WO2017175072A1 - Disruption génique basée sur une navette peptidique - Google Patents

Disruption génique basée sur une navette peptidique Download PDF

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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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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.

Abstract

La présente invention concerne des peptides synthétiques utiles pour accroître l'efficacité de transduction de cargaisons polypeptidiques au niveau du cytosol de cellules eucaryotes cibles. Plus spécifiquement, la présente invention concerne des peptides synthétiques et des agents navettes à base de polypeptides comprenant un domaine de fuite d'endosome (ELD) fonctionnellement lié à un domaine de pénétration des cellules (CPD), ou un ELD fonctionnellement lié à un domaine riche en histidine et un CPD. Cette invention concerne également des compositions, des trousses, des méthodes et des utilisations connexes. L'invention concerne en outre des méthodes d'utilisation des navettes à base de polypeptides décrites pour livrer un facteur de transcription et/ou une nucléase au noyau de cellule eucaryote et des utilisations des cellules dérivées.
PCT/IB2017/000512 2016-04-08 2017-04-07 Disruption génique basée sur une navette peptidique WO2017175072A1 (fr)

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CN108220247A (zh) * 2018-03-20 2018-06-29 杭州史迪姆生物科技有限公司 一种双car-t细胞及其制备方法和应用
WO2019241315A1 (fr) 2018-06-12 2019-12-19 Obsidian Therapeutics, Inc. Constructions régulatrices dérivées de pde5 et procédés d'utilisation en immunothérapie
WO2020086742A1 (fr) 2018-10-24 2020-04-30 Obsidian Therapeutics, Inc. Régulation de protéine accordable par er
WO2020123716A1 (fr) 2018-12-11 2020-06-18 Obsidian Therapeutics, Inc. Il12 liée à la membrane, compositions et procédés de régulation accordable
WO2020185628A1 (fr) 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions de cd40l et procédés de régulation accordable
WO2020185632A1 (fr) 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions d'anhydrase carbonique humaine 2 et procédés de régulation accordable
WO2020252405A1 (fr) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation ajustable
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WO2021040736A1 (fr) 2019-08-30 2021-03-04 Obsidian Therapeutics, Inc. Compositions à base de car cd19 tandem et méthodes d'immunothérapie
WO2021046451A1 (fr) 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions et méthodes de régulation de protéine accordable dhfr
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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 (ko) * 2016-10-12 2019-06-25 펠단 바이오 인코포레이티드 세포외 공간으로부터 표적 진핵 세포의 세포질 및/또는 핵으로 폴리펩타이드 카고를 전달하기 위한 합리적으로-설계된 합성 펩타이드 셔틀제, 이의 용도, 이와 관련된 방법 및 키트
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KR102626671B1 (ko) 2016-10-12 2024-01-18 펠단 바이오 인코포레이티드 세포외 공간으로부터 표적 진핵 세포의 세포질 및/또는 핵으로 폴리펩타이드 카고를 전달하기 위한 합리적으로-설계된 합성 펩타이드 셔틀제, 이의 용도, 이와 관련된 방법 및 키트
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
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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
WO2019241315A1 (fr) 2018-06-12 2019-12-19 Obsidian Therapeutics, Inc. Constructions régulatrices dérivées de pde5 et procédés d'utilisation en immunothérapie
WO2020086742A1 (fr) 2018-10-24 2020-04-30 Obsidian Therapeutics, Inc. Régulation de protéine accordable par er
WO2020123716A1 (fr) 2018-12-11 2020-06-18 Obsidian Therapeutics, Inc. Il12 liée à la membrane, compositions et procédés de régulation accordable
WO2020185632A1 (fr) 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions d'anhydrase carbonique humaine 2 et procédés de régulation accordable
WO2020185628A1 (fr) 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions de cd40l et procédés de régulation accordable
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