CM18-PTD4, (Figure 30C) His-C(LLKK)3C- PTD4, (Figure 30D) PTD4-KALA, (Figure 30E) EB1-PTD4, and (Figure 30F) His-CM18-PTD4-His. The insets in the row of the lower pictures in Figures 30A-30F show the results of corresponding flow cytometry analyses, indicating the percentage of cells exhibiting GFP fluorescence.
Figure 31 shows the results of a transduction efficiency experiment in which GFP-NLS cargo protein was intracellularly delivered using the shuttle His-CM18-PTD4 in THP-1 cells using different Protocols (Protocol A vs C). GFP-NLS transduction efficiency was evaluated by flow cytometry and the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data ("viability (%)") are shown. "Ctrl" corresponds to THP-1 cells exposed to GFP-NLS cargo protein in the absence of a shuttle agent.
Figures 32A-32D show microscopy images of THP-1 cells transduced with GFP-NLS cargo protein using the shuttle His-CM18-PTD4. Images captured under at 4x, 10x and 40x magnifications are shown in Figures 32A-32C, respectively. White triangle windows in Figure 32C indicate examples of areas of co-labelling between cells (bright field) and GFP-NLS fluorescence. Figure 32D shows the results of corresponding flow cytometry analyses, indicating the percentage of cells exhibiting GFP fluorescence.
Figures 33A-33D show microscopy images of THP-1 cells transduced with GFP-NLS cargo protein using the shuttle His-CM18-PTD4. White triangle windows indicate examples of areas of co- labelling between cells (bright field; Figure33A-33B), and GFP-NLS fluorescence (Figure 33C-33D). Figure 33E shows FACS analysis of GFP-positive cells.
Figures 34A-34B show the results of GFP-NLS transduction efficiency experiments in THP-1 cells using the shuttle TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4. The cargo protein/shuttle agents were exposed to the THP-1 cells for 15, 30, 60 or 120 seconds. GFP-NLS transduction efficiency was evaluated by flow cytometry and the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data ("viability (%)") are shown in Figure 34A. In Figure34B, "Relative fluorescence intensity (FL1 -A)" corresponds to the mean of all fluorescence intensities from each cell with a GFP fluorescent signal after GFP-NLS fluorescent protein delivery with the shuttle agent.
Figures 35A-35F show the results of transduction efficiency experiments in which THP-1 cells were exposed daily to GFP-NLS cargo in the presence of a shuttle agent for 2.5 hours. His-CM18- PTD4 was used in Figures 35A-35E, and His-C(LLKK)3C-PTD4 was used in Figure 35F. GFP-NLS transduction efficiency was determined by flow cytometry at Day 1 or Day 3, and the results are expressed as the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data ("viability (%)") in Figures 35A-35C and in Figure 35F.Figure 35D shows the metabolic activity index of the THP-1 cells after 1 , 2, 4, and 24h, and Figure 35E shows the metabolic activity index of the THP-1 cells after 1 to 4 days, for cells exposed to the His-CM18-PTD4 shuttle.
Figure 36 shows a comparison of the GFP-NLS transduction efficiencies in a plurality of different types of cells (e.g., adherent and suspension, as well as cell lines and primary cells) using the shuttle His-CM18-PTD4, as measured by flow cytometry. The results are expressed as the percentage of GFP fluorescent cells ("Pos cells (%)"), as well as corresponding cell viability data ("viability (%)").
Figures 37A-37H show fluorescence microscopy images of different types of cells transduced with GFP-NLS cargo using the shuttle His-CM18-PTD4. GFP fluorescence was visualized by fluorescence microscopy at a 10x magnification. The results of parallel flow cytometry experiments are also provided in the insets (viability and percentage of GFP-fluorescing cells).
Figures 38A-38B show fluorescence microscopy images of primary human myoblasts transduced with GFP-NLS using the shuttle His-CM18-PTD4. Cells were fixed and permeabilized prior to immuno-labelling GFP-NLS with an anti-GFP antibody and a fluorescent secondary antibody. Immuno-labelled GFP is shown in Figure 38A, and this image is overlaid with nuclei (DAPI) labelling in Figure 38B.
Figures 39A-39E show a schematic layout (Figures 39A, 39B and 39C) and sample fluorescence images (D and E) of a transfection plasmid surrogate assay used to evaluate the activity of intracellular^ delivered CRISPR/Cas9-NLS complex. In Figure 39A) At Day 1, cells are transfected with an expression plasmid encoding the fluorescent proteins mCherry™ and GFP, with a STOP codon separating their two open reading frames. Transfection of the cells with the expression plasmid results in only mCherry™ expression as shown in Figure 39D. A CRISPR/Cas9-NLS complex, which has been designed/programmed to cleave the plasmid DNA at the STOP codon, is then delivered intracellular^ to the transfected cells expressing mCherry™, resulting double-stranded cleavage of the plasmid DNA at the STOP codon as shown in Figure 39B In a fraction of the cells, random non-homologous DNA repair of the cleaved plasmid occurs and results in removal of the STOP codon (Figure 39C), and thus GFP expression and fluorescence (Figure 39E). White triangle windows indicate examples of areas of co-labelling of mCherry™ and GFP fluorescence.
Figures 40A-40H show fluorescence microscopy images of HeLa cells expressing mCherry™ and GFP, indicating CRISPR/Cas9-NLS-mediated cleavage of plasmid surrogate DNA. In Figures 40A-40D, HeLa cells were co-transfected with three plasmids: the plasmid surrogate as described in the brief description of Figures 39A-39E, and two other expression plasmids encoding the Cas9-NLS protein and crRNA/tracrRNAs, respectively. CRISPR/Cas9-mediated cleavage of the plasmid surrogate at the STOP codon, and subsequent DNA repair by the cell, enables expression of GFP (Figures40B and 40D) in addition to mCherry™ (Figure40A and 40C). In Figures 40E and 40H, HeLa cells were transfected with the plasmid surrogate and then transduced with an active CRISPR/Cas9-NLS complex using the shuttle His-CM18-PTD4. CRISPR/Cas9-NLS-mediated cleavage of the plasmid surrogate at the STOP codon, and subsequent DNA repair by the cell, enables expression of GFP (Figures 40F and 40H) in addition to mCherry™ (Figures 40E and 40G).
Figure 41A (Lanes A to D)shows the products of a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR/Cas9-mediated cleavage of cellular genomic DNA. HeLa cells were transduced with a CRISPR-Cas9-NLS complex programmed to cleave the PPIB gene. The presence of the cleavage product framed in white boxes 1 and 2, indicates cleavage of the PPIB gene by the CRISPR-Cas9-NLS complex, which was delivered intracellular^ using the shuttle His-C(LLKK)3C-PTD4 (Figure 41-lane B) or with a lipidic transfection agent used as a positive control (lane in Figure 41 D). This cleavage product is absent in negative controls (Figure 41, Lanes A and C).
Figure 41 B shows the products of a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR/Cas9-mediated cleavage of cellular genomic DNA (PPIB DNA sequences). The left picture of the Figure 41 B shows the cleavage product of the amplified PPIB DNA sequence by the CRIPR/Cas9 complex after the delivery of the complex with the shuttle agent His-CM18-PTD4 in HeLa cells. The right picture of the Figure 41 B shows amplified DNA sequence before the T7E1 digestion procedure as a negative control.
Figure 41 C shows the products of a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR/Cas9-mediated cleavage of cellular genomic DNA (PPIB DNA sequences). The left picture of the Figure 41 C shows the amplified PPIB DNA sequence after incubation of the HeLa cells with the Cas9/RNAs complex in presence of a lipidic transfection agent (DharmaFect™ transfection reagent # T-20XX-01) (positive control). The right picture of the Figure 41C shows amplified DNA sequence before the T7E1 digestion procedure as a negative control. Figures 42-44 show the transcriptional activity of THP-1 cells that have been transduced with the transcription factor HOXB4 using different concentrations of the shuttle His-CM18-PTD4 and different cargo/shuttle exposure times. Successful intra-nuclear delivery of HOXB4 was determined by monitoring mRNA levels of a target gene by real-time PCR, and the results are normalized against those in the negative control (HOXB4 without shuttle agent) and expressed as "Fold over control" (left bars). Total cellular RNA (ng/μί) was quantified and used a marker for cell viability (right bars). "0" or "Ctrl" means "no treatment"; "TF" means "Transcription Factor alone"; "FS" means "shuttle alone".
Figures 45A-45D show fluorescence microscopy images of HeLa cells transduced with wild- type HOXB4 cargo using the shuttle His-CM18-PTD4. After a 30-minute incubation to allow transduced HOXB4-WT to accumulate in the nucleus, the cells were fixed, permeabilized and HOXB4-WT was labelled using a primary anti-HOXB4 monoclonal antibody and a fluorescent secondary antibody (Figures 45B and 45D). Nuclei were labelled with DAPI (Figures 45A and 45C). White triangle windows indicate examples of areas of co-labelling between nuclei and HOXB4 - compare Figures 45A vs 45B (x20 magnification), and Figures 45C vs 45D (x40 magnification).
Figures 46A-46B show the products of a DNA cleavage assay separated by agarose gel electrophoresis, which is used to measure CRISPR/Cas9-mediated cleavage of cellular genomic DNA (HPTR sequence) after intracellular delivery of the complex with different shuttle agents. Figure 46A shows the results with the shuttle agents: His-CM18-PTD4, His-CM18-PTD4-His, and His-C(LLKK)3C- PTD4 in HeLa cells. Figure 46B shows the results with His-CM18-PTD4-His and His-CM18-L2-PTD4 in Jurkat cells. Negative controls (lane 4 in Figures 46A and 46B) show amplified HPTR DNA sequence after incubation of the cells with the CRISPR/Cas9 complex without the presence of the shuttle agent. Positive controls (lane 5 in Figures 46A and 46B) show the amplified HPTR DNA sequence after incubation of the cells with the Cas9/RNAs complex in presence of a commercial lipidic transfection agent.
Figure 47 shows the transcriptional activity of THP-1 cells that have been transduced with the transcription factor HOXB4 using the shuttle agents His-CM18-PTD4, TAT-KALA, EB1-PTD4, His- C(LLKK)3C-PTD4 and His-CM18-PTD4-His. Successful intra-nuclear delivery of HOXB4 was determined by monitoring mRNA levels of a target gene by real-time PCR, and the results were normalized against those in the negative control (HOXB4 without shuttle agent) and expressed as "Fold over control" (left bars). Total cellular RNA (ng/μΙ.) was quantified and used a marker for cell viability (right bars). "0" or "Ctrl" means "no treatment"; 'TF" means "Transcription Factor alone"; "FS" means "shuttle alone".
Figures 48A-48D show in vivo GFP-NLS delivery in rat parietal cortex by His-CM18-PTD4. Briefly, GFP-NLS (20 μΜ) was injected in the parietal cortex of rat in presence of the shuttle agent His- CM18-PTD4 (20 μΜ) for 10 min. Dorso-ventral rat brain slices were collected and analysed by fluorescence microscopy at (Figure 48A) 4x, (Figure 48C) 10x and (Figure 48D) 20x magnifications. The injection site is located in the deepest layers of the parietal cortex (PCx). In presence of the His- CM18-PTD4 shuttle agent, the GFP-NLS diffused in cell nuclei of the PCx, of the Corpus Callus (Cc) and of the striatum (Str) (white curves mark limitations between brains structures). Figure 48B shows the stereotaxic coordinates of the injection site (black arrows) from the rat brain atlas of Franklin and Paxinos. The injection of GFP-NLS in presence of His-CM18-PTD4 was performed on the left part of the brain, and the negative control (injection of GFP-NLS alone), was done on the contralateral site. The black circle and connected black lines in Figure 48B show the areas observed in the fluorescent pictures (Figure 48A, 48C and 48D).
Figures 49A-49B depict an example of homologous-directed recombination with 6His-CM18-
PTD4-mediated co-delivery of (Figure 49A) CRISPR/Cas9 or (Figure 49B) CRISPR/Cpfl RNP systems in mammalian cell types. (Figure 49A) After the Cas9-NLS-mediated cleavage of the HPRT gene, a short DNA template (72bp) was inserted in the cut site by HDR. (Figure 49B) After the Cpfl- NLS-mediated cleavage of the DNMT1 gene, a short DNA template (76bp) with an EcoR1 site was inserted in the cut site by HDR.
Figure 50 shows an example of 6His-CM18-PTD4-mediated delivery of CRISPR/Cas9 RNP system in NK cells. Genomic cleavage analysis on agarose gel electrophoresis in NK cells. Cas9-NLS and specific crRNA were used for the cleavage of the HPRT in NK cells.
Figure 51 shows an example of genomic cleavage analysis of multiple exons on the B2M gene with T7E1 assay in HeLa cells after separation by agarose gel electrophoresis. Three CRISPR/Cpfl RNP complexes targeting the exons 1 and 2 of the B2M gene were co-delivered in presence of 6His- CM18-PTD4. Electrophoresis gels show that crRNA-2 (black thick arrows), crRNA-3 (red thick arrows) and crRNA-4 (blue thick arrows) were cleaved by respective CRISPR/Cpfl -crRNA complexes. SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April
7, 2017, is named 49446-702_601_SL.txt and is 81 ,784 bytes in size.
Figure imgf000017_0001
Figure imgf000018_0001
from Yeast cytochrome c
oxidase subunit IV
Mitochondrial signal sequence
53 113 6x histidine tag
from 18S rRNA
Peroxisome signal sequence -
54 114 Cpf1-NLS
PTS1
Nucleolar signal sequence from
55 115 DNMT1 crRNA
BIRC5
Nucleolar signal sequence from
56 116 Short linear DNA template
RECQL4
57 CM18-TAT 117 EcoR1 linear DNA template
58 CM18-Penetratin 118 B2M crRNA-2
59 His-CM18-TAT 119 B2M crRNA-3
60 GFP 120 B2M crRNA-4
DETAILED DESCRIPTION
The present description stems from the surprising discovery that multi-domain synthetic peptides comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD) can significantly increase the transduction efficiency of an independent polypeptide cargo to the cytosol of eukaryotic target cells. In contrast, this increase in transduction efficiency was not found using independent single-domain peptides containing only an ELD, or only a CPD used alone or together (i.e., in a mixture of separate single-domain peptides). Accordingly, in some aspects the present description relates to a polypeptide-based shuttle agent comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD, for use in increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell.
Synthetic peptides and polypeptide-based shuttle agents
As used herein, the term "synthetic" used in expressions such as "synthetic peptide" or
"synthetic polypeptide" is intended to refer to non-naturally occurring molecules that can be produced in vitro (e.g., synthesized chemically and/or produced using recombinant DNA technology). The purities of various synthetic preparations may be assessed by for example high-performance liquid chromatography analysis and mass spectroscopy. Chemical synthesis approaches may be advantageous over cellular expression systems (e.g., yeast or bacteria protein expression systems), as they may preclude the need for extensive recombinant protein purification steps (e.g., required for clinical use). In contrast, longer synthetic polypeptides may be more complicated and/or costly to produce via chemical synthesis approaches and such polypeptides may be more advantageously produced using cellular expression systems. In some embodiments, the peptides or shuttle agent of the present description may be chemically synthesized (e.g., solid- or liquid phase peptide synthesis), as opposed to expressed from a recombinant host cell. In some embodiments, the peptides or shuttle agent of the present description may lack an N-terminal methionine residue. A person of skill in the art may adapt a synthetic peptide or shuttle agent of the present description by using one or more modified amino acids (e.g., non-naturally-occurring amino acids), or by chemically modifying the synthetic peptide or shuttle agent of the present description, to suit particular needs of stability or other needs.
The expression "polypeptide-based" when used here in the context of a shuttle agent of the present description, is intended to distinguish the presently described shuttle agents from non- polypeptide or non-protein-based shuttle agents such as lipid- or cationic polymer-based transduction agents, which are often associated with increased cellular toxicity and may not be suitable for use in human therapy.
As used herein, the expression "increasing transduction efficiency" refers to the ability of a shuttle agent (e.g., a polypeptide-based shuttle agent of the present description) to improve the percentage or proportion of a population of target cells into which a cargo of interest (e.g., a polypeptide cargo) is delivered intracellularly across the plasma membrane. Immunofluorescence microscopy, flow cytometry, and other suitable methods may be used to assess cargo transduction efficiency. In some embodiments, a shuttle agent of the present description may enable a transduction efficiency of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, for example as measure by immunofluorescence microscopy, flow cytometry, FACS, and other suitable methods. In some embodiments, a shuttle agent of the present description may enable one of the aforementioned transduction efficiencies together wish a cell viability of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, for example as measure by the assay described in Example 3.3a, or by another suitable assay known in the art.
As used herein, the term "independent" is generally intended refer to molecules or agents which are not covalently bound to one another. For example, the expression "independent polypeptide cargo" is intended to refer to a polypeptide cargo to be delivered intracellularly that is not covalently bound (e.g., not fused) to a shuttle agent of the present description. In some aspects, having shuttle agents that are independent of (not fused to) a polypeptide cargo may be advantageous by providing increased shuttle agent versatility - e.g., not being required to re-engineer a new fusion protein for different polypeptide cargoes, and/or being able to readily vary the ratio of shuttle agent to cargo (as opposed to being limited to a 1 : 1 ratio in the case of a fusion protein).
In addition to increasing target cell transduction efficiency, shuttle agents of the present description may facilitate the delivery of a cargo of interest (e.g., a polypeptide cargo) to the cytosol of target cells. In this regard, efficiently delivering an extracellular cargo to the cytosol of a target cell using approaches based on cell penetrating peptides can be challenging, as the cargo often becomes trapped in intracellular endosomes after crossing the plasma membrane, which may limit its intracellular availability and may result in its eventual metabolic degradation. For example, use of the protein transduction domain from the HIV-1 Tat protein has been reported to result in massive sequestration of the cargo into intracellular vesicles. In some aspects, shuttle agents of the present description may facilitate the ability of endosomally-trapped cargo to escape from the endosome and gain access to the cytoplasmic compartment. In this regard, the expression "to the cytosol" in the phrase "increasing the transduction efficiency of an independent polypeptide cargo to the cytosol," is intended to refer to the ability of shuttle agents of the present description to allow an intracellular^ delivered cargo of interest to escape endosomal entrapment and gain access to the cytoplasmic compartment. After a cargo of interest has gained access to the cytosol, it may be subsequently targeted to various subcellular compartments (e.g., nucleus, nucleolus, mitochondria, peroxisome). In some embodiments, the expression "to the cytosol" is thus intended to encompass not only cytosolic delivery, but also delivery to other subcellular compartments that first require the cargo to gain access to the cytoplasmic compartment.
As used herein, a "domain" or "protein domain" generally refers to a part of a protein having a particular functionality or function. Some domains conserve their function when separated from the rest of the protein, and thus can be used in a modular fashion. By combining such domains from different proteins of viral, bacterial, or eukaryotic origin, it becomes possible in accordance with the present description to not only design multi-domain polypeptide-based shuttle agents that are able to deliver a cargo intracellular^, but also enable the cargo to escape endosomes and reach the cytoplasmic compartment.
The modular characteristic of many protein domains can provide flexibility in terms of their placement within the shuttle agents of the present description. However, some domains may perform better when engineered at certain positions of the shuttle agent (e.g., at the N- or C-terminal region, or therebetween). The position of the domain within its endogenous protein is sometimes an indicator of where the domain should be engineered within the shuttle agent, and of what type/length of linker should be used. Standard recombinant DNA techniques can be used by the skilled person to manipulate the placement and/or number of the domains within the shuttle agents of the present description in view of the present disclosure. Furthermore, assays disclosed herein, as well as others known in the art, can be used to assess the functionality of each of the domains within the context of the shuttle agents (e.g., their ability to facilitate cell penetration across the plasma membrane, endosome escape, and/or access to the cytosol). Standard methods can also be used to assess whether the domains of the shuttle agent affect the activity of the cargo to be delivered intracellularly. In this regard, the expression "operably linked" as used herein refers to the ability of the domains to carry out their intended function(s) (e.g., cell penetration, endosome escape, and/or subcellular targeting) within the context of the shuttle agents of the present description. For greater clarity, the expression "operably linked" is meant to define a functional connection between two or more domains without being limited to a particular order or distance between same.
In some embodiments, synthetic peptide or polypeptide-based shuttle agent of the present description may comprise a minimum length of 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues and a maximum length of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues. In some embodiments, shorter synthetic peptide or polypeptide-based shuttle agents are particularly advantageous because they may be more easily synthesized and purified by chemical synthesis approaches, which may be more suitable for clinical use (as opposed to recombinant proteins that must be purified from cellular expression systems). While numbers and ranges in the present description are often listed as multiples of 5, the present description should not be so limited. For example, the maximum length described herein should be understood as also encompassing a length of 36, 37, 38...51 , 62, etc., in the present description, and that their non-listing herein is only for the sake of brevity. The same reasoning applies to the % of identities listed herein (e.g., 86%, 87%...93%...), the percentages of histidine residues, etc.
In some embodiments, synthetic peptide or polypeptide-based shuttle agent of the present description may comprise a predicted net charge of at least +5, +6, +7, at least +8, at least +9, at least +10, at least +11 , at least +12, at least +13, at least +14, or at least +15 at physiological pH. These positive charges are generally conferred by the greater presence of positively-charged lysine and/or arginine residues, as opposed to negatively charged aspartate and/or glutamate residues.
In some embodiments, synthetic peptide or polypeptide-based shuttle agent of the present description may be soluble in aqueous solution (e.g., at physiological pH), which facilitates their use in for example cell culture media to delivery cargoes intracellularly to live cells.
In some embodiments, synthetic peptide or polypeptide-based shuttle agent of the present description may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 57-59, 66- 72, or 82-102, or a functional variant thereof having at least 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 % 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to any one of SEQ ID NOs: 57-59, 66-72, or 82-102.
In some embodiments, synthetic peptide or polypeptide-based shuttle agents of the present description may comprise oligomers (e.g., dimers, trimers, etc.) of a synthetic peptide or polypeptide- based shuttle agent as defined herein. Such oligomers may be constructed by covalently binding the same or different types of shuttle agent monomers (e.g., using disulfide bridges to link cysteine residues introduced into the monomer sequences).
In some embodiments, the synthetic peptide or polypeptide-based shuttle agent of the present description may comprise an N-terminal and/or a C-terminal cysteine residue.
Endosome leakage domains (ELDs)
In some aspects, synthetic peptide or polypeptide-based shuttle agents of the present description may comprise an endosome leakage domain (ELD) for facilitating endosome escape and access to the cytoplasmic compartment. As used herein, the expression "endosome leakage domain" refers to a sequence of amino acids which confers the ability of endosomally-trapped macromolecules to gain access to the cytoplasmic compartment. Without being bound by theory, endosome leakage domains are short sequences (often derived from viral or bacterial peptides), which are believed to induce destabilization of the endosomal membrane and liberation of the endosome contents into the cytoplasm. As used herein, the expression "endosomolytic peptide" is intended to refer to this general class of peptides having endosomal membrane-destabilizing properties. Accordingly, in some embodiments, synthetic peptide or polypeptide-based shuttle agents of the present description may comprise an ELD which is an endosomolytic peptide. The activity of such peptides may be assessed for example using the calcein endosome escape assays described in Example 2.
In some embodiments, the ELD may be a peptide that disrupts membranes at acidic pH, such as pH-dependent membrane active peptide (PMAP) or a pH-dependent lytic peptide. For example, the peptides GALA and INF-7 are amphiphilic peptides that form alpha helixes when a drop in pH modifies the charge of the amino acids which they contain. More particularly, without being bound by theory, it is suggested that ELDs such as GALA induce endosomal leakage by forming pores and flip-flop of membrane lipids following conformational change due to a decrease in pH (Kakudo, Chaki et al., 2004, Li, Nicol et al., 2004). In contrast, it is suggested that ELDs such as INF-7 induce endosomal leakage by accumulating in and destabilizing the endosomal membrane (El-Sayed, Futaki et al., 2009). Accordingly in the course of endosome maturation, the concomitant decline in pH causes a change in the conformation of the peptide and this destabilizes the endosome membrane leading to the liberation of the endosome contents. The same principle is thought to apply to the toxin A of Pseudomonas (Varkouhi, Scholte et al., 2011). Following a decline in pH, the conformation of the domain of translocation of the toxin changes, allowing its insertion into the endosome membrane where it forms pores (London 1992, O'Keefe 1992). This eventually favors endosome destabilization and translocation of the complex outside of the endosome. The above described ELDs are encompassed within the ELDs of the present description, as well as other mechanisms of endosome leakage whose mechanisms of action may be less well defined.
In some embodiments, the ELD may be an antimicrobial peptide (AMP) such as a linear cationic alpha-helical antimicrobial peptide (AMP). These peptides play a key role in the innate immune response due to their ability to strongly interact with bacterial membranes. Without being bound by theory, these peptides are thought to assume a disordered state in aqueous solution, but adopt an alpha-helical secondary structure in hydrophobic environments. The latter conformation thought to contribute to their typical concentration-dependent membrane-disrupting properties. When accumulated in endosomes at a certain concentrations, some antimicrobial peptides may induce endosomal leakage.
In some embodiments, the ELD may be an antimicrobial peptide (AMP) such as Cecropin-
A/Melittin hybrid (CM series) peptide. Such peptides are thought to be among the smallest and most effective AMP-derived peptides with membrane-disrupting ability. Cecropins are a family of antimicrobial peptides with membrane-perturbing abilities against both Gram-positive and Gram- negative bacteria. Cecropin A (CA), the first identified antibacterial peptide, is composed of 37 amino acids with a linear structure. Melittin (M), a peptide of 26 amino acids, is a cell membrane lytic factor found in bee venom. Cecropin-melittin hybrid peptides have been shown to produce short efficient antibiotic peptides without cytotoxicity for eukaryotic cells (i.e., non-hemolytic), a desirable property in any antibacterial agent. These chimeric peptides were constructed from various combinations of the hydrophilic N-terminal domain of Cecropin A with the hydrophobic N-terminal domain of Melittin, and have been tested on bacterial model systems. Two 26-mers, CA(1-13)M(1-13) and CA(1-8) M(1 -18) (Boman et al., 1989), have been shown to demonstrate a wider spectrum and improved potency of natural Cecropin A without the cytotoxic effects of melittin.
In an effort to produce shorter CM series peptides, the authors of Andreu et al., 1992 constructed hybrid peptides such as the 26-mer (CA(1-8)M(1-18)), and compared them with a 20-mer (CA(1 -8)M(1 -12)), a 18-mer (CA(1 -8)M(1 -10)) and six 15-mers ((CA(1 -7)M(1 -8), CA(1 -7)M(2-9), CA(1 - 7)M(3-10), CA(1-7)M(4-11), CA(1-7)M(5-12), and CA(1 -7)M(6-13)). The 20 and 18-mers maintained similar activity comparatively to CA(1 -8)M(1-18). Among the six 15-mers, CA(1-7)M(1-8) showed low antibacterial activity, but the other five showed similar antibiotic potency compared to the 26-mer without hemolytic effect. Accordingly, in some embodiments, synthetic peptide or polypeptide-based shuttle agents of the present description may comprise an ELD which is or is from CM series peptide variants, such as those described above.
In some embodiments, the ELD may be the CM series peptide CM18 composed of residues 1- 7 of Cecropin-A (KWKLFKKIGAVLKVLTTG) (SEQ ID NO: 104) fused to residues 2-12 of Melittin (YGRKKRRQRRR) (SEQ ID NO: 105), [C(1-7)M(2-12)]. When fused to the cell penetrating peptide TAT, CM18 was shown to independently cross the plasma membrane and destabilize the endosomal membrane, allowing some endosomally-trapped cargos to be released to the cytosol (Salomone et al., 2012). However, the use of a CM18-TAT11 peptide fused to a fluorophore (atto-633) in some of the author's experiments, raises uncertainty as to the contribution of the peptide versus the fluorophore, as the use of fluorophores themselves have been shown to contribute to endosomolysis - e.g., via photochemical disruption of the endosomal membrane (Erazo-Oliveras et al., 2014).
In some embodiments, the ELD may be CM18 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 1 and having endosomolytic activity.
In some embodiments, the ELD may be a peptide derived from the N terminus of the HA2 subunit of influenza hemagglutinin (HA), which may also cause endosomal membrane destabilization when accumulated in the endosome.
In some embodiments, synthetic peptide or polypeptide-based shuttle agents of the present description may comprise an ELD which is or is from an ELD set forth in Table A, or a variant thereof having endosome escape activity and/or pH-dependent membrane disrupting activity.
Figure imgf000025_0001
VGSSLSCINLDWDVIRDKTKTKIESLKEHG
PIKNKMSESPNKTVSEEKAKQYLEEFHQT
. ALEHPELSELKTVTGTNPVFAGANYAAWA (Uherek, Fominaya et uipntneria toxin VNVAQV|DSETADNLEKTTAALS|LPG|GSV al., 1998, Glover, Ng et in ' MGIADGAVHHNTEEIVAQSIALSSLMVAQA al., 2009)
IPLVGELVDIGFAAYNFVESIINLFQWHNS YNRPAYSPG
(Parente, Nir et al.,
GALA WEAALAEALAEALAEHLAEALAEALEALAA 1990) (Li, Nicol et al.,
2004)
VLAGNPAKHDLDIKPTVISHRLHFPEGGSL AALTAHQACHLPLETFTRHRQPRGWEQL (Fominaya and Wels
PEA EQCGYPVQRLVALYLAARLSWNQVDQVIR 1996) NALASPGSGGDLGEAIREQPEQARLALT
(El-Sayed, Futaki et
INF-7 GLFEAIEGFIENGWEGMIDGWYGC
al., 2009) (Kichler, Mason et al.,
LAH4 KKALLALALHHLAHLALHLALALKKA 2006)
Kichler et al., 2003 (Zhang, Cui et al.,
HGP LLGRRGWEVLKYWWNLLQYWSQEL 7
2006) (Midoux, Kichler et al., H5WYG GLFHAIAHFIHGGWHGLIHGWYG 8
1998) (Lorieau, Louis et al.,
HA2 GLFGAI AGFI ENGWEGMI DGWYG 9
2010) (Amand, Norden et al.,
EB1 LIRLWSHLIHIWFQNRRLKWKKK 10
2012) (Schuster, Wu et al., VSVG KFTIVFPHNQKGNWKNVPSNYHYCP 11
1999)
EGGSLAALTAHQACHLPLETFTRHRQPRG
Pseudomonas WEQLEQCGYPVQRLVALYLAARLSWNQV (Fominaya, Uherek et
12
toxin DQVIRNALASPGSGGDLGEAIREQPEQAR al., 1998)
LALTLAAAESERFVRQGTGNDEAGAANAD
(Tan, Chen et al.,
Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 13
2012) (Wyman, Nicol et al.,
KALA WEAKLAKALAKALAKHLAKALAKALKACEA 14
1997) (Gottschalk, Sparrow
JST-1 GLFEALLELLESLWELLLEA 15
et al., 1996) C(LLKK)3C CLLKKLLKKLLKKC 63 (Luan et al., 2014) G(LLKK)3G GLLKKLLKKLLKKG 64 (Luan et al., 2014)
In some embodiments, shuttle agents of the present description may comprise one or more ELD or type of ELD. More particularly, they can comprise at least 2, at least 3, at least 4, at least 5, or more ELDs. In some embodiments, the shuttle agents can comprise between 1 and 10 ELDs, between 1 and 9 ELDs, between 1 and 8 ELDs, between 1 and 7 ELDs, between 1 and 6 ELDs, between 1 and 5 ELDs, between 1 and 4 ELDs, between 1 and 3 ELDs, etc.
In some embodiments, the order or placement of the ELD relative to the other domains (CPD, histidine-rich domains) within the shuttle agents of the present description may be varied provided the shuttling ability of the shuttle agent is retained.
In some embodiments, the ELD may be a variant or fragment of any one those listed in Table A, and having endosomolytic activity. In some embodiments, the ELD may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 1 -15, 63, or 64, or a sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91 %, 92%, 93%, 94%, or 95% identical to any one of SEQ ID NOs: 1-15, 63, or 64, and having endosomolytic activity. Cell penetration domains (CPDs)
In some aspects, the shuttle agents of the present description may comprise a cell penetration domain (CPD). As used herein, the expression "cell penetration domain" refers to a sequence of amino acids which confers the ability of a macromolecule (e.g., peptide or protein) containing the CPD to be transduced into a cell.
In some embodiments, the CPD may be (or may be from) a cell-penetrating peptide or the protein transduction domain of a cell-penetrating peptide. Cell-penetrating peptides can serve as carriers to successfully deliver a variety of cargos intracellular^ (e.g., polynucleotides, polypeptides, small molecule compounds or other macromolecules/compounds that are otherwise membrane- impermeable). Cell-penetrating peptides often include short peptides rich in basic amino acids that, once fused (or otherwise operably linked) to a macromolecule, mediate its internalization inside cells (Shaw, Catchpole et al., 2008). The first cell-penetrating peptide was identified by analyzing the cell penetration ability of the HIV-1 trans-activator of transcription (Tat) protein (Green and Loewenstein 1988, Vives, Brodin et al., 1997). This protein contains a short hydrophilic amino acid sequence, named 'TAT", which promotes its insertion within the plasma membrane and the formation of pores. Since this discovery, many other cell-penetrating peptides have been described. In this regard, in some embodiments, the CPD can be a cell-penetrating peptide as listed in Table B, or a variant thereof having cell-penetrating activity.
Figure imgf000027_0001
(Mahlum, Mandal et al.,
SP AAVALLPAVLLALLAP 16
2007)
(Green and Loewenstein 1988, Fawell, Seery et al.,
TAT YGRKKRRQRRR 17
1994, Vives, Brodin et al.,
1997)
Penetratin
RQIKIWFQNRRMKWKK 18 (Perez, Joliot et al., 1992) (Antennapedia)
(Elmquist, Lindgren et al., pVEC LLIILRRRIRKQAHAHSK 19
2001)
(El-Andaloussi, Johansson
M918 MVTVLFRRLRIRRACGPPRVRV 20
et al., 2007) (Morris, Depollier et al.,
Pep-1 KETWWETWWTEWSQPKKKRKV 21
2001)
Pep-2 KETWFETWFTEWSQPKKKRKV 22 (Morris, Chaloin et al., 2004)
(Montrose, Yang et al., Xentry LCLRPVG 23
2013)
Arginine stretch RRRRRRRRR 24 (Zhou, Wu et al., 2009) (Hallbrink, Floren et al.,
Transportan WTLNSAGYLLGKINLKALAALAKKIL 25
2001)
SynB1 RGGRLSYSRRRFSTSTGR 26 (Drin, Cottin et al., 2003) SynB3 RRLSYSRRRF 27 (Drin, Cottin et al., 2003) PTD4 YARAAARQARA 65 (Ho et al, 2001)
Without being bound by theory, cell-penetrating peptides are thought to interact with the cell plasma membrane before crossing by pinocytosis or endocytosis. In the case of the TAT peptide, its hydrophilic nature and charge are thought to promote its insertion within the plasma membrane and the formation of a pore (Herce and Garcia 2007). Alpha helix motifs within hydrophobic peptides (such as SP) are also thought to form pores within plasma membranes (Veach, Liu et al., 2004).
In some embodiments, shuttle agents of the present description may comprise one or more CPD or type of CPD. More particularly, they may comprise at least 2, at least 3, at least 4, or at least 5 or more CPDs. In some embodiments, the shuttle agents can comprise between 1 and 10 CPDs, between 1 and 6 CPDs, between 1 and 5 CPDs, between 1 and 4 CPDs, between 1 and 3 CPDs, etc.
In some embodiments, the CPD may be TAT having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 % 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 17 and having cell penetrating activity; or Penetratin having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 % 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 18 and having cell penetrating activity.
In some embodiments, the CPD may be PTD4 having the amino acid sequence of SEQ ID NO: 65, or a variant thereof having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 % 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 65.
In some embodiments, the order or placement of the CPD relative to the other domains (ELD, histidine-rich domains) within the shuttle agents of the present description may be varied provided the shuttling ability of the shuttle agent is retained.
In some embodiments, the CPD may be a variant or fragment of any one those listed in Table
B, and having cell penetrating activity. In some embodiments, the CPD may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65, or a sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91 %, 92%, 93%, 94%, or 95% identical to any one of SEQ ID NOs: 16-27 or 65, and having cell penetrating activity. Histidine-rich domains
In some aspects, the shuttle agents of the present description may comprise a histidine-rich domain. In other embodiments, the shuttle agents of the present description may be combined/used together with a further independent synthetic peptide comprising or consisting essentially of a histidine- rich domain and a CPD (e.g., but lacking an ELD). This latter approach may provide the added advantage of allowing the concentration of the histidine-rich domain to be varied or controlled independently from the concentration of the ELD and the CPD contained in the shuttle agent. Without being bound by theory, the histidine-rich domain may act as a proton sponge in the endosome, providing another mechanism of endosomal membrane destabilization.
In some embodiments, the histidine-rich domain may be a stretch of at least 2, at least 3, at least 4, at least 5, or at least 6 amino acids comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues. In some embodiments, the histidine-rich domain may comprise at least 2, at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, or at least 9 consecutive histidine residues. Without being bound by theory, the histidine-rich domain in the shuttle agent may act as a proton sponge in the endosome through protonation of their imidazole groups under acidic conditions of the endosomes, providing another mechanism of endosomal membrane destabilization and thus further facilitating the ability of endosomally-trapped cargos to gain access to the cytosol. In some embodiments, the histidine-rich domain may be located at the N or C terminus of the synthetic peptide or shuttle agent. In some embodiments, the histidine-rich domain may be located N-terminal or C terminal to the CPD and/or ELD.
In some embodiments, the order or placement of the histidine-rich domain relative to the other domains (CPD, ELD) within the shuttle agents of the present description may be varied provided the shuttling ability of the shuttle agent is retained. In some embodiments, the shuttle agents of the present description may comprise more than one histidine-rich domain (e.g., histidine-rich domains at the amino and carboxyl termini).
Linkers
In some embodiments, suitable linkers (e.g., flexible polypeptide linkers) can be used to operably connect the domains (CPDs, ELDs, or histidine-rich domains) to one another within the context of synthetic peptides and shuttle agents of the present description. In some embodiments, linkers may be formed by adding sequences of small hydrophobic amino acids without rotatory potential (such as glycine) and polar serine residues that confer stability and flexibility. Linkers may be soft and allow the domains of the shuttle agents to move. In some embodiments, prolines may be avoided since they can add significant conformational rigidity. In some embodiments, the linkers may be serine/glycine-rich linkers (e.g., GGS, GGSGGGS (SEQ ID NO: 106), GGSGGGSGGGS (SEQ ID NO: 107), or the like). In some embodiments, the use shuttle agents comprising a suitable linker may be advantageous for delivering an independent polypeptide cargo to suspension cells, rather than to adherent cells.
Cargos
In some aspects, the synthetic peptide or polypeptide-based shuttle agent of the present description may be useful for delivering an independent cargo (e.g., a polypeptide cargo) to the cytosol of a target eukaryotic cell. In some embodiments, the polypeptide cargo may be fused to one or more CPDs to further facilitate intracellular delivery. In some embodiments, the CPD fused to the polypeptide cargo may be the same or different from the CPD of the shuttle agent of the present description. Such fusion proteins may be constructed using standard recombinant technology. In some embodiments, the independent polypeptide cargo may be fused, complexed with, or covalently bound to a second biologically active cargo (e.g., a biologically active polypeptide or compound). Alternatively or simultaneously, the polypeptide cargo may comprise a subcellular targeting domain.
In some embodiments, the polypeptide cargo must be delivered to the nucleus for it to carry out its intended biological effect. One such example is when the cargo is a polypeptide intended for nuclear delivery (e.g., a transcription factor). In this regard, studies on the mechanisms of translocation of viral DNA have led to the identification of nuclear localization signals (NLSs). The NLS sequences are recognized by proteins (importins a and β), which act as transporters and mediators of translocation across the nuclear envelope. NLSs are generally enriched in charged amino acids such as arginine, histidine, and lysine, conferring a positive charge which is partially responsible for their recognition by importins. Accordingly, in some embodiments, the polypeptide cargo may comprise an NLS for facilitating nuclear delivery, such as one or more of the NLSs as listed in Table C, or a variant thereof having nuclear targeting activity. Of course, it is understood that, in certain embodiments, the polypeptide cargo may comprise its natural NLS.
Figure imgf000030_0001
SV40 T-Ag PKKKRKV 29 (Lanford, Kanda et al., 1986)
(Makkerh, Dingwall et al., c-myc PAAKRVKLD 30
1996)
Op-T-NLS SSDDEATADSQHAAPPKKKRKV 31 (Chan and Jans 1999) (Nakanishi, Shum et al.,
Vp3 KKKRK 32
2002)
Nucleoplasmin KRPAATKKAGQAKKKK 33 (Fanara, Hodel et al., 2000)
(Moreland, Langevin et al.,
Histone 2B NILS DGKKRKRSRK 34
1987)
(Kleinschmidt and Seiter
Xenopus N1 VRKKRKTEEESPLKDKDAKKSKQE 35
1988)
(Schreiber, Molinete et al.,
PARP KRKGDEVDGVDECAKKSKK 36
1992)
PDX-1 RRMKWKK 37 (Moede, Leibiger et al., 1999)
QKI-5 RVHPYQR 38 (Wu, Zhou et al., 1999)
(Somasekaram, Jarmuz et al.,
HCDA KRPACTLKPECVQQLLVCSQEAKK 39
1999)
(Moreland, Langevin et al.,
H2B GKKRSKA 40
1987) v-Rel KAKRQR 41 (Gilmore and Temin 1988)
Amida RKRRR 42 (Irie, Yamagata et al., 2000)
RanBP3 PPVKRERTS 43 (Welch, Franke et al., 1999)
Pho4p PYLNKRKGKP 44 (Welch, Franke et al., 1999)
LEF-1 KKKKRKREK 45 (Prieve and Waterman 1999)
TCF-1 KKKRRSREK 46 (Prieve and Waterman 1999)
(Shoya, Kobayashi et al.,
BDV-P PRPRKIPR 47
1998)
TR2 KDCVINKHHRNRCQYCRLQR 48 (Yu, Lee et al., 1998)
SOX9 PRRRK 49 (Sudbeck and Scherer 1997)
Max PQSRKKLR 50 (Kato, Lee et al., 1992)
Once delivered to the cytoplasm, recombinant proteins are exposed to protein trafficking system of eukaryotic cells. Indeed, all proteins are synthetized in the cell's cytoplasm and are then redistributed to their final subcellular localization by a system of transport based on small amino acid sequences recognized by shuttle proteins (Karniely and Pines 2005, Stojanovski, Bohnert et al., 2012). In addition to NLSs, other localization sequences can mediate subcellular targeting to various organelles following intracellular delivery of the polypeptide cargos of the present description. Accordingly, in some embodiments, polypeptide cargos of the present description may comprise a subcellular localization signal for facilitating delivery of the shuttle agent and cargo to specific organelles, such as one or more of the sequences as listed in Table D, or a variant thereof having corresponding subcellular targeting activity.
Figure imgf000031_0001
Mitochondrial signal (Milenkovic, Ramming et al.,
NLVERCFTD 51
sequence from Tim9 2009) Mitochondrial signal
sequence from Yeast (Hurt, Pesold-Hurt et al.,
MLSLRQSIRFFK 52
cytochrome c oxidase subunit 1985)
IV
Mitochondrial signal (Bejarano and Gonzalez
MLISRCKWSRFPGNQR 53
sequence from 18S rRNA 1999)
Peroxisome signal sequence
SKL 54 (Gould, Keller et al., 1989)
- PTS1
Nucleolar signal sequence
MQRKPTIRRKNLRLRRK 55 (Scott, Boisvert et al., 2010) from BIRC5
Nucleolar signal sequence
KQAWKQKWRKK 56 (Scott, Boisvert et al., 2010) from RECQL4
In some embodiments, the cargo can be a biologically active compound such as a biologically active (recombinant) polypeptide (e.g., a transcription factor, a cytokine, or a nuclease) intended for intracellular delivery. As used herein, the expression "biologically active" refers to the ability of a compound to mediate a structural, regulatory, and/or biochemical function when introduced in a target cell.
In some embodiments, the cargo may be a recombinant polypeptide intended for nuclear delivery, such as a transcription factor. In some embodiments, the transcription factor can be HOXB4 (Lu, Feng et al., 2007), NUP98-HOXA9 (Takeda, Goolsby et al., 2006), Oct3/4, Sox2, Sox9, Klf4, c-Myc (Takahashi and Yamanaka 2006), MyoD (Sung, Mun et al., 2013), Pdx1 , Ngn3 and MafA (Akinci, Banga et al., 2012), Blimp-1 (Lin, Chou et al., 2013), Eomes, T-bet (Gordon, Chaix et al., 2012), FOX03A (Warr, Binnewies et al., 2013), NF-YA (Dolfini, Minuzzo et al., 2012), SALL4 (Aguila, Liao et al., 2011), ISL1 (Fonoudi, Yeganeh et al., 2013), FoxA1 (Tan, Xie et al., 2010), Nanog, Esrrb, Lin28 (Buganim et al., 2014) , HIF1-alpha (Lord-Dufour et al., 2009), Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5 (Riddell et al., 2014), or Bcl-6 (lchii, Sakamoto et al., 2004).
In some embodiments, the cargo may be a recombinant polypeptide intended for nuclear delivery, such as a nuclease useful for genome editing technologies. In some embodiments, the nuclease may be an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1 (Zetsche et al., 2015), a zinc-finger nuclease (ZFN), a Transcription activator-like effector nuclease (TALEN) (Cox et al., 2015), a homing endonuclease, a meganuclease, or any combination thereof. Other nucleases not explicitly mentioned here may nevertheless be encompassed in the present description. In some embodiments, the nuclease may be fused to a nuclear localization signal (e.g., Cas9-NLS; Cpfl-NLS; ZFN-NLS; TALEN-NLS). In some embodiments, the nuclease may be complexed with a nucleic acid (e.g., one or more guide RNAs, a crRNA, a tracrRNAs, or both a crRNA and a tracrRNA). In some embodiments, the nuclease may possess DNA or RNA-binding activity, but may lack the ability to cleave DNA.
In some embodiments, the shuttle agents of the present description may be used for intracellular delivery (e.g., nuclear delivery) of one or more CRISPR endonucleases, for example one or more of the CRISPR endonucleases described below.
Type I and its subtypes A, B, C, D, E, F and I, including their respective Cas1 , Cas2, Cas3, Cas4, Cas6, Cas7 and Cas8 proteins, and the signature homologs and subunits of these Cas proteins including Cse1 , Cse2, Cas7, Cas5, and Cas6e subunits in E. coli (type l-E) and Csy1 , Csy2, Csy3, and Cas6f in Pseudomonas aeruginosa (type l-F) (Wiedenheft et al., 2011 ; Makarova et al, 2011). Type II and its subtypes A, B, C, including their respective Cas1 , Cas2 and Cas9 proteins, and the signature homologs and subunits of these Cas proteins including Csn complexes (Makarova et al, 2011). Type III and its subtypes A, B and MTH326-like module, including their respective Cas1 , Cas2, Cas6 and Cas10 proteins, and the signature homologs and subunits of these Cas proteins including Csm and CMR complexes (Makarova et al, 2011). Type IV represents the Csf3 family of Cas proteins. Members of this family show up near CRISPR repeats in Acidithiobacillus ferrooxidans ATCC 23270, Azoarcus sp. (strain EbN1), and Rhodoferax ferrireducens (strain DSM 15236/ATCC BAA-621 T118). In the latter two species, the CRISPR/Cas locus is found on a plasmid. Type V and it subtypes have only recently been discovered and include Cpf1 , C2c1 , and C2c3. Type VI includes the enzyme C2c2, which reported shares little homology to known sequences.
In some embodiments, the shuttle agents of the present description may be used in conjunction with one or more of the nucleases, endonucleases, RNA-guided endonuclease, CRISPR endonuclease described above, for a variety of applications, such as those described herein. CRISPR systems interact with their respective nucleic acids, such as DNA binding, RNA binding, helicase, and nuclease motifs (Marakova et al, 2011 ; Barrangou & Marraffini, 2014). CRISPR systems may be used for different genome editing applications including:
• a Cas-mediated genome editing method conducting to non-homologous end-joining (NHEJ) and/or Homologous-directed recombination (HDR) (Cong et al, 2013);
• a catalytically dead Cas (dCas) that can repress and /or activate transcription initiation when bound to promoter sequences, to one or several crRNA(s) and to a RNA polymerase with or without a complex formation with others protein partners (Bikard et al, 2013);
• a catalytically dead Cas (dCas) that can also be fused to different functional proteins domains as a method to bring enzymatic activities at specific sites of the genome including transcription repression, transcription activation, chromatin remodeling, fluorescent reporter, histone modification, recombinase system acetylation, methylation, ubiquitylation, phosphorylation, sumoylation, ribosylation and citrullination (Gilbert et al, 2013). The person of ordinary skill in the art will understand that the present shuttle agents, although exemplified with Cas9 in the present examples, may be used with other nucleases as described herein. Thus, nucleases such as Cpf1 , Cas9, and variants of such nucleases or others, are encompassed by the present description. It should be understood that, in one aspect, the present description may broadly cover any cargo having nuclease activity, such an RNA-guided endonuclease, or variants thereof (e.g., those that can bind to DNA or RNA, but have lost their nuclease activity; or those that have been fused to a transcription factor).
In some embodiments, the polypeptide cargo may be a cytokine such as a chemokine, an interferon, an interleukin, a lymphokine, or a tumour necrosis factor. In some embodiments, the polypeptide cargo may be a hormone or growth factor. In some embodiments, the cargo may be an antibody (e.g., a labelled antibody). In some embodiments, the cargo can be a detectable label (fluorescent polypeptide or reporter enzyme) that is intended for intracellular delivery, for example, for research and/or diagnostic purposes.
In some embodiments, the cargo may be a globular protein or a fibrous protein. In some embodiments, the cargo may have a molecule weight of any one of about 5, 10, 15, 20, 25, 30, 35, 40, 45, to 50 to about 150, 200, 250, 300, 350, 400, 450, 500 kDa or more. In some embodiments, the cargo may have a molecule weight of between about 20 to 200 kDa.
Non-toxic, metabolizable synthetic peptides and shuttle agents
In some embodiments, synthetic peptides and shuttle agents of the present description may be non-toxic to the intended target eukaryotic cells at concentrations up to 50 μΜ, 45 μΜ, 40 μΜ, 35 μΜ, 30 μΜ, 25 μΜ, 20 μΜ, 15 μΜ, 10 μΜ, 9 μΜ, 8 μΜ, 7 μΜ, 6 μΜ, 5 μΜ, 4 μΜ, 3 μΜ, 2 μΜ, 1 μΜ, 0.5 μΜηι 0.1 μΜ, or 0.05 μΜ. Cellular toxicity of shuttle agents of the present description may be measured using any suitable method. Furthermore, transduction protocols may be adapted (e.g., concentrations of shuttle and/or cargo used, shuttle/cargo exposure times, exposure in the presence or absence of serum), to reduce or minimize toxicity of the shuttle agents, and/or to improve/maximize transfection efficiency.
In some embodiments, synthetic peptides and shuttle agents of the present description may be readily metabolizable by intended target eukaryotic cells. For example, the synthetic peptides and shuttle agents may consist entirely or essentially of peptides or polypeptides, for which the target eukaryotic cells possess the cellular machinery to metabolize/degrade. Indeed, the intracellular half-life of the synthetic peptides and polypeptide-based shuttle agents of the present description is expected to be much lower than the half-life of foreign organic compounds such as fluorophores. However, fluorophores can be toxic and must be investigated before they can be safely used clinically (Alford et al., 2009). In some embodiments, synthetic peptides and shuttle agents of the present description may be suitable for clinical use. In some embodiments, the synthetic peptides and shuttle agents of the present description may avoid the use of domains or compounds for which toxicity is uncertain or has not been ruled out.
Cocktails
In some embodiments, the present description relates to a composition comprising a cocktail of at least 2, at least 3, at least 4, or at least 5 different types of the synthetic peptides or polypeptide- based shuttle agents as defined herein. In some embodiments, combining different types of synthetic peptides or polypeptide-based shuttle agents (e.g., different shuttle agents comprising different types of CPDs) may provide increased versatility for delivering different polypeptide cargos intracellular^. Furthermore, without being bound by theory, combining lower concentrations of different types of shuttle agents may help reduce cellular toxicity associated with using a single type of shuttle agent (e.g., at higher concentrations).
Methods, kits, uses and cells
In some embodiments, the present description relates to a method for increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell. The method may comprise contacting the target eukaryotic cell with the synthetic peptide, polypeptide-based shuttle agent, or composition as defined herein, and the polypeptide cargo. In some embodiments, the synthetic peptide, polypeptide-based shuttle agent, or composition may be pre-incubated with the polypeptide cargo to form a mixture, prior to exposing the target eukaryotic cell to that mixture. In some embodiments, the type of CPD may be selected based on the amino acid sequence of the polypeptide cargo to be delivered intracellular^. In other embodiments, the type of CPD and ELD may be selected to take into account the amino acid sequence of the polypeptide cargo to be delivered intracellularly, the type of cell, the type of tissue, etc.
In some embodiments, the method may comprise multiple treatments of the target cells with the synthetic peptide, polypeptide-based shuttle agent, or composition (e.g., 1 , 2, 3, 4 or more times per day, and/or on a pre-determined schedule). In such cases, lower concentrations of the synthetic peptide, polypeptide-based shuttle agent, or composition may be advisable (e.g., for reduced toxicity). In some embodiments, the cells may be suspension cells or adherent cells. In some embodiments, the person of skill in the art will be able to adapt the teachings of the present description using different combinations of shuttles, domains, uses and methods to suit particular needs of delivering a polypeptide cargo to particular cells with a desired viability.
In some embodiments, the methods of the present description may apply to methods of delivering a polypeptide cargo intracellularly to a cell in vivo. Such methods may be accomplished by parenteral administration or direct injection into a tissue, organ, or system.
In some embodiments, the synthetic peptide, polypeptide-based shuttle agent, or composition, and the polypeptide cargo may be exposed to the target cell in the presence or absence of serum. In some embodiments, the method may be suitable for clinical or therapeutic use.
In some embodiments, the present description relates to a kit for increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell. The kit may comprise the synthetic peptide, polypeptide-based shuttle agent, or composition as defined herein, and a suitable container.
In some embodiments, the target eukaryotic cells may be an animal cell, a mammalian cell, or a human cell. In some embodiments, the target eukaryotic cells may be a stem cell (e.g., embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, peripheral blood stem cells), primary cells (e.g., myoblasts, fibroblasts), or an immune cell (e.g., T cells, NK cells, dendritic cells, antigen presenting cells). In some embodiments, the present description relates to an isolated cell comprising a synthetic peptide or polypeptide-based shuttle agent as defined herein. In some embodiments, the cell may be a protein- induced pluripotent stem cell. It will be understood that cells that are often resistant or not amenable to protein transduction may be interesting candidates for the synthetic peptides or polypeptide-based shuttle agents of the present description.
Peptide shuttle-based cargo protein delivery in eukaryotic cells- therapeutic applications
Disclosed herein are methods for delivering cargo into a eukaryotic cell using a polypeptide- based shuttle as disclosed herein. In a variety of examples, delivery of cargo is performed for therapeutic applications. In such cases, the polypeptide-based shuttles disclosed herein increase the efficiency of 1) translocation across the cell member, 2) escape from the endosome, 3) allowing delivery to the targeted subcellular location. Combined, the increased efficiency of these steps leads to a desired outcome, such as a genetic engineering event by the delivery of a genetic engineering protein such as Cas9, or expression modification by the delivery of proteins such as transcription factors. An additional characteristic of the polypeptide-based shuttles disclosed herein is that they are non-toxic to the host cells and are able to be degraded by the host cell after the delivery function is complete. Disclosed herein are methods for delivering cargo to a cell using a polypeptide-based shuttle as described herein. Additionally disclosed herein are methods for expression modification of target genes. Cargo is any combination of polypeptide, nucleic acid, and/or other molecule. In many examples, the cargo is a polypeptide or protein. Additionally or alternatively, the cargo includes DNA or RNA. Sometime the cargo is a transcription factor or functional fragment thereof. For example, the transcription factor is HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Klf4, c-Myc, MyoD, Pdx1 , Ngn3 and MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1 , FoxA1 , Nanog, Esrrb, Lin28, HI F1 -alpha, Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5, Bcl-6, Sox9, or Yamanaka factors. In certain examples, the transcription factor is HoxB4. In cases where the cargo is a protein or polypeptide, the cargo often comprises a subcellular localization domain. For example, the subcellular localization domain can be a nuclear localization signal, nucleolar localization signal, mitochondrial localization signal, peroxisome localization signal, or cytosol localization signal.
Cargo is delivered into a cell using any polypeptide-based shuttle disclosed herein. In some examples the cell is a eukaryotic cell. Often the eukaryotic cell is a human cell. Alternatively, the cell is a mammalian, animal, plant, archaea, or bacterial cell. In some examples, the cell is a stem cell or stem-cell derived cell. For example, the cell is a hematopoietic stem cell or a megakaryocyte. It will be readily recognize by one of skill in the art that many cell types would be useful in these methods. In some examples, the cells are isolated from patients or human donors. Often the cells are isolated or maintained ex vivo. In other instances, the cells are in vivo, or alternatively, in vitro.
In some embodiments the cargo protein complexed with the polypeptide-based shuttle is intended for delivery to the nucleus. For example, the transcription factor HoxB4 is functional in the nucleus and the transcription factor's inherent NLS and/or an additional NLS ensure deliver of said transcription factor to the nucleus. In some aspects, the polypeptide-based shuttle has a cell penetrating domain (CPD), for transport of HoxB4 across the cell membrane, and an endosome leakage domain (ELD), to reduce sequestration of HoxB4 in the endosome. In such cases, the cargo protein, such as HoxB4, comprises a nuclear localization signal (NLS), to target HoxB4 to the nucleus. In other examples, the cargo comprises a subcellular targeting domain for delivery of the cargo to a desired subcellular localization. For example, a mitochondrial or peroxisome localization domain is used to target cargo to the mitochondria or peroxisome respectively. Other known subcellular targeting domains, including those disclosed herein, are envisioned for use and would be readily recognized by one of skill in the art as being sufficient for use in the methods disclosed herein.
In some cases the cargo protein is a transcription factor which is selected in order to initiate a specific function within the cell. For example, HoxB4 is used for the expansion of hematopoietic stem cells. In such examples, HoxB4 is complexed with a polypeptide-based shuttle as disclosed herein, which is subsequently delivered to the nucleus where HoxB4 functions to expand the HSC population such that the expanded population is greater in number than the starting population.
In some embodiments, the cargo protein is a transcription factor that modifies the expression of target proteins. In some examples, the expression modification leads to cell expansion. In other examples, expression modification leads to cell differentiation. Other outcomes of transcription factor activity are envisioned as well and are well known in the art.
Cells obtained from methods described herein are used, in some embodiments, to treat patients with a condition. In some examples, the condition is a genetic condition. Alternatively or additionally, the condition is a genetic disorder or blood malignancy. In other cases, the condition is ischemic heart failure or an indication requiring a hematopoietic stem cell transplant. In some examples, the condition is thrombocytopenia. In cases where a patient is being treated, the cells used in the methods provided herein are autologous cells isolated from the patient. In other examples, the cells are isolated from a different person. In some embodiments where cells are derived from a person other than the patient, such as a donor, the cells will be further engineered in order to make them immunogenic so they will not be rejected by the patient or cause another sort of adverse effect associated with immunogenicity.
In other embodiments, the cells obtained from methods described herein are subsequently treated further before being used to treat a patient. For example, an expanded hematopoietic stem cell population can be treated with differentiation factors to generate megakaryocytes which are subsequently used to treat a patient in need thereof as described herein. Alternatively, an expanded stem cell population can be differentiated into a desired downstream cell type using methods known in the art.
Alternatively, isolated cells can be treated with transcription factors, such as Yamanaka factors in order to generate induced pluripotent stem cells. In other examples, cells are treated with the transcription factor Sox9 in order to generate chondrocytes.
Following modification of target gene expression by the delivery of protein cargo complexed with a polypeptide-based shuttle as disclosed herein, the generated cells are sometimes further treated. For example, the cells are sometimes further engineered to generate non-immunogenic cells. As an example, expanded hematopoietic stem cell-derived megakaryocytes can be further treated to generate non-immunogenic megakaryocytes. In some embodiments, the non-immunogenic megakaryocytes are generated by disrupting a major histocompatibility complex gene sequences. In such examples, target gene disruption is achieved, for example, by a DNA nuclease. Often the DNA nuclease is an RNA- guided nuclease such as Cas9 or Cpfl In other examples, the RNA-guided nuclease is a Cas protein. Alternatively, the gene disruption is achieve through the activity of TALENs or ZFNs. Disclosed herein are methods for gene disruption. Gene disruption is achieved, in some examples, by the delivery of a DNA-disrupting agent complexed with a polypeptide-based shuttle as disclosed herein. DNA-disrupting agents include proteins or other molecules capable of disrupting a nucleic acid sequence, such as DNA-binding agents, DNA-degrading agents, or DNA-cleaving agents. Some examples of DNA-cleaving agents include RNA-guided nucleases such as Cas proteins. Cas proteins include Cas9 and Cpfl Other DNA-cleaving agents include TALENs or ZFNs. Many other DNA-disrupting agents are known in the art and would be readily recognized as sufficient for methods disclosed herein.
In some embodiments, the gene-disrupting agent is delivered to the nucleus by a polypeptide- based shuttle and NLS as disclosed herein. For example, Cas9 or Cpfl are delivered to the cell and escape the endosome through a complexed interaction with a polypeptide-based shuttle, and are subsequently delivered to the nucleus due to the nuclear localization signal fused to Cas9 or Cpfl as disclosed herein. Alternatively, in some examples, the gene-disrupting agent is small enough or otherwise suited to diffuse into the nucleus without need for an NLS. In some cases, the gene- disrupting agent comprises an inherent NLS, while in other cases the gene-disrupting agent is engineered to comprise an NLS or an additional NLS. Many appropriate nuclear localization signals are known in the art.
Gene-disrupting agents delivered with a polypeptide-based shuttle as disclosed herein are used to target a nucleic acid sequence of interest. For example, Cas9 is guided to a target DNA sequence by an engineered crRNA and corresponding trRNA. Similarly, Cpfl is guided to a target DNA sequence by an engineered crRNA. In these examples, Cas9 or Cpfl cleave the target DNA sequence leading to disruption of the gene product. Cleavage occurs through the generation of a double strand break, or alternatively a single strand break when using a modified Cas9 or Cpfl protein. Disruption of the target DNA sequence occurs through the non-homologous end joining (NJEJ) repair system which imperfectly connects the two exposed ends resulting from the cleavage event, which often leads nucleotides being added or deleted and thereby disrupting the reading frame of the gene product. Additionally or alternatively, a donor sequence lacking homology to the regions flanking the cut site can be incorporated into the cut site by NHEJ. Alternatively, if a donor sequence is provided which shares homology with the sequences flanking the cut site, then disruption occurs by incorporation of the donor sequence through the homology driven recombination (HDR) repair system. In some embodiments, sequences between the two regions matching the homologous regions of the donor sequence are deleted upon incorporation of the donor sequence. In some embodiments, the deleted sequence comprises the target sequence of interest, which often comprises a target gene of target gene fragment of interest. In embodiments, the donor sequence comprises a selective marker, reporter marker, or other exogenous gene of interest. In yet other examples, when using a catalytically dead RNA-guided nuclease, the DNA is not cleaved; instead the dead nuclease binds to the target DNA sequence and blocks transcription from occurring, thereby disrupting production of the gene product. Other methods for targeted gene disruption are well-known in the art and are sufficient for incorporation into the methods provided herein.
Selection of a DNA sequence for targeted disruption depends on the intended outcome. For example, in order to generate non-immunogenic cells, a gene disrupting agent-peptide shuttle complex is used to delete or disrupt a human leukocyte antigen (HLA) sequence or a major histocompatibility complex (MHC) gene sequence. Additionally or alternatively, other immunogenicity target sequences are deleted or disrupted. In some examples, immunogenicity targets are disrupted in megakaryocytes. In other examples, immunogenicity targets are disrupted in stem cells to generate, as an example, universal stem cells. In other examples, immunogenicity targets are deleted or disrupted in T cells or chimeric antigen receptor t cells. Non-immunogenic cells are used for treatment of a variety of diseases. Such diseases include cancer, thrombocytopenia, other blood malignancies, and other genetic diseases.
In some embodiments, disruption or deletion of a target sequence through methods disclosed herein are used for treatment of infection. For example, viral sequences or other infective nucleotide sequences are targeted for disruption or deletion using a DNA-disrupting agent, such as Cas9, complexed with a polypeptide-based shuttle as disclosed herein. In some examples, the viral sequence can be HIV. In other embodiments, a sequence is targeted using methods disclosed herein in order to treat a genetic disease. In some of these cases, the genetic disease is a blood disease.
For some embodiments, cells are eukaryotic cells. In some embodiments, the cells are human. In other examples, the cells are mammalian, animal, plant, or any other eukaryotic cell. When the cells are human, they are often isolated from a patient. In many of these cases, the cells are an autologous population. In other examples, human cells are isolated from a donor. In many of these cases, the isolated cells are an allogenic population. In some aspects, the isolated human cells are stem cells. Stem cells are isolated from a variety of sources, including peripheral blood, bone marrow, and umbilical cord blood. Therapeutic applications- hematopoietic stem cell expansion
A common treatment for patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia, is a hematopoietic stem cell (HSC) transplant. The HSCs to be transplanted can be isolated from the patient (autologous sample) or from a donor (allogenic sample). Autologous samples are often preferred since the patient will not have an adverse immunogenic response or rejection of the sample since it is derived from the patient's own peripheral blood, bone marrow, or umbilical cord blood. The number of HSCs to be transplanted into the patient will affect the prognosis of the transplant, and a threshold population size is often required before a transplant can proceed. Unfortunately, for many patients in need of a HSC transplant, the number of cells isolated from the patient is below this threshold.
Disclosed herein are methods for expanding a HSC population. In some embodiments, the expanded HSC population is subsequently transplanted into a patient in need thereof. In some examples the HSC population is an autologous population from the patient to be treated. In certain cases, the expanded HSC population is above the threshold needed for transplantation.
In some embodiments, HSC expansion is achieved by delivery of a transcription factor involved in HSC expansion. The transcription factor in some embodiments is a Hox family transcription factor. I n specific examples, the transcription factor is HoxB4. For example, HoxB4 is delivered to a HSC nucleus by being complexed with a polypeptide-based shuttle comprising a cell penetrating domain (CPD) and endosome leakage domain (ELD). Furthermore the transcription factor comprises a nuclear localization signal (NLS) as disclosed herein. In other examples, the transcription factor is engineered to comprise an NLS or an additional NLS. The polypeptide-based shuttle is any polypeptide-based shuttle or combination of CPD and ELD disclosed herein. Cargo proteins such as transcription factors destined for the nucleus comprise any NLS sequences disclosed herein or native NLS sequences.
The patient to be treated with the expanded HSC population is in need of a HSC transplantation due to a condition. In a variety of examples, the condition is a malignant condition. Malignant conditions include acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma, neuroblastoma, Ewing sarcoma, myelodysplasia syndrome, glioma, solid tumors, and some genetic diseases, among others. Alternatively, the condition is a non-malignant condition. Non-malignant conditions include thalassemia, sickle cell amenia, aplastic anemia, Fanconi anemia, immune deficiency syndromes, inborn errors of metabolism, and some genetic diseases, among others.
In some examples, expanded stem cell populations are subsequently differentiated into other cells of interest. Differentiation is achieved by contacting the cell population with differentiation factors. Differentiation factors vary depending on the desired cell type and combinations of differentiation factors are well known in the art. In some examples, an expanded HSC population generated using the methods disclosed herein are subsequently differentiated into megakaryocytes. Differentiated cells are subsequently used to treat patients in need thereof. For example, an expanded megakaryocyte population is used in some examples to treat a patient with thrombocytopenia. Therapeutic applications- non-immunogenic megakaryocytes
A common treatment for patients with certain condition is transplantation with specific cells. For example, thrombocytopenia can be treated with transplantation of megakaryocytes. In many cases, autologous samples are not able to be acquired, and therefore allogenic samples from other donors are used in the transplantation. In such cases, immune rejection is a major health risk to the patient.
Disclosed herein are methods for generating non-immunogenic cells. The generated non- immunogenic cells are often subsequently transplanted into patients in need. For example, non- immunogenic megakaryocytes are obtained from methods disclosed herein. Non-immunogenic cells are generated by disruption of immunogenicity targets or genes, for example HLA or MHC genes.
When generating non-immunogenic megakaryocytes, the starting megakaryocytes are be isolated from a patient or donor. Alternatively, HSCs are isolated from a donor or patient and subsequently expanded and differentiated into megakaryocytes using methods disclosed herein.
Therapeutic applications- gene insertion
Disclosed herein are methods of treating a disorder, such as a genetic disease, by inserting a nucleic acid sequence, or a gene, into a target DNA site within a cell using any of the polypeptide shuttles and methods disclosed herein. In some examples, insertion of a nucleic acid sequence or gene results in treatment or amelioration of symptoms of a disease or disorder. In some examples, the genetic disorder is Krabbe Disease, Tay-Sachs Disease, or Hurler Syndrome. In some examples, the gene being inserted is GALC, HEXA, or IDUA. In some examples, the gene being inserted is a normal copy of a gene, and in some of these cases, the normal gene can be inserted into a corresponding abnormal gene locus. In some examples, the insertion site is an abnormal GALC, HEXA, or IDUA gene. In some examples, the insertion site is an albumin gene. In some examples the cell is a eukaryotic cell. In some examples, the cell is a stem cell, hematopoietic cell, central nervous system cell, microgilia cell, neuron, liver cell, hepatocyte, or liver endothelia cell. In some examples, the peptide shuttle and donor gene or donor nucleic acid is delivered ex vivo, or by direct injection. In some examples, direct injection comprises direct CNS injection, or liver intra-arterial injection.
Additional embodiments
Provided herein are methods for delivering cargo protein to a hematopoietic stem cell (HSC) comprising contacting the HSC with a polypeptide-based shuttle complexed with the cargo protein, wherein the polypeptide-based shuttle comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, and wherein the cargo protein and the polypeptide-based shuttle have independent protein backbones. Further provided herein are methods wherein the polypeptide-based shuttle further comprises a histidine rich domain. Further provided herein are methods wherein the cargo protein comprises a subcellular targeting domain Further provided herein are methods wherein the subcellular targeting domain is an organelle localization domain. Further provided herein are methods wherein the organelle localization domain is a nuclear localization signal (NLS). Further provided herein are methods wherein the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity. Further provided herein are methods wherein the cargo protein and the polypeptide-based shuttle are not covalently linked. Further provided herein are methods wherein the cargo protein comprises a transcription factor, or a functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a mammalian Hox family transcription factor, or a functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a human Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises HoxB4, or a functional fragment thereof. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity, and wherein the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof. Further provided herein are methods wherein the polypeptide-based shuttle is non-toxic and/or metabolizable. Further provided herein are methods wherein the HSC is a mammalian HSC. Further provided herein are methods wherein the mammalian HSC is a human HSC.
Provided herein are methods for expanding a hematopoietic stem cell (HSC) population comprising contacting the HSC population having a starting population size with a polypeptide-based shuttle complexed with a cargo protein such that the HSC population expands beyond the starting population size, wherein the polypeptide-based shuttle comprises comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, wherein the cargo protein comprises a subcellular targeting domain, and wherein the polypeptide-based shuttle and the cargo protein have independent protein backbones. Further provided herein are methods wherein the polypeptide-based shuttle further comprises a histidine rich domain. Further provided herein are methods wherein the polypeptide-based shuttle and the cargo protein are not covalently linked. Further provided herein are methods wherein the subcellular targeting domain comprises a nuclear localization signal (NLS). Further provided herein are methods wherein the protein cargo comprises a transcription factor, or a functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a mammalian Hox family transcription factor, or a functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a human Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises HoxB4, or a functional fragment thereof. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity. Further provided herein are methods wherein the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity, and wherein the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof. Further provided herein are methods wherein the HSC population is a mammalian HSC population. Further provided herein are methods wherein the HSC population is a human HSC population. Further provided herein are methods wherein the HSC population is an autologous HSC population. Further provided herein are methods wherein the HSC population is an allogenic HSC population. Further provided herein are methods wherein the HSC population is a CD34+ population. Further provided herein are methods wherein the HSC population is isolated from cord blood. Further provided herein are methods wherein the HSC population is isolated from bone marrow. Further provided herein are methods wherein the HSC population is isolated from peripheral blood. Further provided herein are methods wherein the expanded HSC population is differentiated to produce a differentiated population. Further provided herein are methods further comprising transplantation of the expanded population into a patient in need thereof due to a condition. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood malignancy. Further provided herein are methods wherein the condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are methods wherein the condition is an indication requiring a hematopoietic stem cell transplantation. Further provided herein are methods wherein the differentiated population is an autologous differentiated population. Further provided herein are methods wherein the differentiated population is an allogenic differentiated population. Further provided herein are methods wherein the differentiated population is a megakaryocyte population. Further provided herein are methods of treating a patient with a condition by administering to the patient in need thereof an expanded HSC population obtained by any of the methods disclosed herein. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood disorder. Further provided herein are methods wherein the condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are uses of an expanded HSC population obtained by aby of the methods disclosed herein for treatment of a patient in need thereof due to a condition. Further provided herein are uses wherein the condition is a genetic disease. Further provided herein are uses wherein the condition is a blood disorder. Further provided herein are uses wherein the condition is a malignant condition. Further provided herein are uses wherein the condition is a non-malignant condition.
Provided herein are methods for generating a population of megakaryocytes comprising, contacting a hematopoietic stem cell (HSC) population having a starting population size with a polypeptide-based shuttle complexed with a transcription factor, such that the HSC population expands beyond the starting population size, and contacting the expanded HSC population with differentiating factors such that at least a portion of the HSC population differentiates into megakaryocytes, thereby generating a megakaryocyte population, wherein the polypeptide-based shuttle comprises comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, wherein the transcription factor comprises a nuclear localization signal (NLS), and wherein the polypeptide-based shuttle and the cargo protein have independent protein backbones. Further provided herein are methods wherein the polypeptide-based shuttle further comprises a histidine rich domain. Further provided herein are methods wherein the polypeptide-based shuttle and the transcription factor are not covalently linked. Further provided herein are methods wherein the transcription factor comprises a Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a mammalian Hox family transcription factor, or a functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises a human Hox family transcription factor, or functional fragment thereof. Further provided herein are methods wherein the transcription factor comprises HoxB4, or a functional fragment thereof. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity. Further provided herein are methods wherein the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity, and wherein the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof. Further provided herein are methods wherein the HSC population is human HSC population. Further provided herein are methods wherein the human HSC population is a CD34+ population. Further provided herein are methods wherein the HSC population is isolated from cord blood. Further provided herein are methods wherein the HSC population is isolated from bone marrow. Further provided herein are methods wherein the HSC population is isolated from peripheral blood. Further provided herein are methods further comprising transplantation of the megakaryocyte population into a patient in need thereof due to a condition. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood malignancy. Further provided herein are methods wherein the condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are methods wherein the condition is an indication requiring a hematopoietic stem cell transplant. Further provided herein are methods wherein the condition is thrombocytopenia. Further provided herein are methods wherein the megakaryocyte population is an autologous population. Further provided herein are methods wherein the megakaryocyte population is an allogenic population. Further provided herein are methods wherein the megakaryocyte population is non-immunogenic.
Provided herein are methods for generating a non-immunogenic megakaryocyte comprising contacting a megakaryocyte with a polypeptide-based shuttle complexed with a DNA cleavage protein, such that the DNA cleavage protein cleaves at least one immunogenic target DNA sequence within the megakaryocyte, thereby rendering the at least one immunogenic target DNA sequence non-functional, wherein the polypeptide-based shuttle comprises comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, wherein the DNA cleavage protein comprises a nuclear localization signal (NLS), and wherein the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones. Further provided herein are methods wherein the polypeptide-based shuttle further comprises a histidine rich domain. Further provided herein are methods wherein the polypeptide-based shuttle and the DNA cleavage protein are not covalently linked.
89. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16- 27 or 65, or a variant or functional fragment thereof having cell penetrating activity. Further provided herein are methods wherein the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity, and wherein the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof. Further provided herein are methods wherein the DNA cleavage protein is an RNA-guided nuclease. Further provided herein are methods wherein the RNA-guided nuclease comprises a Cas protein. Further provided herein are methods wherein the Cas protein comprises Cas9, Cpf1 , or a functional fragment thereof. Further provided herein are methods wherein the polypeptide-based shuttle and the RNA-guided nuclease are further complexed with at least one guiding RNA. Further provided herein are methods wherein the RNA-guided nuclease comprises Cas9 and is further complexed with a crRNA and a trRNA. Further provided herein are methods wherein the RNA-guided nuclease comprises Cpf1 and is further complexed with a guiding RNA. Further provided herein are methods wherein the at least one guiding RNA is engineered to target the at least one immunogenic target DNA. Further provided herein are methods wherein the RNA-guided nuclease and at least one guiding RNA contact and cleave the at least one immunogenic target DNA. Further provided herein are methods wherein cleavage of the at least one immunogenic target DNA results in a disrupted immunogenic target gene following DNA repair, such that the immunogenic target gene or gene product is non-functional. Further provided herein are methods wherein the at least one immunogenic target DNA comprises an MHC gene. Further provided herein are methods wherein the at least one immunogenic target DNA comprises a sequence sharing at least 90% identity to a nucleic acid sequence comprised within an MHC gene. Further provided herein are methods further comprising transplantation of the non-immunogenic megakaryocyte population into a patient in need thereof due to a condition. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood malignancy. Further provided herein are methods wherein the condition is a malignant condition. Further provided herein are methods wherein the condition is a non-malignant condition. Further provided herein are methods wherein the condition is an indication requiring a hematopoietic stem cell transplant. Further provided herein are methods wherein the condition is thrombocytopenia. Further provided herein are methods of treating a patient with a condition by administering to the patient in need thereof non-immunogenic cells obtained by any of the methods disclosed herein. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood disorder. Further provided herein are methods wherein the condition is thrombocytopenia. Further provided herein are uses of non- immunogenic cells obtained by any of the methods disclosed herein for treatment of a patient in need thereof due to a condition. Further provided herein are uses wherein the condition is a genetic disease. Further provided herein are uses wherein the condition is a blood disorder. Further provided herein are uses wherein the condition is thrombocytopenia.
Provided herein are methods of modifying gene expression in a cell comprising contacting the cell with a polypeptide-based shuttle complexed with a transcription factor, wherein the polypeptide- based shuttle comprises comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, wherein the transcription factor comprises a subcellular targeting domain and wherein the transcription factor and the polypeptide-based shuttle have independent protein backbones. Further provided herein are methods wherein the polypeptide-based shuttle and the transcription factor are not covalently linked. Further provided herein are methods wherein the polypeptide-based shuttle further comprises a histidine rich domain. Further provided herein are methods wherein the subcellular targeting domain comprises a nuclear localization signal (NLS). Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity. Further provided herein are methods wherein the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the CPD comprises at least one of SEQ ID NOs: 16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity, and wherein the ELD comprises at least one of SEQ ID NOs: 1-15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity. Further provided herein are methods wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof. Further provided herein are methods wherein the transcription factor is a mammalian transcription factor. Further provided herein are methods wherein the transcription factor is a human transcription factor. Further provided herein are methods wherein the cell is within a population. Further provided herein are methods wherein modified gene expression leads to expansion of the cell population. Further provided herein are methods wherein the expanded cell population is transplanted into a patient in need thereof due to a condition. Further provided herein are methods wherein the condition is a genetic disease. Further provided herein are methods wherein the condition is a blood malignancy. Further provided herein are methods wherein the condition is an indication requiring a hematopoietic stem cell transplant. Further provided herein are methods wherein the condition is thrombocytopenia. Further provided herein are methods wherein the cell is a stem cell. Further provided herein are methods wherein the cell is a hematopoietic stem cell. Further provided herein are methods wherein the transcription factor comprises HoxB4, or a functional fragment thereof. Further provided herein are methods wherein the modified gene expression leads to differentiation of the cell population. Further provided herein are methods wherein the cell is a stem cell. Further provided herein are methods wherein the cell is a hematopoietic stem cell.
Provided herein are methods for delivering cargo protein to a hematopoietic stem cell (HSC) comprising contacting the HSC with a polypeptide-based shuttle complexed with the cargo protein, wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs. 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the cargo protein and the polypeptide-based shuttle have independent protein backbones, wherein the cargo protein and the polypeptide-based shuttle are not covalently linked, wherein the cargo protein comprises a HoxB4 transcription factor, or a functional fragment thereof, and wherein the HSC is a human HSC.
Provided herein are methods for expanding a hematopoietic stem cell (HSC) population comprising contacting the HSC population having a starting population size with a polypeptide-based shuttle complexed with a cargo protein such that the HSC population expands beyond the starting population size, wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the polypeptide-based shuttle and the cargo protein have independent protein backbones, wherein the polypeptide-based shuttle and the cargo protein are not covalently linked, wherein the protein cargo comprises a HoxB4 transcription factor, or a functional fragment thereof, wherein the HSC population is a human HSC population. Further provided herein are methods of treating a patient in need of a HSC tranplant by transplanting to the patient in need thereof an expanded HSC population obtained by any of the methods disclosed herein. Further provided herein are uses of an expanded HSC population obtained by any of the methods disclosed herein for treatment of a patient in need of a HSC transplant.
Provided herein are methods for generating a population of megakaryocytes comprising, contacting a hematopoietic stem cell (HSC) population having a starting population size with a polypeptide-based shuttle complexed with a transcription factor, such that the HSC population expands beyond the starting population size, and contacting the expanded HSC population with differentiating factors such that at least a portion of the HSC population differentiates into megakaryocytes, thereby generating a megakaryocyte population, wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the polypeptide-based shuttle and the cargo protein have independent protein backbones, wherein the polypeptide-based shuttle and the transcription factor are not covalently linked, wherein the transcription factor comprises HoxB4, or a functional fragment thereof, wherein the HSC population is human HSC population. Provided herein are methods for generating a non-immunogenic megakaryocyte comprising contacting a megakaryocyte with a polypeptide-based shuttle complexed with a DNA cleavage protein, such that the DNA cleavage protein cleaves at least one immunogenic target DNA sequence within the megakaryocyte, thereby rendering the at least one immunogenic target DNA sequence non-functional, wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof, wherein the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones, wherein the polypeptide-based shuttle and the DNA cleavage protein are not covalently linked, wherein the DNA cleavage protein comprises Cas9 and is further complexed with a crRNA and a trRNA, wherein the crRNA is engineered to target an MHC gene sequence, such that Cas9 contacts and cleaves the MHC gene sequence, and wherein cleavage of the MHC gene sequence results in a disrupted MHC gene following DNA repair, such that the MHC gene or gene product is non-functional, thereby rendering the megakaryocyte non-immunogenic. Further provided herein are methods of treating a patient with thrombocytopenia by administering to the patient in need thereof non-immunogenic megakaryocytes obtained by any of the methods disclosed herein. Further provided herein are uses of non-immunogenic megakaryocytes obtained by any of the methods disclosed herein for treatment of a thrombocytopenia patient in need thereof.
The present description may additionally or alternatively relate to the following aspects:
(1) A synthetic peptide comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD. (2) A polypeptide-based shuttle agent comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), or an ELD operably linked to a histidine-rich domain and a CPD, for use in increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell.
(3) The synthetic peptide or polypeptide-based shuttle agent of (1) or (2), wherein the synthetic peptide or polypeptide-based shuttle agent: (a) comprises a minimum length of 20, 21 , 22, 23,
24, 25, 26, 27, 28, 29, or 30 amino acid residues and a maximum length of 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues; (b) has a predicted net charge of at least +6, +7, +8, +9, +10, +11 , +12, +13, +14, or +15 at physiological pH; (c) is soluble in aqueous solution; or (d) any combination of (a) to (c). (4) The synthetic peptide or polypeptide-based shuttle agent of any one of (1) to (3), wherein: (a) the ELD is or is from: an endosomolytic peptide; an antimicrobial peptide (AMP); a linear cationic alpha-helical antimicrobial peptide; a Cecropin-A/Melittin hybrid (CM series) peptide; pH- dependent membrane active peptide (PAMP); a peptide amphiphile; a peptide derived from the N terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; Diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1 ; VSVG; Pseudomonas toxin; melittin; KALA; JST-1 ; C(LLKK)3C (SEQ ID NO: 63); G(LLKK)3G (SEQ ID NO: 64); or any combination thereof; (b) the CPD is or is from: a cell-penetrating peptide or the protein transduction domain from a cell-penetrating peptide; TAT; PTD4; Penetratin (Antennapedia); pVEC; M918; Pep-1; Pep-2; Xentry; arginine stretch; transportan; SynB1; SynB3; or any combination thereof; (c) the histidine-rich domain is a stretch of at least 3, at least 4, at least 5, or at least 6 amino acids comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues; and/or comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 consecutive histidine residues; or (d) any combination of (a) to (c).
(5) The synthetic peptide or polypeptide-based shuttle agent of any one of (1) to (4), wherein the synthetic peptide or polypeptide-based shuttle agent comprises: (a) an ELD comprising the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64, or a variant or fragment thereof having endosomolytic activity; (b) a CPD comprising the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65, or a variant or fragment thereof having cell penetrating activity; (c) a histidine-rich domain having at least 2, at least 3, at least 4, at least 5, or at least 6 consecutive histidine residues; (d) of any combination of (a) to (c).
(6) The synthetic peptide or polypeptide-based shuttle agent of any one of (1) to (5), wherein the domains are operably linked via one or more linker domains.
(7) The synthetic peptide or polypeptide-based shuttle agent of any one of (1) to (6), wherein the synthetic peptide or polypeptide-based shuttle agent comprises at least two different types of CPDs and/or ELDs.
(8) The synthetic peptide or polypeptide-based shuttle agent of any one of (1) to (7), wherein the synthetic peptide or polypeptide-based shuttle agent comprises: (a) an ELD which is CM18, KALA, or C(LLKK)3C (SEQ ID NO: 63) having the amino acid sequence of SEQ ID NO: 1, 14, or
63, or a variant thereof having at least 85%, 90%, or 95% identity to SEQ ID NO: 1 and having endosomolytic activity; (b) a CPD which is TAT or PTD4 having the amino acid sequence of SEQ ID NO: 17 or 65, or a variant thereof having at least 85%, 90%, or 95% identity to SEQ ID NO: 17 or 65, and having cell penetrating activity; or Penetratin having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least 85%, 90%, or 95% identity to SEQ ID NO: 18 and having cell penetrating activity; (c) a histidine-rich domain comprising at least 6 consecutive histidine residues; or (d) any combination of (a) to (c).
(9) The synthetic peptide or polypeptide-based shuttle agent of any one of (1) to (8), wherein the synthetic peptide or polypeptide-based shuttle agent comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 57-59, 66-73, or 82-102, or a functional variant thereof having at least 85%, 90%, or 95% identity to any one of SEQ ID NOs: 57-59, 66-73, or 82-102. (10) The synthetic peptide or polypeptide-based shuttle agent of any one of (1) to (9), wherein the synthetic peptide or polypeptide-based shuttle agent is non-toxic and/or is metabolizable.
(11) A composition comprising: (a) the synthetic peptide or polypeptide-based shuttle agent as defined in any one of (1) to (10), and a further independent synthetic peptide comprising a histidine-rich domain and a CPD; and/or (b) a cocktail of at least 2, at least 3, at least 4, or at least 5 different types of the synthetic peptides or polypeptide-based shuttle agents as defined in any one of (1) to (10).
(12) Use of the synthetic peptide, polypeptide-based shuttle agent, or composition as defined in any one of (1) to (11), for delivering an independent polypeptide cargo to the cytosol of a target eukaryotic cell.
(13) A method for increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell, the method comprising contacting the target eukaryotic cell with the synthetic peptide, polypeptide-based shuttle agent, or composition as defined in any one of (1) to
(11), and the polypeptide cargo.
(14) A kit for increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell, the kit comprising the synthetic peptide, polypeptide-based shuttle agent, or composition as defined in any one of (1) to (11), and a suitable container.
(15) The synthetic peptide, polypeptide-based shuttle agent, composition, use, method or kit of any one of (1) to (14), for use in increasing the transduction efficiency of a polypeptide cargo to the cytosol of a target eukaryotic cell in the presence of serum.
(16) The synthetic peptide, polypeptide-based shuttle agent, composition, use, method or kit of any one of (2) to (15), wherein the polypeptide cargo: (a) comprises or lacks a CPD or a CPD as defined in (4)(b); (b) is a recombinant protein; (c) comprises a subcellular targeting domain; (d) is complexed with a DNA and/or RNA molecule; or (e) any combination of (a) to (d).
(17) The synthetic peptide, polypeptide-based shuttle agent, composition, use, method or kit of (16), wherein the subcellular targeting domain is: (a) a nuclear localization signal (NLS); (b) a nucleolar signal sequence; (c) a mitochondrial signal sequence; or (d) a peroxisome signal sequence. (18) The synthetic peptide, polypeptide-based shuttle agent, composition, use, method or kit of (17), wherein: (a) the NLS is from: E1 a, T-Ag, c-myc, T-Ag, op-T-NLS, Vp3, nucleoplasm^, histone 2B, Xenopus N1 , PARP, PDX-1 , QKI-5, HCDA, H2B, v-Rel, Amida, RanBP3, Pho4p, LEF-1, TCF-1 , BDV-P, TR2, SOX9, or Max; (b) the nucleolar signal sequence is from BIRC5 or RECQL4; (c) the mitochondrial signal sequence is from Tim9 or Yeast cytochrome c oxidase subunit IV; or (d) the peroxisome signal sequence is from PTS1.
(19) The synthetic peptide, polypeptide-based shuttle agent, composition, use, method or kit of any one of (2) to (18), wherein the polypeptide cargo is a transcription factor, a nuclease, a cytokine, a hormone, a growth factor, or an antibody.
(20) The synthetic peptide, polypeptide-based shuttle agent, composition, use, method or kit of (19), wherein: (a) the transcription factor is: HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Sox9, Klf4, c-Myc, MyoD, Pdx1 , Ngn3, MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1 , FoxA1, Nanog, Esrrb, Lin28, HIF1 -alpha, Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5, Bcl-6, or any combination thereof; and/or the nuclease is: an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1, a zinc-finger nuclease (ZFN), a Transcription activator-like effector nucleases (TALENs), a homing endonuclease, a meganuclease, or any combination thereof.
(21) The synthetic peptide, polypeptide-based shuttle agent, composition, use, method or kit of any one of (1) to (20), for use in cell therapy, genome editing, adoptive cell transfer, and/or regenerative medicine.
(22) The shuttle agent, shuttle system, composition, use, method, or kit of any one of (2) to (21), wherein the target eukaryotic cell is a stem cell, a primary cell, an immune cell, a T cell, an NK cell, or a dendritic cell.
(23) A eukaryotic cell comprising the synthetic peptide or polypeptide-based shuttle agent as defined in any one of (1 ) to (10), or the composition of (11 ).
(24) The eukaryotic cell of (23), wherein said cell further comprises an independent polypeptide cargo delivered intracellular^ by said synthetic peptide or polypeptide-based shuttle agent.
(25) A method for delivering an independent polypeptide cargo to the cytosol of a target eukaryotic cell, said method comprising contacting said target eukaryotic cell with the synthetic peptide or polypeptide-based shuttle agent as defined in any one of (1) to (10), or the composition of (11); and an independent polypeptide cargo to be delivered intracellular^ by said synthetic peptide or polypeptide-based shuttle agent.
(26) The eukaryotic cell of (23) or (24), or the method of (25), wherein said independent polypeptide cargo is as defined in any one of (16) to (20).
(27) The eukaryotic cell of (24) or (26), or the method of (25) or (26), wherein said independent polypeptide cargo is as defined in any one of (16) to (20). (28) The eukaryotic cell of (23), (24), (26) or (27), or the method of (25), (26), or (27), wherein said eukaryotic cell is an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, an NK cell, or a dendritic cell. (29) A method for increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell, said method comprising contacting said target eukaryotic cell with a synthetic peptide and said independent polypeptide cargo, wherein said synthetic peptide:
(a) comprises an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, operably linked to a cell penetrating domain (CPD), wherein said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64;
(b) is not covalently bound to said independent polypeptide cargo;
(c) has an overall length of between 20 and 100 amino acid residues;
(d) has a net charge of at least -+6 at physiological pH; and
(e) is soluble in aqueous solution at physiological pH,
wherein said CPD enables intracellular delivery of said synthetic peptide, and said ELD enables escape of endosomally trapped independent polypeptide cargo to the cytosol of the target eukaryotic cell.
(30) A method for increasing the transduction efficiency and cytosolic delivery of an independent polypeptide cargo in a population of target eukaryotic cells, said method comprising contacting said target eukaryotic cells with said independent polypeptide cargo and a concentration of a synthetic peptide sufficient to increase the percentage or proportion of the population of target eukaryotic cells into which the independent polypeptide is delivered intracellular^ across the plasma membrane, as compared to in the absence of said synthetic peptide, wherein said synthetic peptide:
(a) comprises an endosome leakage domain (ELD), or a variant or fragment thereof having endosomolytic activity, operably linked to a cell penetrating domain (CPD), wherein said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1 -15, 63, or 64;
(b) is not covalently bound to said independent polypeptide cargo;
(c) has an overall length of between 20 and 100 amino acid residues;
(d) has a net charge of at least +6 at physiological pH; and
(e) is soluble in aqueous solution at physiological pH,
wherein said CPD enables intracellular delivery of said synthetic peptide, and said ELD enables escape of endosomally trapped independent polypeptide cargo to the cytosol of the target eukaryotic cells, thereby increasing the transduction efficiency and cytosolic delivery of the independent polypeptide cargo in the population of target eukaryotic cells.
(31) The method of (29) or (30), wherein said synthetic peptide has an overall length of between 20 and 70 amino acid residues.
(32) The method of any one of (29)-(31), wherein said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65, or is a variant or fragment thereof having cell penetrating activity.
(33) The method of any one of (29)-(32), wherein said synthetic peptide further comprises a histidine- rich domain consisting of a stretch of at least 6 amino acids comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues; and/or comprises at least 2, at least 3, at least 4, at least 5, or at least 6 consecutive histidine residues.
(34) The method of any one of (29)-(33), wherein said ELD variant or ELD fragment has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 1-15, 63, or 64.
(35) The method of any one of (29)-(34), wherein said CPD variant or CPD fragment has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 16-27, or 65.
(36) The method of any one of (29)-(35), wherein said ELD and CPD are operably linked via one or more linker domains.
(37) The method of any one of (29)-(36), wherein said synthetic peptide is chemically synthesized without an N-terminal methionine residue.
(38) The method of any one of (29)-(37), wherein the synthetic peptide comprises the amino acid sequence of any one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof having at least 70%, at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID
NOs: 57-59, 66-72, or 82-102.
(39) The method of any one of (29)-(38), wherein said independent polypeptide cargo is a recombinant protein lacking a CPD.
(40) The method of any one of (29)-(39), wherein said independent polypeptide cargo is a transcription factor, a nuclease, a cytokine, a hormone, a growth factor, or an antibody.
(41 ) The method of (40), wherein:
(a) said transcription factor is: HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Sox9, Klf4, c-Myc, MyoD, Pdx1 , Ngn3, MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1 , FoxA1, Nanog, Esrrb, Lin28, HIF1 -alpha, Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5, Bcl-6, or any combination thereof; or
(b) said nuclease is an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1 , a zinc-finger nuclease (ZFNs), a Transcription activator-like effector nucleases (TALENs), a homing endonuclease, or a meganuclease.
(42) The method of any one of (39)-(41 ), wherein said nuclease is Cas9 or Cpf1.
(43) The method of any one of (39)-(42), wherein said nuclease further comprises a guide RNA, a crRNA, a tracrRNA, or both a crRNA and a tracrRNA.
(44) The method of any one of (29)-(43), wherein said independent polypeptide cargo comprises a nuclear localization signal or a further nuclear localization signal.
(45) The method of any one of (29)-(44), wherein said independent polypeptide cargo is a transcription factor or a nuclease.
(46) The method of (45) wherein:
(a) said transcription factor is: HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Sox9, Klf4, c-Myc, MyoD, Pdx1 , Ngn3, MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1 , FoxA1, Nanog, Esrrb, Lin28, HIF1 -alpha, Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5, Bcl-6, or any combination thereof; or
(b) said nuclease is an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1 , a zinc -finger nuclease (ZFNs), a Transcription activator-like effector nucleases (TALENs), a homing endonuclease, or a meganuclease.
(47) The method of (46), wherein said nuclease is Cas9 or Cpf1.
(48) The method of any one of (44)-(47), wherein said nuclease further comprises a guide RNA.
(49) The method of any one of (29)-(48), wherein said cell is stem cell, a primary cell, an immune cell, a T cell, an NK cell, or a dendritic cell.
(50) A method for increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell, said method comprising contacting said target eukaryotic cell with a synthetic peptide and said independent polypeptide cargo, wherein said synthetic peptide: (a) comprises an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), wherein said ELD is an endosomolytic peptide which is, or is derived from: a linear cationic alpha-helical antimicrobial peptide; a Cecropin-A/Melittin hybrid (CM series) peptide; pH-dependent membrane active peptide (PAMP); a peptide amphiphile; a peptide derived from the N terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; Diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1; VSVG; Pseudomonas toxin; melittin; KALA; JST-1 ; C(LLKK)3C (SEQ ID NO: 63); or G(LLKK)3G (SEQ ID NO: 64);
(b) is not covalently bound to said independent polypeptide cargo;
(c) has an overall length of between 20 and 100 amino acid residues;
(d) has a net charge of at least -+6 at physiological pH; and
(e) is soluble in aqueous solution at physiological pH,
wherein said CPD enables intracellular delivery of said synthetic peptide, and said ELD enables escape of endosomally trapped independent polypeptide cargo to the cytosol of the target eukaryotic cell.
(51) A method for increasing the transduction efficiency and cytosolic delivery of an independent polypeptide cargo in a population of target eukaryotic cells, said method comprising contacting said target eukaryotic cells with said independent polypeptide cargo and a concentration of a synthetic peptide sufficient to increase the percentage or proportion of the population of target eukaryotic cells into which the independent polypeptide is delivered intracellular^ across the plasma membrane, as compared to in the absence of said synthetic peptide, wherein said synthetic peptide:
(a) comprises an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), wherein said ELD is an endosomolytic peptide which is, or is derived from: a linear cationic alpha-helical antimicrobial peptide; a Cecropin-A/Melittin hybrid (CM series) peptide; pH-dependent membrane active peptide (PAMP); a peptide amphiphile; a peptide derived from the N terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; Diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1; VSVG; Pseudomonas toxin; melittin; KALA; JST-1 ; C(LLKK)3C; or G(LLKK)3G;
(b) is not covalently bound to said independent polypeptide cargo;
(c) has an overall length of between 20 and 100 amino acid residues;
(d) has a net charge of at least -+6 at physiological pH; and
(e) is soluble in aqueous solution at physiological pH, wherein said CPD enables intracellular delivery of said synthetic peptide, and said ELD enables escape of endosomally trapped independent polypeptide cargo to the cytosol of the target eukaryotic cells,
thereby increasing the transduction efficiency and cytosolic delivery of the independent polypeptide cargo in the population of target eukaryotic cells.
(51) The method of (49) or (50), wherein said CPD is, or is derived from: a cell-penetrating peptide or the protein transduction domain from a cell-penetrating peptide; TAT; PTD4; Penetratin (Antennapedia); pVEC; M918; Pep-1 ; Pep-2; Xentry; arginine stretch; transportan; SynB1; SynB3; or any combination thereof.
(52) The method of any one of (49)-(51), wherein said synthetic peptide further comprises a histidine- rich domain consisting of a stretch of at least 3 amino acids comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues; and/or comprises at least 2, at least 3, at least 4, at least 5, or at least 6 consecutive histidine residues.
(53) The method of any one of (49)-(52), wherein said ELD and CPD are operably linked via one or more linker domains.
(54) The method of any one of (49)-(53), wherein said independent polypeptide cargo is a transcription factor, a nuclease, a cytokine, a hormone, a growth factor, or an antibody.
(55) The method of (54), wherein said transcription factor is: HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Klf4, c-Myc, MyoD, Pdx1 , Ngn3, MafA, Blimp-1 , Eomes, T-bet, FOX03A, NF-YA, SALL4, ISL1,
FoxA1 , Nanog, Esrrb, Lin28, HIF1 -alpha, Hlf, Runx1t1 , Pbx1 , Lmo2, Zfp37, Prdm5, Bcl-6, or any combination thereof.
(56) The method of (54), wherein said nuclease is an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR associated protein 9 (Cas9), Cpf1, a zinc-finger nuclease (ZFNs), a Transcription activator-like effector nucleases (TALENs), a homing endonuclease, or a meganuclease.
(57) The method of (54), wherein said nuclease is Cas9 or Cpf1.
(58) A method for increasing the transduction efficiency of an independent polypeptide cargo to the cytosol of a target eukaryotic cell, said method comprising contacting said target eukaryotic cell with a synthetic peptide and said independent polypeptide cargo which is not covalently bound to said synthetic peptide, wherein said synthetic peptide comprises an endosome leakage domain (ELD) operably linked to a cell penetrating domain, or an ELD operably linked to a CPD and a histidine-rich domain, wherein:
(a) said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64;
(b) said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65; and
(c) said histidine-rich domain comprises at least two consecutive histidine residues.
(59) A method for increasing the transduction efficiency and cytosolic delivery of an independent polypeptide cargo in a population of target eukaryotic cells, said method comprising contacting said target eukaryotic cell with said independent polypeptide cargo and a concentration of a synthetic peptide sufficient to increase the percentage or proportion of the population of target eukaryotic cells into which the independent polypeptide is delivered intracellular^ across the plasma membrane, as compared to in the absence of said synthetic peptide, which is not covalently bound to said synthetic peptide, wherein said synthetic peptide comprises an endosome leakage domain (ELD) operably linked to a cell penetrating domain, or an ELD operably linked to a CPD and a histidine-rich domain, wherein:
(a) said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1 -15, 63, or 64;
(b) said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65; and
(c) said histidine-rich domain comprises at least two consecutive histidine residues, thereby increasing the transduction efficiency and cytosolic delivery of the independent polypeptide cargo in the population of target eukaryotic cells.
(60) A method for delivering a CRISPR associated protein 9 (Cas9) to the nucleus of a target eukaryotic cell, said method comprising contacting said eukaryotic cell with a Cas9 recombinant protein comprising a nuclear localization signal, and a separate synthetic peptide shuttle agent less than 100 residues in length and comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain, or an ELD operably linked to a CPD and a histidine-rich domain, wherein:
(a) said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64;
(b) said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65; and
(c) said histidine-rich domain comprises at least two consecutive histidine residues. (61) A method for delivering a CRISPR associated protein 9 (Cas9) to the nucleus of a population of eukaryotic cells, said method comprising contacting said population of eukaryotic cells with Cas9 recombinant protein comprising a nuclear localization signal, and a separate synthetic peptide shuttle agent less than 100 residues in length and comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain, or an ELD operably linked to a CPD and a histidine-rich domain, wherein:
(a) said ELD comprises the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64;
(b) said CPD comprises the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65; and
(c) said histidine-rich domain comprises at least two consecutive histidine residues, wherein said synthetic peptide is at a concentration sufficient to increase the percentage or proportion of the population of eukaryotic cells into which the independent polypeptide is delivered across the plasma membrane, as compared to in the absence of said synthetic peptide
Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
EXAMPLES
Example 1 :
Materials and Methods
1.1 Materials
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA or Oakville, ON, Canada) or equivalent grade from BioShop Canada Inc. (Mississauga, ON, Canada) or VWR (Ville Mont-Royal, QC, Canada), unless otherwise noted. 1.2 Reagents
Table 1.1 : Reagents
Figure imgf000060_0001
Figure imgf000061_0001
1.3 Cell lines
HeLa, HEK293A, HEK293T, THP-1 , CHO, NIH3T3, CA46, Balb3T3 and HT2 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured following the manufacturer's instructions. Myoblasts are primary human cells kindly provided by Professor J.P. Tremblay (Universite Laval, Quebec, Canada).
Table 1.2: Cell lines and culture conditions
Figure imgf000061_0002
(adherent embryonic 1573 Penicillin 100 units cells) Epithelial Streptomycin l OOpg/mL kidney cells
HEK 293T Human ATCC™ CRL- DMEM 10% FBS L-glutamine 2 mM
(adherent embryonic 3216 Penicillin 100 units cells) Epithelial Streptomycin l OOpg/mL kidney cells
THP-1 Acute ATCC™ TIB202 RPM1 1640 10% FBS 2- mercaptoethanol 0.05
(suspension monocytic mM
cells) leukemia L-glutamine 2 mM
Penicillin 100 units Streptomycin l OOpg/mL
Myoblasts Human (13 Kindly provided MB1 15% FBS ITS 1x, FGF 2 10 ng/mL, (primary months) by Professor JP Dexamethasone adherent myoblasts Tremblay 0.39Mg/mL, cells) BSA 0.5mg/mL,
MB1 85%
CHO Chinese ATCC™ CCL- DMEM 10% FBS L-glutamine 2 mM
(adherent hamster ovary 61 Penicillin 100 units cells) cells Streptomycin 100 pg/mL
NIH3T3 Fibroblasts ATCC™ CRL- DMEM 10% Calf L-glutamine 2 mM
(adherent 1658 serum Penicillin 100 units cells) Streptomycin 100 pg/mL
HT2 T lymphocytes ATCC™ CRL- RPM1 1640 10% FBS 200 lU/mL IL-2
(suspension 1841 β-mercaptoethanol 0.05 cells) mM
L-glutamine 2 mM Penicillin 100 units Streptomycin 100 pg/mL
CA46 Homo sapiens ATCC™ CRL- RPM1 1640 20% FBS L-glutamine 2 mM
(suspension Burkitt's 1648 Penicillin 100 units cells) lymphoma Streptomycin 100 pg/mL
Balb3T3 Fibroblasts ATCC™ CCL- DMEM 10% Calf L-glutamine 2 mM
(adherent 163 serum Penicillin 100 units cells) Streptomycin 100 pg/mL
Jurkat Human T cells ATCC™ TIB- RPM1 1640 10% FBS L-glutamine 2 mM
(suspension 152 Penicillin 100 units cells) Streptomycin 100 pg/mL
FBS: Fetal bovine serum
1.4 Protein purification
Fusion proteins were expressed in bacteria (£. coli BL21 DE3) under standard conditions using an isopropyl β-D- -thiogalactopyranoside (IPTG) inducible vector containing a T5 promoter. Culture media contained 24 g yeast extract, 12 g tryptone, 4 mL glycerol, 2.3 g KH2PO4, and 12.5 g K2HPO4 per liter. Bacterial broth was incubated at 37°C under agitation with appropriate antibiotic (e.g., ampicillin). Expression was induced at optical density (600 nm) between 0.5 and 0.6 with a final concentration of 1 mM IPTG for 3 hours at 30°C. Bacteria were recuperated following centrifugation at 5000 RPM and bacterial pellets were stored at -20°C.
Bacterial pellets were resuspended in Tris buffer (Tris 25 mM pH 7.5, NaCI l OOmM, imidazole 5 mM) with phenylmethylsulfonyl fluoride (PMSF) 1 mM, and lysed by passing 3 times through the homogenizer Panda 2K™ at 1000 bar. The solution was centrifuged at 15000 RPM, 4°C for 30 minutes. Supernatants were collected and filtered with a 0.22 μΜ filtration device.
Solubilized proteins were loaded, using a FPLC (AKTA Explorer 100R), on HisTrap™ FF column previously equilibrated with 5 column volumes (CV) of Tris buffer. The column was washed with 30 column volumes (CV) of Tris buffer supplemented with 0.1 % Triton™ X-114 followed with 30 CV of Tris buffer with imidazole 40 mM. Proteins were eluted with 5 CV of Tris buffer with 350 mM Imidazole and collected. Collected fractions corresponding to specific proteins were determined by standard denaturing SDS-PAGE.
Purified proteins were diluted in Tris 20 mM at the desired pH according to the protein's pi and loaded on an appropriate ion exchange column (Q Sepharose™ or SP Sepharose™) previously equilibrated with 5 CV of Tris 20 mM, NaCI 30 mM. The column was washed with 10 CV of Tris 20 mM, NaCI 30 mM and proteins were eluted with a NaCI gradient until 1 M on 15 CV. Collected fractions corresponding to specific proteins were determined by standard denaturing SDS-PAGE. Purified proteins were then washed and concentrated in PBS 1X on Amicon Ultra™ centrifugal filters 10,000 MWCO. Protein concentration was evaluated using a standard Bradford assay.
1.5 Synthetic peptides and shuttle agents
All peptides used in this study were purchased from GLBiochem (Shanghai, China) and their purities were confirmed by high-performance liquid chromatography analysis and mass spectroscopy. In some cases, chimeric peptides were synthesized to contain a C-terminal cysteine residue to allow the preparation of peptide dimers. These dimeric peptides were directly synthetized with a disulfide bridge between the C-terminal cysteines of two monomers. The amino acid sequences and characteristics of each of the synthetic peptides and shuttle agents tested in the present examples are summarized in Table 1.3.
Table 1.3: Synthetic peptides and shuttle agents tested
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Resu ts computed using t he ProtParam online tool available from ExPA y Bioin formatics
Portal (http://web.expasy.org/cgi-bin/protparam/protparam) MW: Molecular weight
pi: Isoelectric point
Charge: Total number of positively (+) and negatively (-) charged residues Example 2:
Peptide shuttle agents facilitate escape of endosomally-trapped calcein
2.1 Endosome escape assays
Microscopy-based and flow cytometry-based fluorescence assays were developed to study endosome leakage and to determine whether the addition of the shuttle agents facilitates endosome leakage of the polypeptide cargo.
2.1.1 Endosomal leakage visualization by microscopy
Calcein is a membrane-impermeable fluorescent molecule that is readily internalized by cells when administered to the extracellular medium. Its fluorescence is pH-dependent and calcein self- quenches at higher concentrations. Once internalized, calcein becomes sequestered at high concentrations in cell endosomes and can be visualized by fluorescence microscopy as a punctate pattern. Following endosomal leakage, calcein is released to the cell cytoplasm and this release can be visualized by fluorescence microscopy as a diffuse pattern.
One day before the calcein assay was performed, mammalian cells (e.g., HeLa, HEK293A, or myoblasts) in exponential growth phase were harvested and plated in a 24-well plate (80,000 cells per well). The cells were allowed to attach by incubating overnight in appropriate growth media, as described in Example 1. The next day, the media was removed and replaced with 300 μί of fresh media without FBS containing 62.5 pg/mL (100 μΜ) of calcein, except for HEK293A (250 pg/mL, 400 μΜ). At the same time, the shuttle agent(s) to be tested was added at a predetermined concentration. The plate was incubated at 37°C for 30 minutes. The cells were washed with 1x PBS (37°C) and fresh media containing FBS was added. The plate was incubated at 37°C for 2.5 hours. The cells were washed three times and were visualized by phase contrast and fluorescence microscopy (1X81™, Olympus).
A typical result is shown in Figure 1A, in which untreated HEK293A cells loaded with calcein
("100 μΜ calcein") show a low intensity, punctate fluorescent pattern when visualized by fluorescence microscopy (upper left panel in Figure 1A). In contrast, HeLa cells treated with a shuttle agent that facilitates endosomal escape of calcein ("100 μΜ calcein + CM18-TAT 5 μΜ") show a higher intensity, more diffuse fluorescence pattern in a greater proportion of cells (upper right panel in Figure 1A). 2.1.2 Endosomal leakage Quantification by flow cytometry
In addition to microscopy, flow cytometry allows a more quantitative analysis of the endosomal leakage as the fluorescence intensity signal increases once the calcein is released in the cytoplasm. Calcein fluorescence is optimal at physiological pH (e.g., in the cytosol), as compared to the acidic environment of the endosome.
One day before the calcein assay was performed, mammalian cells (e.g., HeLa, HEK293, or myoblasts) in exponential growth phase were harvested and plated in a 96-well plate (20,000 cells per well). The cells were allowed to attach by incubating overnight in appropriate growth media, as described in Example 1. The next day, the media in wells was removed and replaced with 50 μί of fresh media without serum containing 62.5 pg/mL (100 μΜ) of calcein, except for HEK293A (250 g/mL, 400 μΜ). At the same time, the shuttle agent(s) to be tested was added at a predetermined concentration. The plate was incubated at 37°C for 30 minutes. The cells were washed with 1x PBS (37°C) and fresh media containing 5-10% serum was added. The plate was incubated at 37°C for 2.5 hours. The cells were washed with 1x PBS and detached using trypsinization. Trypsinization was stopped by addition of appropriate growth media, and calcein fluorescence was quantified using flow cytometry (Accuri C6, Becton, Dickinson and Company (BD)).
Untreated calcein-loaded cells were used as a control to distinguish cells having a baseline of fluorescence due to endosomally-trapped calcein from cells having increased fluorescence due to release of calcein from endosomes. Fluorescence signal means ("mean counts") were analyzed for endosomal escape quantification. In some cases, the "Mean Factor" was calculated, which corresponds to the fold-increase of the mean counts relative to control (untreated calcein-loaded cells). Also, the events scanned by flow cytometry corresponding to cells (size and granularity) were analyzed. The cellular mortality was monitored with the percentage of cells in the total events scanned. When it became lower than the control, it was considered that the number of cellular debris was increasing due to toxicity and the assay was discarded.
A typical result is shown in Figure 1 B, in which an increase in fluorescence intensity (right-shift) is observed for calcein-loaded HeLa cells treated with a shuttle agent that facilitates endosomal escape ("Calcein 100 μΜ + CM18-TAT 5 μΜ", right panel in Figure 1 B), as compared to untreated calcein- loaded HeLa cells ("Calcein 100 μΜ", left panel in Figure 1 B). The increase in calcein fluorescence is caused by the increase in pH associated with the release of calcein from the endosome (acidic) to the cytoplasm (physiological). 2.2 Results from endosome escape assays
2.2.1 HeLa cells
HeLa cells were cultured and tested in the endosomal escape assays as described in Example 2.1. The results of flow cytometry analyses are summarized below. In each case, the flow cytometry results were also confirmed by fluorescence microscopy (data not shown).
Table 2.1 : CM18-Penetratin-Cys v. Controls in HeLa cells
Figure imgf000068_0002
Table 2.2: CM18-TAT-Cys v. Control in HeLa cells
Figure imgf000068_0001
The results in Tables 2.1 and 2.2 show that treating calcein-loaded HeLa cells with the shuttle agents CM18-Penetratin-Cys and CM18-TAT-Cys (having the domain structure ELD-CPD) results in increased mean cellular calcein fluorescence intensity, as compared to untreated control cells or cells treated with single-domain peptides used alone (CM18, TAT-Cys, Penetratin-Cys) or together (CM18 + TAT-Cys, CM18 + Penetratin-Cys). These results suggest that CM18-Penetratin-Cys and CM18-TAT- Cys facilitate escape of endosomally-trapped calcein, but that single domain peptides (used alone or together) do not. Table 2.3: Dose response of CM18-TAT-Cys in HeLa cells, data from Figure 2
Figure imgf000069_0001
The results in Tables 2.3 (Figure 2), 2.4, and 2.5 (Figure 3) suggest that CM 18-TAT-Cys and CM18-Penetratin-Cys facilitate escape of endosomally-trapped calcein in HeLa cells in a dose- dependent manner. In some cases, concentrations of CM18-TAT-Cys or CM18-Penetratin-Cys above 10 μΜ were associated with an increase in cell toxicity in HeLa cells. Table 2.6: Dimers v. monomers of CM18-TAT-Cys and CM18-Penetratin-Cys in HeLa cells
Figure imgf000070_0001
The results in Table 2.6 and 2.7 suggest that shuttle peptide dimers (which are molecules comprising more than one ELD and CPD) are able to facilitate calcein endosomal escape levels that are comparable to the corresponding monomers.
2.2.3 HEK293A cells
To examine the effects of the shuttle agents on a different cell line, HEK293A cells were cultured and tested in the endosomal escape assays as described in Example 2.1. The results of flow cytometry analyses are summarized below in Table 2.8 and in Figure 1 B.
Table 2.8: CM18-TAT-Cys in HEK293A cells
Figure imgf000070_0002
The results in Table 2.8 and in Figure 1 B show that treating calcein-loaded HEK293A cells with the shuttle agent CM18-TAT-Cys results in increased mean cellular calcein fluorescence intensity, as compared to untreated control cells. 2.2.2 Myoblasts
To examine the effects of the shuttle agents on primary cells, primary myoblast cells were cultured and tested in the endosomal escape assays as described in Example 2.1. The results of flow cytometry analyses are summarized below in Tables 2.9 and 2.10, and in Figure 4. In each case, the flow cytometry results were also confirmed by fluorescence microscopy.
Table 2.9: Dose response of CM18-TAT-Cys in primary myoblasts, data from Figure 4
Figure imgf000071_0001
The results in Table 2.9 (shown graphically in Figure 4) and Table 2.10 suggest that CM18-
TAT-Cys facilitates escape of endosomally-trapped calcein in a dose-dependent manner in primary myoblasts. Concentrations of CM18-TAT-Cys above 10 μΜ were associated with an increase in cell toxicity in myoblast cells, as for HeLa cells. Table 2.11: Monomers v. dimers CM18-TAT-Cys and CM18-Penetratin-Cys in primary myoblasts
Figure imgf000071_0002
The results in Table 2.11 suggest that shuttle peptide dimers are able to facilitate calcein endosomal escape levels that are comparable to the corresponding monomers in primary myoblasts.
Example 3:
Peptide shuttle agents increase GFP transduction efficiency
3.1 Protein transduction assay
One day before the transduction assay was performed, mammalian cells (e.g., HEK293, CHO, HeLa, THP-1 , and myoblasts) in exponential growth phase were harvested and plated in a 96-well plate (20,000 cells per well). The cells were incubated overnight in appropriate growth media containing FBS (see Example 1). The next day, in separate sterile 1.5 mL tubes, cargo protein at 0.5 to 10 μΜ (GFP, TAT-GFP, GFP-NLS, or FITC-labeled anti-tubulin antibody) was pre-mixed (pre-incu bated) for 10 min at 37°C with shuttle agents (0.5 to 5 μΜ) in 50 L of fresh medium without serum (unless otherwise specified). GFP, GFP-NLS and TAT-GFP are recombinant proteins developed and produced by Feldan (see Example 3.4 below). FITC-labeled anti-tubulin antibody was purchased from Abeam (ab64503). The media in wells was removed and the cells were washed three times with freshly prepared phosphate buffered saline (PBS) previously warmed at 37°C. The cells were incubated with the cargo protein/shuttle agent mixture at 37°C for 5 or 60 min. After the incubation, the cells were quickly washed three times with freshly prepared PBS and/or heparin (0.5 mg/mL) previously warmed at 37°C. The washes with heparin were required for human THP-1 blood cells to avoid undesired cell membrane-bound protein background in subsequent analyses (microscopy and flow cytometry). The cells were finally incubated in 50 μί of fresh medium with serum at 37°C before analysis.
3.2 Fluorescence microscopy analysis
The delivery of fluorescent protein cargo in cytosolic and nuclear cell compartments was observed with an Olympus IX70™ microscope (Japan) equipped with a fluorescence lamp (Model U- LH100HGAPO) and different filters. The Olympus filter U-MF2™ (C54942-Exc495/Em510) was used to observe GFP and FITC-labeled antibody fluorescent signals. The Olympus filter HQ-TR™ (V-N41004- Exc555-60/Em645-75) was used to observe mCherry™ and GFP antibody fluorescent signals. The Olympus filter U-MWU2™ (Exc330/Em385) was used to observe DAPI or Blue Hoechst fluorescent signals. The cells incubated in 50 L of fresh medium were directly observed by microscopy (Bright- field and fluorescence) at different power fields (4x to 40x). The cells were observed using a CoolSNAP-PRO™ camera (Series A02D874021) and images were acquired using the Image-Proplus™ software. 3.2a Cell immuno-labelling
Adherent cells were plated on a sterile glass strip at 1.5x105 cells per well in a 24-plate well and incubated overnight at 37°C. For fixation, cells were incubated in 500 L per well of formaldehyde (3.7% v/v) for 15 minutes at room temperature, and washed 3 times for 5 minutes with PBS. For permeabilization, cells were incubated in 500 L per well of Triton™ X-100 (0.2%) for 10 minutes at room temperature, and washed 3 times for 5 minutes with PBS. For blocking, cells were incubated in 500 [il per well of PBS containing 1 % BSA (PBS/BSA) for 60 minutes at room temperature. Primary mouse monoclonal antibody was diluted PBS/BSA (1 %). Cells were incubated in 30 L of primary antibody overnight at 4°C. Cells were washed 3 times for 5 minutes with PBS. Secondary antibody was diluted in PBS/BSA (1 %) and cells were incubated in 250 L of secondary antibody 30 minutes at room temperature in the dark. Cells were washed 3 times for 5 minutes with PBS. Glass strips containing the cells were mounted on microscope glass slides with 10 L of the mounting medium Fluoroshield™ with DAPI. 3.3 Flow cytometry analysis:
The fluorescence of GFP was quantified using flow cytometry (Accuri C6, Becton, Dickinson and Company (BD)). Untreated cells were used to establish a baseline in order to quantify the increased fluorescence due to the internalization of the fluorescent protein in treated cells. The percentage of cells with a fluorescence signal above the maximum fluorescence of untreated cells, "mean %" or "Pos cells (%)", is used to identify positive fluorescent cells. "Relative fluorescence intensity (FL1 -A)" corresponds to the mean of all fluorescence intensities from each cell with a fluorescent signal after fluorescent protein delivery with the shuttle agent. Also, the events scanned by flow cytometry corresponding to cells (size and granularity) were analyzed. The cellular toxicity (% cell viability) was monitored comparing the percentage of cells in the total events scanned of treated cells comparatively to untreated cells.
3.3a Viability analysis
The viability of cells was assessed with a rezazurine test. Rezazurine is a sodium salt colorant that is converted from blue to pink by mitochondrial enzymes in metabolically active cells. This colorimetric conversion, which only occurs in viable cells, can be measured by spectroscopy analysis in order to quantify the percentage of viable cells. The stock solution of rezazurine was prepared in water at 1 mg/100 mL and stored at 4°C. 25 L of the stock solution was added to each well of a 96-well plate, and cells were incubated at 37°C for one hour before spectrometry analysis. The incubation time used for the rezazurine enzymatic reaction depended on the quantity of cells and the volume of medium used in the wells.
3.4 Construction and amino acid sequence of GFP
The GFP-encoding gene was cloned in a T5 bacterial expression vector to express a GFP protein containing a 6x histidine tag (SEQ ID NO: 113) and a serine/glycine rich linker in the N-terminal end, and a serine/glycine rich linker and a stop codon (-) at the C-terminal end. Recombinant GFP protein was purified as described in Example 1.4. The sequence of the GFP construct was:
MHHHHHHGGGGSGGGGSGGASTGTGIRMVSKGEELFTGWPILVELDGDVNG
HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPD HMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGI DFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLG MDELYKGGSGGGSGGGSGWIRASSGGREIS- [SEQ ID NO: 60]
(MW= 31.46 kDa; pl=6.19)
Serine/glycine rich linkers are in bold
GFP sequence is underlined 3.5 GFP transduction by CM18-TAT-Cys in HeLa cells: Fluorescence microscopy
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein was co-incubated with 0, 3 or 5 μΜ of CM18-TAT, and then exposed to HeLa cells for 1 hour. The cells were observed by bright field and fluorescence microscopy as described in Example 3.2. The results presented in Figure 5 show that GFP was delivered intracellular^ to HeLa cells in the presence of the shuttle agent CM18-TAT.
3.6 GFP transduction by shuttle agents in HeLa cells: Dose responses (CM18-TAT-Cys, dCM18-TAT-Cys, GFP) and cell viability
HeLa cells were cultured and tested in the protein transduction assay described in Examples 3.1 -3.3. Briefly, GFP recombinant protein was co-incubated with different concentrations of CM18-TAT- Cys or dimerized CM18-TAT-Cys (dCM18-TAT-Cys), and then exposed to HeLa cells for 1 hour. The results are shown in Table 3.1 and Figures 6A-6B. Table 3.1 : Dose response (CM18-TAT) and cell viability, data from Figures 6A and 6B
Figure imgf000075_0001
Table 3.1 and Figure 6A show the results of flow cytometry analysis of the fluorescence intensity of HeLa cells transduced with GFP (5 μΜ) without or with 5, 3, 1 , and 0.5 μΜ of CM18-TAT- Cys. Corresponding cellular toxicity data are presented in Table 3.1 and in Figure 6B. These results suggest that the shuttle agent CM18-TAT-Cys increases the transduction efficiency of GFP in a dose- dependent manner.
Table 3.2: Dose response (GFP), data from Figures 7A and 7B
Figure imgf000075_0002
Table 3.2 and Figures 7A-7B show the results of flow cytometry analysis of the fluorescence intensity of HeLa cells transduced with different concentrations of GFP (1 to 10 μΜ) without or with 5 μΜ of CM18-TAT-Cys (Figure 7A) or 2.5 μΜ dCM18-TAT-Cys (Figure 7B). 3.7 GFP transduction in HeLa cells: Dose responses of CM18-TAT-Cys and CM18- Penetratin-Cys, and dimers thereof
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein (5 μΜ) was co-incubated with different concentrations and combinations of CM18-TAT-Cys, CM18-Penetratin-Cys, and dimers of each (dCM18-TAT-Cys, dCM18- Penetratin-Cys), and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 3.3 and Figure 8, as well as in Table 3.4 and Figure 9.
Table 3.3: Data in Figure 8
Figure imgf000076_0001
The results in Table 3.3 and Figure 8 show that the transduction efficiency of GFP is increased in HeLa cells using the shuttle agents CM18-TAT-Cys and dCM18-TAT-Cys (see bars "1" and "2" in Figure 8). Although no GFP intracellular delivery was observed using CM18-Penetratin-Cys or dCM18- Penetratin-Cys alone (see bars "3" or "4" in Figure 8), combination of CM18-TAT-Cys with CM18- Penetratin-Cys (monomer or dimer) improved GFP protein delivery (see four right-most bars in Figure 8).
Table 3.4: Data in Figure 9
The results in Table 3.4 and Figure 9 show that the transduction efficiency of GFP is increased in HeLa cells using the shuttle agents CM18-TAT-Cys and dCM18-TAT-Cys (see bars "1" and "2" in Figure 9). Although no GFP intracellular delivery was observed using CM18-Penetratin-Cys or dCM18- Penetratin-Cys alone (see bars "3" or "4" in Figure 9), combination of CM18-TAT-Cys with CM18- Penetratin-Cys (monomer or dimer) improved GFP protein delivery (see four right-most bars in Figure 9).
3.8 GFP transduction by shuttle agents in HeLa cells: Controls
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein (5 μΜ) was co-incubated with 5 μΜ of each of the following peptide(s): TAT-Cys; CM18; Penetratin-Cys; TAT-Cys + CM18; Penetratin-Cys + CM18; and CM18- TAT-Cys, and then exposed to HeLa cells for 1 hour. GFP fluorescence was visualized by bright field and fluorescence microscopy. The microscopy results (data not shown) showed that GFP was successfully delivered intracellularly using CM18-TAT-Cys. However, GFP was not successfully delivered intracellularly using single-domain peptides used alone (CM18, TAT-Cys, Penetratin-Cys) or together (CM18 + TAT-Cys, CM18 + Penetratin-Cys). These results are consistent with those presented in Tables 2.1 and 2.2 with respect to the calcein endosome escape assays.
Example 4:
Peptide shuttle agents increase TAT-GFP transduction efficiency
The experiments in Example 3 showed the ability of shuttle agents to deliver GFP intracellularly. The experiments presented in this example show that the shuttle agents can also increase the intracellular delivery of a GFP cargo protein that is fused to a CPD (TAT-GFP). 4.1 Construction and amino acid sequence of TAT-GFP
Construction was performed as described in Example 3.4, except that a TAT sequence was cloned between the 6x histidine tag (SEQ ID NO: 113) and the GFP sequences. The 6x histidine tag (SEQ ID NO: 113), TAT, GFP and a stop codon (-) are separated by serine/glycine rich linkers. The recombinant TAT-GFP protein was purified as described in Example 1.4. The sequence of the TAT- GFP construct was: MHHHHHHGGGGSGGGGSGGASTGTGRKKRRQRRRPPQGGGGSGGGGSGGGTG
IRMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM ADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQS ALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGGSGGGSGWIRASSG GREIS-
[SEQ ID NO: 61]
(MW= 34.06 kDa ; pl=8.36)
TAT sequence is underlined
Serine/glycine rich linkers are in bold
4.2 TAT-GFP transduction by CM18-TAT-Cys in HeLa cells: Visualisation by fluorescence microscopy
HeLa cells were cultured and tested in the protein transduction assay described in Example
3.1. Briefly, TAT-GFP recombinant protein (5 μΜ) was co-incubated with 3 μΜ of CM18-TAT-Cys and then exposed to HeLa cells for 1 hour. Cells and GFP fluorescence were visualized by bright field and fluorescence microscopy (as described in Example 3.2) at 10x and 40x magnifications, and sample results are shown in Figure 10. The microscopy results revealed that in the absence of CM18-TAT- Cys, TAT-GFP shows a low intensity, endosomal distribution as reported in the literature. In contrast, TAT-GFP is delivered to the cytoplasm and to the nucleus in the presence of the shuttle agent CM18- TAT-Cys. Without being bound by theory, the TAT peptide itself may act as a nuclear localization signal (NLS), explaining the nuclear localization of TAT-GFP. These results show that CM18-TAT-Cys is able to increase TAT-GFP transduction efficiency and allow endosomally-trapped TAT-GFP to gain access to the cytoplasmic and nuclear compartments.
4.3 TAT-GFP transduction by CM18-TAT-Cys in HeLa cells: Dose responses and viability of cells transduced
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, TAT-GFP-Cys recombinant protein (5 μΜ) was co-incubated with different concentrations of CM18-TAT-Cys (0, 0.5, 1 , 3, or 5 μΜ) and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 4.3 and Figure 11 A. Corresponding cellular toxicity data are presented in Figure 11 B. Table 4.3: Data from Figure 11A and 11 B
Figure imgf000079_0001
uorescence was more use an a so nuc ear, as con rme y uorescence microscopy.
Example 5:
Peptide shuttle agents increase GFP-NLS transduction efficiency and nuclear localization
The experiments in Examples 3 and 4 showed the ability of shuttle agents to deliver GFP and TAT-GFP intracellularly. The experiments presented in this example show that the shuttle agents can facilitate nuclear delivery of a GFP protein cargo fused to a nuclear localization signal (NLS).
5.1 Construction and amino acid sequence of GFP-NLS
Construction was performed as described in Example 3.4, except that an optimized NLS sequence was cloned between the GFP sequence and the stop codon (-). The NLS sequence is separated from the GFP sequence and the stop codon by two serine/glycine rich linkers. The recombinant GFP-NLS protein was purified as described in Example 1.4. The sequence of the GFP- NLS construct was:
MHHHHHHGGGGSGGGGSGGASTGIRMVSKGEELFTGWPILVELDGDVNGHK
FSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDF KEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMD ELYKGGSGGGSGGGSGWIRASSGGRSSDDEATADSQHAAPPKKKRKVGGSGG GSGGGSGGGRGTEIS- [SEQ ID NO: 62]
(MW = 34.85 kDa; pi = 6.46)
NLS sequence is underlined
Serine/glycine rich linkers are in bold 5.2 Nuclear delivery of GFP-NLS by CM18-TAT-Cys in HeLa cells in 5 minutes: Visualisation by fluorescence microscopy
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP-NLS recombinant protein (5 μΜ) was co-incubated with 5 μΜ of CM18-TAT-Cys, and then exposed to HeLa cells. GFP fluorescence was visualized by bright field and fluorescence microscopy after 5 minutes (as described in Example 3.2) at 10x, 20x and 40x magnifications, and sample results are shown in Figure 12. The microscopy results revealed that GFP-NLS is efficiently delivered to the nucleus in the presence of the shuttle agent CM18-TAT-Cys, after only 5 minutes of incubation.
5.3 GFP-NLS transduction by CM18-TAT-Cys in HeLa cells: Dose responses and viability of cells transduced
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. GFP-NLS recombinant protein (5 μΜ) was co-incubated with 0, 0.5, 1 , 3, or 5 μΜ of CM18-TAT- Cys, and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 5.1 and Figure 13A. Corresponding cellular toxicity data are presented in Figure 13B.
Table 5.1 : Data from Figure 13A and 13B
Figure imgf000080_0001
These results show that CM 18-TAT-Cys is able to increase GFP-NLS transduction efficiency in HeLa cells in a dose-dependent manner.
5.4 GFP-NLS transduction by CM18-TAT-Cys, CM18-Penetratin-Cys, and dimers thereof in HeLa cells
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. GFP-NLS recombinant protein (5 μΜ) was co-incubated with different concentrations and combinations of CM18-TAT-Cys, CM18-Penetratin-Cys, and dimers of each (dCM18-TAT-Cys, dCM18- Penetratin-Cys), and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Tables 5.2 and 5.3, and in Figures 14 and 15.
Table 5.2: Data in Figure 14
Figure imgf000081_0001
The results in Tables 5.2 and 5.3 and Figures 14 and 15 show that the transduction efficiency of GFP-NLS is increased in HeLa cells using the shuttle agents CM18-TAT-Cys and dCM18-TAT-Cys (see bars "1 " and "2" in Figures 14 and 15). Although no GFP-NLS intracellular delivery was observed using CM18-Penetratin-Cys or dCM18-Penetratin-Cys alone (see bars "3" and "4" in Figures 14 and 15), combination of CM18-TAT-Cys with CM18-Penetratin-Cys (monomer or dimer) improved GFP-NLS intracellular delivery (see four right-most bars in Figures 14 and 15).
5.5 GFP-NLS transduction by shuttle agents in HeLa cells: 5 min v. 1 h incubation; with or without FBS
HeLa cells were cultured and tested in the protein transduction assay described in Example
3.1. GFP-NLS recombinant protein (5 μΜ) was co-incubated with either CM18-TAT-Cys (3.5 μΜ) alone or with dCM18-Penetratin-Cys (1 μΜ). Cells were incubated for 5 minutes or 1 hour in plain DMEM media ("DMEM") or DMEM media containing 10% FBS ("FBS"), before being subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 5.4, and in Figure 16. Cells that were not treated with shuttle agent or GFP-NLS ("ctrl"), and cells that were treated with GFP- NLS without shuttle agent ("GFP-NLS 5 μΜ") were used as controls.
Table 5.4: Data in Figure 16
Figure imgf000082_0001
Figure imgf000082_0002
Figure imgf000083_0001
The results in Table 5.4 and Figure 16 show that the addition of even a relatively low amount of the dimer dCM18-Penetratin-Cys (1 μΜ; "dCM18pen") to the CM18-TAT-Cys monomer improved GFP-NLS transduction efficiency. Interestingly, intracellular GFP-NLS delivery was achieved in as little as 5 minutes of incubation, and delivery was still achievable (although reduced) in the presence of FBS.
5.6 GFP-NLS transduction by shuttle agents in THP-1 suspension cells
The ability of the shuttle agents to deliver GFP-NLS intracellular^ was tested in THP-1 cells, which is an acute monocytic leukemia cell line that grows in suspension. THP-1 cells were cultured (see Example 1) and tested in the protein transduction assay described in Example 3.1. GFP-NLS recombinant protein (5 μΜ) was co-incubated with or without 1 μΜ CM18-TAT-Cys, and exposed to the THP-1 cells for 5 minutes, before being subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 5.5 and in Figure 17A. Corresponding cellular toxicity data are presented in Figure 17B.
Table 5.5: Data in Figure 17A and 17B
Figure imgf000083_0002
The results in Table 5.5 and Figures 17A-17B demonstrate the ability of the shuttle agents to deliver protein cargo intracellular^ to a human monocytic cell line grown in suspension.
Example 6:
Peptide shuttle agents increase transduction efficiency of an FITC-labeled anti-tubulin antibody
The experiments in Examples 3-5 showed the ability of shuttle agents to increase the transduction efficiency of GFP, TAT-GFP, and GFP-NLS. The experiments presented in this example show that the shuttle agents can also deliver a larger protein cargo: an FITC-labeled anti-tubulin antibody. The FITC-labeled anti-tubulin antibody was purchased from (Abeam, ab64503) and has an estimated molecular weight of 150 KDa. The delivery and microscopy protocols are described in Example 3. 6.1 Transduction of a functional antibody by CM18-TAT-Cys in HeLa cells: Visualization by microscopy
FITC-labeled anti-tubulin antibody (0.5 μΜ) was co-incubated with 5 μΜ of CM18-TAT-Cys and exposed to HeLa cells for 1 hour. Antibody delivery was visualized by bright field (20x) and fluorescence microscopy (20x and 40x). As shown in Figure 18, fluorescent tubulin fibers in the cytoplasm were visualized, demonstrating the functionality of the antibody inside the cell.
6.2 Transduction of a functional antibody by CM18-TAT-Cys, CM18-Penetratin-Cys, and dimers in HeLa cells: Flow cytometry
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. FITC-labeled anti-tubulin antibody (0.5 μΜ) was co-incubated with 3.5 μΜ of CM18-TAT-Cys, CM18-Penetratin-Cys or dCM18-Penetratin-Cys, or a combination of 3.5 μΜ of CM18-TAT-Cys and 0.5 μΜ of dCM18-Penetratin-Cys, and exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 6.1 and Figure 19A. Corresponding cellular toxicity data are presented in Figure 19B.
Table 6.1 : Data from Figure 19A and 19B
Figure imgf000084_0001
The results in Table 6.1 and Figures 18A-18C and 19A-19B show that both CM18-TAT-Cys and CM18-Penetratin-Cys facilitate intracellular delivery of an FITC-labeled anti-tubulin antibody. In contrast to the results with GFP, TAT-GFP, and GFP-NLS in Examples 3-5, CM18-Penetratin-Cys was able to deliver the antibody cargo intracellularly when used alone (without CM18-TAT-Cys). However, combination of CM18-TAT-Cys and dCM18-Penetratin-Cys allowed for higher intracellular delivery as compared with CM18-TAT-Cys alone, and with less cell toxicity as compared to CM18-Penetratin-Cys and dCM18-Penetratin-Cys (see Figure 19A and 19B).
Example 7:
CM18-TAT-Cys enables intracellular plasmid DNA delivery but poor plasmid expression
The ability of the CM 18-TAT-Cys shuttle agent to deliver plasmid DNA intracellularly was tested in this example on HEK293A cells using a plasmid encoding GFP.
7.1 Transfection assay in HEK293A cells
One day before the transfection assay was performed, mammalian cells (HEK293A) in exponential growth phase were harvested and plated in a 24-well plate (50,000 cells per well). The cells were incubated overnight in appropriate growth media containing FBS. The next day, in separate sterile 1.5 mL tubes, pEGFP labeled with a Cy5™ fluorochrome was mixed for 10 min at 37°C with CM18- TAT-Cys (0.05, 0.5, or 5 μΜ) in fresh PBS at a final 100 μί volume. The media in wells was removed and the cells were quickly washed three times with PBS and 500 L of warm media without FBS was added. The pEGFP and CM 18-TAT-Cys solution was added to the cells and incubated at 37°C for 4 hours. After the incubation, cells were washed with PBS and fresh media containing FBS was added. Cells were incubated at 37°C before being subjected to flow cytometry analysis as described in Example 3.
7.2 Plasmid DNA delivery with CM18-TAT-Cys
Plasmid DNA (pEGFP) was labeled with a Cy5™ dye following the manufacturer's instructions (Mirus Bio LLC). Cy5™ Moiety did not influence transfection efficiency when compared to unlabelled plasmid using standard transfection protocol (data not shown). Flow cytometry analysis allowed quantification of Cy5™ emission, corresponding to DNA intracellular delivery, and GFP emission, corresponding to successful nuclear delivery, DNA transcription and protein expression. The results are shown in Table 7.1 and in Figure 20. Table 7.1 : Data from Figure 20
Figure imgf000086_0001
The results shown in Table 7.1 and in Figure 20 show that CM18-TAT-Cys was able to increase the intracellular delivery the plasmid DNA when used at 0.05, 0.5 and 5 μΜ concentrations, as compared to cell incubated with DNA alone ("pEGFP-Cy5"). However, no expression of GFP was detected in the cells, which suggests that very little of the plasmid DNA gained access to the cytoplasmic compartment, allowing nuclear localization. Without being bound by theory, it is possible that the plasmid DNA was massively sequestered in endosomes, preventing escape to the cytoplasmic compartment. Salomone et al., 2013 reported the use of a CM18-TAT11 hybrid peptide to deliver plasmid DNA intracellular^. They used the luciferase enzyme reporter assay to assess transfection efficiency, which may not be ideal for quantifying the efficiency of cytoplasmic/nuclear delivery, as the proportion of plasmid DNA that is successfully released from endosomes and delivered to the nucleus may be overestimated due to the potent activity of the luciferase enzyme. In this regard, the authors of Salomone et al., 2013 even noted that the expression of luciferase occurs together with a massive entrapment of (naked) DNA molecules into vesicles, which is consistent with the results shown in Table 7.1 and in Figure 20.
Example 8:
Addition of a histidine-rich domain to shuttle agents further improves GFP-NLS transduction efficiency
8.1 GFP-NLS transduction by His-CM18-TAT-Cys in HeLa cells: Visualization by microscopy
GFP-NLS (5 μΜ; see Example 5) was co-incubated with 5 μΜ of CM18-TAT-Cys or His- CM18-TAT and exposed to HeLa cells for 1 hour. Nuclear fluorescence of intracellular^ delivered GFP- NLS was confirmed by fluorescence microscopy (data not shown), indicating successful delivery of GFP-NLS to the nucleus.
8.2 GFP-NLS transduction by His-CM18-TAT in HeLa cells: Flow cytometry
HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. GFP-NLS (5 μΜ) was co-incubated with 0, 1 , 3, or 5 μΜ of CM18-TAT-Cys or His-CM18-TAT, and exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 8.1 and Figure 21 A. Corresponding cellular toxicity data are presented in Figure 21 B.
Table 8.1 : Data from Figure 21 A and 21 B
Figure imgf000087_0001
Figure imgf000087_0002
Strikingly, the results in Table 8.1 and in Figures 21A-21 B show that His-CM18-TAT was able to increase GFP-NLS protein transduction efficiency by about 2-fold at 3 μΜ and 5 μΜ concentrations, as compared to CM18-TAT-Cys. These results suggest that adding a histidine-rich domain to a shuttle agent comprising an ELD and CPD, may significantly increase its polypeptide cargo transduction efficiency. Alternatively or in parallel, combining the shuttle agents with a further independent synthetic peptide containing a histidine-rich domain fused to a CPD (but lacking an ELD) may provide a similar advantage for protein transduction, with the added advantage of allowing the concentration of the histidine-rich domain to be varied or controlled independently from the concentration of the shuttle agent. Without being bound by theory, the histidine-rich domain may act as a proton sponge in the endosome, providing another mechanism of endosomal membrane destabilization. Example 9:
His-CM18-PTD4 increases transduction efficiency and nuclear delivery of GFP-NLS, mCherry™-
NLS and FITC-labeled anti-tubulin antibody
9.1 Protein transduction protocols
Protocol A: Protein transduction assay for delivery in cell culture medium
One day before the transduction assay was performed, cells in exponential growth phase were harvested and plated in a 96-well plate (20,000 cells per well). The cells were incubated overnight in appropriate growth media containing FBS (see Example 1). The next day, in separate sterile 1.5-mL tubes, cargo protein at the desired concentration was pre-mixed (pre-incubated) for 10 min at 37°C with the desired concentration of shuttle agents in 50 L of fresh serum-free medium (unless otherwise specified). The media in wells was removed and the cells were washed one to three times (depending on the type of cells used) with PBS previously warmed at 37°C. The cells were incubated with the cargo protein/shuttle agent mixture at 37°C for the desired length of time. After the incubation, the cells were washed three times with PBS and/or heparin (0.5 mg/mL) previously warmed at 37°C. The washes with heparin were used for human THP-1 blood cells to avoid undesired cell membrane-bound protein background in subsequent analyses (microscopy and flow cytometry). The cells were finally incubated in 50 L of fresh medium with serum at 37°C before analysis. Protocol B: Protein transduction assay for adherent cells in PBS
One day before the transduction assay was performed, cells in exponential growth phase were harvested and plated in a 96-well plate (20,000 cells per well). The cells were incubated overnight in appropriate growth media containing serum (see Example 1). The next day, in separate sterile 1.5-mL tubes, shuttle agents were diluted in sterile distilled water at room temperature (if the cargo is or comprised a nucleic acid, nuclease-free water was used). Cargo protein(s) were then added to the shuttle agents and, if necessary, sterile PBS was added to obtain the desired concentrations of shuttle agent and cargo in a sufficient final volume to cover the cells (e.g., 10 to 100 L per well for a 96-well plate). The shuttle agent/cargo mixture was then immediately used for experiments. At least three controls were included for each experiment, including: (1) shuttle agent alone (e.g., at highest concentration tested); (2) cargo alone; and (3) without any cargo or shuttle agent. The media in wells was removed, cells were washed once with PBS previously warmed at 37°C, and the shuttle agent/cargo mixture was then added to cover all cells for the desired length of time. The shuttle agent/cargo mixture in wells was removed, the cells were washed once with PBS, and fresh complete medium was added. Before analysis, the cells were washed once with PBS and fresh complete medium was added.
Protocol C: Protein transduction assay for suspension cells in PBS
One day before the transduction assay was performed, suspension cells in exponential growth phase were harvested and plated in a 96-well plate (20,000 cells per well). The cells were incubated overnight in appropriate growth media containing serum (see Example 1). The next day, in separate sterile 1.5-mL tubes, shuttle agents were diluted in sterile distilled water at room temperature (if the cargo is or comprised a nucleic acid, nuclease-free water was used). Cargo protein(s) were then added to the shuttle agents and, if necessary, sterile PBS or cell culture medium (serum-free) was added to obtain the desired concentrations of shuttle agent and cargo in a sufficient final volume to resuspend the cells (e.g., 10 to 100 μί per well in a 96-well plate). The shuttle agent/cargo mixture was then immediately used for experiments. At least three controls were included for each experiment, including: (1) shuttle agent alone (e.g., at highest concentration tested); (2) cargo alone; and (3) without any cargo or shuttle agent. The cells were centrifuged for 2 minutes at 400g, the medium was then removed and the cells were resuspended in PBS previously warmed at 37°C. The cells were centrifuged again 2 minutes at 400g, the PBS removed, and the cells were resuspended in the shuttle agent/cargo mixture. After the desired incubation time, 100 μί of complete medium was added directly on the cells. Cells were centrifuged for 2 minutes at 400g and the medium was removed. The pellet was resuspended and washed in 200 μί of PBS previously warmed at 37°C. After another centrifugation, the PBS was removed and the cells were resuspended in 100 μί of complete medium. The last two steps were repeated one time before analysis.
9.2 GFP-NLS transduction by His-CM18-PTD4 in HeLa cells using Protocol A or B: Flow cytometry
To compare the effects of different protocols on shuttle agent transduction efficiency, HeLa cells were cultured and tested in the protein transduction assays using Protocol A or B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co-incubated with 10 μΜ of His-CM18-PTD4 and exposed to HeLa cells for 1 hour using Protocol A, or was co-incubated with 35 μΜ of His-CM18-PTD4 and exposed to HeLa cells for 10 seconds using Protocol B. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 9.1 and Figure 22A. ("Pos cells (%)" is the percentage of cells emanating a GFP signal). Table 9.1 : Comparison of Protein Transduction Protocols A and B: Data from Figure 22A
Figure imgf000090_0001
The above results show that higher protein transduction efficiency for the cargo GFP-NLS using the shuttle agent His-CM18-PTD4 was obtained using Protocol B, as compared to Protocol A.
9.3 GFP-NLS transduction by His-CM18-PTD4 in HeLa cells using Protocol B: Flow cytometry
A dose response experiment was performed to evaluate the effect of His-CM18-PTD4 concentration on protein transduction efficiency. HeLa cells were cultured and tested in the protein transduction assay described in Protocol B of Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co-incubated with 0, 50, 35, 25, or 10 μΜ of His-CM18-PTD4, and then exposed to HeLa cells for 10 seconds. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 9.2 and Figure 22B.
Table 9.2: Dose response of shuttle agent using Protocol B: Data from Figure 22B
Figure imgf000090_0002
The above results show that His-CM18-PTD4 is able to increase GFP-NLS transduction efficiency in HeLa cells in a dose-dependent manner.
9.4 GFP-NLS transduction by His-CM18-PTD4 in HeLa cells using Protocol B: Visualization by microscopy
GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co-incubated with 35 μΜ of His- CM18-PTD4, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. The cells were then subjected to fluorescence microscopy analysis as described in Examples 3.2 and 3.2a.
For the sample results shown in Figures 23A-23D and 24A-24B, GFP fluorescence of the HeLa cells was immediately visualized by bright field and fluorescence microscopy at 4x, 20x and 40x magnifications after the final washing step.
In Figures 23A-23D, the upper panels in Figures 23A, 23B and 23C show nuclei labelling (DAPI) at 4x, 20x and 40x magnifications, respectively, while the lower respective panels show corresponding GFP-NLS fluorescence. In Figure 23C, white triangle windows indicate examples of areas of co-labelling between nuclei (DAPI) and GFP-NLS signals. In Figure 23D, the upper and bottom panels show sample bright field images of the HeLa cells, and the middle panel shows the results of a corresponding FACS analysis (performed as described in Example 3.3), which indicates the percentage of cells in a 96-plate with a GFP signal. No significant GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
Figures 24A-24Bshows bright field (Figure 24A) and fluorescent images (Figure 24B). The inset in Figure 24B shows the results of a corresponding FACS analysis (performed as described in Example 3.3), which indicates the percentage of cells in a 96-plate well with a GFP signal. No significant GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
For the sample results shown in Figures 25A-25B, the HeLa cells were fixed, permeabilized and subjected to immuno-labelling as described in Example 3.2a before visualization by fluorescence microscopy as described in Example 3.2. GFP-NLS was labelled using a primary mouse monoclonal anti-GFP antibody (Feldan, #A017) and a secondary goat anti-mouse Alexa™-594 antibody (Abeam #150116). The upper panels in Figures 25A-25B show nuclei labelling (DAPI), and the lower respective panels show corresponding labelling for GFP-NLS. Figures 25A and 25B show sample images at 20x and 40x magnifications, respectively. White triangle windows indicate examples of areas of co-labelling between nuclei and GFP-NLS. No significant GFP-NLS labelling was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
Figure 26A-26C shows sample images captured with confocal microscopy at 63x magnification of living cells. Figure 26A shows a bright field image, while Figure 26B shows the corresponding fluorescent GFP-NLS. Figure 26C is an overlay between the images in Figures 26A and 26B. No significant GFP-NLS fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown). 9.4a FTIC-labeled anti-tubulin antibody transduction by His-CM18-PTD4 in HeLa cells using Protocol B: Visualization by microscopy
FITC-labeled anti-tubulin antibody (0.5 μΜ; Abeam, ab64503) was co-incubated with 50 μΜ of His-CM18-PTD4, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. The cells were then subjected to fluorescence microscopy analysis as described in Examples 3.2 and 3.2a, wherein the FITC fluorescence of the anti-tubulin antibody in the HeLa cells was immediately visualized by bright field and fluorescence microscopy at 20x magnification after the final washing step. No significant FITC fluorescence was observed in negative control samples (i.e., cells exposed to the FITC-labeled anti-tubulin antibody without any shuttle agent; data not shown).
Overall, the results in Examples 9.4 and 9.4a show that GFP-NLS and FITC-labeled anti- tubulin antibody cargos are successfully transduced and delivered to the nucleus and/or the cytosol of HeLa cells in the presence of the shuttle agent His-CM18-PTD4.
9.5 GFP-NLS kinetic transduction by His-CM18-PTD4 in HeLa cells: Visualization by microscopy
GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co-incubated with 50 μΜ of His- CM18-PTD4, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. After a washing step, the GFP fluorescence of the HeLa cells was immediately visualized by fluorescence microscopy (Example 3.2) at 20x magnification after different intervals of time. Typical results are shown in Figures 27A to 27D, in which fluorescence microscopy images were captured after 45, 75, 100, and 120 seconds (see Figures 27A, 27B, 27C and 27D, respectively).
As shown in Figure 27A, diffuse cellular GFP fluorescence was generally observed after 45 seconds, with areas of lower GFP fluorescence in the nucleus in many cells. These results suggest predominantly cytoplasmic and low nuclear distribution of the GPF-NLS delivered intracellular^ via the shuttle agent after 45 seconds. Figures 27B to 27D show the gradual redistribution of GFP fluorescence to the cell nuclei at 75 seconds (Figure 27B), 100 seconds (Figure 27C), and 120 seconds (Figure 27D) following exposure to the His-CM18-PTD4 shuttle agent and GFP-NLS cargo. No significant cellular GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
The results in Example 9.5 show that GFP-NLS is successfully delivered to the nucleus of
HeLa cells in the presence of the shuttle agent His-CM18-PTD4 by 2 minutes. 9.6 GFP-NLS and mCherry™-NLS co-transduction by His-CM18-PTD4 in HeLa cells: Visualization by microscopy
mCherry™-NLS recombinant protein was constructed, expressed and purified from a bacterial expression system as described in Example 1.4. The sequence of the mCherry™-NLS recombinant protein was:
MHHHHHHGGGGSGGGGSGGASTGIRMVSKCEEDNMAI IKEFMRFKVHMEG SVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK AYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGWTVTQDSSLQDGEFIY KVKLRGTNFPSDGQVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDG GHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGR HSTGGMDELYKGGSGGGSGGGSGWIRASSGGRSSDDEATADSQHAAPPKK KRKVGGSGGGSGGGSGGGRGTEIS [SEQ ID NO: 73]
(MW = 34.71 kDa; pi = 6.68)
NILS sequence is underlined
Serine/glycine rich linkers are in bold
GFP-NLS recombinant protein (5 μΜ; see Example 5.1) and mCherry™-NLS recombinant protein (5 μΜ) were co-incubated together with 35 μΜ of His-CM18-PTD4, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. After washing steps, the cells were immediately visualized by bright field and fluorescence microscopy at 20x magnifications as described in Example 3.2. Sample results are shown in Figure 28A-28D, in which corresponding images showing bright field (Figure 28A), DAPI fluorescence (Figure 28B), GFP-NLS fluorescence (Figure 28C), and mCherry™-NLS fluorescence (Figure 28D) are shown. White triangle windows indicate examples of areas of co-labelling between GFP-NLS and mCherry™ fluorescence signals in cell nuclei. No significant cellular GFP or mCherry™ fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS or mCherry™ without any shuttle agent; data not shown).
These results show that GFP-NLS and mCherry™-NLS are successfully delivered together to the nucleus in HeLa cells in the presence of the shuttle agent His-CM18-PTD4. 9.7 GFP-NLS transduction by His-CM18-PTD4 in THP-1 suspension cells: Flow cytometry
The ability of the His-CM18-PTD4 to deliver GFP-NLS in the nuclei of suspension cells was tested using THP-1 cells. THP-1 cells were cultured and tested in the protein transduction assays using Protocols A and C as described in Example 9.1. GFP-NLS (5 μΜ; see Example 5.1) was co-incubated with 1 μΜ of His-CM18-PTD4 and exposed to THP-1 cells for 1 hour (Protocol A), or was co-incubated with 5 μΜ of His-CM18-PTD4 and exposed to THP-1 cells for 15 seconds (Protocol C). The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 9.3 and in Figure 31. Table 9.3: Data from Figure 31
Figure imgf000094_0001
9.8 GFP-NLS transduction by His-CM18-PTD4 in THP-1 cells: Visualization by microscopy
GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co-incubated with 5 μΜ of His- CM18-PTD4, and then exposed to THP-1 cells for 15 seconds using Protocol C as described in Example 9.1. The cells were subjected to microscopy visualization as described in Example 3.2.
For the sample results shown in Figure 32A-32D, GFP fluorescence of the HeLa cells was immediately visualized by bright field (upper panels in Figures 32A-32C) and fluorescence (lower panels in Figures 32A-32C) microscopy at 4x, 10x and 40x magnifications (Figures 32A-32C, respectively) after the final washing step. White triangle windows in Figure 32C indicate examples of areas of co-labelling between bright field and fluorescence images. Figure 32D shows typical results of a corresponding FACS analysis (performed as described in Example 3.3), which indicates the percentage of cells in a 96-plate well with a GFP signal. Additional results are shown in Figure 33A- 33D, in which Figures 33A and 33B show bright field images, and Figures 33C and 33D show corresponding fluorescence images. White triangle windows indicate examples of areas of co-labelling between Figures 33A and 33C, as well as Figures 33B and 33D. The right-most panel shows typical results of a corresponding FACS analysis (performed as described in Example 3.3), which indicates the percentage of cells in a 96-plate well with a GFP signal.
The results in this example show that GFP-NLS is successfully delivered intracellular^ in THP- 1 cells in the presence of the shuttle agent His-CM18-PTD4.
Example 10:
Different multi-domain shuttle agents, but not single-domain peptides, successfully transduce
GFP-NLS in HeLa and THP-1 cells
10.1 GFP-NLS transduction by different shuttle agents in HeLa cells: Flow cytometry
HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co- incubated with 50 μΜ of different shuttle agents and exposed to the HeLa cells for 10 seconds. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.1 and Figure 29A. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any shuttle agent.
Table 10.1 : Data from Figure 29A
Figure imgf000095_0001
10.2 GFP-NLS transduction by different shuttle agents with varying incubation times in HeLa cells: Flow cytometry
HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co- incubated with 10 μΜ of TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4 for 1 , 2, or 5 minutes. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.2 and Figure 29B. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any shuttle agent. Table 10.2: Data from Figure 29B
Figure imgf000096_0001
10.3 GFP-NLS transduction by TAT-KALA, His-CM18-PTD4 and His-C(LLKK)3C-PTD4 with varying incubation times in HeLa cells: Flow cytometry
HeLa cells were cultured and tested in the protein transduction assays using Protocol C as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co- incubated with 5 μΜ of TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4 for 1 , 2, or 5 minutes. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.3 and Figure 29C. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any shuttle agent.
Table 10.3: Data from Figure 29C
Figure imgf000096_0002
10.4 GFP-NLS transduction by different shuttle agents in HeLa cells: Flow cytometry
HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co- incubated with 50 μΜ of different shuttle agents (see Table 1.3 for amino acid sequences and properties) and exposed to the HeLa cells for 10 seconds. The cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Tables 10.3a & 10.3b and Figures 29E & 29F. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any shuttle agent.
Table 10.3a: Data from Figure 29E
Figure imgf000097_0001
HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co- incubated with 10 μΜ of TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4 for 1 , 2, or 5 minutes. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Tables 10.3c & 10.3b and Figures 29G and 29H. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any shuttle agent.
Table 10.3c: Data from Figure 29G
Figure imgf000098_0001
Table 10.3d: Data from Figure 29H
Figure imgf000098_0002
The shuttle agent CM18-PTD4 was used as a model to demonstrate the modular nature of the individual protein domains, as well as their ability to be modified. More particularly, the presence or absence of: an N-terminal cysteine residue ("Cys"); different flexible linkers between the ELD and CPD domains (11": GGS; 12": GGSGGGS (SEQ ID NO: 106); and 13": GGSGGGSGGGS (SEQ ID NO: 107)) and different lengths, positions, and variants to histidine-rich domains; were studied.
HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co- incubated with 20 μΜ of different shuttle peptide variants (see Table 1.3 for amino acid sequences and properties) of the shuttle agent His-CM18-PTD4 for 1 minute. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.3e and Figure 29I. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any shuttle agent.
Table 10.3e: Data from Figure 29I
Figure imgf000099_0001
These results show that variations in a given shuttle (e.g., CM18-PTD4) may be used to modulate the degree of transduction efficiency and cell viability of the given shuttle. More particularly, the addition of an N-terminal cysteine residue to CM18-PTD4 (see Cys-CM18-PTD4), decreased GFP- NLS transduction efficiency by 11 % (from 47.6% to 36.6%), but increased cell viability from 33.9% to 78.7%. Introduction of flexible linker domains (L1 , L2, and L3) of different lengths between the CM18 and PTD4 domains did not result in a dramatic loss of transduction efficiency, but increased cell viability (see CM18-L1-PTD4, CM18-L2-PTD4, and CM18-L3-PTD4). Finally, variations to the amino acid sequences and/or positions of the histidine-rich domain(s) did not result in a complete loss of transduction efficiency and cell viability of His-CM18-PTD4 (see 3His-CM18-PTD4, 12His-CM18-PTD4, HA-CM18-PTD4, 3HA-CM18-PTD4, CM18-His-PTD4, and His-CM18-PTD4-His). Of note, adding a second histidine-rich domain at the C terminus of His-CM18-PTD4 (i.e., His-CM18-PTD4-His) increased transduction efficiency from 60% to 68% with similar cell viability.
10.5 Lack of GFP-NLS transduction by single-domain peptides or a His-CPD peptide in HeLa cells: Flow cytometry
HeLa cells were cultured and tested in the protein transduction assays using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co- incubated with 50 μΜ of different single-domain peptides (TAT; PTD4; Penetratin; CM18; C(LLKK)3C (SEQ ID NO: 63); KALA) or the two-domain peptide His-PTD4 (lacking an ELD), and exposed to the HeLa cells for 10 seconds. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 10.4 and Figure 29D. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any single- domain peptide or shuttle agent.
Table 10.4: Data from Figure 29D
Figure imgf000100_0001
Figure imgf000101_0001
These results show that the single-domain peptides TAT, PTD4, Penetratin, CM18, C(LLKK)3C (SEQ ID NO: 63), KALA, or the two-domain peptide His-PTD4 (lacking an ELD), are not able to successfully transduce GFP-NLS in HeLa cells.
10.6 GFP-NLS transduction by TAT-KALA, His-CM18-PTD4, His-C(LLKK)3C-PTD4, PTD4- KALA, EB1-PTD4, and His-CM18-PTD4-His in HeLa cells: Visualization by microscopy
GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co-incubated with 50 μΜ of shuttle agent, and then exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. The cells were visualized by microscopy as described in Example 3.2, after an incubation time of 2 minutes.
For the sample results shown in Figures 30A-30F, GFP fluorescence of the HeLa cells was immediately visualized by bright field (bottom row panels in Figures 30A-30F) and fluorescence (upper and middle row panels in Figures 30A-30F) microscopy at 20x or 40x magnifications after the final washing step. The results with the shuttle agents TAT-KALA, His-CM18-PTD4, and His-C(LLKK)3C- PTD4 are shown in Figures 30A, 30B and 30C, respectively. The results with the shuttle agents PTD4- KALA, EB1 -PTD4, and His-CM18-PTD4-His are shown in Figures 30D, 30E and 30F, respectively. The insets in the bottom row panels in Figures 30A-30F show the results of corresponding FACS analyses (performed as described in Example 3.3), which indicates the percentage of cells in a 96-plate well with a GFP signal. No significant cellular GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
10.7 GFP-NLS transduction by TAT-KALA, His-CM18-PTD4 and His-C(LLKK)3C-PTD4 with varying incubation times in THP-1 cells: Flow cytometry
THP-1 cells were cultured and tested in the protein transduction assays using Protocol C as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co- incubated with 1 μΜ of TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4 for 15, 30, 60, or 120 seconds. After the final washing step, the cells were subjected to flow cytometry analysis as described in Example 3.3. The mean percentages of cells emanating a GFP signal ("Pos cells (%)") are shown in Table 10.4 and in Figure 34A. The mean fluorescence intensity is shown in Table 10.5 and Figure
34B. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any shuttle agent.
Table 10.4: Data from Figure 34A
Figure imgf000102_0001
Figure imgf000102_0002
Table 10.5: Data from Figure 34B
Figure imgf000102_0003
Example 11 :
Repeated daily treatments with low concentrations of shuttle agent in the presence of serum results in GFP-NLS transduction in THP-1 cells
11.1 GFP-NLS transduction with His-CM18-PTD4 or His-C(LLKK)3C-PTD4 in THP-1 cells: Flow cytometry
THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1, but with the following modifications. GFP-NLS recombinant protein (5, 2.5, or 1 μΜ; see Example 5.1) was co-incubated with 0.5 or 0.8 μΜ of His-CM18-PTD4, or with 0.8 μΜ of His-C(LLKK)3C-PTD4, and then exposed to THP-1 cells each day for 150 min in the presence of cell culture medium containing serum. Cells were washed and subjected to flow cytometry analysis as described in Example 3.3 after 1 or 3 days of repeated exposure to the shuttle agent/cargo. The results are shown in Table 11.1 and in Figures 35A, 35B, 35C and 35F. The negative control ("Ctrl") corresponds to cells that were incubated with GFP-NLS recombinant protein (5 μΜ) without any shuttle agent.
Table 11.1 : Data from Figures 35A, 35B, 35C and 35F
Figure imgf000103_0001
The viability of THP-1 cells repeatedly exposed to His-CM18-PTD4 and GFP-NLS was determined as described in Example 3.3a. The results are shown in Tables 11.2 and 11.3 and in Figures 35D and 35E. The results in Table 11.2 and Figure 35D show the metabolic activity index of the THP-1 cells after 1 , 2, 4, and 24h, and the results in Table 11.3 and Figure 35E show the metabolic activity index of the THP-1 cells after 1 to 4 days.
Table 11.2: Data from Figure 35D
Figure imgf000104_0001
Figure imgf000104_0002
The results in Example 11 show that repeated daily (or chronic) treatments with relatively low concentrations of His-CM18-PTD4 or His-C(LLKK)3C-PTD4 in the presence of serum result in intracellular delivery of GFP-NLS in THP-1 cells. The results also suggest that the dosages of the shuttle agents and the cargo can be independently adjusted to improve cargo transduction efficiency and/or cell viability. Example 12:
His-CM18-PTD4 increases transduction efficiency and nuclear delivery of GFP-NLS in a plurality of cell lines
12.1 GFP-NLS transduction with His-CM18-PTD4 in different adherent & suspension cells: Flow cytometry
The ability of the shuttle agent His-CM18-PTD4 to deliver GFP-NLS to the nuclei of different adherent and suspension cells using Protocols B (adherent cells) or C (suspension cells) as described in Example 9.1 was examined. The cell lines tested included: HeLa, Balb3T3, HEK 293T, CHO, NIH3T3, Myoblasts, Jurkat, THP-1 , CA46, and HT2 cells, which were cultured as described in Example 1. GFP-NLS (5 μΜ; see Example 5.1) was co-incubated with 35 μΜ of His-CM18-PTD4 and exposed to adherent cells for 10 seconds (Protocol B), or was co-incubated with 5 μΜ of His-CM18-PTD4 and exposed to suspension cells for 15 seconds (Protocol C). Cells were washed and subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 12.1 and Figure 36. "Pos cells (%)" is the mean percentages of all cells that emanate a GFP signal.
Table 12.1 : Data from Figure 36
Figure imgf000105_0001
12.2 GFP-NLS transduction with His-CM18-PTD4 in several adherent and suspension cells: visualization by microscopy
GFP-NLS recombinant protein (5 μΜ; see Example 5.1) was co-incubated with 35 μΜ of His- CM18-PTD4 and exposed to adherent cells for 10 seconds using Protocol A, or was co-incubated with 5 μΜ of His-CM18-PTD4 and exposed to suspension cells for 15 seconds using Protocol B, as described in Example 9.1. After washing the cells, GFP fluorescence was visualized by bright field and fluorescence microscopy. Sample images captured at 10x magnifications showing GFP fluorescence are shown in Figures 37A-37H for (Figure 37A) 293T, (Figure 37B) Balb3T3, (Figure 37C) CHO, (Figure 37D) Myoblasts, (Figure 37E) Jurkat, (Figure 37F) CA46, (Figure 37G) HT2, and (Figure 37H) NIH3T3 cells. The insets show corresponding flow cytometry results performed as described in Example 3.3, indicating the percentage of GFP-NLS-positive cells. No significant cellular GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
Nuclear localization of the GFP-NLS was further confirmed in fixed and permeabilized myoblasts using cell immuno-labelling as described in Example 3.2a. GFP-NLS was labeled using a primary mouse monoclonal anti-GFP antibody (Feldan, #A017) and a secondary goat anti-mouse Alexa™-594 antibody (Abeam #150116). Nuclei were labelled with DAPI. Sample results for primary human myoblast cells are shown in Figures 38A-38B, in which GFP immuno-labelling is shown in Figure 38A, and an overlay of the GFP immuno-labelling and DAPI labelling is shown in Figure 38B. No significant cellular GFP labelling was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).
The microscopy results revealed that GFP-NLS is successfully delivered to the nucleus of all the tested cells using the shuttle agent His-CM18-PTD4.
Example 13:
His-CM18-PTD4 enables transduction of a CRISPR/Cas9-NLS system and genome editing
Hela cells
13.1 Cas9-NLS recombinant protein
Cas9-NLS recombinant protein was constructed, expressed and purified from a bacterial expression system as described in Example 1.4. The sequence of the Cas9-NLS recombinant protein produced was:
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYID GGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA
GSPAIKKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV NIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL WAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDL11
KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS I TGLYETRIDLSQLGGDGGRSSDDEATADSQHAAPPKKKRKVGGSGGGSGGGS GGGRHHHHHH [SEQ ID NO: 74]
(MW = 162.9 kDa; pi = 9.05)
NILS sequence is underlined
Serine/glycine rich linkers are in bold 13.2 Transfection plasmid surrogate assay
This assay enables one to visually identify cells that have been successfully delivered an active CRISPR/Cas9 complex. As shown in Figure 39A, the assay involves transfecting cells with an expression plasmid DNA encoding the fluorescent proteins mCherry™ and GFP, with a STOP codon separating their two open reading frames. Transfection of the cells with the expression plasmid results in mCherry™ expression, but no GFP expression (Figure 39B). A CRISPR/Cas9 complex, which has been designed/programmed to cleave the plasmid DNA at the STOP codon, is then delivered intracellular^ to the transfected cells expressing mCherry™ (Figure 39D). Successful transduction of an active CRISPR/Cas9 complex results in the CRISPR/Cas9 complex cleaving the plasmid DNA at the STOP codon (Figure 39C). In a fraction of the cells, random non-homologous DNA repair of the cleaved plasmid occurs and results in removal of the STOP codon, and thus GFP expression and fluorescence (Figure 39E).
On Day 1 of the transfection plasmid surrogate assay, DNA plasmids for different experimental conditions (250 ng) are diluted in DMEM (50 L) in separate sterile 1.5-mL tubes, vortexed and briefly centrifuged. In separate sterile 1.5-mL tubes, Fastfect™ transfection reagent was diluted in DMEM (50 [il) with no serum and no antibiotics at a ratio of 3:1 (3 μί of Fastfect™ transfection reagent for 1 g of DNA) and then quickly vortexed and briefly centrifuged. The Fastfect™/DMEM mixture was then added to the DNA mix and quickly vortexed and briefly centrifuged. The Fastfect™/DMEM/DNA mixture is then incubated for 15-20 min at room temperature, before being added to the cells (100 L per well). The cells are then incubated at 37°C and 5% CO2 for 5h. The media is then changed for complete medium (with serum) and further incubated at 37°C and 5% CO2 for 24-48h. The cells are then visualized under fluorescent microscopy to view the mCherry™ signal.
13.3 His-CM18-PTD4 - mediated CRISPR/Cas9-NLS system delivery and cleavage of plasmid DNA
RNAs (crRNA & tracrRNA) were designed to target a nucleotide sequence of the EMX1 gene, containing a STOP codon between the mCherry™ and GFP coding sequences in the plasmid of Example 13.2. The sequences of the crRNA and tracrRNA used were as follows:
- crRNA [SEQ ID NO: 75] :
5 '-GAGUCCGAGCAGAAGAAGAAGUUUUAGAGCUAUGCUGUUUUG-3 '
- tracrRNA [SEQ ID NO: 76] :
5 '-
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU
CGGUGCU-3 '
HeLa cells were cultured and subjected to the transfection plasmid surrogate assay as described in Example 13.2). On Day 1 , the HeLa cells were transfected with a plasmid surrogate encoding the mCherry™ protein as shown in Figure 39A. On Day 2, a mix of Cas9-NLS recombinant protein (2 μΜ; see Example 13.1) and RNAs (crRNA & tracrRNA; 2 μΜ; see above) were co-incubated with 50 μΜ of His-CM18-PTD4, and the mixture (CRISPR/Cas9 complex) was exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. Double-stranded plasmid DNA cleavage by the CRISPR/Cas9 complex at the STOP codon between the mCherry™ and GFP coding sequences (Figure 39B), and subsequent non-homologous repair by the cell in some cases results in removal of the STOP codon (Figure 39C), thereby allowing expression of both the mCherry™ and GFP fluorescent proteins in the same cell on Day 3 (Figure 39D-39E). White triangle windows in Figures 39D and 39E indicate examples of areas of co-labelling between mCherry™ and GFP.
As a positive control for the CRISPR/Cas9-NLS system, HeLa cells were cultured and co- transfected with three plasmids: the plasmid surrogate (as described in Example 13.2) and other expression plasmids encoding the Cas9-NLS protein (Example 13.1) and the crRNMracrRNAs (Example 13.3). Typical fluorescence microscopy results are shown in Figure 40A to 40D. Figures 40A and 40B show cells 24 hours post-transfection, while Figures 40C and 40D show cells 72 hours post-transfection.
Figure 40E-40H shows the results of a parallel transfection plasmid surrogate assay performed using 35 μΜ of the shuttle His-CM18-PTD4, as described for Figure 39A-39E. Figures 40E and 40F show cells 24 hours post-transduction, while Figures 40G and 40H show cells 48 hours post- transduction. Figures 40E and 40G show mCherry™ fluorescence, and Figures 40F and 40H show GFP fluorescence, the latter resulting from removal of the STOP codon by the transduced CRISPR/Cas9-NLS complex and subsequent non-homologous repair by the cell. No significant cellular GFP fluorescence was observed in negative control samples (i.e., cells exposed to CRISPR/Cas9-NLS complex without any shuttle agent; data not shown).
13.4 T7E1 assay
The T7E1 assay was performed with the Edit-R™ Synthetic crRNA Positive Controls (Dharmacon #U-007000-05) and the T7 Endonuclease I (NEB, Cat #M0302S). After the delivery of the CRISPR/Cas9 complex, cells were lysed in 100 μΙ_ of Phusion™ High-Fidelity DNA polymerase (NEB #M0530S) laboratory with additives. The cells were incubated for 15-30 minutes at 56°C, followed by deactivation for 5 minutes at 96°C. The plate was briefly centrifuged to collect the liquid at bottom of the wells. 50-μΙ. PCR samples were set up for each sample to be analyzed. The PCR samples were heated to 95°C for 10 minutes and then slowly (>15 minutes) cooled to room temperature. PCR product (~5 μί.) was then separated on an agarose gel (2%) to confirm amplification. 15 of each reaction was incubated with T7E1 nuclease for 25 minutes at 37 °C. Immediately, the entire reaction volume was run with the appropriate gel loading buffer on an agarose gel (2%).
13.5 His-CM18-PTD4 and His-C(LLKK)3C-PTD4 -mediated CRISPR/Cas9-NLS system delivery and cleavage of genomic PPIB sequence
A mix composed of a Cas9-NLS recombinant protein (25 nM; Example 13.1) and crRNA/tracrRNA (50 nM; see below) targeting a nucleotide sequence of the PPIB gene were co- incubated with 10 μΜ of His-CM18-PTD4 or His-C(LLKK)3C-PTD4, and incubated with HeLa cells for 16h in medium without serum using Protocol A as described in Example 9.1.
The sequences of the crRNA and tracrRNAs constructed and their targets were:
- Feldan tracrR A [SEQ ID NO: 77] :
5 '-
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU
CGGUGCU-3 '
- PPIB crRNA [SEQ ID NO: 78] :
5' -GUGUAUUUUGACCUACGAAUGUUUUAGAGCUAUGCUGUUUUG-3 '
- Dharmacon tracrRNA [SEQ ID NO: 79] :
5 '-
AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC
GGUGCUUUUUUU-3 ' After 16h, HeLa cells were washed with PBS and incubated in medium with serum for 48h. HeLa cells were harvested to proceed with the T7E1 protocol assay as described in Example 13.4.
Figure 41A shows an agarose gel with the PPIB DNA sequences after PCR amplification. Lane A shows the amplified PPIB DNA sequence in HeLa cells without any treatment (i.e., no shuttle or Cas9/RNAs complex). Lanes B: The two bands framed in white box #1 are the cleavage product of the PPIB DNA sequence by the CRIPR/Cas9 complex after the delivery of the complex with the shuttle His- C(LLKK)3C-PTD4. Lane C: These bands show the amplified PPIB DNA sequence after incubation of the HeLa cells with the Cas9/RNAs complex without shuttle (negative control). Lane D: The bands framed in white box #2 show the amplified PPIB DNA sequence after incubation of the HeLa cells with the Cas9/RNAs complex in presence of a lipidic transfection agent (DharmaFect™ tranfection reagent # T-20XX-01) (positive control). Similar results were obtained using the shuttle His-CM18-PTD4 (data not shown).
Figure 41 B shows an agarose gel with the PPIB DNA sequences after PCR amplification. The left panel in Figure 41 B shows the cleavage product of the amplified PPIB DNA sequence by the CRIPR/Cas9 complex after the delivery of the complex with the shuttle agent His-CM18-PTD4 in HeLa cells. The right panel Figure 41 B shows amplified DNA sequence before the T7E1 digestion procedure as a negative control.
Figure 41 C shows an agarose gel with the PPIB DNA sequences after PCR amplification. The left panel Figure 41 C shows the amplified PPIB DNA sequence after incubation of the HeLa cells with the Cas9/RNAs complex in presence of a lipidic transfection agent (DharmaFect™ transfection reagent # T-20XX-01) (positive control). The right panel Figure 41 C shows amplified DNA sequence before the T7E1 digestion procedure as a negative control.
These results show that the shuttle agents His-CM18-PTD4 and His-C(LLKK)3C-PTD4 successfully deliver a functional CRISPR/Cas9 complex to the nucleus of HeLa cells, and that this delivery results in CRISPR/Cas9-mediated cleavage of genomic DNA.
13.6 CRISPR/Cas9-NLS system delivery by different shuttle agents, and cleavage of genomic HPTR sequence in HeLa and Jurkat cells
A mix composed of a Cas9-NLS recombinant protein (2.5 μΜ; Example 13.1) and crRNA/tracrRNA (2 μΜ; see below) targeting a nucleotide sequence of the HPTR gene were co- incubated with 35 μΜ of His-CM18-PTD4, His-CM18-PTD4-His, His-C(LLKK)3C-PTD4, or EB1-PTD4, and incubated with HeLa or Jurkat cells for 2 minutes in PBS using Protocol B as described in Example 9.1. The sequences of the crRNA and tracrRNAs constructed and their targets were:
- Feldan tracrR A [SEQ ID NO: 77] :
5 '-
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU
CGGUGCU-3 '
- HPRT crRNA [SEQ ID NO: 103] :
5' - AAUUAUGGGGAUUACUAGGAGUUUUAGAGCUAUGCU-3 '
After 2 minutes, cells were washed with PBS and incubated in medium with serum for 48h. Cells were harvested to proceed with the T7E1 protocol assay as described in Example 13.4. Figures 46A-46B shows an agarose gel with the HPTR DNA sequences after PCR amplification and the cleavage product of the amplified HPTR DNA sequence by the CRISPR/Cas9 complex after the delivery of the complex with the different shuttle agents. Figure 46A shows the results with the shuttle agents: His-CM18-PTD4, His-CM18-PTD4-His, and His-C(LLKK)3C-PTD4 in HeLa cells. Figure 46B shows the results with His-CM18-PTD4 and His-CM18-L2-PTD4 in Jurkat cells. Negative controls (lanes 4) show amplified HPTR DNA sequence after incubation of the cells with the CRISPR/Cas9 complex without the presence of the shuttle agent. Positive controls (lane 5 in Figures 46A and 46B) show the amplified HPTR DNA sequence after incubation of the cells with the Cas9/RNAs complex in presence of a lipidic transfection agent (Lipofectamine® RNAiMAX™ Transfection Reagent ThermoFisher Product No. 13778100)..
These results show that different polypeptide shuttle agents of the present description may successfully deliver a functional CRISPR/Cas9 complex to the nucleus of HeLa and Jurkat cells, and that this delivery results in CRISPR/Cas9-mediated cleavage of genomic DNA.
13.7 CRISPR/Cpf1 -NLS system delivery by shuttle agents, cleavage of genomic target sequence in HeLa cells.
A mix composed of a Cpf1 -NLS recombinant protein (1.33 μΜ) and crRNA (2 μΜ; see below) targeting a nucleotide sequence of the DNMT1 gene was co-incubated with different concentrations of His-CM18-PTD4 and incubated with HeLa cells for 2 min in PBS using Protocol A as described in Example 9.1.
The sequence of the Cpf1-NLS recombinant protein produced was:
MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKP I IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHEN ALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIF TRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQ LLGGISREAGTEKIKGLNEVLNLAIQKNDETAHI IASLPHRFIPLFKQILSD RNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNS IDLTHI
FISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHE DINLQEI ISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILK SQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNY ATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQK GRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTT PILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKW IDFTRDFLSKYTKTTS IDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAE KEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTS IK LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNH RLSHDLSDEARALLPNVITKEVSHEI IKDRRFTSDKFFFHVPITLNYQAANS PSKFNQRVNAYLKEHPETPI IGIDRGERNLIYITVIDSTGKILEQRSLNTIQ QFDYQKKLDNREKERVAARQAWSWGTIKDLKQGYLSQVIHEIVDLMIHYQA
λΑΑLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL
NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHE SRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNE TQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDG SNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVC FDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRNGGRSSDDEATADSQHAAPPKKKRKVGGSGGGSGGGSGGGRHHHHH
H [SEQ ID NO: 114]
(MW = 155.7kDa; pi =8.34)
NLS sequence is underlined
Serine/glycine rich linkers are in bold
13.8 His-CM18-PTD4 - mediated CRISPR/Cas9-NLS and CRISPR/Cpf1-NLS systems delivery, cleavage of respective genomic HPRT and DNMT1 targets and homologous-directed recombination with specific linear DNA templates A mix composed of a Cas9-NLS recombinant protein (2.5 μΜ; Example 13.1), guide RNAs
(crRNA & tracrRNA) (2 μΜ; see below) targeting a nucleotide sequence of the HPTR gene and a short DNA template (72 nucleotides), containing 30 nucleotide homology arms each side, that was specifically designed for insertion in HPRT gene, were all co-incubated with 35 μΜ of His-CM18-PTD4 and incubated with HeLa cells for 48h in medium with serum using Protocol A as described in Example 9.1.
A mix composed of a Cpf1 -NLS recombinant protein (1.33 μΜ; Example 13.7), a guide RNAs (crRNA) (2 μΜ; see below) targeting a nucleotide sequence of the DNMT1 gene and a short DNA template (76 nucleotides), containing 30 nucleotide homology arms each side with an EcoR1 site, that was specifically designed for insertion in DNMT1 gene, were all co-incubated with 35 μΜ of His-CM18- PTD4 and incubated with HeLa cells for 48h in medium with serum using Protocol A as described in Example 9.1.
The sequences of the guide RNAs and the DNA template constructed and their targets were: - Feldan tracrR A [SEQ ID NO: 77] :
5 '-
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU
CGGUGCU-3 '
- DNMTl crRNA [SEQ ID NO: 115] :
5' - AAU UUCUACUGUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC -3'
- HPRT crRNA [SEQ ID NO: 103] :
5' - AAUUAUGGGGAUUACUAGGAGUUUUAGAGCUAUGCU-3 '
- Short linear DNA template (insertion in HPRT gene) (SEQ ID NO: 116)
5'-
TGAAATGGAGAGCTAAATTATGGGGATTACAAGCTTGATAGCGAAGGGGCAGCAATGAGTTG ACACTACAGA-3'
- EcoRl linear DNA template (insertion in DNMTl gene) (SEQ ID NO: 117)
5' -
AGTACGTTAATGTTTCCTGATGGTCCATGTCTGTTGAATTCACTCGCCTGTCAAGTGGCGTG ACACCGGGCGTGTT-3' 2 minutes after co-incubation the mix, HeLa cells were washed with PBS and incubated in medium with serum for 48h. HeLa cells were harvested to proceed with the T7E1 protocol assay as described in Example 13.4. Figures 49A-49B show agarose gels with PCR amplification of the HPTR or the DNMT1 DNA sequences and the cleavage products of the amplified HPTR and DNMT1 DNA sequences after His-CM18-PTD4-mediated delivery of (upper panel of Figure 49A) the CRISPR/Cas9 complex and (upper panel of Figure 49B) the CRISPR/Cpfl complex in HeLa cells, (bottom panel of Figure 49A) PCR amplification of the DNA template delivered in HeLa cells in the HPRT gene, (bottom panel of Figure 49B) PCR amplification of the DNMT1 gene from HeLa cells extracts after the genomic insertion of the DNA template in this gene, and exposure of the DNMT1 sequence to the restriction enzyme Ecorl Negative controls show amplified HPTR or DNMT1 DNA sequences after incubation of the cells with the CRISPR/Cas9-NLS or CRISPR/Cpfl -NLS complex and respective DNA template without the presence of a His-CM18-PTD4. Positive controls show the amplified HPTR or DNMT1 DNA sequence after incubation of the cells with the Cas9/RNAs or Cpf1/RNAs complex in presence of the lipid transfection agent Lipofectamine CRISPRMax (product #B25642). Dotted arrows indicate the bands corresponding to the target gene, and thick black arrows indicate the bands corresponding to the cleavage products of this target gene, which indicate the successful delivery of functional CRISPR RNP systems for genome editing. An imaging software was used to quantify the relative signal intensities of each of the different bands directly on gels. The sum of all three bands (one gene target and two cleavage products) in a given lane corresponds to 100% of the signal, and the numerical value in italics at the bottom of each lane is the sum of the relative signals (%) of the two cleavage product bands (thick solid arrows). This imaging software has been used for the quantification of relative signal intensities of each of the different bands in all figures with genome editing results.
13.9 His-CM18-PTD4 - mediated CRISPR/Cas9-NLS systems delivery and cleavage of genomic HPRT target in NK cells
A mix composed of a Cas9-NLS recombinant protein (2.5 μΜ; Example 13.1) and guide RNAs (crRNA & tracrRNA) (2 μΜ; see below) targeting a nucleotide sequence of the HPTR gene were co- incubated with 4 μΜ of His-CM18-PTD4 and incubated with NK cells for 2 minutes in PBS using Protocol C as described in Example 9.1.
The sequences of the guide RNAs and the DNA template constructed and their targets were:
- Feldan tracrRNA [SEQ ID NO: 77] :
5 '-
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU I CGGUGCU-3'
- HPRT crRNA [SEQ ID NO: 103] :
5' - AAUUAUGGGGAUUACUAGGAGUUUUAGAGCUAUGCU-3 '
2 minutes after co-incubation with the mix, NK cells were washed with PBS and incubated in medium with serum for 48h. NK cells were harvested to proceed with the T7E1 protocol assay as described in Example 13.4. Figure 50 shows agarose gels with the HPTR DNA sequences after PCR amplification and the cleavage product of the amplified HPTR DNA sequence by the CRISPR/Cas9 complex after the delivery of the complex with His-CM18-PTD4 in NK cells. Negative controls show amplified HPTR or DNMT1 DNA sequences after incubation of the cells with the CRISPR/Cas9-NLS or CRISPR/Cpfl -NLS complex and respective DNA template without the presence of a His-CM18-PTD4. Positive control shows the amplified HPTR sequence after incubation of the cells with the Cas9/RNAs complex in presence of the lipidic transfection agent Lipofectamine CRISPRMax (product # B25642).
13.10 His-CM18-PTD4 - mediated CRISPR/Cpf1-NLS systems multiple delivery and cleavage of genomic B2M exons targets in HeLa cells
A mix composed of a Cpf1 -NLS recombinant protein (1 μΜ; Example 13.7) and three guide RNAs (crRNA2, crRNA3 and crRNA4) (1.2 μΜ; see below) targeting exon 1 (crRNA2) and exon2 (crRNAs 3 & 4) of the B2M gene were co-incubated with 20 μΜ of His-CM18-PTD4 and incubated with HeLa cells for 2 minutes in PBS using Protocol B as described in Example 9.1. After 2 minutes, HeLa cells were washed with PBS and incubated in medium with serum for 48h. HeLa cells were harvested to proceed with the T7E1 protocol assay as described in Example 13.4. Figure 51 shows agarose gels with the B2M sequence after PCR amplification and the cleavage products of the amplified (upper panel) B2M exons 1 and 2 sequences by the three CRISPR/Cpf1 complexes after their delivery in presence of His-CM18-PTD4 in HeLa cells (upper panels). Figure 51 also shows agarose gels with the B2M sequence after PCR amplification of the B2M exons 1 and 2 sequences before enzymatic T7E1 assay (bottom panels). Exons 1 and 2 have been cleaved in presence of respective CRISPR/Cpf1 -NLS complexes or in presence of the three CRISPR/Cpf1 -NLS complexes. Indels (%) indicated successful genome editing although the presence of non-specific complexes reduced gene cutting. It indicated that His-CM18-PTD4 can deliver a limited amount of each CRISPR complex in cells and/or each complex competes for cell entrance. Negative controls show amplified B2M exonl sequence after incubation of the cells without any CRISPR/Cpf1 -NLS complex. The sequences of the crRNA used was as follows:
- B2M crR A-2 [SEQ ID NO: 118] :
5' - AAUUUCUACUGUUGUAGAUAUAUAAGUGGAGGCGUCGCG -3'
- B2M crRNA-3 [SEQ ID NO: 119] :
5' - AAUUUCUACUGUUGUAGAUAUCCAUCCGACAUUGAAGUU -3'
- B2M crRNA-4 [SEQ ID NO: 120] :
5' - AAUUUCUACUCUUGUAGAUCCGAUAU UCCUCAGGUACUCCA -3'
Example 14:
His-CM18-PTD4 enables transduction of the transcription factor H0XB4 in THP-1 cells
14.1 H0XB4-WT recombinant protein
Human H0XB4 recombinant protein was constructed, expressed and purified from a bacterial expression system as described in Example 1.4. The sequence of the H0XB4-WT recombinant protein produced was:
MHHHHHHMAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRE SSFQPEAGFGRRAACTVQRYPPPPPPPPPPGLSPRAPAPPPAGALLPEPGQR CEAVSSSPPPPPCAQNPLHPSPSHSACKEPWYPWMRKVHVSTVNPNYAGGE PKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSERQIKIWFQNR RMKWKKDHKLPNTKIRSGGAAGSAGGPPGRPNGGPRAL [SEQ ID NO:
80]
(MW = 28.54 kDa; pi =9.89)
The initiator methionine and the 6x Histidine tag (SEQ ID NO: 113) are shown in bold. 14.2 Real-Time Polymerase Chain Reaction (rt-PCR)
Control and treated cells are transferred to separate sterile 1.5-mL tubes and centrifuged for 5 minutes at 300g. The cell pellets are resuspended in appropriate buffer to lyse the cells. RNAase-free 70% ethanol is then added followed by mixing by pipetting. The lysates are transferred to an RNeasy™ Mini spin column and centrifuged 30 seconds at 13000 RPM. After several washes with appropriate buffers and centrifugation steps, the eluates are collected in sterile 1.5-mL tubes on ice, and the RNA quantity in each tube is then quantified with a spectrophotometer. For DNase treatment, 2 g of RNA is diluted in 15 L of RNase-free water. 1.75 μΐ of 10X DNase buffer and 0.75 μΐ of DNase is then added, followed by incubation at 37°C for 15 minutes. For reverse transcriptase treatment, 0.88 L of EDTA (50 nM) is added, followed by incubation at 75°C for 5 minutes. In a PCR tube, 0.5 g of DNase- treated RNA is mixed with 4 L of iScript™ Reverse transcription Supermix (5X) and 20 L of nuclease-free water. The mix is incubated in a PCR machine with the following program: 5 min at 25°C, 30 min at 42°C and 5 min at 85°C. Newly synthesized cDNA is transferred in sterile 1.5-mL tubes and diluted in 2 μί of nuclease-free water. 18 L per well of a qPCR machine (CFX-96™) mix is then added in a PCR plate for analysis.
14.3 HOXB4-WT transduction by His-CM18-PTD4 in THP-1 cells: Dose responses and viability
THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were plated at 30 000 cells/well one day before transduction. HOXB4-WT recombinant protein (0.3, 0.9, or 1.5 μΜ; Example 14.1) was co-incubated with different concentrations of His-CM18-PTD4 (0, 0.5, 7.5, 0.8 or 1 μΜ) and then exposed to THP-1 cells for 2.5 hours in the presence of serum. The cells were subjected to real time-PCR analysis as described in Example 14.2 to measure the mRNA levels of a target gene as a marker for HOXB4 activity, which was then normalized to the target gene mRNA levels detected in the negative control cells (no treatment), to obtain a "Fold over control" value. Total RNA levels (ng/μΙ.) were also measured as a marker for cell viability. Results are shown in Table 14.1 and Figure 42.
Table 14.1 : Data from Figure 42
Figure imgf000116_0001
His-CM18-PTD4
THP-1 1 0 2.7 ± 0.3 252 ± 10.7 alone ("FS")
0.3 2.7 ± 0.6 255 ± 3.9
His-CM18-PTD4 +
THP-1 0.5 0.9 4.3 ± 2.1 239 ± 17.5 HOXB4-WT
1.5 3.8 ± 0.7 269 ± 6.4
0.3 4.2 ± 1.2 248 ± 28
His-CM18-PTD4 +
THP-1 0.75 0.9 5.7 ± 2.5 245 ± 31 HOXB4-WT
1.5 7.5 ± 2.8 230 ± 3.3
0.3 9.1 ± 2.7 274 ± 4.4
His-CM18-PTD4 +
THP-1 0.8 0.9 16.4 ± 1.7 272 ± 12.5 HOXB4-WT
1.5 22.7 ± 3.2 282 ± 4.7
0.3 10.2 ± 2.5 280 ± 11.3
His-CM18-PTD4 +
THP-1 0.9 0.9 18.7 ± 3.1 281 ± 9.2 HOXB4-WT
1.5 26.1 ± 3.5 253 ± 7.1
0.3 10.5 ± 0.7 184 ± 12.3
His-CM18-PTD4 +
THP-1 1 0.9 17 ± 3.7 168 ± 16.2 HOXB4-WT
1.5 24.5 ± 3.9 154 ± 4.7
These results show that exposing THP-1 cells to a mixture of the shuttle agent His-CM18-PTD4 and the transcription factor HOXB4-WT for 2.5 hours in the presence of serum results in a dose- dependent increase in mRNA transcription of the target gene. These results suggest that HOXB4-WT is successfully delivered in an active form to the nucleus of THP-1 cells, where it can mediate transcriptional activation.
14.4 HOXB4-WT transduction by His-CM18-PTD4 in THP-1 cells: Time course and viability (0 to 48 hours)
THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were plated at 30 000 cells/well one day before the first time course experiment. HOXB4-WT recombinant protein (1.5 μΜ; Example 14.1) was co-incubated with His-CM18-PTD4 (0.8 μΜ) and then exposed to THP-1 cells for 0, 2.5, 4, 24 or 48 hours in presence of serum. The cells were subjected to real time-PCR analysis as described in Example 14.2 to measure mRNA levels of a target gene as a marker for HOXB4 activity, which was then normalized to the target gene mRNA levels detected in the negative control cells (no treatment), to obtain a "Fold over control" value. Total RNA levels (ng/μΙ.) were also measured as a marker for cell viability. Results are shown in Table 14.2 and Figure 43. Table 14.2: Data from Figure 43
Figure imgf000118_0002
14.5 HOXB4-WT transduction by His-CM18-PTD4 in THP-1 cells: Time course and viability (0 to 4 hours)
THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were plated at 30 000 cells/well one day before the first time course experiment. HOXB4-WT recombinant protein (0.3 μΜ; Example 14.1) was co-incubated with His-CM18-PTD4 (0.8 μΜ) and then exposed to THP-1 cells for 0, 0.5, 1 , 2, 2.5, 3 or 4 hours in presence of serum. The cells were subjected to real time-PCR analysis as described in Example 14.2 to measure mRNA levels of a target gene as a marker for HOXB4 activity, which was then normalized to target gene mRNA levels detected in the negative control cells (no treatment), to obtain a "Fold over control" value. Total RNA levels (ng/μί.) were also measured as a marker for cell viability. Results are shown in Table 14.3 and Figure 44.
Table 14.3: Data from Figure 44
Figure imgf000118_0001
H0XB4-WT
THP-1 0.8 0 2.5 h 1 ± 0.14 264 ± 12.3 alone ("TF")
4 h 1.2 ± 0.1 198 ± 6.0
3 h 1.3 ± 0.21 268 ± 12.5
His-CM18- 2.5 h 2 ± 0.3 275 ± 4.7
PTD4 + THP-1 0.8 0.3 2 h 2.2 ± 0.2 269 ± 12.5
HOXB4-WT 1 9.7 ± 2.6 268 ± 3.9
0.5 23.1 ± 2.0 266 ± 17.5
0 4 ± 0.5 217 ± 6.4
14.6 HOXB4-WT transduction by His-CM18-PTD4 in HeLa cells: immuno-labelling and visualization by microscopy
Recombinant HOXB4-WT transcription factor (25 μΜ; Example 14.1) was co-incubated with 35 μΜ of His-CM18-PTD4 and exposed to HeLa cells for 10 seconds using Protocol B as described in Example 9.1. After a 30-minute incubation to allow transduced HOXB4-WT to accumulate in the nucleus, the cells were fixed, permeabilized and immuno-labelled as described in Example 3.2a. HOXB4-WT was labelled using a primary mouse anti-HOXB4 monoclonal antibody (Novus Bio #NBP2- 37257) diluted 1/500, and a secondary anti-mouse antibody Alexa™-594 (Abeam #150116) diluted 1/1000. Nuclei were labelled with DAPI. The cells were visualized by bright field and fluorescence microscopy at 20x and 40x magnifications as described in Example 3.2, and sample results are shown in Figures 45A-45D. Co-localization was observed between nuclei labelling (Figures 45A and 45C) and HOXB4-WT labelling (Figures 45B and 45D), indicating that HOXB4-WT was successfully delivered to the nucleus after 30 min in the presence of the shuttle agent His-CM18-PTD4. White triangle windows show examples of areas of co-localization between the nuclei (DAPI) and HOXB4-WT immuno-labels.
14.7 HOXB4-WT transduction by different shuttle agents in THP-1 cells: Dose responses and viability
THP-1 cells were cultured and tested in the protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were plated at 30 000 cells/well one day before the first time course experiment. HOXB4-WT recombinant protein (1.5 μΜ; Example 14.1) co-incubated with the shuttle agents His-CM18-PTD4, TAT-KALA, EB1-PTD4, His-C(LLKK)3C-PTD4 and His-CM18- PTD4-His at 0.8 μΜ, and then exposed to THP-1 cells for 2.5 hours in presence of serum. The cells were subjected to real time-PCR analysis as described in Example 14.2 to measure mRNA levels of a target gene as a marker for HOXB4 activity, which was then normalized to target gene mRNA levels detected in the negative control cells (no treatment), to obtain a "Fold over control" value. Total RNA levels (ng/μί) were also measured as a marker for cell viability. Results are shown in Table 14.4 and Figure 47.
Table 14.4: Data from Figure 47
Figure imgf000120_0001
Example 15:
In vivo GFP-NLS delivery in rat parietal cortex by His-CM18-PTD4
The ability of the shuttle agent His-CM18-PTD4 to deliver GFP-NLS in vivo in the nuclei of rat brain cells was tested.
In separate sterile 1.5-mL tubes, shuttle agent His-CM18-PTD4 was diluted in sterile distilled water at room temperature. GFP-NLS, used as cargo protein, was then added to the shuttle agent and, if necessary, sterile PBS was added to obtain the desired concentrations of shuttle agent and cargo in a sufficient final volume for injection in rat brain (e.g., 5 μί per each injection brain site). The shuttle agent/cargo mixture was then immediately used for experiments. One negative control was included for the experiment, which corresponds to the injection of the GFP-NLS alone.
Bilateral injections were performed in the parietal cortex of three rats. In the left parietal cortex (ipsilateral), a mix composed of the shuttle agent (20 μΜ) and the GFP-NLS (20 μΜ) was injected, and in the right parietal cortex (contralateral), only the GFP-NLS (20 μΜ) was injected as a negative control. For surgical procedures, mice were anesthetized with isoflurane. Then the animal was placed in a stereotaxic frame, and the skull surface was exposed. Two holes were drilled at the appropriate sites to allow bilateral infusion of the shuttle/cargo mix or GFP-NLS alone (20 μΜ) with 5-μί Hamilton syringe. Antero-posterior (AP), lateral (L), and dorso-ventral (DV) coordinates were taken relative to the bregma: (a) AP 0.48 mm, L ± 3 mm, V - 5 mm; (b) AP - 2 mm, L ± 1.3 mm, V - 1.5 mm; (c) AP - 2.6 mm, L ± 1.5 mm, V - 1.5 mm. The infused volume of the shuttle/cargo mix or cargo alone was 5 \ l per injection site and the injection was performed for 10 minutes. After that, experimenter waited 1 min before removing the needle from the brain. All measures were taken before, during, and after surgery to minimize animal pain and discomfort. Animals were sacrificed by perfusion with paraformaldehyde (4%) 2 h after surgery, and brain were collected and prepared for microcopy analysis. Experimental procedures were approved by the Animal Care Committee in line with guidelines from the Canadian Council on Animal Care.
Dorso-ventral rat brain slices were collected and analysed by fluorescence microscopy and results are shown in Figure 48A-48D at (Figure 48A) 4x, (Figure 48C) 10x and (Figure 48D) 20x magnifications. The injection site is located in the deepest layers of the parietal cortex (PCx). In the presence of the His-CM18-PTD4 shuttle, the GFP-NLS diffused in cell nuclei of the PCx, of the Corpus Callus (Cc) and of the striatum (Str) (White curves mean limitations between brains structures). Figure 48B shows the stereotaxic coordinates of the injection site (black arrows) from the rat brain atlas of Franklin and Paxinos. The injection of GFP-NLS in presence of His-CM18-PTD4 was performed on the left part of the brain, and the negative control (an injection of GFP-NLS alone), was done on the contralateral site. The black circle and connected black lines in Figure 48B show the areas observed in the fluorescent pictures (Figures 48A, 48C and 48D).
This experiment demonstrated the cell delivery of the cargo GFP-NLS after its stereotaxic injection in the rat parietal cortex in the presence of the shuttle agent His-CM18-PTD4. Results show the delivery of the GFP-NLS in the nucleus of cells from the deeper layers of the parietal cortex (injection site) to the corpus callus and the dorsal level of the striatum (putamen). In contrast, the negative control in which GFP-NLS is only detectable locally around the injection site. This experiment shows that shuttle agent induced nuclear delivery of the cargo in the injection site (parietal cortex) and its diffusion through both neighboring brain areas (corpus callus and striatum rat brain). The following examples are prophetic examples.
Example 16
Inefficient cargo protein delivery to a hematopoietic stem cell
A hematopoietic stem cells (HSCs) are derived from mesoderm and are the cell that give rise to all blood cells through the process of haematopoiesis. HSC populations are transplanted into patients to treat certain blood malignancies and a threshold population size is required for successful treatment outcomes. Often, isolated HSC population sizes are sub-optimal or have inadequate cell counts.
The transcription factor HoxB4 is involved in HSC expansion. Therefore, HoxB4 is selected for delivery to the HSC nuclease with the goal of HSC population expansion in order to generate an adequate population size for treatment of a patient in need of a HSC transplantation. To this end, HoxB4 comprising a fused cell permeable domain (TAT) is cloned, expressed, and purified. The TAT-HoxB4 fusion protein is added to a population of ex vivo HSCs isolated from the patient. Following translocation across the cell membrane, the HoxB4 protein is sequestered in the endosome and the majority is eventually degraded. Therefore, very little HoxB4 protein reaches the nucleus and cell expansion occurs at a low frequency due to the inefficient delivery of HoxB4. As a result, the patient undergoes a transplantation with a sub-optimal HSC population size.
Example 17
Peptide shuttle-based cargo protein delivery to a hematopoietic stem cell
HSCs are isolated from the patient described in Example 16 and maintained ex vivo. HoxB4 is complexed with a peptide shuttle as disclosed herein and is added to the ex vivo population of HSCs. The peptide shuttle has a cell penetrating domain (CPD), which aids in transport of HoxB4 across the cell membrane, and an endosome leakage domain (ELD), which ensures HoxB4 does not become sequestered in the endosome. HoxB4 comprises a nuclear localization signal (NLS), which targets HoxB4 to the nucleus, where HoxB4 functions to initiate expansion of the population. Following translocation across the HSC cell membrane, the HoxB4-peptide shuttle complex successfully escapes the endosome and is targeted to the nucleus. As a result, the HSC population expands and surpasses the threshold needed for transplantation. The expanded HSC population is transplanted into the patient.
Example 18
Peptide shuttle-based expansion of megakaryocytes
Blood malignancies such as thrombocytopenia can be treated with megakaryocytes, which are platelet-producing cells derived from hematopoietic stem cells. The success of megakaryocyte transplantation is effected by the population size and immunogenicity of the cells to be transplanted.
A patient has thrombocytopenia and is need of a megakaryocyte transplantation. HSCs are isolated from the patient and maintained ex vivo. As described in Example 17, HoxB4 is complexed with a peptide shuttle as disclosed herein and is added to the ex vivo population of HSCs. The peptide shuttle has a cell penetrating domain (CPD), which aids in transport of HoxB4 across the cell membrane, and an endosome leakage domain (ELD), which ensures HoxB4 does not become sequestered in the endosome. HoxB4 comprises a nuclear localization signal (NLS), which targets HoxB4 to the nucleus, where HoxB4 functions to initiate differentiation of the HSC to a megakaryocyte. Following translocation across the HSC cell membrane, the HoxB4-peptide shuttle complex successfully escapes the endosome and is targeted to the nucleus. As a result, the HSC population expands.
Following HSC population expansion, the cells are treated with differentiation factors which initiate differentiation of the HSCs into megakaryocytes. Because of the expanded starting HSC population, the number of generated megakaryocytes surpasses the threshold needed for transplantation. The expanded HSC population-derived megakaryocytes are transplanted into the patient.
Example 19
Peptide shuttle-based production of non-immunogenic megakaryocytes
A patient similar to the one described in Example 18 has thrombocytopenia and is need of a megakaryocyte transplantation. An attempt to isolate HSCs from the patient is unsuccessful, therefore donor HSCs from a different person are obtained. These non-autologous HSCs are expanded with a HoxB4-peptide shuttle complex as described in Examples 16-17.
Following HSC population expansion, the cells are treated with differentiation factors which initiate differentiation of the HSCs into megakaryocytes. Because of the expanded starting HSC population, the number of generated megakaryocytes surpasses the threshold needed for transplantation. However, the megakaryocytes are immunogenic to the patient and will be rejected during transplantation.
In order to generate non-immunogenic megakaryocytes, a Cas9-peptide shuttle complex is used to delete a major histocompatibility complex (MHC) gene from the cells. The peptide shuttle is the same as that described in Examples 16-17 and contains a cell penetrating domain and an endosome leakage domain. Cas9 is engineered to comprise a nuclear localization signal. The Cas9-peptide shuttle complex also contains a crRNA engineered to target the MHC DNA sequence and a corresponding trRNA.
Following translocation across the HSC cell membrane, the Cas9-peptide shuttle complex successfully escapes the endosome and is targeted to the nucleus. Within the nucleus, the crRNA and trRNA guide Cas9 to the MHC DNA target sequence, followed by Cas9-based cleavage of the target sequence. Cleavage of the target sequence leads to disruption of the MHC gene product and results in non-immunogenic megakaryocytes.
The non-immunogenic megakaryocytes are transplanted into the patient.

Claims

CLAIMS:
1. A method of editing a genome of one or more eukaryotic cells comprising contacting a population of eukaryotic cells with a polypeptide-based shuttle and a DNA cleavage protein, such that the DNA cleavage protein is delivered to the nucleus and binds to at least one target DNA sequence, thereby editing the genome, wherein the polypeptide-based shuttle comprises a cell penetrating domain (CPD), or a variant or functional fragment thereof having cell penetrating activity, and an endosome leakage domain (ELD), or a variant or functional fragment thereof having endosomolytic activity, and wherein the polypeptide-based shuttle and the DNA cleavage protein have independent protein backbones.
2. The method of claim 1 , wherein the polypeptide-based shuttle is present at a concentration sufficient to increase the percentage or proportion of the population eukaryotic cells into which the DNA cleavage protein is delivered across the plasma membrane, as compared to in the absence of said polypeptide-based shuttle.
3. The method of claim 1 or 2, wherein the polypeptide-based shuttle further comprises a histidine rich domain.
4. The method of any one of claims 1-3, wherein the CPD comprises at least one of SEQ ID NOs:
16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity.
5. The method of any one of claims 1-4, wherein the ELD comprises at least one of SEQ ID NOs:
1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
6. The method of any one of claims 1 -5, wherein the DNA-cleavage protein comprises a
subcellular targeting domain, optionally wherein the subcellular targeting domain comprises a nuclear localization signal (NLS).
7. The method of claim 6, wherein the NLS comprises at least one of SEQ ID NO. 28-50.
8. The method of any one of claims 1-7, wherein the CPD comprises at least one of SEQ ID NOs:
16-27 or 65, or a variant or functional fragment thereof having cell penetrating activity, and the ELD comprises at least one of SEQ ID NOs: 1 -15, 63, or 64, or a variant or functional fragment thereof having endosomolytic activity.
9. The method of any one of claims 1-7, wherein the polypeptide-based shuttle comprises at least one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof.
10. The method of any one of claims 1-9, wherein the DNA cleavage protein comprises an RNA- guided nuclease, optionally wherein the RNA-guided nuclease comprises a Cas protein.
11. The method of claim 10, wherein the Cas protein comprises a Type I, Type II, Type III, Type IV, Type V, or a Type VI Cas protein or protein complex or wherein the Cas protein comprises Cas9, Cpf1 , or at least one functional fragment or derivative thereof.
12. The method of any one of claims 1-11 , wherein the population of eukaryotic cells is additionally contacted with at least one guiding RNA.
13. The method of claim 12, wherein the RNA-guided nuclease comprises Cas9 and wherein the at least one guiding RNA comprises a crRNA and a trRNA, wherein the crRNA is engineered to hybridize with the at least one target DNA sequence, wherein the trRNA is engineered to hybridize with the crRNA, and wherein the crRNA and the trRNA form a complex with Cas9, thereby targeting Cas9 to the at least one target DNA sequence such that the at least one target DNA sequence is cleaved by Cas9.
14. The method of claim 13, wherein the crRNA and trRNA have independent phosphodiester backbones or wherein the crRNA and trRNA share a common phosphodiester backbone
15. The method of claim 12, wherein the RNA-guided nuclease comprises Cpfl and wherein the at least one guiding RNA comprises a crRNA, wherein the crRNA is engineered to hybridize with the at least one target DNA sequence, and wherein the crRNA forms a complex with Cpf1 , thereby targeting Cpfl to the at least one target DNA sequence such that the at least one target DNA sequence is cleaved by Cpfl .
16. The method of any one of claims 1-15, wherein binding to at least one target further comprises cleavage of the at least one target DNA sequence, such that cleavage creates a double strand break which is repaired by a nonhomologous end joining (NHEJ) repair mechanism, thereby editing the at least one target DNA molecule, or wherein binding to at least one target further comprises cleavage of the at least one target DNA sequence, such that cleavage creates a double strand break which is repaired by a homology- directed repair mechanism which incorporates a sequence of a donor polynucleotide into the at least one target DNA sequence, thereby editing the target DNA molecule, or wherein the Cas9 protein is a nickase variant of Cas9 which comprises at least one mutation in at least one of a RuvC domain and a HNH domain such binding to at least one target further comprises cleavage of only one strand of the at least one target DNA sequence.
17. The method of any one of claims 1-16, wherein the method comprises editing the at least one target DNA sequence by insertion of a sequence for a donor polynucleotide into the cleaved strand of the at least one target DNA sequence.
18. The method of any one of claims 1-17, wherein the method comprises editing the at least one target DNA sequence by a homology directed repair mechanism which incorporates a sequence of a donor polynucleotide into the at least one target DNA sequence, thereby editing the at least one target DNA molecule.
19. The method of claim 17 or 18, wherein incorporation of a sequence of a donor polynucleotide results in insertion, deletion, or substitution of one or more nucleotides.
20. The method of any one of claims 10-19, wherein the RNA-guided nuclease is multiplexed with at least two guiding RNAs, such that at least two target DNA sequences are cleaved.
21. The method of claim 10, wherein the Cas protein is catalytically dead such that the Cas protein binds but does not cleave the at least one target DNA sequence.
22. The method of claim 21 , wherein binding of the catalytically dead Cas protein blocks functional transcription the at least one target DNA sequence.
23. The method of any one of claims 1-22, wherein the population of eukaryotic cells is a
mammalian cell population, a human cell population, a T cell population, a megakaryocyte population, a NK cell population, a stem cell population, or a hematopoietic stem cell population.
24. The method of any one of claims 1-23, wherein the target DNA sequence is implicated in a genetic disease, such that the genome editing treats said genetic disease.
25. The method of any one of claims 1-23, wherein the target DNA sequence is implicated in an infection, such that the genome editing treats said infection.
26. The method of any one of claims 1-23, wherein the target DNA sequence is implicated in immunogenicity, such that the genome editing deletes or ameliorates the immunogenicity.
27. The method of claim 26, wherein the target DNA sequence comprises a human leukocyte
antigen gene complex sequence or a major histocompatibility complex gene sequence.
28. The method of any of claims 26-27, wherein the non-immunogenic cells comprise universal stem cells or CAR-T cells.
29. A method of treating a patient with a condition by administering to the patient in need thereof non-immunogenic cells obtained by the method of any one of claims 1-28, optionally wherein the condition is a genetic disease, a blood disorder, a malignant condition, a non-malignant condition, or thrombocytopenia.
30. Use of non-immunogenic cells obtained by the method of any one of claims 1 -28 for treatment of a patient in need thereof due to a condition, optionally wherein the condition is a genetic disease, a blood disorder, a malignant condition, a non-malignant condition, or
thrombocytopenia..
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018068135A1 (en) * 2016-10-12 2018-04-19 Feldan Bio Inc. Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same
CN108220247A (en) * 2018-03-20 2018-06-29 杭州史迪姆生物科技有限公司 A kind of double CAR-T cells and its preparation method and application
WO2019241315A1 (en) 2018-06-12 2019-12-19 Obsidian Therapeutics, Inc. Pde5 derived regulatory constructs and methods of use in immunotherapy
WO2020086742A1 (en) 2018-10-24 2020-04-30 Obsidian Therapeutics, Inc. Er tunable protein regulation
WO2020123716A1 (en) 2018-12-11 2020-06-18 Obsidian Therapeutics, Inc. Membrane bound il12 compositions and methods for tunable regulation
WO2020185632A1 (en) 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Human carbonic anhydrase 2 compositions and methods for tunable regulation
WO2020185628A1 (en) 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Cd40l compositions and methods for tunable regulation
WO2020252404A1 (en) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Ca2 compositions and methods for tunable regulation
WO2020252405A1 (en) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Ca2 compositions and methods for tunable regulation
WO2021040736A1 (en) 2019-08-30 2021-03-04 Obsidian Therapeutics, Inc. Tandem cd19 car-based compositions and methods for immunotherapy
WO2021046451A1 (en) 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions and methods for dhfr tunable protein regulation
WO2021142376A1 (en) 2020-01-08 2021-07-15 Obsidian Therapeutics, Inc. Compositions and methods for tunable regulation of transcription
WO2022094309A1 (en) * 2020-10-30 2022-05-05 Arbor Biotechnologies, Inc. Compositions comprising an rna guide targeting b2m and uses thereof
US11446398B2 (en) 2016-04-11 2022-09-20 Obsidian Therapeutics, Inc. Regulated biocircuit systems
AU2019275071B2 (en) * 2018-05-24 2022-12-15 Sirnaomics, Inc. Composition and methods of controllable co-coupling polypeptide nanoparticle delivery system for nucleic acid therapeutics
US11629170B2 (en) 2016-10-12 2023-04-18 Feldan Bio Inc. Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same
EP3956348A4 (en) * 2019-04-18 2023-07-19 Feldan Bio Inc. Peptide-based non-proteinaceous cargo delivery

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015089462A1 (en) * 2013-12-12 2015-06-18 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for genome editing
WO2016161516A1 (en) * 2015-04-10 2016-10-13 Feldan Bio Inc. 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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015089462A1 (en) * 2013-12-12 2015-06-18 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for genome editing
WO2016161516A1 (en) * 2015-04-10 2016-10-13 Feldan Bio Inc. 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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
RAMAKRISHNA ET AL.: "Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA", GENOME RESEARCH, vol. 24, no. 6, 2 April 2014 (2014-04-02), pages 1020 - 1027, XP055128944, ISSN: 1088-9051 *
SALOMONE ET AL.: "A novel chimeric cell penetrating peptide with membrane-disruptive properties for efficient endosomal escape", JOURNAL OF CONTROLLED RELEASE, vol. 163, no. 3, 11 October 2012 (2012-10-11), pages 293 - 303, XP055321151, ISSN: 0168-3659 *
SALOMONE ET AL.: "In Vitro Efficient Transfection by CM18-Tat1 1 Hybrid Peptide: A New Tool for Gene -Delivery Applications", PLOS ONE, vol. 8, no. 7, 29 July 2013 (2013-07-29), pages 1 - 11, XP055291411, ISSN: 1932-6203 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11446398B2 (en) 2016-04-11 2022-09-20 Obsidian Therapeutics, Inc. Regulated biocircuit systems
AU2017341736B2 (en) * 2016-10-12 2022-09-08 Feldan Bio Inc. Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same
KR20190072559A (en) * 2016-10-12 2019-06-25 펠단 바이오 인코포레이티드 Rationally-designed synthetic peptide shuttles for delivery of a polypeptide cargo from the extracellular space to the cytoplasm and / or nucleus of the target eukaryotic cell, its use, methods and kits related thereto
WO2018068135A1 (en) * 2016-10-12 2018-04-19 Feldan Bio Inc. Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same
KR102626671B1 (en) 2016-10-12 2024-01-18 펠단 바이오 인코포레이티드 Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargo from the extracellular space to the cytoplasm and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits related thereto
US11629170B2 (en) 2016-10-12 2023-04-18 Feldan Bio Inc. Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same
CN108220247A (en) * 2018-03-20 2018-06-29 杭州史迪姆生物科技有限公司 A kind of double CAR-T cells and its preparation method and application
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