WO2024159069A1 - Icosahedral phage derived particles - Google Patents

Icosahedral phage derived particles Download PDF

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Publication number
WO2024159069A1
WO2024159069A1 PCT/US2024/013053 US2024013053W WO2024159069A1 WO 2024159069 A1 WO2024159069 A1 WO 2024159069A1 US 2024013053 W US2024013053 W US 2024013053W WO 2024159069 A1 WO2024159069 A1 WO 2024159069A1
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pdp
moiety
coat protein
bases
cell
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PCT/US2024/013053
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French (fr)
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Lavi Erisson
Uyanga TSEDEV
Jonathan Y. HSU
Alex J. WANG
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Gensaic, Inc.
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Publication of WO2024159069A1 publication Critical patent/WO2024159069A1/en

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10223—Virus like particles [VLP]
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10241—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10242—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

  • nucleic acid delivery systems are being developed in an attempt to facilitate the targeted delivery of nucleic acid payloads.
  • certain viral vectors such as adenoviral vectors, adeno-associated viral vectors (AAVs), and lentiviral vectors, have been used to deliver therapeutic nucleic acid payloads to certain tissues and/or cells.
  • AAVs adeno-associated viral vectors
  • lentiviral vectors have been used to deliver therapeutic nucleic acid payloads to certain tissues and/or cells.
  • phage derived particles derived from icosahedral phage (e.g., T7 phage) with improved properties, as well as methods of making and using such PDPs.
  • a PDP is a particle in which a non-phage nucleic acid payload is encapsulated by phage-derived coat proteins.
  • the nucleic acid payload of a PDP substantially lacks the genome from which the PDP is derived (e.g., the nucleic acid payload does not encode the phage-derived coat proteins in which it is encapsulated).
  • the PDP does not comprise nucleic acid sequences encoding at least 50% (e.g., 60%, 70%, 80%, 90%, 95%) of the genome of the phage from which the PDP is derived. In some embodiments, the PDP does not comprise a nucleic acid sequence encoding a phage coat protein. In certain embodiments, the PDP comprises no more than 2000 bases of the genome of the phage from which the PDP was derived.
  • PDPs provided herein possess advantageous properties compared to existing viral vectors making them particularly useful, for example, in the treatment and/or prevention of one or more diseases.
  • the PDPs provided herein exhibit improved: cell or tissue specificity, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics, and/or mammalian expression as compared to existing phage derived vectors.
  • the PDPs provided herein comprise phage coat proteins encapsulating a nucleic acid payload, wherein the nucleic acid payload comprises a sequence encoding an agent that treats a disease when delivered to a cell of a subject in need thereof.
  • the PDPs are derived from a T7 phage (bacteriophage T7).
  • the PDPs further comprise a phage coat protein displaying a cell-targeting moiety specific for a cell type.
  • the phage coat protein displaying the cell-targeting moiety is a 10A, and/or 10B coat (a/k/a capsid) protein and/or a derivative or fragment thereof.
  • the PDPs provided herein are designed to inhibit the expression of a gene (e.g., a disease-associated gene).
  • a gene e.g., a disease-associated gene
  • such PDPs would comprise a nucleic acid payload that encodes an inhibitory RNA and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that targets the gene.
  • the PDPs provided herein are designed to enhance the expression of a therapeutic peptide.
  • such PDPs would comprise a nucleic acid payload that encodes the therapeutic peptide and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate insertion of a sequence encoding the therapeutic peptide into the genome of a cell (e.g., into a safe harbor locus in the cell, such as a safe harbor locus listed in Table 2).
  • the PDPs provided herein are designed to inhibit the expression of a gene (e.g., a disease-associated gene) in a cell and enhance expression of a therapeutic peptide (e.g., a peptide that treats and/or prevents a disease) in a cell.
  • a gene e.g., a disease-associated gene
  • a therapeutic peptide e.g., a peptide that treats and/or prevents a disease
  • the gene for which expression is inhibited is a mutant gene associated with a disease, while the therapeutic peptide is the peptide encoded by the wild-type version of that gene.
  • such PDPs would comprise a nucleic acid payload that comprises a sequence that encodes an inhibitory RNA and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that targets the gene; and (2) a sequence that encodes the therapeutic peptide and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate insertion of a sequence encoding the therapeutic peptide into the genome of the cell (e.g., into the mutant gene locus and/or a safe harbor locus in the cell, such as a safe harbor locus listed in Table 2).
  • a gene editing system disclosed herein e.g., a CRISPR/Cas system
  • the PDPs provided herein are designed to modify the sequence of a gene in a cell (e.g., to change the sequence of a gene from a disease- associated sequence to a sequence that is not associated with a disease).
  • such PDPs would comprise a nucleic acid payload that encodes one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate modification of the gene in the genome of the endothelial cell (e.g., converting mutant version of a gene into a wild-type version of a gene.
  • the PDPs provided herein to provide a double stranded DNA homology directed repair (HDR) donor comprising: a first homology arm region, an insert region, and a second homology arm region.
  • the double stranded DNA HDR donor improves homology directed repair efficiency and reduces homologyindependent integration in a programmable nuclease system.
  • the programmable nuclease system comprises one or more of transcription activator-like effector nucleases (TALENs), zinc fingers (ZFNs), or clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas.
  • TALENs transcription activator-like effector nucleases
  • ZFNs zinc fingers
  • CRISPR clustered, regularly interspaced, short palindromic repeat
  • the PDPs provided herein comprise moieties that impart certain beneficial properties upon the PDP.
  • the PDPs comprise moieties that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived.
  • such moi eties are displayed on one or more of the phage coat proteins of the PDP (e.g., a 10A and/or 10B coat protein).
  • such moi eties are covalently attached to the phage coat protein.
  • such moieties are non-covalently attached to the phage coat protein.
  • the phage coat protein displaying the moiety is a fusion protein comprising the phage coat protein and the moiety.
  • the PDPs comprise modified coat proteins (e.g., modified 10A and/or 10B coat proteins) that impart certain beneficial properties upon the PDP.
  • the PDPs comprise modified coat proteins that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived.
  • the PDPs provided herein can accommodate larger nucleic acid payloads than commonly used viral vectors.
  • the PDPs provided herein can accommodate at least 300 bases (e.g., at least 325 bases, at least 350 bases, at least 375 bases, at least 400 bases, at least 425 bases, at least 450 bases, at least 475 bases, at least 500 bases, at least 525 bases, at least 550 bases, at least 575 bases, at least 600 bases, at least 625 bases, at least 650 bases, at least 675 bases, at least 700 bases, at least 725 bases, at least 750 bases, at least 775 bases, at least 800 bases at least 825 bases, at least 850 bases, at least 875 bases, at least 900 bases, at least 925 bases, at least 950 bases, at least 975 bases, at least 1000 bases, at least 1025 bases, at least 1050 bases, at least 1075 bases, at least 1100, at least 1125 bases, at least 1150 bases, at least 1175
  • the PDPs provided herein comprises a larger nucleic acid payloads than commonly used viral vectors can accommodate.
  • the PDPs provided herein comprise at least 300 bases (e.g., at least 325 bases, at least 350 bases, at least 375 bases, at least 400 bases, at least 425 bases, at least 450 bases, at least 475 bases, at least 500 bases, at least 525 bases, at least 550 bases, at least 575 bases, at least 600 bases, at least 625 bases, at least 650 bases, at least 675 bases, at least 700 bases, at least 725 bases, at least 750 bases, at least 775 bases, at least 800 bases at least 825 bases, at least 850 bases, at least 875 bases, at least 900 bases, at least 925 bases, at least 950 bases, at least 975 bases, at least 1000 bases, at least 1025 bases, at least 1050 bases, at least 1075 bases, at least 1100, at least 1125 bases, at least 1150 bases, at least
  • the PDPs provided herein comprise a payload of non-phage DNA that makes up at least 30% 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of its genome.
  • the PDPs provided herein comprise no more than 300 bases (e.g., no more than 325 bases, no more than 350 bases, no more than 375 bases, no more than 400 bases, no more than 425 bases, no more than 450 bases, no more than 475 bases, no more than 500 bases, no more than 525 bases, no more than 550 bases, no more than 575 bases, no more than 600 bases, no more than 625 bases, no more than 650 bases, no more than 675 bases, no more than 700 bases, no more than 725 bases, no more than 750 bases, no more than 775 bases, no more than 800 bases no more than 825 bases, no more than 850 bases, no more than 875 bases, no more than 900 bases, no more than 925 bases, no more than 950 bases, no
  • the PDPs provided herein have improved cellular internalization compared to the phage from which the PDP was derived.
  • the PDP comprises a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an internalization moiety.
  • the internalization moiety acts via membrane penetration.
  • the internalization moiety acts via membrane endocytosis.
  • the PDP is internalized by a mechanism selected from macropinocytosis, phagocytosis, clathrin-mediated, caveolin- mediated, interaction of hydrophilic lipid membrane and fusogenic moi eties, interaction with hydrophobic portion of lipid membrane, and hydrophobic cloaking.
  • the internalization moiety is selected from the moieties listed in Table 26.
  • the PDPs provided herein have improved endosomal escape compared to the phage from which the PDP was derived.
  • the PDP comprises a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an endosomal escape moiety.
  • the endosomal escape moiety is an endosomal escape peptide (e.g., a Tat peptide, a H5WYG peptide, INF7 peptide, or PCI peptide).
  • the endosomal escape moiety acts via proton sponge and/or osmotic disruption.
  • the endosomal escape moiety acts via compartment membrane disruption.
  • the endosomal escape moiety acts via membrane pore formation.
  • the endosomal escape moiety is selected from a moiety listed in Table 27.
  • the PDPs provided herein have improved nuclear shuttling compared to the phage from which the PDP was derived.
  • the PDP comprises a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying a nuclear localization moiety.
  • the nuclear localization moiety is a nuclear localization signal (NLS) (e.g., an NLS peptide from SV40 T antigen, an optimized SV40 NLS, an optimized short M9 (osM9), a c-Myc NLS, a nucleoplasmin NLS, or a heptamer NLS peptide).
  • NLS nuclear localization signal
  • the nuclear localization moiety acts via direct transport (nuclear pore entry) of the PDP into the nucleus. In some embodiments, the nuclear localization moiety acts via indirect transport (nuclear membrane translocation) of the PDP into the nucleus. In some embodiments, the nuclear localization moiety is selected from a moiety listed in Table 28.
  • the PDPs provided herein have improved immune evasion compared to the phage from which the PDP was derived.
  • the PDP comprises a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to enhance immune evasion of the PDP.
  • the phage coat protein is modified such that the PDP avoids neutralizing antibodies and/or immune cell uptake.
  • the phage coat protein is modified to reduce antibody epitope recognition, to reduce T cell epitope recognition, and/or to reduce surface charge.
  • the phage coat protein is modified to display an immune evasion moiety.
  • the PDP elicits a reduced immune response when administered to a subject as compared to the immune response that occurs when a phage from which the PDP was derived is administered to a subject.
  • the phage coat protein modification to enhance immune evasion is selected from the modifications listed in Table 29.
  • the PDPs provided herein have improved pharmacokinetic and/or pharmacodynamic properties compared to the phage from which the PDP was derived.
  • the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to extend circulation half-life of the PDP.
  • the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to increase stability of the PDP in circulation.
  • the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to reduce degradation of the PDP.
  • the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to reduce clearance of the PDP.
  • the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to kidney localization of the PDP.
  • the PDPs provided herein comprise a coat protein (e.g., 10A and/or 10B coat protein) modified to display pharmacokinetics or pharmacodynamics enhancing moiety.
  • the phage coat protein modification is selected from the modifications listed in Table 30.
  • the nucleic acid payload is a linear double stranded DNA (dsDNA) construct.
  • the dsDNA payload comprises DNA secondary structures that enhance expression in a mammalian system.
  • the dsDNA construct comprises a DNA sequence element that enhance expression and/or specificity in mammalian cells (e.g., DNA sequence element is selected from the DNA sequence elements listed in Table 1).
  • systems for producing the PDPs described herein from a prokaryotic host comprising (i) a phage vector comprising a packaging signal for replication of the vector into the nucleic acid payload of the PDP, wherein the nucleic acid payload is a linear double stranded DNA (dsDNA); and (ii) a second vector comprising nucleic acid sequences encoding the phage coat proteins of the PDP.
  • a method of making a PDP provided herein comprising delivering into a prokaryotic cell such a system and culturing the prokaryotic cell under conditions such that it produces the PDP.
  • icosahedral phage-derived particles comprising a nucleic acid payload (e.g., transgenes encoding a therapeutic protein, inhibitory nucleic acids, and/or gene editing (e.g., CRISPR/Cas) systems).
  • a nucleic acid payload e.g., transgenes encoding a therapeutic protein, inhibitory nucleic acids, and/or gene editing (e.g., CRISPR/Cas) systems.
  • the PDPs provided herein possess advantageous properties compared to existing viral vectors making them particularly useful for the treatment and/or prevention of certain diseases.
  • the PDPs provided herein exhibit improved: cell or tissue specificity, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics, and/or mammalian expression as compared to existing phage derived vectors.
  • therapeutic methods comprising administering one or more of the PDP compositions described to a subject in need thereof, as well as systems and methods of making
  • administering broadly refers to a route of administration of a composition (e.g., a therapeutic composition) to a subject.
  • routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection.
  • Administration by injection includes intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration.
  • a therapeutic composition described herein can be administered in any form by any effective route, including but not limited to oralf, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intraarterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial.
  • transdermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.
  • a therapeutic composition described herein is administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.
  • a therapeutic composition described herein is administered orally or intravenously.
  • a therapeutic composition described herein is administered intranasally.
  • a therapeutic composition described herein is administered orally.
  • antibody may refer to both an intact antibody and an antigen binding fragment thereof.
  • Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), singlechain antibodies and antigen-binding antibody fragments.
  • antigen binding fragment and “antigen-binding portion” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to an antigen.
  • binding fragments encompassed within the term "antigen-binding fragment” of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody.
  • These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.
  • binding refers to an association, which may be a stable association, between two molecules, e.g., between a polypeptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
  • CDR complementarity determining region
  • CDRL1, CDRL2 and CDRL3 three CDRs are present in a light chain variable region
  • CDRH1, CDRH2 and CDRH3 three CDRs are present in a heavy chain variable region.
  • CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions.
  • the CDR3 sequences, and particularly CDRH3 are the most diverse and therefore have the strongest contribution to antibody specificity.
  • CDRs There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated by reference in its entirety); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), incorporated by reference in its entirety).
  • cross-species sequence variability i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated by reference in its entirety
  • crystallographic studies of antigen-antibody complexes Chothia et al., Nature, 342:877 (1989), incorporated by reference in its entirety.
  • the “reverse complement” of a specific nucleic acid sequence is has a nucleic acid sequence that is able to for a Watson/Crick base pair with the specific nucleic acid sequence.
  • the term “consists essentially of’ means limited to the recited elements and/or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.
  • the term “effective dose” is the amount of the therapeutic composition that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, with the least toxicity to the subject.
  • epitope means a protein determinant capable of specific binding to an antibody or T cell receptor.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.
  • RNA transcripts are processed and translated into polypeptides.
  • gene refers to a nucleic acid sequence (e.g., DNA or RNA) that encodes a molecule (e.g., a protein).
  • a gene is a double-stranded DNA molecule that encodes a protein.
  • a gene generally comprises coding DNA sequences (e.g., exons), non-coding DNA sequences (e.g., introns), and one or more promoters or other regulatory element that controls gene expression.
  • An organism’s entire set of genes is referred as its genome.
  • heterologous DNA and “heterologous RNA” refer to nucleotides that are not endogenous (native) to the cell or part of the genome in which they are present. Generally heterologous DNA or RNA is added to a cell by transduction, infection, transfection, transformation or the like, as further described below. Such nucleotides generally include at least one coding sequence, but the coding sequence need not be expressed.
  • heterologous DNA may refer to a “heterologous coding sequence” or a “transgene”.
  • the term “host cell”, as used herein refers to a cell which has been transduced, infected, transfected or transformed with a vector.
  • the vector may be a plasmid, a viral particle, a phage, etc.
  • the culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. It will be appreciated that the term “host cell” refers to the original transduced, infected, transfected or transformed cell and progeny thereof.
  • hybridize or “hybridization” refer to the hydrogen bonding of complementary DNA and/or RNA sequences to form a duplex molecule.
  • hybridization takes place under conditions that can be adjusted to a level of stringency that reduces or even prevents base-pairing between a first oligonucleotide primer or oligonucleotide probe and a target sequence, if the complementary sequences are mismatched by as little as one base-pair.
  • “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et aL, Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., etal., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.
  • locus refers to a fixed position on a chromosome, such as the position of a gene or marker (z.e., genetic marker). A variant of a similar DNA sequence located at a given locus is called an allele.
  • the locus is a safe harbor locus.
  • safe harbor locus refers to a position on the chromosome (z.e., locus) that can tolerate the insertion, deletion, and/or mutation of the nucleic acid sequence in the safe harbor locus without perturbing the endogenous activity of the gene or risking the integrity of the host genome.
  • a gene is located in a safe harbor locus (z.e., a safe harbor gene).
  • the gene is the chemokine (C-C motif) receptor 5 (CC ?5) gene.
  • the gene is the adeno-associated virus site 1 (AAVS1) gene.
  • the safe harbor locus is the Rosa26 locus (e.g., from mice, or the human ortholog). Safe harbor genes are described in, e.g., Papapetrou EP and Schambach A (2016) Gene insertion into genomic safe harbors for human gene therapy. Mol Ther 24(4), 678-684, which is incorporated by reference herein.
  • a safe harbor locus is used as a recording locus as described herein.
  • mutation refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the nucleic acid and amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).
  • nucleic acid refers to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides.
  • polymeric nucleic acids e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides).
  • nucleic acid refers to an oligonucleotide chain comprising three or more individual nucleotide residues.
  • oligonucleotide and polynucleotide can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides).
  • nucleic acid encompasses RNA as well as single and/or double-stranded DNA.
  • Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, sRNAi, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule.
  • a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides.
  • nucleic acid examples include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone.
  • Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated.
  • a nucleic acid is or comprises natural nucleosides (e.g adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine); nucleoside analogs (e.g, 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-
  • operably linked as used herein relative to a recombinant DNA construct or vector means nucleotide components of the recombinant DNA construct or vector are functionally related to one another for operative control of a selected coding sequence.
  • “operably linked” DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous.
  • protein refers to a polymer of amino acid residues linked together by peptide (amide) bonds.
  • the terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long.
  • a protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins.
  • One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex.
  • a protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide.
  • a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
  • fusion protein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins.
  • One protein may be located at the amino-terminal (N- terminal) portion of the fusion protein or at the carboxy -terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively.
  • a protein may comprise different domains, for example, a nucleic acid programmable DNA binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein.
  • a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent.
  • a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA.
  • Any of the proteins provided herein may be produced by any method known in the art.
  • the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker.
  • promoter refers to a control region of a nucleic acid sequence (e.g., within a plasmid) at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled.
  • a promoter may also contain subregions to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors.
  • a promoter controls the expression of a nucleic acid sequence (e.g., a gene) that is operably linked to the promoter.
  • a promoter is located on the same strand and upstream of the nucleic acid sequence (e.g., gene) that is operably linked to the promoter. In general, promoters are between 100-1000 base pairs long.
  • the promoter is a promoter suitable for use in a prokaryotic system (z.e., a bacterial promoter). In some embodiments, the promoter is a promoter suitable for use in a eukaryotic system (z.e., a eukaryotic promoter). In some embodiments, the promoter is a promoter suitable for use in a mammalian (e.g., human) system (z.e., a mammalian promoter). In some embodiments, the promoter is induced by a stimulus (z.e., an inducible promoter).
  • the stimulus is a small molecule, a protein, a peptide, an amino acid, a metabolite, an inorganic molecule, an organometallic molecule, an organic molecule, a drug or drug candidate, a sugar, a lipid, a metal, a nucleic acid, a molecule produced during the activation of an endogenous or an exogenous signaling cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism, change in pH, or change in oxidation/reduction state.
  • the stimulus is a light.
  • the stimulus is a virus.
  • the stimulus is a small molecule.
  • the stimulus is an antibiotic. In some embodiments, the stimulus is anhydrotetracycline, tanespimycin, tunicamycin, or doxycycline. In some embodiments, the stimulus is a sugar. In some embodiments, the stimulus is arabinose, rhamnose, or IPTG.
  • repressor refers to a DNA- or RNA-binding protein that binds to a repressor binding site (e.g., an operator, a promoter, or a silencing sequence) to inhibit the expression of one or more genes.
  • a DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the downstream nucleic acid sequence (e.g., gene) operably linked to the promoter into messenger RNA and consequent expression of the protein encoded by the gene.
  • An inducer z.e., a molecule that initiates the gene expression, can interact with the repressor protein and detach it from the operator (e.g., the promoter).
  • the repressor is a LacI repressor that represses the expression of a nucleic acid sequence operably linked to a lactose-inducible (e.g, an IPTG-inducible) promoter.
  • the repressor is a tetracycline repressor (TetR) that represses the expression of a nucleic acid sequence operably linked to a tetracycline-inducible promoter.
  • TetR tetracycline repressor
  • Additional suitable repressor systems will be apparent to those of ordinary skill in the art based on this disclosure and knowledge in the field, and are within the scope of the present disclosure.
  • small molecule is art-recognized and refers to a composition which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu.
  • Small molecules may be, for example, nucleic acids, peptides, polypeptides, peptide nucleic acids, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules.
  • Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays described herein.
  • small organic molecule refers to a small molecule that is often identified as being an organic or medicinal compound and does not include molecules that are exclusively nucleic acids, peptides or polypeptides.
  • specific binding refers to the ability of an antibody to bind to a predetermined antigen or the ability of a polypeptide to bind to its predetermined binding partner.
  • an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 10' 7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10-fold less, at least 100-fold less or at least 1000-fold less than its affinity for binding to a non-specific and unrelated antigen/binding partner (e.g, BSA, casein).
  • specific binding applies more broadly to a two component system where one component is a protein, lipid, or carbohydrate or combination thereof and engages with the second component which is a protein, lipid, carbohydrate or combination thereof in a specific way.
  • subject refers to any animal.
  • a subject or a patient described as “in need thereof’ refers to one in need of a treatment and/or prevention for a disease.
  • Mammals i.e., mammalian animals
  • mammals include humans, laboratory animals (e.g, primates, rats, mice), livestock (e.g, cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents).
  • the subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
  • a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
  • transgene refers to any nucleic acid molecule that is introduced into a cell, that may be intermittently termed herein as a recipient cell.
  • the resultant cell after receiving a transgene may be referred to a transgenic cell.
  • a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism or cell, or may represent a gene homologous to an endogenous gene of the organism or cell.
  • transgenes include any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacks an expression control element, such as a promoter that drives transcription).
  • Transcripts and encoded polypeptides may be collectively referred to as "gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening.
  • a PDP is a particle in which a non-phage nucleic acid payload is encapsulated by phage-derived coat proteins.
  • the nucleic acid payload of a PDP substantially lacks the genome from which the PDP is derived.
  • the PDP does not comprise nucleic acid sequences encoding at least 50% (e.g., 60%, 70%, 80%, 90%, 95%) of the genome of the phage from which the PDP is derived.
  • the PDP does not comprise a nucleic acid sequence encoding one or more phage coat proteins (e.g., a 10A and/or 10B coat protein). In some embodiments, the PDP does not comprise a nucleic acid sequence encoding any phage coat proteins. In certain embodiments, the PDP comprises no more that 2000 bases of the genome of the phage from which the PDP was derived.
  • PDPs provided herein possess advantageous properties compared to existing viral vectors making them particularly useful, for example, in the treatment and/or prevention of various diseases.
  • the PDPs provided herein exhibit improved: cell or tissue specificity, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics, and/or mammalian expression as compared to existing phage derived vectors.
  • the PDPs provided herein comprise phage coat proteins encapsulating a nucleic acid payload, wherein the nucleic acid payload comprises a sequence encoding an agent that treats a disease when delivered to a cell of a subject in need thereof.
  • the PDPs are derived from an icosahedral phage (e.g., T7 phage).
  • the icosahedral bacteriophage is T7.
  • Bacteriophage T7 is an icosahedral virus (of family Podoviridae) that infects most strains of E. coli. It exhibits a lytic life cycle and destroys its host following infection. T7 phage exhibits a rapid life cycle of 17 minutes at 37C and can produce upwards of 10 13 particles in just one hour of growth.
  • the wild-type T7 phage has a diameter of 55 nm and packages a genome of ⁇ 40 kb.
  • the wild-type T7 phage has two capsid proteins, 10A (a/k/a gplOA) and 10B (a/k/a gplOB), products of gene 70, which make up the 415 total proteins on a single capsid at a ratio of 90/10, respectively.
  • the proportion of 10A and 10B may vary and does not affect the integrity of phage particles.
  • Capsid protein 10B is the result of a frame shift at the end of the 10A coding frame.
  • the PDPs disclosed herein comprise one or more modified coat proteins that impart beneficial properties upon the PDP (e.g., improved cell targeting, improved internalization, improved endosomal escape, improved, nuclear shuttling, improved immune evasion, improved pharmacokinetics, improved pharmacodynamics, improved transgene expression).
  • beneficial properties e.g., improved cell targeting, improved internalization, improved endosomal escape, improved, nuclear shuttling, improved immune evasion, improved pharmacokinetics, improved pharmacodynamics, improved transgene expression.
  • any of the two of the coat proteins — 10 A, 10B — can be modified to provide such beneficial properties.
  • any combination of the coat proteins can be modified to provide such beneficial properties.
  • the coat protein(s) are modified to display functional moieties disclosed herein (e.g., cell-targeting moieties, nuclear localization moieties, endosomal escape moieties, internalization moieties). In some embodiments, such moieties are displayed on 10A and/or 10B capsid proteins.
  • the wild type 10A gene is expressed as a 345 amino acid polypeptide and is the major capsid protein of the icosahedral T7 capsid. It is incorporated into the capsid at approximately a 90/10 ratio relative to the 10B minor capsid protein, where there are a total of 415 capsid proteins per single T7 capsid.
  • a DNA sequence encoding an exemplary T7 10A capsid protein is provided as SEQ ID NO: 1, as follows: atggctagcatgactggtggacagcaaatgggtactaaccaaggtaaaggtgtagttgctgctggagataaactggcgttgttctt gaaggtatttggcggtgaagtcctgactgcgttcgctcgtacctccgtgaccacttctcgccacatggtacgttccatctccagcgg taaatccgctcagttccctgttctgggtcgcactcaggcagcgtatctggctccgggcgagaacctcgacgataaacgtaggac atcaaacacaccgagaaggtaatcaccattgacggtctctgacggac atcaa
  • amino acid sequence of an exemplary T7 10A capsid protein is provided as SEQ ID No: 2, as follow:
  • the wild type 10B gene is expressed as a 398 amino acid polypeptide and is the minor capsid protein of the icosahedral T7 capsid. It is incorporated into the capsid at approximately a 10/90 ratio relative to the 10A major capsid protein, where there are a total of 415 capsid proteins per single T7 capsid.
  • a DNA sequence encoding an exemplary T7 10B capsid protein is provided as SEQ ID NO: 3, as follows: atggctagcatgactggtggacagcaaatgggtactaaccaaggtaaaggtgtagttgctgctggagataaactggcgttgttctt gaaggtatttggcggtgaagtcctgactgcgttcgctcgtacctccgtgaccacttctcgccacatggtacgttccatctccagcgg taaatccgctcagttccctgttctgggtcgcactcaggcagcgtatctggctccgggcgagaacctcgacgataaacgtaggac atcaaacacaccgagaaggtaatcaccattgacggtctctgacggac atcaa
  • amino acid sequence of an exemplary T7 10B capsid protein is provided as SEQ ID NO: 4, as follows:
  • the PDPs provided herein comprise modified coat proteins (e.g., modified 10A and/or 10B coat proteins) that impart certain beneficial properties upon the PDP.
  • the PDPs comprise modified coat proteins that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived.
  • the PDPs provided herein comprise moieties that impart certain beneficial properties upon the PDP.
  • the PDPs comprise moieties that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived.
  • such moieties are displayed on one or more of the phage coat proteins of the PDP (e.g., a 10A and/or 10B coat protein).
  • such moieties are covalently attached to the phage coat protein.
  • such moieties are non-covalently attached to the phage coat protein.
  • the phage coat protein displaying the moiety is a fusion protein comprising the phage coat protein and the moiety.
  • the nucleic acid payload of the PDPs provided herein is typically in the form of linear double-stranded DNA (dsDNA).
  • dsDNA linear double-stranded DNA
  • the phages and/or PDPs disclosed herein use borrowed and/or altered T7 origins of replication to allow for the packaging of foreign sequences (i.e., the PDP payload) within a phage or PDP body.
  • a template phagemid with a T7 origin insert is coinfected with a T7 helper phage that will express all the necessary phage assembly proteins to yield PDPs carrying the sequence from the template phagemid.
  • PDPs can be produced in bacterial culture at high yields and purified for acquisition of desired dsDNA sequences and structures.
  • PDP nucleic acid payload carries an expression cassette that encodes the fusion-coat protein to be displayed and allows for two-gene display systems (i.e., type 10A+10A or type 10B+10B).
  • the helper phage or helper plasmid bears a defective T7 origin of replication or packaging signal, which allows the preferential packaging of the PDP nucleic acid payload over the helper system.
  • the PDP nucleic acid payload lacks sequences coding one or more of the phage capsid proteins.
  • the dsDNA in the PDP comprises an origin of replication for enabling replication of a double-stranded vector inside a prokaryotic host.
  • the origin of replication enables high copy number replication of the vector inside the host.
  • the origin of replication comprises a pUC ori.
  • the DNA sequence of one embodiment of the pUC ori is represented herein as follows:
  • a PDP can be engineered to have a desired surface charge.
  • the highest priority barrier to cargo delivery is the degradation of the phage by the proteasome.
  • Endosomal escape strategies such as cationic charge on the phage surface is shown to drive reporter gene transduction (20-30%) and suggests that directed or rational mutagenesis of the phage capsids will yield transducing variants.
  • the negative charge of the T7 bacteriophage head may play a crucial role in the extracellular barriers to phage due to the generation of high non-specific binding to positively charged molecules (around 35% of proteins in the human proteome).
  • a genetic approach may be used to change the C-term of the 10A/10B coat proteins of the PDP provided herein into a mixture of anionic and cationic terminal groups by introducing a short charged neutralizing peptide termed AKAS (Ala-Lys-Ala-Ser).
  • AKAS Al-Lys-Ala-Ser
  • the coat protein of the PDP may comprise at least one modification which neutralizes the negative charge of the surface of the phage, and, in some embodiments results in a net positive charge at physiological pH.
  • the modification may comprise a tetrapeptide comprising the amino acid sequence AKAS located in the C-terminal of the 10A/10B coat proteins.
  • the targeted PDP comprises a recombinant targeted-bacteriophage and a cationic polymer, wherein the complex has a net positive charge.
  • the PDP-polymer complex provides a hybrid vector platform, for use in a wide range of gene therapy applications.
  • the cationic polymer is any polymeric compound having a net positive charge at physiological pH.
  • the polymer may comprise a plurality of positively charged repeating units.
  • the cationic polymer may be selected from a group consisting of: chitosan; poly-D-lysine (PDL); diethylaminoethyl (DEAE); diethylaminoethyl-dextran (DEAE.DEX); polyethyleneimine (PEI); polybrene; protamine sulphate; and a cationic lipid.
  • the cationic lipid may be selected from the group consisting of Fugene®, Lipofectamine®, and DOTAP (N-[l-(2,3- Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate).
  • the molecular weight of the cationic polymer may be at least 4 kD, 6 kD, 10 kD, 50 kD, 100 kD or 500 kD.
  • the molecular weight of the polymer is about 500 kD for PDL, 70-150 kD for DEAE.DEX, 750 kD for PEI, 4 kD-6 kD for polybrene, 5.1 kD for protamine sulfate.
  • the inventors believe that the lower the molecular weight of the cationic polymer, the more polymer is required in the complex in order to convert the negative charge of the PDP such that it is positive at physiological pH.
  • the cationic polymer may comprise DEAE, more preferably DEAE.DEX.
  • the polymer comprises PDL.
  • the cationic polymer may comprise a combination of any of the polymers described herein, such as DEAE.DEX and PDL.
  • the complex may comprise a weight weight ratio of about 50 ng-500 ng polymer: 1 pg PDP, more preferably about 100 ng-400 ng polymer: 1 pg PDP, and even more preferably about 130 ng-320 ng polymer: 1 pg PDP.
  • the complex preferably comprises a weightweight ratio of about 50 ng- 300 ng polymer: 1 pg PDP, more preferably about 100 ng-200 ng polymer: 1 pg PDP, and even more preferably about 120 ng-150 ng polymer: 1 pg PDP.
  • the complex preferably comprises a weight: weight ratio of about 100 ng-500 ng polymer: 1 pg PDP, more preferably about 200 ng-400 ng polymer: 1 pg PDP, and even more preferably about 250 ng-350 ng polymer: 1 pg PDP.
  • the PDPs provided herein comprise modified coat proteins (e.g., modified 10A and/or 10B coat proteins) that impart certain beneficial properties upon the PDP.
  • the PDPs comprise modified coat proteins that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived.
  • the PDPs provided herein comprise a modified 10A coat protein.
  • the modified 10A coat protein comprises an amino acid sequence that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to a sequence of a wildtype 10A coat protein.
  • the PDP comprises a modified 10A coat protein that comprises an amino acid sequence that has no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type 10A coat protein sequence.
  • the PDP comprises a modified 10A coat protein that comprises an amino acid sequence that has at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type 10A coat protein sequence.
  • the PDP comprises a modified 10A coat protein that comprises an amino acid sequence that includes deletions of at least 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids compared to a wild-type 10A coat protein sequence.
  • the PDP comprises a modified 10A coat protein that comprises an amino acid sequence that includes additions of at least 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids compared to a wild-type 10A coat protein sequence.
  • a PDP comprising the modified 10A coat protein has improved improve targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared an identical PDP but comprising a wild-type 10A coat protein instead of the modified 10A coat protein.
  • the PDPs provided herein comprise a modified 10B coat protein.
  • the modified 10B coat protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to a sequence of a wild-type 10B coat protein.
  • the PDP comprises a modified 10B coat protein that comprises an amino acid sequence that has no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type 10B coat protein sequence.
  • the PDP comprises a modified 10B coat protein that comprises an amino acid sequence that has at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type 10B coat protein sequence.
  • the PDP comprises a modified 10B coat protein that comprises an amino acid sequence that includes deletions of at least 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids compared to a wild-type 10B coat protein sequence.
  • the PDP comprises a modified 10B coat protein that comprises an amino acid sequence that includes additions of at least 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids compared to a wild-type 10B coat protein sequence.
  • a PDP comprising the modified 10B coat protein has improved improve targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared an identical PDP but comprising a wild-type 10B coat protein instead of the modified 10B coat protein.
  • the PDPs provided herein comprise moieties that impart certain beneficial properties upon the PDP.
  • the PDPs comprise moieties that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived.
  • such moieties are displayed on one or more of the phage coat proteins of the PDP (e.g., a 10A and/or 10B coat protein).
  • such moieties are covalently attached to the phage coat protein.
  • such moieties are non-covalently attached to the phage coat protein.
  • the phage coat protein displaying the moiety is a fusion protein comprising the phage coat protein and the moiety, linkers and conjugation methods
  • the coat protein is 10A and/or 10B coat protein (e.g., 10A and/or 10B coat protein configured to display a moiety provided herein).
  • the moiety is a domain of a coat protein fusion protein (e.g., a 10A and/or 10B coat protein fusion protein).
  • constructing a targeted bacteriophage with more than one 10A/10B major coat protein provides the choice to use one 10A/10B major coat protein for the display of large foreign peptide or proteins while keeping the wild type 10A/10B intact, which may be important to achieve efficient phage assembly and subsequently high titers of the phage vector, (ii) allows the display of a considerable copy number of the peptide in order to yield a phage that displays hundreds or even thousands of functional foreign peptides, and (iii) offers the possibility of the simultaneous display of two different functional peptides on the capsid of a single bacteriophage particle. It will be appreciated that a foreign peptide or protein is one that is not normally or naturally expressed by the phage, i.e., it can be heterologous.
  • the coat proteins of the PDP may be conjugated to a moiety using heterobifunctional crosslinking reagents.
  • heterobifunctional linkers including, but not limited to N-succinimidyl 3-(2-pyridyl dithio) propionate (SPDP), m- maleimidobenzoyl-N-hydroxysulfosuccinimida ester, and N-succinimidyl-(4-iodoacetyl) amino-benzoate.
  • Such heterobifunctional crosslinking reagents can also be used to link the drug to the coat proteins of the bacteriophage via a linker such as a peptide, a polypeptide, a peptide derivative, an oligonucleotide, a lipid, a glycolipid, an oligosaccharide and the like.
  • a linker such as a peptide, a polypeptide, a peptide derivative, an oligonucleotide, a lipid, a glycolipid, an oligosaccharide and the like.
  • linkers disclosed herein may also comprise non-covalent bonds either within the linker, or between the linker and the coat protein and/or the moiety to be linked.
  • linkers may be conjugated to the coat protein by means of an avidin/biotin complex.
  • the PDP may be modified to express on its surface avidin or a portion thereof that selectively binds to biotin with the requisite binding affinity. Modification of the PDP to express avidin is easily accomplished by inserting the nucleic acid encoding avidin or a functionally active portion thereof into a helper plasmid used in the making of the PDP such that the avidin or avidin portion is expressed on the PDP surface (e.g., on a coat protein).
  • an avidin-expressing PDP which serves as an intermediate for attachment of a biotinylated moiety to the PDP surface.
  • avidin or a functionally active portion thereof can be chemically coupled to the PDP surface using standard cross-linking chemistries, such as those described above.
  • the avidin-labeled PDP permits non-covalent, yet high affinity, attachment of pre-selected biotinylated moieties to the bacteriophage surface.
  • the PDP can be biotinylated and an avidin-labeled moiety provided herein, can be used to form the PDPs described herein.
  • conjuggated thus explicitly includes both covalent and non-covalent links between the PDP and the moieties.
  • the PDP provided herein may be conjugated to multiple different moieties modulating different PDP properties (e.g., PDP targeting, immunogenicity, endosomal escape, nuclear localization, pharmacokinetics, and/or pharmacodynamics).
  • the PDP may be pegylated (i.e., conjugated to polyethylene glycol), resulting in reduced immunogenicity.
  • conjugations may be done prior or following the conjugation of other moieties, and typically involve a different conjugation method than that used for conjugating the first moiety to the PDP.
  • a first moiety may be conjugated to an amino group of a coat protein while a second moiety may be conjugated to a thiol group engineered into the coat protein.
  • dual conjugation chemistries may also be used for conjugating a plurality of moieties to the PDP.
  • different moieties can be displayed on different moiety/coat protein fusion proteins.
  • the linker is a branched linker or a dendrimer.
  • dendrimer refers to a three-dimensionally branched, multi-branched compound, and generally refers to all of hyper-branched polymer having a low regularity and dendrimers having a high regularity.
  • a branched linker suitable for linking moieties to the PDP coat proteins by means of chemical conjugation contains at least two reactive residues that may be used for conjugation. The residues may be selected from amine, carboxyl, hydroxyl and sulfhydryl residues.
  • the branched linker has high water solubility, and is thus useful for conjugating hydrophobic drugs.
  • the linker is an aminoglycoside.
  • the aminoglycoside antibiotics are highly hydrophilic substances, which are naturally produced by the actinomycetes. Most of the molecules in the group comprise multiple amino sugars. The aminoglycosides vary in the form and quanta of amine residues within the molecules, which range between 1-7 amine residues per molecule. The chemical structure of the aminoglycoside antibiotic drug kanamycin, which comprises three amine sugars and four amine residues.
  • Amplification of the carrying capacity of the PDP may be facilitated by chemical conjugation of a single amine from an aminoglycoside molecule to a carboxyl residue of the carrier, thereby converting the single carboxyl residue to an amine branched linker.
  • suitable aminoglycoside molecules have two or more reactive residues.
  • exemplary aminoglycosides include, but are not limited to, hygromycin, kanamycin, gentamycin, amikacin, neomycin, pardomycin, tobramycin and viomycin.
  • amine conjugation methods well-known in the art may be used to conjugate aminoglycosides to the PDP carrier and to the drug molecules, including, but not limited to, NHS chemistry, paranitophenyl phosphate (PNP) chemistry, isothiocyanate chemistry and N-(3-dimethylaminopropyl)-N'- ethycarbodiimide (EDC) chemistry.
  • NHS paranitophenyl phosphate
  • EDC N-(3-dimethylaminopropyl)-N'- ethycarbodiimide
  • the linker, moiety, and/or a component thereof may be displayed on the PDP as a result of genetic modification.
  • a peptide linker and/or moiety may be fused to a coat protein of a PDP disclosed herein, to which other moieties may be linked by means of chemical conjugation or genetic modification.
  • a peptide comprising a moiety e.g, a moiety disclosed herein
  • the peptide comprising a moiety can be fused to the N’ of a coat protein of the phage (e.g., 10A, 10B), such that the moiety is displayed by the PDP.
  • the PDP surface can be modified to include a poly-glycine (polyG) motif (e.g., a GGGGG docking site, where G(n) can vary from 1-10).
  • a sortase enzyme can catalyze the formation of a peptide bond between the C-terminal LPXTG amino acid motif, where X is any amino acid, of a desired moiety and the N-terminal of a poly-glycine motif, to functionalize the PDP surface with the desired moiety.
  • the PDP surface can be modified to include an LPXTG amino acid motif and the desired moiety can contain the G(n) sequence for the sortase conjugation reaction.
  • icosahedral e.g., T7
  • PDPs phage derived particles
  • the PDPs provided herein are useful for the delivery of nucleic acid payloads to mammalian cells.
  • the nucleic acid payload of the PDPs provided herein is a linear double stranded DNA (dsDNA) construct comprising a non-phage sequence encoding a therapeutic agent to be delivered to a cell of interest.
  • the nucleic acid payload of a PDP substantially lacks the genome of the phage from which the PDP is derived (e.g., the nucleic acid payload does not encode the phage-derived coat proteins in which it is encapsulated).
  • the PDP does not comprise nucleic acid sequences encoding at least 50% (e.g., 60%, 70%, 80%, 90%, 95%) of the phage genome of the phage from which the PDP is derived.
  • the PDP does not comprise a nucleic acid sequence encoding a phage coat protein. In certain embodiments, the PDP comprises no more that 300 bases of the genome of the phage from which the PDP was derived.
  • a nucleic acid sequence delivered by a PDP provided herein comprises a sequence to be expressed
  • the application envisages the use of codon-optimized sequences.
  • An example of a codon optimized sequence may be a sequence optimized for expression in a eukaryote, e.g., humans (i.e., being optimized for expression in humans), or for another eukaryote, animal or mammal.
  • Codon optimization for a host species other than human, or for codon optimization for specific organs is known.
  • the coding sequence encoding a protein may be codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.
  • Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codons e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons
  • Codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell may generally reflect the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database" available on the internet at www.kazusa.orjp/codon/ and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
  • nucleic acid payloads of the PDPs are modified to (1) increase the cargo-carrying capacity by using minimal sequence requirements for packaging and (2) improve the levels, kinetics, and durability of transgene expression following cellular transduction.
  • nucleic acid payload of the PDPs provided herein is a linear double stranded DNA (dsDNA) construct.
  • payload dsDNA construct comprises a DNA sequence element that enhance expression and/or specificity in mammalian cells.
  • the DNA sequence element is selected from a promoter, an enhancer, a silencer, an insulator, an untranslated region, and a microRNA binding site.
  • the payload dsDNA construct comprises a coding region that is codon optimized for mammalian expression.
  • the dsDNA construct comprises a terminator sequence.
  • the episomal expression of the dsDNA construct is durable in slow and/or non-dividing cells (e.g., lasting at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days).
  • the dsDNA construct encodes a transgene (e.g., a transgene encoding a therapeutic agent provided herein).
  • transgene refers to any nucleic acid molecule that is introduced into a cell, that may be intermittently termed herein as a recipient cell. The resultant cell after receiving a transgene may be referred to a transgenic cell.
  • a transgene may include a gene that is partly or entirely heterologous (z.e., foreign) to the transgenic organism or cell, or may represent a gene homologous to an endogenous gene of the organism or cell.
  • transgenes include any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacks an expression control element, such as a promoter that drives transcription).
  • Transcripts and encoded polypeptides may be collectively referred to as "gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • the dsDNA construct comprises promoters, e.g., to drive expression of a therapeutic agent described herein.
  • promoter refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell.
  • promoters used in the polynucleotide constructs of the disclosure include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene.
  • a promoter may be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation.
  • These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription.
  • a "tissue-specific promoter” initiates transcription only in one or a few particular tissue types, whereas a “non-specific promoter” is one that is capable of initiating transcription in nearly all tissue types.
  • a “constitutive promoter” is a promoter that is capable of driving expression of a coding sequence in most or all environmental conditions.
  • the cytomegalovirus (CMV) promoter is a constitutive promoter that is often used when continual expression is desired.
  • an "inducible promoter” is one that initiates transcription only under particular environmental conditions, developmental conditions, or drug or chemical conditions.
  • Exemplary inducible promoter may be a doxycycline or a tetracycline inducible promoter.
  • Tetracycline regulated promoters may be both tetracycline inducible or tetracycline repressible, called the tet-on and tet-off systems.
  • tet regulated systems rely on two components, i.e., a tetracycline-controlled regulator (also referred to as transactivator) (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of a downstream cDNA, in a tetracycline-dependent manner.
  • tTA is a fusion protein containing the repressor of the TnlO tetracycline-resistance operon of Escherichia coli and a carboxyl-terminal portion of protein 16 of herpes simplex virus (VP 16).
  • the tTA-dependent promoter consists of a minimal RNA polymerase II promoter fused to tet operator (tetO) sequences (an array of seven cognate operator sequences). This fusion converts the tet repressor into a strong transcriptional activator in eukaryotic cells.
  • tetO tet operator
  • tTA binds to the tetO sequences, allowing transcriptional activation of the tTA-dependent promoter.
  • tTA cannot interact with its target and transcription does not occur.
  • tet system that uses tTA is termed /e -OFF, because tetracycline or doxycycline allows transcriptional down-regulation.
  • tet-ON a mutant form of tTA, termed rtTA, has been isolated using random mutagenesis.
  • rtTA is not functional in the absence of doxycycline but requires the presence of the ligand for transactivation.
  • termination sequence refers to a nucleic acid sequence which is recognized by the polymerase of a host cell and results in the termination of transcription.
  • the termination sequence is a sequence of DNA that, at the 3' end of a natural or synthetic gene, provides for termination of mRNA transcription or both mRNA transcription and ribosomal translation of an upstream open reading frame.
  • Prokaryotic termination sequences commonly comprise a GC-rich region that has a two-fold symmetry followed by an AT-rich sequence.
  • a commonly used termination sequence is the T7 termination sequence.
  • termination sequences are known in the art and may be employed in the nucleic acid constructs of the present invention, including the TINT3, TL13, TL2, TRI, TR2, and T6S termination signals derived from the bacteriophage lambda, and termination signals derived from bacterial genes, such as the trp gene of E. coli.
  • polyadenylation sequence (also referred to as a "poly A site” or “poly A sequence”) refers to a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, as transcripts lacking a poly A tail are typically unstable and rapidly degraded.
  • the poly A signal utilized in an expression vector disclosed herein may be “heterologous” or "endogenous". An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome.
  • a heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene, e.g., coding sequence for a protein.
  • a commonly used heterologous poly A signal is the SV40 poly A signal.
  • the SV40 poly A signal is contained on a 237 bp BamFE/BclI restriction fragment and directs both termination and polyadenylation.
  • Another commonly used heterologous poly A signal is derived from the bovine growth hormone (BGH) gene; the BGH poly A signal is also available on a number of commercially available vectors.
  • BGH bovine growth hormone
  • the poly A signal from the Herpes simplex virus thymidine kinase (HSV tk) gene is also used as a poly A signal on a number of commercial expression vectors.
  • the polyadenylation signal facilitates the transportation of the RNA from within the cell nucleus into the cytosol as well as increases cellular halflife of such an RNA.
  • ccsDNA construct comprises a the DNA sequence element is selected from the DNA sequence elements listed in Table 1.
  • the PDPs provided herein are designed to inhibit the expression of a gene (e.g., a disease-associated gene) in a cell, for example, wherein reduction of such a gene in a cell would have a therapeutic effect.
  • a gene e.g., a disease-associated gene
  • such PDPs would comprise a nucleic acid payload that encodes an inhibitory RNA disclosed herein and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that targets the gene.
  • the PDPs provided herein are designed to enhance the expression of a peptide (e.g., a therapeutic peptide) in a cell (e.g., a peptide whose expression treats and/or prevents a disease).
  • a peptide e.g., a therapeutic peptide
  • such PDPs would comprise a nucleic acid payload that encodes the peptide and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate insertion of a sequence encoding the peptide into the genome of the cell.
  • the sequence encoding a peptide is inserted into safe harbor locus in the cell. Exemplary safe harbor loci are listed in Table 2.
  • the PDPs provided herein are designed to inhibit the expression of a gene (e.g., a disease-associated gene) in a cell and enhance expression of a peptide (e.g., a therapeutic peptide) in a cell.
  • a gene e.g., a disease-associated gene
  • a peptide e.g., a therapeutic peptide
  • the gene for which expression is inhibited is a mutant gene associated with a disease, while the peptide is the peptide encoded by the wild-type version of that gene.
  • such PDPs would comprise a nucleic acid payload that comprises a sequence that encodes an inhibitory RNA and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that targets the gene; and (2) a sequence that encodes the peptide and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate insertion of a sequence encoding the peptide into the genome of the cell (e.g., into the mutant gene locus and/or a safe harbor locus in the cell, such as a safe harbor locus listed in Table 2).
  • a gene editing system disclosed herein e.g., a CRISPR/Cas system
  • the PDPs provided herein are designed to modify the sequence of a gene in a cell.
  • such PDPs would comprise a nucleic acid payload that encodes one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate modification of the gene in the genome of the cell (e.g., converting mutant version of a gene into a wild-type version of a gene).
  • a gene editing system e.g., a CRISPR/Cas system
  • PDPs able to be targeted to specific cells and/or tissues.
  • PDPs that display a ligand, antigen-binding, or other targeting moiety endowing them with specificity towards target molecules, cells, tissues and/or other biological structures.
  • These PDPs can be used to deliver internal nucleic acid payloads and/or external conjugated moieties (e.g., conjugated through a labile/non labile linker or directly) to specific cells and/or tissues and are thus useful as targeted delivery vessels for the treatment of a disease.
  • the PDPs comprise a phage coat protein displaying a cell-targeting moiety specific for a cell type selected from erythrocytes, granulocytes, agranulocytes, platelets, neurons, neuroglial cells, skeletal muscle cells, cardiac muscle cells, smooth muscle cells, chondrocytes, lymphocytes, osteoblasts, osteoclasts, osteocytes, lining cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, epithelial cells (e.g.
  • PDPs disclosed herein may display a targeting moiety that selectively binds a target molecule on a target cell and/or in a target tissue.
  • displaying a “targeting moiety” as used herein encompasses targeting moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both.
  • the targeting moiety and its manner of expression and/or linkage is designed to facilitate the PDP to selectively bind a target cell.
  • This term further includes a targeting moiety comprising a moiety conjugated to the PDP that binds non-covalently to a second target or molecule capable of binding the target cell.
  • the targeting moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs.
  • Targeting moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
  • An isolated nucleic acid sequence encoding a targeting moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof.
  • a nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional targeting moiety of the present invention.
  • a nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
  • a targeting moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
  • the targeting moiety may be any biological or synthetic substance endowed with specific binding properties towards a selected target cell and/or tissue.
  • targeting moieties may be antibody -based moieties, including, but not limited to: monoclonal antibodies, polyclonal antibodies, and antibody fragments such as recombinant antibody fragments, single-chain antibodies (scFv), single antibody variable domains, and the like (Borrebaeck, 1995; Lo, 2003).
  • Single-chain antibodies are small recognition units consisting of the variable regions of the immunoglobulin heavy (VH) and light (VL) chains which are connected by a synthetic linker sequence.
  • Single antibody domain proteins (dAbs) are minimized antibody fragments comprising either an individual VL domain or an individual VH domain.
  • Antibodies may be generated via any one of several known methods, which may employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries, or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique.
  • Antibody fragments may be obtained using methods well known in the art, including, but not limited to by proteolytic hydrolysis of the antibody or by expression in E.
  • coli or mammalian cells e.g., Chinese hamster ovary (CHO) cell culture or other protein expression systems
  • DNA encoding the fragment (Fab')2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent e.g., a thiol reducing agent
  • optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages e.g., a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • An Fv is composed of paired heavy chain variable and light chain variable domains. This association may be non- covalent.
  • the variable domains may be linked to generate a single-chain Fv by an intermolecular disulfide bond, or alternately such chains may be cross-linked by chemicals such as glutaraldehyde.
  • the Fv is a singlechain Fv.
  • Single-chain Fvs are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker.
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains.
  • techniques may be utilized to change a “murine” antibody to a “human” antibody, without altering the binding specificity of the antibody.
  • the targeting moiety may be a peptide endowed with binding specificity towards the target cell (linear, circularly constrained or cyclic) or a short peptide selected from a library of short peptide sequences that is endowed with binding specificity towards the target cell (Kay et al., 1996). Methods for constructing libraries and using them for screening for ligands having an affinity to a selected target molecule or cell are known in the art.
  • the targeting moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to selectively bind a target molecule on a target cell.
  • the ligand may include known ligands of cell surface receptors, or any natural or synthetic derivative thereof.
  • the targeting moiety is chosen according to the target cell and/or tissue that is to be targeted.
  • targeting moieties are chosen such that they are internalized by the target cell upon binding the target molecule, thereby enabling the internalization of the PDP.
  • Methods of constructing and selecting for internalizing phages are known in the art (see, for example, Becerril et al., 1999, Kassner et al., 1999, Poul and Marks, 1999, Larocca and Baird, 2001, Larocca et al., 2001, Urbanelli et al., 2001).
  • the targeting moieties used in the compositions and/or methods disclosed herein do not necessarily retain any of their in vivo biological activities, other than binding a target molecule on a target cell and/or tissue. However, it may be desirable in certain contexts that a ligand exerts certain of its biological activities.
  • the targeting moiety may act as an agonist, or alternatively as an antagonist, upon binding a cell surface receptor.
  • the targeting moiety displayed by the PDP disclosed herein is selected so as to facilitate selective binding of the conjugate to a target cell and/or tissue involved in a disease or disorder in a subject in need thereof (e.g., a disease or disorder disclosed herein).
  • Aptamers are another class of binding agent that can be used as a targeting moiety.
  • Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g., membrane lipid rafts).
  • Aptamers that specifically bind a marker of the cell are useful in the methods of the invention.
  • Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No. 5,660,985; No. 5,567,588; No. 5,683,867; No. 5,637,459; and No. 6,011,020.
  • the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics.
  • aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups (e.g., 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
  • the PDPs provided herein are designed to target immune cells. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of the function of the targeted immune cell would have a therapeutic effect.
  • the targeted immune cell is a neutrophil.
  • the PDPs provided herein comprise a neutrophil-targeting moiety specific for a neutrophil surface antigen.
  • the neutrophil-targeting moiety can be specific for any protein expressed on the surface of a neutrophil. Exemplary neutrophil surface antigens are listed in Table 3.
  • the neutrophil-targeting moiety comprises an antibody or antibody fragment specific for a neutrophil surface antigen (e.g., an antigen listed in Table 3).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the neutrophil-targeting moiety comprises an aptamer specific for a neutrophil surface antigen (e.g., an antigen listed in Table 3).
  • the neutrophil-targeting moiety comprises a ligand that binds to a neutrophil surface antigen.
  • the targeted immune cell is an eosinophil.
  • the PDPs provided herein comprise an eosinophil-targeting moiety specific for a eosinophil surface antigen.
  • the eosinophil-targeting moiety can be specific for any protein expressed on the surface of an eosinophil. Exemplary eosinophil surface antigens are listed in Table 4.
  • the eosinophil-targeting moiety comprises an antibody or antibody fragment specific for an eosinophil surface antigen (e.g., an antigen listed in Table 4).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the eosinophil-targeting moiety comprises an aptamer specific for an eosinophil surface antigen (e.g., an antigen listed in Table 4).
  • the eosinophil-targeting moiety comprises a ligand that binds to an eosinophil surface antigen.
  • the targeted immune cell is a basophil.
  • the PDPs provided herein comprise a basophil-targeting moiety specific for a basophil surface antigen.
  • the basophil-targeting moiety can be specific for any protein expressed on the surface of a basophil. Exemplary basophil surface antigens are listed in Table 5.
  • the basophil-targeting moiety comprises an antibody or antibody fragment specific for a basophil surface antigen (e.g., an antigen listed in Table 5 or a biologically active fragment thereof).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the basophil-targeting moiety comprises an aptamer specific for a basophil surface antigen (e.g., an antigen listed in Table 5 or a biologically active fragment thereof).
  • the basophil-targeting moiety comprises a ligand that binds to a basophil surface antigen.
  • the targeted immune cell is a lymphocyte (e.g., a T cell, a B cell, a natural killer (NK) cell)).
  • a lymphocyte e.g., a T cell, a B cell, a natural killer (NK) cell
  • the targeted lymphocyte is a T cell.
  • the targeted T cell is a CD4 helper T cell (e.g., a Thl cell, a Th2 cell, a Thl7 cell, a Th9 cell, a Tfh cell, a Th22 cell).
  • the targeted T cell is a cytotoxic CD8 T cell (e.g., a Tel cell, a Tc2 cell, a Tc9 cell, a Tcl7 cell).
  • the targeted T cell is a naive T cell (Tn).
  • the targeted T cell is an effector T cell (Teff).
  • the targeted T cell is a memory T cell (e.g., a central memory T cell (Tern), an effector memory T cell (Tern), a tissue-resident memory T cell (Trm), a stem cell memory T cell (Tscm)).
  • the targeted T cell is a regulatory T cell (Treg).
  • the targeted T cell is a natural killer T cell (NKT).
  • a combination of the above T cell sub types are targeted.
  • the lymphocyte is a B cell.
  • the targeted B cell is a naive B cell.
  • the targeted B cell is a memory B cell.
  • the targeted B cell is a plasmablast.
  • the targeted B cell is a plasma cell.
  • the targeted B cell is a lymphoplasmacytoid cell.
  • the targeted B cell is a B-2 cell.
  • the targeted B cell is a B-l cell.
  • the targeted B cell is a regulatory B cell (Breg). In some embodiments, a combination of the above B cell sub types are targeted.
  • the lymphocyte is a natural killer (NK) cell.
  • the PDPs provided herein comprise a lymphocytetargeting moiety specific for a lymphocyte surface antigen.
  • the lymphocyte-targeting moiety can be specific for any protein expressed on the surface of a lymphocyte.
  • Exemplary lymphocyte surface antigens are listed in Table 6. Table 6: Exemplary Lymphocyte Surface Markers
  • the lymphocyte-targeting moiety comprises an antibody or antibody fragment specific for a lymphocyte surface antigen (e.g., an antigen listed in Table 6).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the lymphocyte-targeting moiety comprises an aptamer specific for a lymphocyte surface antigen (e.g., an antigen listed in Table 6).
  • the lymphocyte-targeting moiety comprises a ligand that binds to a lymphocyte surface antigen.
  • the targeted immune cell is a monocyte.
  • the PDPs provided herein comprise a monocyte-targeting moiety specific for a monocyte surface antigen.
  • the monocyte-targeting moiety can be specific for any protein expressed on the surface of a monocyte. Exemplary monocyte surface antigens are listed in Table 7.
  • the monocyte-targeting moiety comprises an antibody or antibody fragment specific for a monocyte surface antigen (e.g., an antigen listed in Table 7).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the monocyte-targeting moiety comprises an aptamer specific for a monocyte surface antigen (e.g., an antigen listed in Table 7).
  • the monocyte-targeting moiety comprises a ligand that binds to a monocyte surface antigen.
  • the targeted immune cell is a macrophage.
  • the PDPs provided herein comprise a macrophage-targeting moiety specific for a macrophage surface antigen.
  • the macrophagetargeting moiety can be specific for any protein expressed on the surface of a macrophage. Exemplary macrophage surface antigens are listed in Table 8.
  • the macrophage-targeting moiety comprises an antibody or antibody fragment specific for a macrophage surface antigen (e.g., an antigen listed in Table 8).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the macrophage-targeting moiety comprises an aptamer specific for a macrophage surface antigen (e.g., an antigen listed in Table 8).
  • the macrophage-targeting moiety comprises a ligand that binds to a macrophage surface antigen.
  • the targeted immune cell is a dendritic cell.
  • the PDPs provided herein comprise a dendritic cell-targeting moiety specific for a dendritic cell surface antigen.
  • the dendritic cell-targeting moiety can be specific for any protein expressed on the surface of a dendritic cell. Exemplary dendritic cell surface antigens are listed in Table 9.
  • the dendritic cell-targeting moiety comprises an antibody or antibody fragment specific for a dendritic cell surface antigen (e.g., an antigen listed in Table 9).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the dendritic cell-targeting moiety comprises an aptamer specific for a dendritic cell surface antigen (e.g., an antigen listed in Table 9).
  • the dendritic cell-targeting moiety comprises a ligand that binds to a dendritic cell surface antigen.
  • the PDPs provided herein comprise a neuronal cell-targeting moiety specific for a neuronal cell surface antigen.
  • the targeted neuronal cell is a neuroepithelial cell, a radial glia, an intermediate progenitor, an immature neuron, an oligodendrocyte precursor cell, a mature oligodendrocyte, a schwann cell, an astrocyte, a microglia cell,, an Ml microglia cell, a M2 microglia cell, a mature neuron, a glutamatergic neuron, a GABAergic neuron, a domaminergic neuron, a serotonergic neuron, and/or a cholinergic neuron.
  • neuronal cell-targeting moiety can be specific for any protein expressed on the surface of a neuronal cell.
  • Exemplary neuronal cell surface antigens are listed in Table 10.
  • the neuronal cell-targeting moiety comprises an antibody or antibody fragment specific for a neuronal cell surface antigen (e.g., an antigen listed in Table 10).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the neuronal cell-targeting moiety comprises an aptamer specific for a neuronal cell surface antigen (e.g., an antigen listed in Table 10).
  • the neuronal cell-targeting moiety comprises a ligand that binds to a neuronal cell surface antigen.
  • the PDPs provided herein comprise an epithelial cell-targeting moiety specific for a epithelial cell surface antigen.
  • the epithelial cell-targeting moiety can be specific for any protein expressed on the surface of an epithelial cell. Exemplary epithelial cell surface antigens are listed in Table 11.
  • the epithelial cell-targeting moiety comprises an antibody or antibody fragment specific for an epithelial cell surface antigen (e.g., an antigen listed in Table 11).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the epithelial cell-targeting moiety comprises an aptamer specific for an epithelial cell surface antigen (e.g., an antigen listed in Table 11).
  • the epithelial cell-targeting moiety comprises a ligand that binds to an epithelial cell surface antigen.
  • the PDPs provided herein comprise an adipocytetargeting moiety specific for a adipocyte surface antigen.
  • the adipocyte-targeting moiety can be specific for any protein expressed on the surface of an adipocyte. Exemplary adipocyte surface antigens are listed in Table 12.
  • the adipocyte-targeting moiety comprises an antibody or antibody fragment specific for an adipocyte surface antigen (e.g., an antigen listed in Table 12).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the adipocyte-targeting moiety comprises an aptamer specific for an adipocyte surface antigen (e.g., an antigen listed in Table 12).
  • the adipocyte -targeting moiety comprises a ligand that binds to an adipocyte surface antigen.
  • the PDPs provided herein comprise an hepatocyte-targeting moiety specific for a hepatocyte surface antigen.
  • the hepatocyte-targeting moiety can be specific for any protein expressed on the surface of an hepatocyte. Exemplary hepatocyte surface antigens are listed in Table 13.
  • the hepatocyte-targeting moiety comprises an antibody or antibody fragment specific for an hepatocyte surface antigen (e.g., an antigen listed in Table 13).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the hepatocyte-targeting moiety comprises an aptamer specific for an hepatocyte surface antigen (e.g., an antigen listed in Table 13).
  • the hepatocyte-targeting moiety comprises a ligand that binds to an hepatocyte surface antigen.
  • the PDPs provided herein comprise a fibroblasttargeting moiety specific for a fibroblast surface antigen.
  • the fibroblast-targeting moiety can be specific for any protein expressed on the surface of a fibroblast. Exemplary fibroblast surface antigens are listed in Table 14.
  • the fibroblast-targeting moiety comprises an antibody or antibody fragment specific for a fibroblast surface antigen (e.g., an antigen listed in Table 14).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the fibroblast-targeting moiety comprises an aptamer specific for a fibroblast surface antigen (e.g., an antigen listed in Table 14).
  • the fibroblast-targeting moiety comprises a ligand that binds to a fibroblast surface antigen.
  • the PDPs provided herein comprise a pancreatic cell-targeting moiety specific for a pancreatic cell surface antigen.
  • the pancreatic cell-targeting moiety can be specific for any protein expressed on the surface of a pancreatic cell. Exemplary pancreatic cell surface antigens are listed in Table 15.
  • the pancreatic cell-targeting moiety comprises an antibody or antibody fragment specific for a pancreatic cell surface antigen (e.g., an antigen listed in Table 15).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the pancreatic cell- targeting moiety comprises an aptamer specific for a pancreatic cell surface antigen (e.g., an antigen listed in Table 15).
  • the pancreatic cell-targeting moiety comprises a ligand that binds to a pancreatic cell surface antigen.
  • the PDPs provided herein comprise an osteoblasttargeting moiety specific for a osteoblast surface antigen.
  • the osteoblast-targeting moiety can be specific for any protein expressed on the surface of an osteoblast. Exemplary osteoblast surface antigens are listed in Table 16.
  • the osteoblast-targeting moiety comprises an antibody or antibody fragment specific for an osteoblast surface antigen (e.g., an antigen listed in Table 16).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the osteoblast-targeting moiety comprises an aptamer specific for an osteoblast surface antigen (e.g., an antigen listed in Table 16).
  • the osteoblast-targeting moiety comprises a ligand that binds to an osteoblast surface antigen.
  • the PDPs provided herein comprise a stem celltargeting moiety specific for a stem cell surface antigen.
  • the stem cell-targeting moiety can be specific for any protein expressed on the surface of a stem cell. Exemplary stem cell surface antigens are listed in Table 17.
  • the stem cell-targeting moiety comprises an antibody or antibody fragment specific for a stem cell surface antigen (e.g., an antigen listed in Table 17).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the stem cell-targeting moiety comprises an aptamer specific for a stem cell surface antigen (e.g., an antigen listed in Table 17).
  • the stem cell-targeting moiety comprises a ligand that binds to a stem cell surface antigen.
  • the PDPs provided herein comprise a skeletal muscle cell-targeting moiety specific for a skeletal muscle cell surface antigen.
  • the skeletal muscle cell-targeting moiety can be specific for any protein expressed on the surface of a skeletal muscle cell. Exemplary skeletal muscle cell surface antigens are listed in Table 18.
  • the skeletal muscle cell-targeting moiety comprises an antibody or antibody fragment specific for a skeletal muscle cell surface antigen (e.g., an antigen listed in Table 18).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the skeletal muscle celltargeting moiety comprises an aptamer specific for a skeletal muscle cell surface antigen (e.g., an antigen listed in Table 18).
  • the skeletal muscle cell -targeting moiety comprises a ligand that binds to a skeletal muscle cell surface antigen.
  • the PDPs provided herein are designed to target endothelial cells (e.g., vascular endothelial cell). Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of endothelial cells (e.g., vascular endothelial cell) function would have a therapeutic effect.
  • endothelial cells e.g., vascular endothelial cell
  • the PDPs provided herein comprise an endothelial cell-targeting moiety specific for an endothelial cell surface antigen.
  • the endothelial cell-targeting moiety can be specific for any protein expressed on the surface of an endothelial cell (e.g., a vascular endothelial cell.
  • the endothelial cell-targeting moiety comprises an antibody or antibody fragment specific for an endothelial cell surface antigen (e.g., an antigen listed in Table 19).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the endothelial celltargeting moiety comprises an aptamer specific for an endothelial cell surface antigen (e.g., an antigen listed in Table 19).
  • the endothelial cell-targeting moiety comprises a ligand that binds to an endothelial cell surface antigen.
  • the ligand is P-selectin glycoprotein ligand 1, CD44, or E-selectin ligand-1.
  • the PDPs provided herein are designed to target cardiomyocytes. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of cardiomyocyte function would have a therapeutic effect.
  • the PDPs provided herein comprise a cardiomyocyte-targeting moiety specific for a cardiomyocyte surface antigen.
  • the cardiomyocyte-targeting moiety can be specific for any protein expressed on the surface of a cardiomyocyte. Exemplary cardiomyocyte surface antigens are listed in Table 20.
  • the cardiomyocyte-targeting moiety comprises an antibody or antibody fragment specific for a cardiomyocyte surface antigen (e.g., an antigen listed in Table 20).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the cardiomyocyte- targeting moiety comprises an aptamer specific for a cardiomyocyte surface antigen (e.g., an antigen listed in Table 20).
  • the cardiomyocyte-targeting moiety comprises a ligand that binds to a cardiomyocyte surface antigen.
  • RPE Retinal Pigment Epithelium
  • the PDPs provided herein are designed to target retinal pigment epithelium (RPE) cells.
  • RPE retinal pigment epithelium
  • Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of RPE cell function would have a therapeutic effect.
  • the PDPs provided herein comprise an RPE celltargeting moiety specific for a RPE cell surface antigen.
  • the RPE cell-targeting moiety can be specific for any protein expressed on the surface of an RPE cell. Exemplary RPE cell surface antigens are listed in Table 21.
  • the RPE cell-targeting moiety comprises an antibody or antibody fragment specific for an RPE cell surface antigen (e.g., an antigen listed in Table 21).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the RPE cell-targeting moiety comprises an aptamer specific for an RPE cell surface antigen (e.g., an antigen listed in Table 21).
  • the RPE cell-targeting moiety comprises a ligand that binds to an RPE cell surface antigen.
  • the PDPs provided herein are designed to target chondrocytes. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of chondrocyte function would have a therapeutic effect.
  • the PDPs provided herein comprise a chondrocyte-targeting moiety specific for a chondrocyte surface antigen.
  • the chondrocyte-targeting moiety can be specific for any protein expressed on the surface of an chondrocyte. Exemplary chondrocyte surface antigens are listed in Table 22.
  • the chondrocyte-targeting moiety comprises an antibody or antibody fragment specific for a chondrocyte surface antigen (e.g., an antigen listed in Table 22).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the chondrocyte-targeting moiety comprises an aptamer specific for a chondrocyte surface antigen (e.g., an antigen listed in Table 22).
  • the chondrocyte-targeting moiety comprises a ligand that binds to a chondrocyte surface antigen.
  • the PDPs provided herein are designed to target keratinocytes. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of keratinocyte function would have a therapeutic effect.
  • the PDPs provided herein comprise a keratinocyte-targeting moiety specific for a keratinocyte surface antigen.
  • the keratinocyte-targeting moiety can be specific for any protein expressed on the surface of an keratinocyte. Exemplary keratinocyte surface antigens are listed in Table 23.
  • the keratinocyte-targeting moiety comprises an antibody or antibody fragment specific for a keratinocyte surface antigen (e.g., an antigen listed in Table 23).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the keratinocyte-targeting moiety comprises an aptamer specific for a keratinocyte surface antigen (e.g., an antigen listed in Table 23).
  • the keratinocyte-targeting moiety comprises a ligand that binds to a keratinocyte surface antigen.
  • the PDPs provided herein are designed to target neuroglia cells. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of neuroglia cell function would have a therapeutic effect.
  • the PDPs provided herein comprise a neuroglia cell-targeting moiety specific for a neuroglia cell surface antigen.
  • the neuroglia cell-targeting moiety can be specific for any protein expressed on the surface of a neuroglia cell. Exemplary neuroglia cell surface antigens are listed in Table 24.
  • the neuroglia cell-targeting moiety comprises an antibody or antibody fragment specific for a neuroglia cell surface antigen (e.g., an antigen listed in Table 24).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the neuroglia celltargeting moiety comprises an aptamer specific for a neuroglia cell surface antigen (e.g., an antigen listed in Table 24).
  • the neuroglia cell-targeting moiety comprises a ligand that binds to a neuroglia cell surface antigen.
  • the PDPs provided herein are designed to target melanocytes. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of melanocyte function would have a therapeutic effect.
  • the PDPs provided herein comprise a melanocytetargeting moiety specific for a melanocyte surface antigen.
  • the melanocyte-targeting moiety can be specific for any protein expressed on the surface of an melanocyte. Exemplary melanocyte surface antigens are listed in Table 25.
  • the melanocyte-targeting moiety comprises an antibody or antibody fragment specific for a melanocyte surface antigen (e.g., an antigen listed in Table 25).
  • the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
  • the melanocyte-targeting moiety comprises an aptamer specific for a melanocyte surface antigen (e.g., an antigen listed in Table 25).
  • the melanocyte-targeting moiety comprises a ligand that binds to a melanocyte surface antigen.
  • PDPs that have improved cellular uptake compared to the phage from which they are derived.
  • PDPs that display an internalization moiety endowing them improved cellular uptake.
  • These PDPs can be used to deliver internal nucleic acid payloads and/or external conjugated moieties (e.g., conjugated through a labile/non labile linker or directly) into cells and are thus useful as delivery vessels for the treatment and/or prevention of a disease.
  • the PDPs comprise a phage coat protein displaying a internalization moiety.
  • PDPs disclosed herein may display a internalization moiety that facilitates internalization of the PDP into a mammalian cell.
  • the term displaying a “internalization moiety” as used herein encompasses internalization moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both.
  • the internalization moiety and its manner of expression and/or linkage is designed to facilitate internalization of the PDP into a cell.
  • This term further includes a internalization moiety comprising a moiety conjugated to the PDP that binds non-covalently to a second target or molecule capable of binding the target cell.
  • the internalization moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs.
  • Internalization moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
  • An isolated nucleic acid sequence encoding a internalization moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof.
  • a nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional internalization moiety of the present invention.
  • a nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
  • An internalization moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
  • the internalization moiety may be any biological or synthetic substance that facilitates internalization of the PDP.
  • internalization moieties may be antibody-based moieties, including, but not limited to: monoclonal antibodies, polyclonal antibodies, and antibody fragments such as recombinant antibody fragments, single-chain antibodies (scFv), single antibody variable domains, and the like (Borrebaeck, 1995; Lo, 2003).
  • Single-chain antibodies are small recognition units consisting of the variable regions of the immunoglobulin heavy (VH) and light (VL) chains which are connected by a synthetic linker sequence.
  • Single antibody domain proteins (dAbs) are minimized antibody fragments comprising either an individual VL domain or an individual VH domain.
  • Antibodies may be generated via any one of several known methods, which may employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries, or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique.
  • Antibody fragments may be obtained using methods well known in the art, including, but not limited to by proteolytic hydrolysis of the antibody or by expression in E.
  • coli or mammalian cells e.g., Chinese hamster ovary (CHO) cell culture or other protein expression systems
  • DNA encoding the fragment (Fab')2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent e.g., a thiol reducing agent
  • optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages e.g., a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • An Fv is composed of paired heavy chain variable and light chain variable domains. This association may be non- covalent.
  • the variable domains may be linked to generate a single-chain Fv by an intermolecular disulfide bond, or alternately such chains may be cross-linked by chemicals such as glutaraldehyde.
  • the Fv is a singlechain Fv.
  • Single-chain Fvs are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker.
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains.
  • techniques may be utilized to change a “murine” antibody to a “human” antibody, without altering the binding specificity of the antibody.
  • the internalization moiety may be a peptide that facilitates PDP internalization into a mammalian cells. Methods for constructing libraries and using them for screening for ligands having a particular functional property are known in the art.
  • the internalization moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to facilitate internalization of a PDP into a mammalian cell.
  • the ligand may include known ligands of cell surface receptors, or any natural or synthetic derivative thereof.
  • Aptamers are another class of binding agent that can be used as an internalization moiety.
  • Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g, membrane lipid rafts).
  • Aptamers that specifically bind a marker of the cell e.g., a cell surface moiety or receptor
  • Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No.
  • the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics.
  • aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups (e.g, 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
  • the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an internalization moiety.
  • the internalization moiety is covalently attached to the phage coat protein displaying the internalization moiety.
  • the internalization moiety is non-covalently attached to the phage coat protein displaying the internalization moiety.
  • the internalization moiety is a fusion protein comprising the phage coat protein and the internalization moiety.
  • the internalization moiety acts via membrane penetration.
  • the internalization moiety is selected from a cell-penetrating peptide (CPP), transbody, cationic polymer, sugar, lipid, inorganic small molecule, and a mammalian viral particle (e.g. AAV).
  • the internalization moiety acts via membrane endocytosis.
  • the internalization moiety is selected from a high- affinity antibody (e.g. mAb, scFv, VHH), peptide sequence, endogenous ligands (e.g. glucose; growth hormones), sugars (e.g. beta-galNAc), lipids, nucleotide sequence (e.g. aptamers).
  • the PDP can be internalized by any mechanism.
  • the PDP is internalized by a mechanism selected from macropinocytosis, phagocytosis, clathrin-mediated, caveolin-mediated, interaction of hydrophilic lipid membrane and fusogenic moieties, interaction with hydrophobic portion of lipid membrane, and hydrophobic cloaking.
  • the internalization of the PDP is improved compared to the internalization of the phage type from which the PDP is derived.
  • internalization of the PDP is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more efficient that internalization of the phage type from which the PDP is derived.
  • internalization can be measured by any means known in the art. For example, in some embodiments internalization is measured via flow cytometry, western blotting, immunofluore scent staining, density gradient separation of cellular compartments, ELISA, or other published assay.
  • internalization moiety is selected from the moieties listed in Table 26.
  • PDPs that have improved endosomal escape compared to the phage from which they are derived.
  • PDPs that display an endosomal escape moiety endowing them improved endosomal escape.
  • These PDPs can be used to deliver internal nucleic acid payloads and/or external conjugated moieties (e.g., conjugated through a labile/non labile linker or directly) into cells and are thus useful as delivery vessels for the treatment and/or prevention of a disease.
  • the PDPs comprise a phage coat protein displaying an endosomal escape moiety.
  • PDPs disclosed herein may display an endosomal escape moiety that facilitates endosomal escape of the PDP in a mammalian cell.
  • the term displaying a “endosomal escape moiety” as used herein encompasses endosomal escape moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both.
  • the endosomal escape moiety and its manner of expression and/or linkage is designed to facilitate endosomal escape of the PDP in a cell..
  • PDP is generated using a helper plasmid encoding a coat protein (e.g., a 10A and/or 10B coat protein) modified to display an endosomal escape moiety.
  • the genetic modification may result in the display of an endosomal escape moiety on the PDP coat.
  • the genetic modification is in the form of an endosomal escape moiety-coding DNA sequence fused to a gene coding for a coat protein of the PDP (e.g., a 10A and/or 10B coat protein).
  • endosomal escape moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs.
  • Endosomal escape moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
  • An isolated nucleic acid sequence encoding an endosomal escape moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof.
  • a nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • PCR polymerase chain reaction
  • Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional endosomal escape moiety of the present invention.
  • a nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
  • An endosomal escape moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
  • the endosomal escape moiety may be any biological or synthetic substance that facilitates endosomal escape of the PDP.
  • endosomal escape moieties may be antibody -based moieties, including, but not limited to: monoclonal antibodies, polyclonal antibodies, and antibody fragments such as recombinant antibody fragments, single-chain antibodies (scFv), single antibody variable domains, and the like (Borrebaeck, 1995; Lo, 2003).
  • Single-chain antibodies are small recognition units consisting of the variable regions of the immunoglobulin heavy (VH) and light (VL) chains which are connected by a synthetic linker sequence.
  • Single antibody domain proteins (dAbs) are minimized antibody fragments comprising either an individual VL domain or an individual VH domain.
  • Antibodies may be generated via any one of several known methods, which may employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries, or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique.
  • Antibody fragments may be obtained using methods well known in the art, including, but not limited to by proteolytic hydrolysis of the antibody or by expression in E.
  • coli or mammalian cells e.g., Chinese hamster ovary (CHO) cell culture or other protein expression systems
  • DNA encoding the fragment (Fab')2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent e.g., a thiol reducing agent
  • optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages e.g., a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • An Fv is composed of paired heavy chain variable and light chain variable domains. This association may be non- covalent.
  • the variable domains may be linked to generate a single-chain Fv by an intermolecular disulfide bond, or alternately such chains may be cross-linked by chemicals such as glutaraldehyde.
  • the Fv is a singlechain Fv.
  • Single-chain Fvs are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker.
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains.
  • techniques may be utilized to change a “murine” antibody to a “human” antibody, without altering the binding specificity of the antibody.
  • the endosomal escape moiety may be a peptide that facilitates PDP endosomal escape in a mammalian cells. Methods for constructing libraries and using them for screening for ligands having a particular functional property are known in the art.
  • the endosomal escape moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to facilitate endosomal escape of a PDP in a mammalian cell.
  • Aptamers are another class of binding agent that can be used as an endosomal escape moiety.
  • Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g., membrane lipid rafts).
  • Aptamers that specifically bind a marker of the cell e.g., a cell surface moiety or receptor
  • Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No.
  • the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics.
  • aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups e.g., 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
  • the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an endosomal escape moiety.
  • the endosomal escape moiety is covalently attached to the phage coat protein displaying the endosomal escape moiety.
  • the endosomal escape moiety is non-covalently attached to the phage coat protein displaying the endosomal escape moiety.
  • the endosomal escape moiety is a fusion protein comprising the phage coat protein and the endosomal escape moiety.
  • the endosomal escape moiety is an endosomal escape peptide.
  • endosomal escape peptides include, H5WYG peptides, INF7 peptides, and PCI peptides.
  • the endosomal escape moiety is selected from an endosomal-escape peptide (EEP), transbody, cationic polymer, sugar, lipid, inorganic small molecule, mammalian viral particle (e.g. AAV), and nucleotides.
  • EEP endosomal-escape peptide
  • transbody cationic polymer
  • sugar lipid
  • inorganic small molecule e.g. AAV
  • nucleotides e.g. AAV
  • the endosomal escape moiety can act via any mechanism. In some embodiments, the endosomal escape moiety acts via proton sponge and/or osmotic disruption. In certain embodiments, the endosomal escape moiety acts via compartment membrane disruption. In some embodiments, the endosomal escape moiety acts via membrane pore formation.
  • the endosomal escape moiety is an endosomal escape peptide (EEP).
  • EEPs are known for their potential to promote escape of vectors from endosomes by inducing disruption of endosomes (endosmolytic peptides) or by fusion with the endosomal membranes (fusogenic peptides). Accordingly, in one embodiment, the EEP is an endosmolytic peptide. In another embodiment, the EEP is a fusogenic peptide.
  • EEPs may include the H5WYG peptide, the INF7 peptide, and the PCI peptide, or functional fragments or variants thereof.
  • the endosomal escape of the PDP is improved compared to the endosomal escape of the phage from which the PDP was derived.
  • endosomal escape of the PDP is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more efficient than endosomal escape of the phage type from which the PDP is derived.
  • endosomal escape can be measured by any means known in the art.
  • endosomal escape is measured via flow cytometry, western blotting, immunofluore scent staining, density gradient separation of cellular compartments, or ELISA.
  • the endosomal escape moiety is selected from a moiety listed in Table 27.
  • the rate of transport of the bacteriophage of the invention to the nucleus may also represent another rate-limiting step to transgene expression.
  • PDPs that have improved nuclear shuttling compared to the phage from which they are derived.
  • PDPs that display a nuclear localization moiety endowing them improved nuclear shuttling. These PDPs can be used to deliver internal nucleic acid payloads into the nuclei of cells and are thus useful as delivery vessels for the treatment of a disease.
  • the PDPs comprise a phage coat protein displaying a nuclear localization moiety.
  • PDPs disclosed herein may display a nuclear localization moiety that facilitates nuclear shuttling of the PDP in a mammalian cell.
  • the term displaying a “nuclear localization moiety” as used herein encompasses nuclear localization moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both.
  • the nuclear localization moiety and its manner of expression and/or linkage is designed to facilitate nuclear shuttling of the PDP into the nucleus of a cell.
  • PDP is generated using a helper plasmid encoding a coat protein (e.g., a 10A and/or 10B coat protein) modified to display an nuclear localization moiety.
  • the genetic modification may result in the display of a nuclear localization moiety on the PDP coat.
  • the genetic modification is in the form of an nuclear localization moiety-coding DNA sequence fused to a gene coding for a coat protein of the PDP (e.g., a 10A and/or 10B coat protein).
  • the nuclear localization moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs.
  • Nuclear localization moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
  • An isolated nucleic acid sequence encoding a nuclear localization moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof.
  • a nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • PCR polymerase chain reaction
  • Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional nuclear localization moiety of the present invention.
  • a nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
  • a nuclear localization moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
  • the nuclear localization moiety may be a peptide that facilitates PDP nuclear shuttling in a mammalian cells. Methods for constructing libraries and using them for screening for ligands having a particular functional property are known in the art.
  • the nuclear localization moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to facilitate nuclear shuttling of a PDP in a mammalian cell.
  • Aptamers are another class of binding agent that can be used as an nuclear localization moiety.
  • Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g., membrane lipid rafts).
  • Aptamers that specifically bind a marker of the cell e.g., a cell surface moiety or receptor
  • Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No.
  • the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics.
  • aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups (e.g., 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
  • the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an nuclear localization moiety.
  • the nuclear localization moiety is covalently attached to the phage coat protein displaying the nuclear localization moiety.
  • the nuclear localization moiety is non-covalently attached to the phage coat protein displaying the nuclear localization moiety.
  • the nuclear localization moiety is a fusion protein comprising the phage coat protein and the nuclear localization moiety.
  • the nuclear localization moiety is a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • examples of NLS include, but are not limited to, SV40 T antigen, an optimized SV40 NLS, an optimized short M9 (osM9), a c-Myc NLS, a nucleoplasmin NLS, or a heptamer NLS peptide.
  • the nuclear localization moiety is selected from a nuclear localization signal peptide (NLS), transbody, cationic polymer, sugar, lipid, inorganic small molecule, mammalian viral particle (e.g. AAV), and nucleotides.
  • the nuclear localization moiety can act via any mechanism.
  • the nuclear localization moiety acts via direct transport (nuclear pore entry) of the PDP into the nucleus.
  • the nuclear localization moiety acts via indirect transport (nuclear membrane translocation) of the PDP into the nucleus.
  • the PDP has improved nuclear localization in mammalian cells compared to the nuclear localization of the phage from which the PDP was derived.
  • nuclear localization of the PDP is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more more efficient than nuclear localization of the phage type from which the PDP is derived.
  • nuclear localization can be measured by any means known in the art.
  • nuclear localization is measured via flow cytometry, western blotting, immunofluorescent staining, density gradient separation of cellular compartments, or ELISA.
  • the nuclear localization moiety is selected from a moiety listed in Table 28. Table 28. Exemplary Nuclear Localization Moieties.
  • PDPs that have immune evasion compared to the phage from which they are derived. These PDPs can be used to deliver internal nucleic acid payloads to cells in a subject while eliciting a reduced immune response (or without eliciting an immune response) in the subject and are thus useful as delivery vessels for the treatment and/or prevention of a disease.
  • the PDP comprises a phage coat protein that is modified to enhance immune evasion of the PDP (e.g., a modified 10A and/or 10B coat protein).
  • the phage coat protein is modified such that the PDP avoids neutralizing antibodies and/or immune cell uptake.
  • the modified phage coat protein is modified to reduce antibody epitope recognition, to reduce T cell epitope recognition, and/or to reduce surface charge.
  • the modified phage coat protein is modified to display an immune evasion moiety.
  • the term displaying a “immune evasion moiety” as used herein encompasses immune evasion moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both.
  • the immune evasion moiety and its manner of expression and/or linkage is designed to reduce or eliminate the immune response elicited by the PDP when it is administered to a subject.
  • PDP is generated using a helper plasmid encoding a coat protein (e.g., a 10A and/or 10B coat protein) modified to display an immune evasion moiety.
  • the genetic modification may result in the display of a immune evasion moiety on the PDP coat.
  • the genetic modification is in the form of an immune evasion moiety-coding DNA sequence fused to a gene coding for a coat protein of the PDP (e.g., a 10A and/or 10B coat protein).
  • the immune evasion moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs.
  • Immune evasion moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
  • An isolated nucleic acid sequence encoding a immune evasion moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof.
  • a nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • PCR polymerase chain reaction
  • Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional immune evasion moiety of the present invention.
  • a nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
  • An immune evasion moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
  • the immune evasion moiety may be any biological or synthetic substance that reduces the immune response generated by the PDP when it is administered to a subject.
  • the endosomal escape moiety may be a peptide that inhibits an immune response by a mammalian subject. Methods for constructing libraries and using them for screening for ligands having a particular functional property are known in the art.
  • the immune evasion moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to inhibit the immune response elicited by a PDP when administered to a subject.
  • Aptamers are another class of binding agent that can be used as an immune evasion moiety.
  • Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g., membrane lipid rafts).
  • Aptamers that specifically bind a marker of the cell are useful in the methods of the invention.
  • Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No. 5,660,985; No. 5,567,588; No. 5,683,867; No. 5,637,459; and No. 6,011,020.
  • the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics.
  • aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups (e.g., 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
  • the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an immune evasion moiety.
  • the immune evasion moiety is covalently attached to the phage coat protein displaying the immune evasion moiety.
  • the immune evasion moiety is non-covalently attached to the phage coat protein displaying the immune evasion moiety.
  • the immune evasion moiety is a fusion protein comprising the phage coat protein and the immune evasion moiety.
  • the immune evasion moiety is an antiinflammatory signal.
  • anti-inflammatory signals include, but are not limited to IL 10 and other “anti-inflammatory” cytokines
  • the immune evasion moiety is a mammalian selfprotein signal.
  • mammalian self-protein signals include, but are not limited to, CD47, NKR-P1B- R, PD-1, FcyRIIB, CD22, and Siglec-G.
  • the immune evasion moiety is a capsid masking moiety.
  • capsid masking moieties include, but are not limited to, polymers (e.g. PEG), sugars (e.g. glycoproteins), lipids, and protein corona (e.g. serum proteins).
  • the PDP provided herein elicits a reduced immune response when administered to a subject as compared to the immune response that occurs when a phage from which the PDP was derived is administered to a subject.
  • the reduced immune response includes reduced production of anti-PDP antibodies, reduced B cell activation, reduced B cell proliferation, reduced T cell activation, reduced T cell proliferation, reduced expression of inflammatory cytokines (e.g., IFNy, TNFa, IL12, etc.).
  • the immune response is measured via flow cytometry, western blotting, immunofluorescent staining, density gradient separation of cellular compartments, ELISA, multiplexed cytokine measurement (e.g. Luminex), or other published assay.
  • the phage coat protein modification to enhance immune evasion is selected from the modifications listed in Table 29.
  • the PDPs provided herein contain phage coat proteins that are modified to improve pharmacokinetic and/or pharmacodynamics properties of the PDP.
  • the one or more pharmacokinetic parameters that are improved include, but are not limited to, area under the plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), agent half-life (t’ ), and volume of distribution at steady state (Vss).
  • the PDP comprises a phage coat protein that is modified to extend circulation half-life of the PDP.
  • the PDP comprises a phage coat protein that is modified to increase stability of the PDP in circulation.
  • the PDP comprises a phage coat protein that is modified to reduce degradation of the PDP. In some embodiments, the PDP comprises a phage coat protein that is modified to reduce clearance of the PDP. In some embodiments, the PDP comprises a phage coat protein that is modified to kidney localization of the PDP. In some embodiments, the PDP comprises a phage coat protein that is modified to reduce off-target binding of the PDP. In some embodiments, the PDP comprises the modified phage coat protein is a 10A and/or 10B coat protein and/or a derivative or fragment thereof.
  • the modified phage coat protein is modified to display pharmacokinetics or pharmacodynamics enhancing moiety.
  • the pharmacokinetics or pharmacodynamics moiety and its manner of expression and/or linkage is designed to facilitate improved pharmacokinetics or pharmacodynamics of the PDP when administered to a subject.
  • PDP is generated using a helper plasmid encoding a coat protein (e.g., a 10A and/or 10B coat protein) modified to display an pharmacokinetics or pharmacodynamics moiety.
  • the genetic modification may result in the display of a pharmacokinetics or pharmacodynamics moiety on the PDP coat.
  • the genetic modification is in the form of an pharmacokinetics or pharmacodynamics moiety-coding DNA sequence fused to a gene coding for a coat protein of the PDP (e.g., a 10A and/or 10B coat protein).
  • the pharmacokinetics or pharmacodynamics moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs.
  • Pharmacokinetics or pharmacodynamics moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
  • the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an pharmacokinetics or pharmacodynamics enhancing moiety.
  • a phage coat protein e.g., a 10A and/or 10B coat protein
  • the pharmacokinetics or pharmacodynamics enhancing moiety is covalently attached to the phage coat protein displaying pharmacokinetics or pharmacodynamics enhancing moiety.
  • the pharmacokinetics or pharmacodynamics enhancing moiety is non- covalently attached to the phage coat protein displaying the pharmacokinetics or pharmacodynamics enhancing moiety.
  • the pharmacokinetics or pharmacodynamics enhancing moiety is a fusion protein comprising the phage coat protein and the pharmacokinetics or pharmacodynamics enhancing moiety.
  • the pharmacokinetics or pharmacodynamics enhancing moiety is selected from a peptide sequence, polymer, sugar, lipid, inorganic small molecule, and nucleotides.
  • the phage coat protein modification that enhances the pharmacokinetics or pharmacodynamics of the PDP is selected from the modifications listed in Table 30.
  • Apps of the present disclosure encompass but are not limited to methods and compositions related to expression of an exogenous nucleic acid in a cell that is delivered by a PDP described herein (i.e., as part of the PDP’s nucleic acid payload).
  • the exogenous nucleic acid delivered by the PDP is configured for stable integration in the genome of a cell.
  • the stable integration of the exogenous nucleic acid may be at specific targets within the genome of the cell.
  • the exogenous nucleic acid comprises one or more coding sequences.
  • An exogenous nucleic acid can refer to a nucleic acid that was not originally in a cell and is added from outside the cell, irrespective of whether it comprises a sequence that may already be present in the cell endogenously.
  • An exogenous nucleic acid may be a DNA or an RNA molecule or a hybrid thereof.
  • An exogenous nucleic acid may comprise a sequence encoding a transgene.
  • An exogenous nucleic acid may encode a recombinant protein.
  • provided herein are methods and compositions for delivery inside a cell, including stable incorporation of one or more nucleic acids, comprising nucleic acid sequences encoding one or more proteins.
  • a method of delivering a composition inside a cell the composition comprising one or more nucleic acid sequences encoding one or more proteins (e.g., one or more therapeutic proteins described herein).
  • the nucleic acid payloads of the PDPs provided herein comprise nucleic acid sequences encoding one or more proteins for expression in a cell (e.g., a therapeutic protein disclosed herein).
  • the nucleic acid sequence is designed for stable expression of the one or more proteins or polypeptides encoded by the recombinant nucleic acid.
  • the stable expression is achieved by incorporation of the nucleic acid sequence within the genome of the cell.
  • the nucleic acid payload of a PDP provided herein encodes one or more components of a gene editing system.
  • Gene editing systems allow for the targeted modification of a cell’s genome by inserting, deleting and/or modifying sequences within the genome.
  • the gene editing system is a CRISPR/Cas system.
  • CRISPR/Sy stems include at least a Cas nuclease (or nucleic acid encoding a Cas nuclease) and a guide RNA (gRNA) that targets the Cas nuclease to a particular sequence in a cell genome.
  • the Cas nuclease is a Cas9 nuclease.
  • a CRISPR/Cas system can also include a template sequence to be inserted into the cell genome at the position targeted by the gRNA.
  • CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids).
  • CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids.
  • CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).
  • crRNA CRISPR RNA
  • type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein.
  • tracrRNA serves as a guide for ribonuclease 3 -aided processing of pre- crRNA.
  • the Cas9/crRNA/tracrRNA complex endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer.
  • the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically.
  • DNA-binding and cleavage typically requires protein and both RNAs.
  • single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species (see, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.
  • Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.
  • Cas9 nuclease sequences and structures are well known to those of ordinary skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al.. J.
  • Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes S. thermophiles, Geobaciullus stearothermophilus, Corynebacterium ulcerous, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquisl, Listeria innocua, Campylobacter jejuni, or Neisseria meningitidis.
  • the Cas9 ortholog is an saCas9 domain, an spCas9 domain comprising one or more mutations to alter the PAM specificity, or a Cpfl domain.
  • Additional suitable Cas9 nucleases and sequences will be apparent to those of ordinary skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
  • a Cas9 nuclease comprises one or more mutations that inactive the DNA cleavage domain, that is, the Cas9 is a nickase and/or a nuclease-inactivated Cas9 protein.
  • proteins comprising fragments of Cas9 are encoded by the PDP nucleic acid payload.
  • a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9.
  • proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.”
  • a Cas9 variant shares homology to Cas9, or a fragment thereof.
  • a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9.
  • the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9.
  • the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA- cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9.
  • a fragment of Cas9 e.g., a gRNA binding domain or a DNA- cleavage domain
  • the fragment is 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%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.
  • the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
  • Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1. In some embodiments, Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2, (nucleotide); and Uniprot Reference Sequence: Q99ZW2.
  • Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC 017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP 820832.1), Geobacillus stearothermophilus (NCBI Ref: NZ_CP008934.1);
  • Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof.
  • a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all.
  • Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC 017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1
  • nucleic acid programmable DNA binding protein refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid (e.g, gRNA), that guides the napDNAbp to a specific nucleic acid sequence, for example, by hybridizing to the target nucleic acid sequence.
  • a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence is has complementary to the guide RNA.
  • the napDNAbp is a class 2 microbial CRISPR-Cas effector.
  • the napDNAbp is a Cas9 domain, for example, a nuclease active Cas9, a Cas9 nickase (Cas9n), or a nuclease inactive Cas9 (dCas9).
  • nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g, dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, and Argonaute. It should be appreciated, however, that nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind RNA.
  • the napDNAbp may be associated with a nucleic acid that guides the napDNAbp to an RNA.
  • Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they may not be specifically described in this disclosure.
  • nucleic acid programmable DNA binding protein refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence, for example, by hybridizing to the target nucleic acid sequence.
  • a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence is has complementary to the guide RNA.
  • the napDNAbp is a class 2 microbial CRISPR-Cas effector.
  • the napDNAbp is a Cas9 domain, for example, a nuclease active Cas9, a Cas9 nickase (Cas9n), or a nuclease inactive Cas9 (dCas9).
  • nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, and Argonaute. It should be appreciated, however, that nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind RNA.
  • the napDNAbp may be associated with a nucleic acid that guides the napDNAbp to an RNA.
  • Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they may not be specifically described in this disclosure.
  • the napDNAbp is an “RNA-programmable nuclease” or “RNA-guided nuclease.”
  • RNA-programmable nuclease or “RNA-guided nuclease.”
  • the terms are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA(s) that is not a target for cleavage.
  • an RNA-programmable nuclease when in a complex with an RNA, may be referred to as a nuclease:RNA complex.
  • the bound RNA(s) is referred to as a guide RNA (gRNA).
  • Guide RNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.
  • Guide RNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is also used to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules.
  • sgRNAs single-guide RNAs
  • gRNAs that exist as a single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (z.e., directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein.
  • domain (2) corresponds to a sequence known as a tracrRNA and comprises a stem-loop structure.
  • domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al.. Science 337:816-821 (2012), the entire contents of which is incorporated herein by reference.
  • Other examples of gRNAs e.g., those including domain 2 can be found in International Patent Application PCT/US2014/054252, filed September 5, 2014, published as Patent Publication No. WO2015/035139, published March 12, 2015, entitled “Switchable Cas9 Nucleases And Uses Thereof,” and International Patent Application PCT/US2014/054247, filed September 5, 2014, published as Patent Publication No.
  • a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.”
  • an extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein.
  • the gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex.
  • the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (also known as Csnl) from Streptococcus pyogenes (see, c.g, “Complete genome sequence of an Ml strain of Streptococcus pyogenes”' Ferretti J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc.
  • Cas9 also known as Csnl
  • RNA-programmable nucleases e.g., Cas9
  • Cas9 RNA-programmable nucleases
  • Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong et al., Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W.Y.
  • the PDPs disclosed herein comprise a nucleic acid payload encoding a gene editing system comprising one or more base editors comprising one or more nucleic acid effector domains.
  • base editor refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA).
  • a base e.g., A, T, C, G, or U
  • a nucleic acid sequence e.g., DNA or RNA.
  • the base editor is capable of deaminating a base within a nucleic acid.
  • the base editor is capable of deaminating a base within a DNA molecule.
  • the base editor is capable of deaminating a cytosine (C) in DNA.
  • the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein fused to a nucleic acid editing domain. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a Cas9 domain (e.g., dCas9 or Cas9n), CasX, CasY, Cpfl, C2cl, C2c2, C2c3, or Argonaute protein fused to a cytidine deaminase.
  • Cas9 domain e.g., dCas9 or Cas9n
  • the base editor comprises a Cas9 nickase (Cas9n) fused to an cytidine deaminase domain.
  • the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to a cytidine deaminase domain.
  • the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain, or a dISN domain.
  • the base editor comprises a CasX protein fused to a cytidine deaminase domain.
  • the base editor comprises a CasY protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a Cpfl protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a C2cl protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a C2c2 protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a C2c3 protein fused to a cytidine deaminase domain.
  • the base editor comprises an Argonaute protein fused to a cytidine deaminase domain.
  • Base editors have been described, e.g., in Patent Publication No. W02017/070632, published April 27, 2017, entitled “Nucleobase Editors and Uses Thereof’, in Patent Publication No. W02018/027078, published February 8, 2018, entitled “Adenosine Base Editors and Uses Thereof’, in Patent Publication No. WO2018/165629, published September 13, 2018, entitled “Cytosine to Guanine Base Editor”, and in Patent Publication No. W02018/176009, published September 27, 2018, entitled “Nucleobase Editors Comprising Nucleic Acid Progrrammable DNA Binding Proteins”; the entire contents of each of which are incorporated by reference herein.
  • deaminase or “deaminase domain,” as used herein, refers to a protein or enzyme that catalyzes a deamination reaction.
  • the deaminase or deaminase domain is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.
  • the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism, that does not occur in nature.
  • the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism.
  • the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively.
  • the deaminase or deaminase domain is a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil.
  • the cytidine deaminase catalyzes the hydrolytic deamination of cytidine or cytosine in deoxyribonucleic acid (DNA).
  • the cytidine deaminase or cytidine deaminase domain is a naturally- occurring cytidine deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the cytidine deaminase or cytidine deaminase domain is a variant of a naturally-occurring cytidine deaminase from an organism that does not occur in nature.
  • the cytidine deaminase or cytidine deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring cytidine deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.
  • the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine.
  • the deaminase or deaminase domain is an adenosine deaminase, catalyzing the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively.
  • the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA).
  • the adenosine deaminases may be from any organism, such as a bacterium.
  • the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism.
  • the deaminase or deaminase domain does not occur in nature.
  • the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase.
  • the adenosine deaminase is from a bacterium, such as E.coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus.
  • the adenosine deaminase is a TadA deaminase.
  • the TadA deaminase is an E. coli TadA deaminase (ecTadA).
  • the TadA deaminase is a truncated E. coli TadA deaminase.
  • the truncated ecTadA may be missing one or more N-terminal amino acids relative to a full-length ecTadA.
  • the truncated ecTadA may be missing 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine.
  • effector domains may be used in place of any of the deaminases or deaminase domains provided herein.
  • an “effector domain” refers to a molecule (e.g., a protein) that regulates a biological activity and/or is capable of modifying a biological molecule (e.g., a protein, or a nucleic acid such as DNA or RNA).
  • the effector domain is a protein.
  • the effector domain is capable of modifying a protein (e.g., a histone).
  • the effector domain is capable of modifying DNA (e.g., genomic DNA).
  • the effector domain is capable of modifying RNA (e.g., mRNA).
  • the effector molecule is a nucleic acid editing domain.
  • the effector molecule is capable of regulating an activity of a nucleic acid (e.g., transcription, and/or translation).
  • Exemplary effector domains include, without limitation, a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.
  • the base editor is capable of deaminating an adenosine (A) in DNA.
  • the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein fused to a nucleic acid editing domain.
  • the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase domain.
  • the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to one or more adenosine deaminase domains.
  • the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to two adenosine deaminase domains.
  • the base editor comprises a Cas9 (e.g., dCas9 and Cas9n), CasX, CasY, Cpfl, C2cl, C2c2, C2c3, or Argonaute protein fused to an adenosine deaminase domain.
  • the base editor comprises a Cas9 nickase (Cas9n) fused to an adenosine deaminase domain.
  • the base editor comprises a Cas9 nickase (Cas9n) fused to two adenosine deaminase domains.
  • the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase domain.
  • the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to two adenosine deaminase domains.
  • the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain, or a dISN domain.
  • the base editor comprises a CasX protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a CasY protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a Cpfl protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a C2cl protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a C2c2 protein fused to one or more adenosine deaminase domains.
  • the base editor comprises a C2c3 protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises an Argonaute protein fused to one or more adenosine deaminase domains.
  • the nucleic acid effector domain may be any protein, enzyme, or polypeptide (or functional fragment thereof) which is capable of modifying a DNA or RNA molecule.
  • Nucleobase modification moieties can be naturally occurring, or can be recombinant.
  • a nucleobase modification moiety can include one or more DNA repair enzymes, for example, and an enzyme or protein involved in base excision repair (BER), nucleotide excision repair (NER), homology-dependnent recombinational repair (HR), non-homologous end-joining repair (NHEJ), microhomology end-joining repair (MMEJ), mismatch repair (MMR), direct reversal repair, or other known DNA repair pathway.
  • BER base excision repair
  • NER nucleotide excision repair
  • HR homology-dependnent recombinational repair
  • NHEJ non-homologous end-joining repair
  • MMEJ microhomology end-joining repair
  • MMR mismatch repair
  • a nucleobase modification moiety can have one or more types of enzymatic activities, including, but not limited to endonuclease activity, polymerase activity, ligase activity, replication activity, proofreading activity.
  • Nucleobase modification moieties can also include DNA or RNA-modifying enzymes and/or mutagenic enzymes, such as, DNA methylases and deaminating enzymes (i.e., deaminases, including cytidine deaminases and adenosine deaminases, all defined above), which deaminate nucleobases leading in some cases to mutagenic corrections by way of normal cellular DNA repair and replication processes.
  • nucleic acid effector domain (c.g, a DNA effector domain or an RNA effector domain) as used herein may also refer to a protein or enzyme capable of making one or more modifications (e.g., deamination of a cytidine residue) to a nucleic acid (e.g., DNA or RNA).
  • nucleic acid editing domains include, but are not limited to a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.
  • the nucleic acid editing domain is a deaminase (e.g., an adenosie deaminase and/or a cytidine deaminase).
  • the PDP comprises a nucleic acid sequence encoding a fusion protein comprising a nucleic acid binding domain and a deaminase domain.
  • adenosine deaminase e.g, an adenosine deaminase that deaminates deoxyadenosine in DNA as described herein
  • a domain e.g., a Cas9 or a Cpfl protein
  • the deamination of an adenosine by an adenosine deaminase can lead to a point mutation, thereby editing the nucleic acid.
  • the adenosine may be converted to an inosine residue, which typically base pairs with a cytosine residue.
  • Such fusion proteins are useful for targeted editing of nucleic acid sequences.
  • Such fusion proteins may be used for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re-introduced into the same or another subject; and for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a subject.
  • the nucleobase editors provided herein can be made by fusing together one or more protein domains, thereby generating a fusion protein.
  • the fusion proteins provided herein comprise one or more features that improve the base editing activity (e.g, efficiency, selectivity, and specificity) of the fusion proteins.
  • the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity.
  • the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9).
  • dCas9 nuclease activity
  • nCas9 Cas9 nickase
  • the presence of the catalytic residue e.g., H840 maintains the activity of the Cas9 to cleave the nonedited (e.g., non-deaminated) strand containing a T opposite the targeted A.
  • Mutation of the catalytic residue (e.g., DIO to A10) of Cas9 prevents cleavage of the edited strand containing the targeted A residue.
  • Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a T to C change on the non-edited strand.
  • adenosine deaminases Some aspects of the disclosure provide adenosine deaminases. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA.
  • the adenosine deaminase may be derived from any suitable organism (e.g., E. coli).
  • the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA).
  • mutations in ecTadA e.g., mutations in ecTadA.
  • One of skill in the art will be able to identify the corresponding residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues.
  • adenosine deaminase e.g., having homology to ecTadA
  • the adenosine deaminase is from a prokaryote.
  • the adenosine deaminase is from a bacterium.
  • the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.
  • the fusion protein comprises a Cas9 nickase fused to a deaminase, e.g., a cytidine deaminase (rAPOBECl) which converts a DNA base cytosine to uracil.
  • a deaminase e.g., a cytidine deaminase (rAPOBECl) which converts a DNA base cytosine to uracil.
  • rAPOBECl cytidine deaminase
  • the fusion protein comprises a nuclease-inactive Cas9 fused to a deaminase and further fused to a UGI domain (uracil DNA glycosylase inhibitor, which prevents the subsequent U:G mismatch from being repaired back to a C:G base pair).
  • UGI domain uracil DNA glycosylase inhibitor, which prevents the subsequent U:G mismatch from being repaired back to a C:G base
  • the catalytic His residue at position 840 in the Cas9 HNH domain of BE2 can be restore (resulting in “BE3” as described in the literature), which nicks only the non-edited strand, simulating newly synthesized DNA and leading to the desired U:A product.
  • the dCas9 is any dCas9 disclosed or described in PCT/US2017/045381 (published as WO 2018/027078), which is incorporated herein by reference in its entirety.
  • NBEs nucleobase editors
  • BEs base editors
  • base editors encompasses any base editor known or described in the art at the time of this filing, but also the improved base editors described herein.
  • the base editors known in the state of the art which may be modified by the methods and strategies described herein to improve editing efficiency include, for example, BE1, BE2, BE3, or BE4.
  • the nucleic acid editing domain comprises a deaminase. In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase. In other embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
  • APOBEC apolipoprotein B mRNA-editing complex
  • the deaminase is an APOBEC 1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4 deaminase.
  • the deaminase is an activation-induced deaminase (AID).
  • the deaminase is a Lamprey CDA1 (pmCDAl) deaminase.
  • the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is from a human. In some embodiments the deaminase is from a rat.
  • nucleic-acid editing domains e.g., deaminases and deaminase domains
  • the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).
  • second protein comprises a nucleic acid editing domain.
  • the nucleic acid editing domain can catalyze a C to U base change.
  • the nucleic acid editing domain is a deaminase domain.
  • the deaminase is a cytidine deaminase or a cytidine deaminase.
  • the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
  • APOBEC apolipoprotein B mRNA-editing complex
  • the deaminase is an APOBEC 1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3 A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase.
  • the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase.
  • AID activation-induced deaminase
  • the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBECl.
  • a nucleic acid programmable DNA binding protein is a Cas9 domain.
  • the Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase.
  • the Cas9 domain is a nuclease active domain.
  • the Cas9 domain may be a Cas9 domain that cuts both strands of a duplexed nucleic acid (e.g., both strands of a duplexed DNA molecule).
  • the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9).
  • the dCas9 domain may bind to a duplexed nucleic acid molecule e.g., via a gRNA molecule) without cleaving either strand of the duplexed nucleic acid molecule Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure.
  • the Cas9 domain is a Cas9 nickase.
  • the Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule e.g., a duplexed DNA molecule).
  • the Cas9 nickase cleaves the target strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired to (complementary to) a gRNA e.g., an sgRNA) that is bound to the Cas9.
  • Cas9 domains that have different PAM specificities.
  • Cas9 proteins such as Cas9 from S. pyogenes (spCas9)
  • spCas9 require a canonical NGG PAM sequence to bind a particular nucleic acid region, where the “N” in “NGG” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the G is guanine. This may limit the ability to edit desired bases within a genome.
  • the base editing fusion proteins provided herein need to be positioned at a precise location, for example, where a target base is within a 4 base region (e.g, a “deamination window”), which is approximately 15 bases upstream of the PAM.
  • a deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base region.
  • any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g, NGG) PAM sequence.
  • Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B.
  • fusion proteins comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an adenosine deaminase.
  • any of the fusion proteins provided herein are base editors.
  • the napDNAbp is a Cas9 domain, a Cpfl domain, a CasX domain, a CasY domain, a C2cl domain, a C2c2 domain, aC2c3 domain, or an Argonaute domain.
  • the napDNAbp is any napDNAbp provided herein.
  • the Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein.
  • any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the adenosine deaminases provided herein.
  • fusion proteins that comprise a nucleic acid programmable DNA binding protein (napDNAbp) and at least two adenosine deaminase domains.
  • adenosine deaminases e.g., in cis or in trans
  • dimerization of adenosine deaminases may improve the ability (e.g., efficiency) of the fusion protein to modify a nucleic acid base, for example to deaminate adenine.
  • any of the fusion proteins may comprise 2, 3, 4 or 5 adenosine deaminase domains.
  • any of the fusion proteins provided herein comprise two adenosine deaminases.
  • any of the fusion proteins provided herein contain only two adenosine deaminases.
  • the adenosine deaminases are the same.
  • the adenosine deaminases are any of the adenosine deaminases provided herein.
  • the adenosine deaminases are different.
  • the first adenosine deaminase is any of the adenosine deaminases provided herein
  • the second adenosine is any of the adenosine deaminases provided herein, but is not identical to the first adenosine deaminase.
  • Some aspects of the disclosure provide methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to correct a point mutation in a target gene.
  • the disclosure provides methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to generate an A to G and/or T to C mutation in a target gene.
  • the disclosure provides method for deaminating an adenosine nucleobase (A) in a target gene, the method comprising contacting the target gene with a base editor and a guide RNA bound to the base editor, where the guide RNA comprises a guide sequence that is complementary to a target nucleic acid sequence in the target gene.
  • the target gene comprises a C to T or G to A mutation.
  • the C to T or G to A mutation in the target gene impairs function of the target protein encoded by the target gene.
  • the C to T or G to A mutation in the target gene impairs function of the target protein encoded by the target gene by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%.
  • deaminating an adenosine (A) nucleobase complementary to the T corrects the C to T or G to A mutation in the target gene.
  • the C to T or G to A mutation in the target gene leads to a Cys (C) to Tyr (Y) mutation in the target protein encoded by the target gene.
  • deaminating the adenosine nucleobase complementary to the T corrects the Cys to Tyr mutation in the target protein.
  • the guide sequence of the gRNA comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleic acids that are 100% complementary to a target nucleic acid sequence of the target gene.
  • the base editor nicks the target sequence that is complementary to the guide sequence.
  • a PDP comprising a nucleic acid sequence encoding a transcription activator- like effector nuclease (TALEN).
  • TALEN transcription activator- like effector nuclease
  • TALE Transcriptional Activator-Like Effector
  • RVD Repeat Variable Diresidue
  • the RVD motif determines binding specificity to a nucleic acid sequence and can be engineered according to methods well known to those of skill in the art to specifically bind a desired DNA sequence.
  • the simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
  • TALEN Transcriptional Activator-Like Element Nuclease
  • TALEN-induced double-strand breaks can result in targeted mammalian gene knockout through non-homologous end joining (NHEJ) or targeted genomic sequence replacement through homology-directed repair (HDR) using an exogenous DNA template.
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • the nuclease domain also sometimes referred to as a nucleic acid cleavage domain is a non-specific cleavage domain, e.g., a FokI nuclease domain.
  • the nuclease domain is monomeric and must dimerize or multimerize in order to cleave a nucleic acid.
  • Homo- or heterodimerization or multimerization of TALEN monomers typically occurs via binding of the monomers to binding sequences that are in sufficiently close proximity to allow dimerization, e.g., to sequences that are proximal to each other on the same nucleic acid molecule (e.g., the same double-stranded nucleic acid molecule).
  • a TALEN is provided herein that comprises a canonical N-terminal domain, a TALE repeat array, a modified C-terminal domain, and a nuclease domain.
  • a TALEN is provided herein that comprises a modified N-terminal domain, a TALE repeat array, a canonical C-terminal domain, and a nuclease domain.
  • a TALEN is provided herein that comprises a modified N-terminal domain, a TALE repeat array, a modified C-terminal domain, and a nuclease domain.
  • the nuclease domain is a FokI nuclease domain.
  • the FokI nuclease domain is a homodimeric FokI domain, or a Fokl-EL, Fokl-KK, Fokl-ELD, or Fokl-KKR domain.
  • the TALEN cleaves the target sequence upon dimerization. In some embodiments, a TALEN provided herein cleaves a target site within an allele that is associated with a disease or disorder. In some embodiments, the TALEN cleaves a target site the cleavage of which results in the treatment or prevention of a disease or disorder.
  • the canonical N-terminal domain and/or the canonical C-terminal domain is modified to replace an amino acid residue that is positively charged at physiological pH with an amino acid residue that is not charged or is negatively charged to arrive at the isolated N-terminal and/or C-terminal domain provided herein.
  • the modification includes the replacement of a positively charged residue with a negatively charged residue.
  • the modification includes the replacement of a positively charged residue with a neutral (uncharged) residue.
  • the modification includes the replacement of a positively charged residue with a residue having no charge or a negative charge.
  • the net charge of the isolated N-terminal domain and/or of the isolated C- terminal domain provided herein is less than or equal to +10, less than or equal to +9, less than or equal to +8, less than or equal to +7, less than or equal to +6, less than or equal to +5, less than or equal to +4, less than or equal to +3, less than or equal to +2, less than or equal to +1, less than or equal to 0, less than or equal to -1, less than or equal to -2, less than or equal to -3, less than or equal to -4, or less than or equal to -5, or less than or equal to -10 at physiological pH.
  • the net charge of the isolated N- terminal domain and/or of the isolated C-terminal domain is between +5 and -5, between +2 and -7, between 0 and -5, between 0 and -10, between -1 and -10, or between -2 and -15 at physiological pH. In some embodiments, the net charge of the isolated N- terminal TALE domain and/or of the isolated C-terminal TALE domain is negative. In some embodiments, an isolated N-terminal TALE domain and an isolated C-terminal TALE domain are provided and the net charge of the isolated N-terminal TALE domain and of the isolated C-terminal TALE domain, together, is negative.
  • the net charge of the isolated N-terminal TALE domain and/or of the isolated C-terminal TALE domain is neutral or slightly positive (e.g., less than +2 or less than +1 at physiological pH).
  • an isolated N-terminal TALE domain and an isolated C-terminal TALE domain are provided, and the net charge of the isolated N-terminal TALE domain and of the isolated C-terminal TALE domain, together, is neutral or slightly positive (e.g., less than +2 or less than +1 at physiological pH).
  • the isolated N-terminal domain and/or the isolated C- terminal domain provided herein comprise(s) an amino acid sequence that differs from the respective canonical domain sequence in that at least one cationic amino acid residue of the canonical domain sequence is replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH.
  • at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 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 cationic amino acid(s) is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH in the isolated N-terminal domain and/or in the isolated C-terminal domain provided.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 cationic amino acid(s) is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH in the isolated N-terminal domain and/or in the isolated C-terminal domain.
  • the cationic amino acid residue is arginine (R), lysine (K), or histidine (H). In some embodiments, the cationic amino acid residue is R or H. In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is glutamine (Q), glycine (G), asparagine (N), threonine (T), serine (S), aspartic acid (D), or glutamic acid (E). In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is Q.
  • At least one lysine or arginine residue is replaced with a glutamine residue in the isolated N-terminal domain and/or in the isolated C-terminal domain.
  • an isolated N-terminal TALE domain is provided that is a truncated version of the canonical N-terminal domain.
  • an isolated C-terminal TALE domain is provided that is a truncated version of the canonical C-terminal domain.
  • the truncated N-terminal domain and/or the truncated C-terminal domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the canonical domain. In some embodiments, the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues.
  • the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 residues.
  • an isolated N-terminal TALE domain and/or an isolated C-terminal domain is provided herein that is/are truncated and comprise(s) one or more amino acid replacement(s).
  • the PDPs provided herein comprise a payload encoding interfering nucleic acid molecules that selectively target a mRNA encoded by a gene whose expression is to be suppressed.
  • Interfering nucleic acids generally include a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence.
  • Interfering RNA molecules include, but are not limited to, antisense molecules, siRNA molecules, single-stranded siRNA molecules, miRNA molecules and shRNA molecules.
  • the interfering nucleic acid molecule is double-stranded RNA.
  • the double-stranded RNA molecule may have a
  • shRNA molecules can contain hairpins derived from microRNA molecules.
  • an RNAi vector can be constructed by cloning the interfering RNA sequence into a pCAG-miR30 construct containing the hairpin from the miR30 miRNA.
  • RNA interference molecules may include DNA residues, as well as RNA residues.
  • the interfering nucleic acid molecule is a siRNA molecule.
  • siRNA molecules should include a region of sufficient homology to the target region, and be of sufficient length in terms of nucleotides, such that the siRNA molecule down-regulate target RNA.
  • ribonucleotide or nucleotide can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.
  • the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.
  • Each strand of an siRNA molecule can be equal to or less than 35, 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the strand is at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. In some embodiments, siRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, such as one or two 3' overhangs, of 2-3 nucleotides.
  • a “small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • shRNAs are about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, or are about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded shRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, or about 18-22, 19-20, or 19-21 base pairs in length).
  • shRNA duplexes may comprise 3’ overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides on the antisense strand and/or 5 ’-phosphate termini on the sense strand.
  • the shRNA comprises a sense strand and/or antisense strand sequence of from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), or from about 19 to about 40 nucleotides in length (e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length), or from about 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).
  • Non-limiting examples of shRNA include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a doublestranded polynucleotide molecule with a hairpin secondary structure having self- complementary sense and antisense regions.
  • the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.
  • miRNAs represent a large group of small RNAs produced naturally in organisms, some of which regulate the expression of target genes. miRNAs are formed from an approximately 70 nucleotide single-stranded hairpin precursor transcript by Dicer. miRNAs are not translated into proteins, but instead bind to specific messenger RNAs, thereby blocking translation. In some instances, miRNAs base-pair imprecisely with their targets to inhibit translation.
  • antisense oligonucleotide compounds are provided herein.
  • the degree of complementarity between the target sequence and antisense targeting sequence is sufficient to form a stable duplex.
  • the region of complementarity of the antisense oligonucleotides with the target RNA sequence may be as short as 8-11 bases, but can be 12-15 bases or more, e.g., 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers in between these ranges.
  • An antisense oligonucleotide of about 14-15 bases is generally long enough to have a unique complementary sequence.
  • antisense oligonucleotides may be 100% complementary to the target sequence, or may include mismatches, e.g., to improve selective targeting of allele containing the disease-associated mutation, as long as a heteroduplex formed between the oligonucleotide and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo.
  • certain oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 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%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence.
  • Oligonucleotide backbones that are less susceptible to cleavage by nucleases are discussed herein.
  • Mismatches are typically less destabilizing toward the end regions of the hybrid duplex than in the middle.
  • the number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • methods of manufacturing a PDP are provided.
  • the assembly of the T7 is now well enough understood to allow for manipulations that have had significant impact on the ease of dsDNA isolation and E. coli infection/transformation of foreign sequences carried by phage.
  • Phagemid systems and helper phage systems have borrowed or altered T7 origins of replication that allow for the packaging of foreign sequences within a phage body.
  • a template phagemid with a T7 origin insert is co-infected with a helper phage that will express all the necessary phage assembly proteins to yield PDP carrying the sequence from the template phagemid.
  • helper phage that will express all the necessary phage assembly proteins to yield PDP carrying the sequence from the template phagemid.
  • the dsDNA sequences of interest may be directly integrated into different areas of the T7 phage genome and packaged into T7 phage particles.
  • the dsDNA sequences of interest may be placed at any site of the T7 genome that does not disrupt any protein coding genes, sequence elements related to genome replication, and/or sequence elements related to T7 packaging.
  • packageable dsDNA are generated with chosen sizes (i.e. 100s to 1000s base-pairs), which are much shorter than the ⁇ 40 kb observed in wildtype T7 phage.
  • These packagable genomes known as phagemids, can be of varying lengths and contain the T7 phage packaging signal, T7 origin of replication, and terminal repeats required for replication, but none of the T7 phage protein genes.
  • the T7 phage particles may preferentially package the phagemid over the wild-type T7 phage genome by sequence manipulation of the T7 packaging signal in the wild-type T7 phage genome.
  • compositions comprising a PDP of the present disclosure are provided herein. Such compositions further comprise a pharmaceutically acceptable carrier and can be supplied a sterile pharmaceutical composition. This composition can be in any suitable form (depending upon the desired method of administering it to a patient).
  • Administration of the pharmaceutical composition may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral, or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • the most suitable route for administration in any given case will depend on the particular PDP, the subject, and the nature and severity of the disease and the physical condition of the subject.
  • compositions can be conveniently presented in unit dose forms containing a predetermined amount of a PDP of the disclosure per dose.
  • a unit can contain for example but without limitation 5 mg to 5 g, for example 10 mg to 1 g, or 20 to 50 mg.
  • Pharmaceutically acceptable carriers for use in the disclosure can take a wide variety of forms depending, e.g., on the condition to be treated or route of administration.
  • Therapeutic formulations of the PDPs of the disclosure can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the PDPs having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are referred to herein as “carriers”), i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be nontoxic to the recipients at the dosages and concentrations employed.
  • Buffering agents help to maintain the pharmaceutical composition’s pH in the range that approximates physiological conditions. They can be present at concentration ranging from about 2 mM to about 50 mM.
  • Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof such as citrate buffers (e.g, monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g, succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid
  • Preservatives can be added to retard microbial growth, and can be added in amounts ranging from 0.2%4% (w/v).
  • Suitable preservatives for use with the present disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3 -pentanol.
  • Isotonicifiers sometimes known as “stabilizers” can be added to ensure isotonicity of liquid compositions of the present disclosure and include polyhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low
  • Non-ionic surfactants or detergents can be added to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein.
  • Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.).
  • Non- ionic surfactants can be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, for example about 0.07 mg/ml to about 0.2 mg/ml.
  • Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.
  • bulking agents e.g., starch
  • chelating agents e.g., EDTA
  • antioxidants e.g., ascorbic acid, methionine, vitamin E
  • cosolvents e.g., ascorbic acid, methionine, vitamin E
  • the formulation herein can also contain a second therapeutic agent in addition to the PDP of the disclosure.
  • the dosing schedule for subcutaneous administration can vary from once a month to daily depending on a number of clinical factors, including the type of disease, severity of disease, and the patient's sensitivity to the PDP.
  • the dosage of a PDP of the disclosure to be administered will vary according to the particular PDP, the type of disease, the subject, and the nature and severity of the disease, the physical condition of the subject, the therapeutic regimen (e.g., whether a second therapeutic agent is used), and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art.

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Abstract

Provided herein are T7 phage derived particles (PDFs) with beneficial properties and methods of use thereof.

Description

ICOSAHEDRAL PHAGE DERIVED PARTICLES
CROSS-REFERENCE TO RELATED APPLICATIONS
[1] This application claims the benefit of U.S. Provisional Application No. 63/441,547, filed on January 27, 2023, the content of which is hereby incorporated by reference in its entirety.
BACKGROUND
[2] A wide range of nucleic acid delivery systems are being developed in an attempt to facilitate the targeted delivery of nucleic acid payloads. For example, certain viral vectors, such as adenoviral vectors, adeno-associated viral vectors (AAVs), and lentiviral vectors, have been used to deliver therapeutic nucleic acid payloads to certain tissues and/or cells.
[3] However, despite their promise, currently available viral vectors have a number of shortcomings. For example, the amount of exogenous nucleic acid that can be packaged into current viral vectors is typically quite limited, severely limiting the size of the nucleic acid payload they can effectively deliver. Moreover, many currently used viral vectors are themselves immunogenic and thus can trigger immune reactions that lessen or eliminate the desired therapeutic results. What is more, even when current viral vectors are designed to specifically recognize certain cells or tissues, off-target events (e.g., delivery of the nucleic payload to an unintended recipient cell) are often common and can lead to complications in treatment and adverse events, up to and including death. Lastly, currently used viral vectors are difficult to manufacture at scale due to production in mammalian systems. This creates a significant barrier for its utility in many disease indications.
[4] Thus, there is a great need for improved systems for delivering therapeutic nucleic acid payloads.
SUMMARY
[5] In certain aspects, provided herein are phage derived particles (PDPs) derived from icosahedral phage (e.g., T7 phage) with improved properties, as well as methods of making and using such PDPs. As used herein, a PDP is a particle in which a non-phage nucleic acid payload is encapsulated by phage-derived coat proteins. Typically, the nucleic acid payload of a PDP substantially lacks the genome from which the PDP is derived (e.g., the nucleic acid payload does not encode the phage-derived coat proteins in which it is encapsulated). In some embodiments, the PDP does not comprise nucleic acid sequences encoding at least 50% (e.g., 60%, 70%, 80%, 90%, 95%) of the genome of the phage from which the PDP is derived. In some embodiments, the PDP does not comprise a nucleic acid sequence encoding a phage coat protein. In certain embodiments, the PDP comprises no more than 2000 bases of the genome of the phage from which the PDP was derived.
[6] In certain embodiments, PDPs provided herein possess advantageous properties compared to existing viral vectors making them particularly useful, for example, in the treatment and/or prevention of one or more diseases. For example, in certain embodiments, the PDPs provided herein exhibit improved: cell or tissue specificity, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics, and/or mammalian expression as compared to existing phage derived vectors.
[7] In certain aspects, the PDPs provided herein comprise phage coat proteins encapsulating a nucleic acid payload, wherein the nucleic acid payload comprises a sequence encoding an agent that treats a disease when delivered to a cell of a subject in need thereof. In some embodiments, the PDPs are derived from a T7 phage (bacteriophage T7). In certain embodiments, the PDPs further comprise a phage coat protein displaying a cell-targeting moiety specific for a cell type. In some embodiments, the phage coat protein displaying the cell-targeting moiety is a 10A, and/or 10B coat (a/k/a capsid) protein and/or a derivative or fragment thereof.
[8] In certain embodiments, the PDPs provided herein are designed to inhibit the expression of a gene (e.g., a disease-associated gene). In certain embodiments, such PDPs would comprise a nucleic acid payload that encodes an inhibitory RNA and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that targets the gene.
[9] In certain embodiments, the PDPs provided herein are designed to enhance the expression of a therapeutic peptide. In certain embodiments, such PDPs would comprise a nucleic acid payload that encodes the therapeutic peptide and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate insertion of a sequence encoding the therapeutic peptide into the genome of a cell (e.g., into a safe harbor locus in the cell, such as a safe harbor locus listed in Table 2). [10] In certain embodiments, the PDPs provided herein are designed to inhibit the expression of a gene (e.g., a disease-associated gene) in a cell and enhance expression of a therapeutic peptide (e.g., a peptide that treats and/or prevents a disease) in a cell. For example, in some embodiments, the gene for which expression is inhibited is a mutant gene associated with a disease, while the therapeutic peptide is the peptide encoded by the wild-type version of that gene. In certain embodiments, such PDPs would comprise a nucleic acid payload that comprises a sequence that encodes an inhibitory RNA and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that targets the gene; and (2) a sequence that encodes the therapeutic peptide and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate insertion of a sequence encoding the therapeutic peptide into the genome of the cell (e.g., into the mutant gene locus and/or a safe harbor locus in the cell, such as a safe harbor locus listed in Table 2).
[11] In certain embodiments, the PDPs provided herein are designed to modify the sequence of a gene in a cell (e.g., to change the sequence of a gene from a disease- associated sequence to a sequence that is not associated with a disease). In certain embodiments, such PDPs would comprise a nucleic acid payload that encodes one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate modification of the gene in the genome of the endothelial cell (e.g., converting mutant version of a gene into a wild-type version of a gene.
[12] In certain embodiments, the PDPs provided herein to provide a double stranded DNA homology directed repair (HDR) donor comprising: a first homology arm region, an insert region, and a second homology arm region. In another aspect, the double stranded DNA HDR donor improves homology directed repair efficiency and reduces homologyindependent integration in a programmable nuclease system. In another aspect, the programmable nuclease system comprises one or more of transcription activator-like effector nucleases (TALENs), zinc fingers (ZFNs), or clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas.
[13] In certain embodiments, the PDPs provided herein comprise moieties that impart certain beneficial properties upon the PDP. For example, in certain embodiments, the PDPs comprise moieties that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived. In certain embodiments, such moi eties are displayed on one or more of the phage coat proteins of the PDP (e.g., a 10A and/or 10B coat protein). In some embodiments, such moi eties are covalently attached to the phage coat protein. In certain embodiments, such moieties are non-covalently attached to the phage coat protein. In some embodiments, the phage coat protein displaying the moiety is a fusion protein comprising the phage coat protein and the moiety.
[14] In certain embodiments, the PDPs comprise modified coat proteins (e.g., modified 10A and/or 10B coat proteins) that impart certain beneficial properties upon the PDP. For example, in certain embodiments, the PDPs comprise modified coat proteins that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived.
[15] In certain embodiments, the PDPs provided herein can accommodate larger nucleic acid payloads than commonly used viral vectors. In certain embodiments, the PDPs provided herein can accommodate at least 300 bases (e.g., at least 325 bases, at least 350 bases, at least 375 bases, at least 400 bases, at least 425 bases, at least 450 bases, at least 475 bases, at least 500 bases, at least 525 bases, at least 550 bases, at least 575 bases, at least 600 bases, at least 625 bases, at least 650 bases, at least 675 bases, at least 700 bases, at least 725 bases, at least 750 bases, at least 775 bases, at least 800 bases at least 825 bases, at least 850 bases, at least 875 bases, at least 900 bases, at least 925 bases, at least 950 bases, at least 975 bases, at least 1000 bases, at least 1025 bases, at least 1050 bases, at least 1075 bases, at least 1100, at least 1125 bases, at least 1150 bases, at least 1175 bases, at least 1200, at least 1225 bases, at least 1250 bases, at least 1275 bases, at least 1300, at least 1325 bases, at least 1350 bases, at least 1375 bases, at least 1400, at least 1425 bases, at least 1450 bases, at least 1475 bases, at least 1500, at least 1600 bases, at least 1700 bases, at least 1800 bases, at least 1900 bases, at least 2000 bases, at least 2500 bases, at least 3000 bases, at least 3500 bases, at least 4000 bases, at least 4500 bases, at least 5000 bases, at least 5500 bases, at least 6000 bases, at least 6500 bases, at least 3000 bases, at least 3500 bases, at least 3000 bases, at least 3500 bases, at least 7000 bases, at least 7500 bases, at least 8000 bases, at least 8500 bases, at least 9000 bases, at least 9500 bases, at least 10000 bases, at least 10500 bases, at least 11000 bases, at least 11500 bases, at least 12000 bases, at least 12500 bases, at least 13000 bases, at least 13500 bases, at least 14000 bases, at least 14500 bases, at least 15000 bases, at least 15500 bases, at least 16000 bases, at least 16500 bases, at least 17000 bases, at least 17500 bases, at least 18000 bases, at least 18500 bases, at least 19000 bases, at least 19500 bases, at least 20000 bases, at least 20500 bases, at least 21000 bases, at least 21500 bases, at least 22000 bases, at least 22500 bases, at least 23000 bases, at least 23500 bases, at least 24000 bases, at least 24500 bases, at least 25000 bases, at least 25500 bases, at least 26000 bases, at least 26500 bases, at least 27000 bases, at least 27500 bases, at least 28000 bases, at least 28500 bases, at least 29000 bases, at least 29500 bases, at least 30000 bases, at least 30500 bases, at least 31000 bases, at least 31500 bases, at least 32000 bases, at least 32500 bases, at least 33000 bases, at least 33500 bases, at least 34000 bases, at least 34500 bases, at least 35000 bases, at least 35500 bases, at least 36000 bases, at least 36500 bases, at least 37000 bases, at least 37500 bases, at least 38000 bases, at least 38500 bases, at least 39000 bases, at least 39500 bases, at least 40000 bases, or more) of non-phage DNA in its payload. In some embodiments, the PDPs provided herein can accommodate a payload of non-phage DNA that makes up at least 30% 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of its genome.
[16] In certain embodiments, the PDPs provided herein comprises a larger nucleic acid payloads than commonly used viral vectors can accommodate. In certain embodiments, the PDPs provided herein comprise at least 300 bases (e.g., at least 325 bases, at least 350 bases, at least 375 bases, at least 400 bases, at least 425 bases, at least 450 bases, at least 475 bases, at least 500 bases, at least 525 bases, at least 550 bases, at least 575 bases, at least 600 bases, at least 625 bases, at least 650 bases, at least 675 bases, at least 700 bases, at least 725 bases, at least 750 bases, at least 775 bases, at least 800 bases at least 825 bases, at least 850 bases, at least 875 bases, at least 900 bases, at least 925 bases, at least 950 bases, at least 975 bases, at least 1000 bases, at least 1025 bases, at least 1050 bases, at least 1075 bases, at least 1100, at least 1125 bases, at least 1150 bases, at least 1175 bases, at least 1200, at least 1225 bases, at least 1250 bases, at least 1275 bases, at least 1300, at least 1325 bases, at least 1350 bases, at least 1375 bases, at least 1400, at least 1425 bases, at least 1450 bases, at least 1475 bases, at least 1500, at least 1600 bases, at least 1700 bases, at least 1800 bases, at least 1900 bases, at least 2000 bases, at least 2500 bases, at least 3000 bases, at least 3500 bases, at least 4000 bases, at least 4500 bases, at least 5000 bases, at least 5500 bases, at least 6000 bases, at least 6500 bases, at least 3000 bases, at least 3500 bases, at least 3000 bases, at least 3500 bases, at least 7000 bases, at least 7500 bases, at least 8000 bases, at least 8500 bases, at least 9000 bases, at least 9500 bases, at least 10000 bases, at least 10500 bases, at least 11000 bases, at least 11500 bases, at least 12000 bases, at least 12500 bases, at least 13000 bases, at least 13500 bases, at least 14000 bases, at least 14500 bases, at least 15000 bases, at least 15500 bases, at least 16000 bases, at least 16500 bases, at least 17000 bases, at least 17500 bases, at least 18000 bases, at least 18500 bases, at least 19000 bases, at least 19500 bases, at least 20000 bases, at least 20500 bases, at least 21000 bases, at least 21500 bases, at least 22000 bases, at least 22500 bases, at least 23000 bases, at least 23500 bases, at least 24000 bases, at least 24500 bases, at least 25000 bases, at least 25500 bases, at least 26000 bases, at least 26500 bases, at least 27000 bases, at least 27500 bases, at least 28000 bases, at least 28500 bases, at least 29000 bases, at least 29500 bases, at least 30000 bases, at least 30500 bases, at least 31000 bases, at least 31500 bases, at least 32000 bases, at least 32500 bases, at least 33000 bases, at least 33500 bases, at least 34000 bases, at least 34500 bases, at least 35000 bases, at least 35500 bases, at least 36000 bases, at least 36500 bases, at least 37000 bases, at least 37500 bases, at least 38000 bases, at least 38500 bases, at least 39000 bases, at least 39500 bases, at least 40000 bases, or more) of non-phage DNA in its payload. In some embodiments, the PDPs provided herein comprise a payload of non-phage DNA that makes up at least 30% 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of its genome. [17] In certain embodiments, the PDPs provided herein comprise no more than 300 bases (e.g., no more than 325 bases, no more than 350 bases, no more than 375 bases, no more than 400 bases, no more than 425 bases, no more than 450 bases, no more than 475 bases, no more than 500 bases, no more than 525 bases, no more than 550 bases, no more than 575 bases, no more than 600 bases, no more than 625 bases, no more than 650 bases, no more than 675 bases, no more than 700 bases, no more than 725 bases, no more than 750 bases, no more than 775 bases, no more than 800 bases no more than 825 bases, no more than 850 bases, no more than 875 bases, no more than 900 bases, no more than 925 bases, no more than 950 bases, no more than 975 bases, no more than 1000 bases, no more than 1025 bases, no more than 1050 bases, no more than 1075 bases, no more than 1100, no more than 1125 bases, no more than 1150 bases, no more than 1175 bases, no more than 1200, no more than 1225 bases, no more than 1250 bases, no more than 1275 bases, no more than 1300, no more than 1325 bases, no more than 1350 bases, no more than 1375 bases, no more than 1400, no more than 1425 bases, no more than 1450 bases, no more than 1475 bases, no more than 1500, no more than 1600 bases, no more than 1700 bases, no more than 1800 bases, no more than 1900 bases, no more than 2000 bases, no more than 2500 bases, no more than 3000 bases, no more than 3500 bases, no more than 4000 bases, no more than 4500 bases, no more than 5000 bases, no more than 5500 bases, no more than 6000 bases, no more than 6500 bases, no more than 3000 bases, no more than 3500 bases, no more than 3000 bases, no more than 3500 bases, no more than 7000 bases, no more than 7500 bases, no more than 8000 bases, no more than 8500 bases, no more than 9000 bases, no more than 9500 bases, no more than 10000 bases, no more than 10500 bases, no more than 11000 bases, no more than 11500 bases, no more than 12000 bases, no more than 12500 bases, no more than 13000 bases, no more than 13500 bases, no more than 14000 bases, no more than 14500 bases, no more than 15000 bases, no more than 15500 bases, no more than 16000 bases, no more than 16500 bases, no more than 17000 bases, no more than 17500 bases, no more than 18000 bases, no more than 18500 bases, no more than 19000 bases, no more than 19500 bases, no more than 20000 bases, no more than 20500 bases, no more than 21000 bases, no more than 21500 bases, no more than 22000 bases, no more than 22500 bases, no more than 23000 bases, no more than 23500 bases, no more than 24000 bases, no more than 24500 bases, no more than 25000 bases, no more than 25500 bases, no more than 26000 bases, no more than 26500 bases, no more than 27000 bases, no more than 27500 bases, no more than 28000 bases, no more than 28500 bases, no more than 29000 bases, no more than 29500 bases, no more than 30000 bases, no more than 30500 bases, no more than 31000 bases, no more than 31500 bases, no more than 32000 bases, no more than 32500 bases, no more than 33000 bases, no more than 33500 bases, no more than 34000 bases, no more than 34500 bases, no more than 35000 bases, no more than 35500 bases, no more than 36000 bases, no more than 36500 bases, no more than 37000 bases, no more than 37500 bases, no more than 38000 bases, no more than 38500 bases, no more than 39000 bases, no more than 39500 bases, no more than 40000 bases, or more) of non-phage DNA in its payload. In some embodiments, the PDPs provided herein comprise a payload of non- phage DNA that makes up no more than 30% 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of its genome.
[18] In certain embodiments, the PDPs provided herein have improved cellular internalization compared to the phage from which the PDP was derived. In certain embodiments, the PDP comprises a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an internalization moiety. In some embodiments, the internalization moiety acts via membrane penetration. In certain embodiments, the internalization moiety acts via membrane endocytosis. In some embodiments, the PDP is internalized by a mechanism selected from macropinocytosis, phagocytosis, clathrin-mediated, caveolin- mediated, interaction of hydrophilic lipid membrane and fusogenic moi eties, interaction with hydrophobic portion of lipid membrane, and hydrophobic cloaking. In some embodiments, the internalization moiety is selected from the moieties listed in Table 26.
[19] In certain embodiments, the PDPs provided herein have improved endosomal escape compared to the phage from which the PDP was derived. In certain embodiments, the PDP comprises a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an endosomal escape moiety. In some embodiments, the endosomal escape moiety is an endosomal escape peptide (e.g., a Tat peptide, a H5WYG peptide, INF7 peptide, or PCI peptide). In certain embodiments, the endosomal escape moiety acts via proton sponge and/or osmotic disruption. In some embodiments, the endosomal escape moiety acts via compartment membrane disruption. In some embodiments, the endosomal escape moiety acts via membrane pore formation. In certain embodiments, the endosomal escape moiety is selected from a moiety listed in Table 27.
[20] In certain embodiments, the PDPs provided herein have improved nuclear shuttling compared to the phage from which the PDP was derived. In certain embodiments, the PDP comprises a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying a nuclear localization moiety. In some embodiments, the nuclear localization moiety is a nuclear localization signal (NLS) (e.g., an NLS peptide from SV40 T antigen, an optimized SV40 NLS, an optimized short M9 (osM9), a c-Myc NLS, a nucleoplasmin NLS, or a heptamer NLS peptide). In certain embodiments, the nuclear localization moiety acts via direct transport (nuclear pore entry) of the PDP into the nucleus. In some embodiments, the nuclear localization moiety acts via indirect transport (nuclear membrane translocation) of the PDP into the nucleus. In some embodiments, the nuclear localization moiety is selected from a moiety listed in Table 28.
[21] In certain embodiments, the PDPs provided herein have improved immune evasion compared to the phage from which the PDP was derived. In certain embodiments, the PDP comprises a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to enhance immune evasion of the PDP. In certain embodiments, the phage coat protein is modified such that the PDP avoids neutralizing antibodies and/or immune cell uptake. In some embodiments, the phage coat protein is modified to reduce antibody epitope recognition, to reduce T cell epitope recognition, and/or to reduce surface charge. In some embodiments, the phage coat protein is modified to display an immune evasion moiety. In some embodiments, the PDP elicits a reduced immune response when administered to a subject as compared to the immune response that occurs when a phage from which the PDP was derived is administered to a subject. In certain embodiments, the phage coat protein modification to enhance immune evasion is selected from the modifications listed in Table 29.
[22] In certain embodiments, the PDPs provided herein have improved pharmacokinetic and/or pharmacodynamic properties compared to the phage from which the PDP was derived. In some embodiments, the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to extend circulation half-life of the PDP. In some embodiments, the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to increase stability of the PDP in circulation. In some embodiments, the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to reduce degradation of the PDP. In some embodiments, the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to reduce clearance of the PDP. In certain embodiments, the PDPs provided herein comprise a phage coat protein (e.g., 10A and/or 10B coat protein) that is modified to kidney localization of the PDP. In some embodiments, the PDPs provided herein comprise a coat protein (e.g., 10A and/or 10B coat protein) modified to display pharmacokinetics or pharmacodynamics enhancing moiety. In some embodiments, the phage coat protein modification is selected from the modifications listed in Table 30.
[23] In some embodiments, the nucleic acid payload is a linear double stranded DNA (dsDNA) construct. In certain embodiments, the dsDNA payload comprises DNA secondary structures that enhance expression in a mammalian system, In some embodiments, the dsDNA construct comprises a DNA sequence element that enhance expression and/or specificity in mammalian cells (e.g., DNA sequence element is selected from the DNA sequence elements listed in Table 1).
[24] In certain aspects, provided herein are systems for producing the PDPs described herein from a prokaryotic host comprising (i) a phage vector comprising a packaging signal for replication of the vector into the nucleic acid payload of the PDP, wherein the nucleic acid payload is a linear double stranded DNA (dsDNA); and (ii) a second vector comprising nucleic acid sequences encoding the phage coat proteins of the PDP. In certain aspects, provided herein is a method of making a PDP provided herein, the method comprising delivering into a prokaryotic cell such a system and culturing the prokaryotic cell under conditions such that it produces the PDP.
DETAILED DESCRIPTION
General
[25] Provided herein are icosahedral phage-derived particles (PDPs) comprising a nucleic acid payload (e.g., transgenes encoding a therapeutic protein, inhibitory nucleic acids, and/or gene editing (e.g., CRISPR/Cas) systems). Notably, in certain embodiments the PDPs provided herein possess advantageous properties compared to existing viral vectors making them particularly useful for the treatment and/or prevention of certain diseases. For example, in certain embodiments, the PDPs provided herein exhibit improved: cell or tissue specificity, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics, and/or mammalian expression as compared to existing phage derived vectors. Also provided herein are therapeutic methods comprising administering one or more of the PDP compositions described to a subject in need thereof, as well as systems and methods of making the PDPs provided herein.
Definitions
[26] As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents.
[27] The term “about” when used before a numerical value indicates that the value may vary within a reasonable range, such as within ± 10%, ± 5% or ± 1% of the stated value.
[28] “Administration” broadly refers to a route of administration of a composition (e.g., a therapeutic composition) to a subject. Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Administration by injection includes intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration. A therapeutic composition described herein can be administered in any form by any effective route, including but not limited to oralf, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intraarterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial. In certain embodiments, a therapeutic composition described herein is administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously. In another embodiment, a therapeutic composition described herein is administered orally or intravenously. In another embodiment, a therapeutic composition described herein is administered intranasally. In another embodiment, a therapeutic composition described herein is administered orally.
[29] As used herein, the term “antibody” may refer to both an intact antibody and an antigen binding fragment thereof. Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), singlechain antibodies and antigen-binding antibody fragments.
[30] The terms “antigen binding fragment” and “antigen-binding portion” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.
[31] The term “binding” or “interacting” refers to an association, which may be a stable association, between two molecules, e.g., between a polypeptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
[32] The terms “CDR”, and its plural “CDRs”, refer to a complementarity determining region (CDR) of an antibody or antibody fragment, which determine the binding character of an antibody or antibody fragment. In most instances, three CDRs are present in a light chain variable region (CDRL1, CDRL2 and CDRL3) and three CDRs are present in a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. Among the various CDRs, the CDR3 sequences, and particularly CDRH3, are the most diverse and therefore have the strongest contribution to antibody specificity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated by reference in its entirety); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), incorporated by reference in its entirety).
[33] As used herein, two nucleic acid sequences “complement” one another or are “complementary” to one another if they base pair one another at each position. Thus, the “reverse complement” of a specific nucleic acid sequence is has a nucleic acid sequence that is able to for a Watson/Crick base pair with the specific nucleic acid sequence.
[34] As used herein, the term “consists essentially of’ (or “consisting essentially of’) means limited to the recited elements and/or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.
[35] The term “effective dose” is the amount of the therapeutic composition that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, with the least toxicity to the subject.
[36] The term “epitope” means a protein determinant capable of specific binding to an antibody or T cell receptor. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.
[37] The term “expression” encompasses the processes by which nucleic acids (e.g., DNA) are transcribed to produce RNA, and (where applicable) RNA transcripts are processed and translated into polypeptides.
[38] The term “gene” as used herein refers to a nucleic acid sequence (e.g., DNA or RNA) that encodes a molecule (e.g., a protein). In general, a gene is a double-stranded DNA molecule that encodes a protein. A gene generally comprises coding DNA sequences (e.g., exons), non-coding DNA sequences (e.g., introns), and one or more promoters or other regulatory element that controls gene expression. An organism’s entire set of genes is referred as its genome.
[39] The terms “heterologous DNA” and “heterologous RNA” refer to nucleotides that are not endogenous (native) to the cell or part of the genome in which they are present. Generally heterologous DNA or RNA is added to a cell by transduction, infection, transfection, transformation or the like, as further described below. Such nucleotides generally include at least one coding sequence, but the coding sequence need not be expressed. The term “heterologous DNA” may refer to a “heterologous coding sequence” or a “transgene”.
[40] The term “host cell”, as used herein refers to a cell which has been transduced, infected, transfected or transformed with a vector. The vector may be a plasmid, a viral particle, a phage, etc. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. It will be appreciated that the term “host cell” refers to the original transduced, infected, transfected or transformed cell and progeny thereof.
[41] As used herein, the terms "hybridize" or "hybridization" refer to the hydrogen bonding of complementary DNA and/or RNA sequences to form a duplex molecule. As used herein, hybridization takes place under conditions that can be adjusted to a level of stringency that reduces or even prevents base-pairing between a first oligonucleotide primer or oligonucleotide probe and a target sequence, if the complementary sequences are mismatched by as little as one base-pair.
[42] “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et aL, Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., etal., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).
[43] The term “locus” (plural “loci”) as used herein refers to a fixed position on a chromosome, such as the position of a gene or marker (z.e., genetic marker). A variant of a similar DNA sequence located at a given locus is called an allele. In some embodiments, the locus is a safe harbor locus. The term “safe harbor locus” refers to a position on the chromosome (z.e., locus) that can tolerate the insertion, deletion, and/or mutation of the nucleic acid sequence in the safe harbor locus without perturbing the endogenous activity of the gene or risking the integrity of the host genome. In some embodiments, a gene is located in a safe harbor locus (z.e., a safe harbor gene). In some embodiments, the gene is the chemokine (C-C motif) receptor 5 (CC ?5) gene. In some embodiments, the gene is the adeno-associated virus site 1 (AAVS1) gene. In some embodiments, the safe harbor locus is the Rosa26 locus (e.g., from mice, or the human ortholog). Safe harbor genes are described in, e.g., Papapetrou EP and Schambach A (2016) Gene insertion into genomic safe harbors for human gene therapy. Mol Ther 24(4), 678-684, which is incorporated by reference herein. In some embodiments, a safe harbor locus is used as a recording locus as described herein.
[44] The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the nucleic acid and amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). [45] The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, sRNAi, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides.
[46] Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine); nucleoside analogs (e.g, 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-A-phosphoramidite linkages).
[47] The term “operably linked” as used herein relative to a recombinant DNA construct or vector means nucleotide components of the recombinant DNA construct or vector are functionally related to one another for operative control of a selected coding sequence. Generally, “operably linked” DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous.
[48] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N- terminal) portion of the fusion protein or at the carboxy -terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid programmable DNA binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
[49] The term “promoter” as used herein refers to a control region of a nucleic acid sequence (e.g., within a plasmid) at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain subregions to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. In some embodiments, a promoter controls the expression of a nucleic acid sequence (e.g., a gene) that is operably linked to the promoter. A promoter is located on the same strand and upstream of the nucleic acid sequence (e.g., gene) that is operably linked to the promoter. In general, promoters are between 100-1000 base pairs long. In some embodiments, the promoter is a promoter suitable for use in a prokaryotic system (z.e., a bacterial promoter). In some embodiments, the promoter is a promoter suitable for use in a eukaryotic system (z.e., a eukaryotic promoter). In some embodiments, the promoter is a promoter suitable for use in a mammalian (e.g., human) system (z.e., a mammalian promoter). In some embodiments, the promoter is induced by a stimulus (z.e., an inducible promoter). In some embodiments, the stimulus is a small molecule, a protein, a peptide, an amino acid, a metabolite, an inorganic molecule, an organometallic molecule, an organic molecule, a drug or drug candidate, a sugar, a lipid, a metal, a nucleic acid, a molecule produced during the activation of an endogenous or an exogenous signaling cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism, change in pH, or change in oxidation/reduction state. In some embodiments, the stimulus is a light. In some embodiments, the stimulus is a virus. In some embodiments, the stimulus is a small molecule. In some embodiments, the stimulus is an antibiotic. In some embodiments, the stimulus is anhydrotetracycline, tanespimycin, tunicamycin, or doxycycline. In some embodiments, the stimulus is a sugar. In some embodiments, the stimulus is arabinose, rhamnose, or IPTG. [50] The term “repressor” as used herein refers to a DNA- or RNA-binding protein that binds to a repressor binding site (e.g., an operator, a promoter, or a silencing sequence) to inhibit the expression of one or more genes. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the downstream nucleic acid sequence (e.g., gene) operably linked to the promoter into messenger RNA and consequent expression of the protein encoded by the gene. An inducer, z.e., a molecule that initiates the gene expression, can interact with the repressor protein and detach it from the operator (e.g., the promoter). In some embodiments, the repressor is a LacI repressor that represses the expression of a nucleic acid sequence operably linked to a lactose-inducible (e.g, an IPTG-inducible) promoter. In some embodiments, the repressor is a tetracycline repressor (TetR) that represses the expression of a nucleic acid sequence operably linked to a tetracycline-inducible promoter. Additional suitable repressor systems will be apparent to those of ordinary skill in the art based on this disclosure and knowledge in the field, and are within the scope of the present disclosure.
[51] The term “small molecule” is art-recognized and refers to a composition which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu. Small molecules may be, for example, nucleic acids, peptides, polypeptides, peptide nucleic acids, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays described herein. The term “small organic molecule” refers to a small molecule that is often identified as being an organic or medicinal compound and does not include molecules that are exclusively nucleic acids, peptides or polypeptides.
[52] As used herein, “specific binding” refers to the ability of an antibody to bind to a predetermined antigen or the ability of a polypeptide to bind to its predetermined binding partner. Typically, an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 10'7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10-fold less, at least 100-fold less or at least 1000-fold less than its affinity for binding to a non-specific and unrelated antigen/binding partner (e.g, BSA, casein). Alternatively, specific binding applies more broadly to a two component system where one component is a protein, lipid, or carbohydrate or combination thereof and engages with the second component which is a protein, lipid, carbohydrate or combination thereof in a specific way.
[53] The terms “subject” or “patient” refers to any animal. A subject or a patient described as “in need thereof’ refers to one in need of a treatment and/or prevention for a disease. Mammals (i.e., mammalian animals) include humans, laboratory animals (e.g, primates, rats, mice), livestock (e.g, cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents). For example, the subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
[54] The term "transgene" refers to any nucleic acid molecule that is introduced into a cell, that may be intermittently termed herein as a recipient cell. The resultant cell after receiving a transgene may be referred to a transgenic cell. A transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism or cell, or may represent a gene homologous to an endogenous gene of the organism or cell. In some cases, transgenes include any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacks an expression control element, such as a promoter that drives transcription). Transcripts and encoded polypeptides may be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[55] As used herein, the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening.
Icosahedral Phage Derived Particles (PDP)
[56] In certain aspects, provided herein are icosahedral (e.g., T7) phage derived particles (PDPs) with improved properties, as well as methods of making and using such PDPs. As used herein, a PDP is a particle in which a non-phage nucleic acid payload is encapsulated by phage-derived coat proteins. Typically, the nucleic acid payload of a PDP substantially lacks the genome from which the PDP is derived. In some embodiments, the PDP does not comprise nucleic acid sequences encoding at least 50% (e.g., 60%, 70%, 80%, 90%, 95%) of the genome of the phage from which the PDP is derived. In some embodiments, the PDP does not comprise a nucleic acid sequence encoding one or more phage coat proteins (e.g., a 10A and/or 10B coat protein). In some embodiments, the PDP does not comprise a nucleic acid sequence encoding any phage coat proteins. In certain embodiments, the PDP comprises no more that 2000 bases of the genome of the phage from which the PDP was derived.
[57] In certain embodiments, PDPs provided herein possess advantageous properties compared to existing viral vectors making them particularly useful, for example, in the treatment and/or prevention of various diseases. For example, in certain embodiments, the PDPs provided herein exhibit improved: cell or tissue specificity, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics, and/or mammalian expression as compared to existing phage derived vectors.
[58] In certain aspects, the PDPs provided herein comprise phage coat proteins encapsulating a nucleic acid payload, wherein the nucleic acid payload comprises a sequence encoding an agent that treats a disease when delivered to a cell of a subject in need thereof. In some embodiments, the PDPs are derived from an icosahedral phage (e.g., T7 phage). In certain embodiments, the icosahedral bacteriophage is T7.
[59] Bacteriophage T7 is an icosahedral virus (of family Podoviridae) that infects most strains of E. coli. It exhibits a lytic life cycle and destroys its host following infection. T7 phage exhibits a rapid life cycle of 17 minutes at 37C and can produce upwards of 1013 particles in just one hour of growth. The wild-type T7 phage has a diameter of 55 nm and packages a genome of ~40 kb. The wild-type T7 phage has two capsid proteins, 10A (a/k/a gplOA) and 10B (a/k/a gplOB), products of gene 70, which make up the 415 total proteins on a single capsid at a ratio of 90/10, respectively. In some embodiments, the proportion of 10A and 10B may vary and does not affect the integrity of phage particles. Capsid protein 10B is the result of a frame shift at the end of the 10A coding frame.
[60] In certain embodiments, the PDPs disclosed herein comprise one or more modified coat proteins that impart beneficial properties upon the PDP (e.g., improved cell targeting, improved internalization, improved endosomal escape, improved, nuclear shuttling, improved immune evasion, improved pharmacokinetics, improved pharmacodynamics, improved transgene expression). In some embodiments, any of the two of the coat proteins — 10 A, 10B — can be modified to provide such beneficial properties. In certain embodiments, any combination of the coat proteins can be modified to provide such beneficial properties. In certain embodiments, the coat protein(s) are modified to display functional moieties disclosed herein (e.g., cell-targeting moieties, nuclear localization moieties, endosomal escape moieties, internalization moieties). In some embodiments, such moieties are displayed on 10A and/or 10B capsid proteins.
[61] The wild type 10A gene is expressed as a 345 amino acid polypeptide and is the major capsid protein of the icosahedral T7 capsid. It is incorporated into the capsid at approximately a 90/10 ratio relative to the 10B minor capsid protein, where there are a total of 415 capsid proteins per single T7 capsid.
[62] A DNA sequence encoding an exemplary T7 10A capsid protein is provided as SEQ ID NO: 1, as follows: atggctagcatgactggtggacagcaaatgggtactaaccaaggtaaaggtgtagttgctgctggagataaactggcgttgttctt gaaggtatttggcggtgaagtcctgactgcgttcgctcgtacctccgtgaccacttctcgccacatggtacgttccatctccagcgg taaatccgctcagttccctgttctgggtcgcactcaggcagcgtatctggctccgggcgagaacctcgacgataaacgtaaggac atcaaacacaccgagaaggtaatcaccattgacggtctcctgacggctgacgttctgatttatgatattgaggacgcgatgaacca ctacgacgttcgctctgagtatacctctcagttgggtgaatctctggcgatggctgcggatggtgcggttctggctgagattgccg gtctgtgtaacgtggaaagcaaatataatgagaacatcgagggcttaggtactgctaccgtaattgagaccactcagaacaaggc cgcacttaccgaccaagttgcgctgggtaaggagattattgcggctctgactaaggctcgtgcggctctgaccaagaactatgttc cggctgctgaccgtgtgttctactgtgacccagatagctactctgcgattctggcagcactgatgccgaacgcagcaaactacgct gctctgattgaccctgagaagggttctatccgcaacgttatgggctttgaggttgtagaagttccgcacctcaccgctggtggtgct ggtaccgctcgtgagggcactactggtcagaagcacgtcttccctgccaataaaggtgagggtaatgtcaaggttgctaaggac aacgttatcggcctgttcatgcaccgctctgcggtaggtactgttaagctgcgtgacttggctctggagcgcgctcgccgtgctaa cttccaagcggaccagattatcgctaagtacgcaatgggccacggtggtcttcgcccagaagctgctggtgcagtggttttcaaa gtggagtaa
[63] The amino acid sequence of an exemplary T7 10A capsid protein is provided as SEQ ID No: 2, as follow:
MASMTGGQQMGTNQGKGVVAAGDKLALFLKVFGGEVLTAFARTSVTTSRHMV RSISSGKSAQFPVLGRTQAAYLAPGENLDDKRKDIKHTEKVITIDGLLTADVLIYD IEDAMNHYDVRSEYTSQLGESLAMAADGAVLAEIAGLCNVESKYNENIEGLGTA TVIETTQNKAALTDQVALGKEIIAALTKARAALTKNYVPAADRVFYCDPDSYSAI LAALMPNAANYAALIDPEKGSIRNVMGFEVVEVPHLTAGGAGTAREGTTGQKH VFPANKGEGNVKVAKDNVIGLFMHRSAVGTVKLRDLALERARRANFQADQIIAK YAMGHGGLRPEAAGAVVFKVE
[64] The wild type 10B gene is expressed as a 398 amino acid polypeptide and is the minor capsid protein of the icosahedral T7 capsid. It is incorporated into the capsid at approximately a 10/90 ratio relative to the 10A major capsid protein, where there are a total of 415 capsid proteins per single T7 capsid.
[65] A DNA sequence encoding an exemplary T7 10B capsid protein is provided as SEQ ID NO: 3, as follows: atggctagcatgactggtggacagcaaatgggtactaaccaaggtaaaggtgtagttgctgctggagataaactggcgttgttctt gaaggtatttggcggtgaagtcctgactgcgttcgctcgtacctccgtgaccacttctcgccacatggtacgttccatctccagcgg taaatccgctcagttccctgttctgggtcgcactcaggcagcgtatctggctccgggcgagaacctcgacgataaacgtaaggac atcaaacacaccgagaaggtaatcaccattgacggtctcctgacggctgacgttctgatttatgatattgaggacgcgatgaacca ctacgacgttcgctctgagtatacctctcagttgggtgaatctctggcgatggctgcggatggtgcggttctggctgagattgccg gtctgtgtaacgtggaaagcaaatataatgagaacatcgagggcttaggtactgctaccgtaattgagaccactcagaacaaggc cgcacttaccgaccaagttgcgctgggtaaggagattattgcggctctgactaaggctcgtgcggctctgaccaagaactatgttc cggctgctgaccgtgtgttctactgtgacccagatagctactctgcgattctggcagcactgatgccgaacgcagcaaactacgct gctctgattgaccctgagaagggttctatccgcaacgttatgggctttgaggttgtagaagttccgcacctcaccgctggtggtgct ggtaccgctcgtgagggcactactggtcagaagcacgtcttccctgccaataaaggtgagggtaatgtcaaggttgctaaggac aacgttatcggcctgttcatgcaccgctctgcggtaggtactgttaagctgcgtgacttggctctggagcgcgctcgccgtgctaa cttccaagcggaccagattatcgctaagtacgcaatgggccacggtggtcttcgcccagaagctgctggtgcagtggttttcaaa gtggagtaatgctgggggtggcctcaacggtcgctgctagtcccgaagaggcgagtgttacttcaacagaagaaaccttaacgc cagcacaggaggccgcacgcacccgcgctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaat aa
[66] The amino acid sequence of an exemplary T7 10B capsid protein is provided as SEQ ID NO: 4, as follows:
MASMTGGQQMGTNQGKGVVAAGDKLALFLKVFGGEVLTAFARTSVTTSRHMV RSISSGKSAQFPVLGRTQAAYLAPGENLDDKRKDIKHTEKVITIDGLLTADVLIYD IEDAMNHYDVRSEYTSQLGESLAMAADGAVLAEIAGLCNVESKYNENIEGLGTA TVIETTQNKAALTDQVALGKEIIAALTKARAALTKNYVPAADRVFYCDPDSYSAI LAALMPNAANYAALIDPEKGSIRNVMGFEVVEVPHLTAGGAGTAREGTTGQKH VFPANKGEGNVKVAKDNVIGLFMHRSAVGTVKLRDLALERARRANFQADQIIAK YAMGHGGLRPEAAGAVVFQSGVMLGVASTVAASPEEASVTSTEETLTPAQEAA RTRAANKARKEAELAAATAEQ
[67] In certain embodiments, the PDPs provided herein comprise modified coat proteins (e.g., modified 10A and/or 10B coat proteins) that impart certain beneficial properties upon the PDP. For example, in certain embodiments, the PDPs comprise modified coat proteins that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived. [68] In certain embodiments, the PDPs provided herein comprise moieties that impart certain beneficial properties upon the PDP. For example, in certain embodiments, the PDPs comprise moieties that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived. In certain embodiments, such moieties are displayed on one or more of the phage coat proteins of the PDP (e.g., a 10A and/or 10B coat protein). In some embodiments, such moieties are covalently attached to the phage coat protein. In certain embodiments, such moieties are non-covalently attached to the phage coat protein. In some embodiments, the phage coat protein displaying the moiety is a fusion protein comprising the phage coat protein and the moiety.
[69] The nucleic acid payload of the PDPs provided herein is typically in the form of linear double-stranded DNA (dsDNA). In certain embodiments, the phages and/or PDPs disclosed herein use borrowed and/or altered T7 origins of replication to allow for the packaging of foreign sequences (i.e., the PDP payload) within a phage or PDP body.
[70] In certain embodiments, a template phagemid with a T7 origin insert is coinfected with a T7 helper phage that will express all the necessary phage assembly proteins to yield PDPs carrying the sequence from the template phagemid. These PDPs can be produced in bacterial culture at high yields and purified for acquisition of desired dsDNA sequences and structures. In certain embodiments, PDP nucleic acid payload carries an expression cassette that encodes the fusion-coat protein to be displayed and allows for two-gene display systems (i.e., type 10A+10A or type 10B+10B). With PDP systems, the helper phage or helper plasmid bears a defective T7 origin of replication or packaging signal, which allows the preferential packaging of the PDP nucleic acid payload over the helper system. In certain embodiments, the PDP nucleic acid payload lacks sequences coding one or more of the phage capsid proteins.
[71] Preferably, the dsDNA in the PDP comprises an origin of replication for enabling replication of a double-stranded vector inside a prokaryotic host. In some embodiments, the origin of replication enables high copy number replication of the vector inside the host. In some embodiments, the origin of replication comprises a pUC ori. The DNA sequence of one embodiment of the pUC ori is represented herein as follows:
TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC
AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT
CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTT C7\AG7\ACTCTGTAGCACCGCCTACATACCTCGCTCTGCT7\ATCCTGTTACCAGTGGCTGC TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGAC CTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT TGAGCGTCGATTTTTGTGAT GCTCGTCAGGGGGGCGGAGCCTATGGAAA ( SEQ ID NO . 5 )
[72] A PDP can be engineered to have a desired surface charge. In engineering the T7 coat for efficient mammalian cell penetration, the highest priority barrier to cargo delivery is the degradation of the phage by the proteasome. Endosomal escape strategies such as cationic charge on the phage surface is shown to drive reporter gene transduction (20-30%) and suggests that directed or rational mutagenesis of the phage capsids will yield transducing variants.
[73] The negative charge of the T7 bacteriophage head may play a crucial role in the extracellular barriers to phage due to the generation of high non-specific binding to positively charged molecules (around 35% of proteins in the human proteome). Without being bound by theory, and purely for exemplary purposes, a genetic approach may be used to change the C-term of the 10A/10B coat proteins of the PDP provided herein into a mixture of anionic and cationic terminal groups by introducing a short charged neutralizing peptide termed AKAS (Ala-Lys-Ala-Ser). The incorporation of the AKAS peptide into the coat protein results in neutralisation of the negative charge, and may even result in a net positive charge on the phage. Thus, the coat protein of the PDP may comprise at least one modification which neutralizes the negative charge of the surface of the phage, and, in some embodiments results in a net positive charge at physiological pH. The modification may comprise a tetrapeptide comprising the amino acid sequence AKAS located in the C-terminal of the 10A/10B coat proteins. In some embodiments, the targeted PDP comprises a recombinant targeted-bacteriophage and a cationic polymer, wherein the complex has a net positive charge. Without being bound by theory, incubating a PDP with a cationic polymer results in the formation of PDP-polymer complex having a net positive charge under physiological pH, which enhances the ability of the PDP to perform target-specific cellular transduction, for example when administered systemically. Therefore, the PDP-polymer complex provided herein provides a hybrid vector platform, for use in a wide range of gene therapy applications. [74] In some embodiments, the cationic polymer is any polymeric compound having a net positive charge at physiological pH. For example, the polymer may comprise a plurality of positively charged repeating units. The cationic polymer may be selected from a group consisting of: chitosan; poly-D-lysine (PDL); diethylaminoethyl (DEAE); diethylaminoethyl-dextran (DEAE.DEX); polyethyleneimine (PEI); polybrene; protamine sulphate; and a cationic lipid. For example, the cationic lipid may be selected from the group consisting of Fugene®, Lipofectamine®, and DOTAP (N-[l-(2,3- Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate).
[75] The molecular weight of the cationic polymer may be at least 4 kD, 6 kD, 10 kD, 50 kD, 100 kD or 500 kD. For example, the molecular weight of the polymer is about 500 kD for PDL, 70-150 kD for DEAE.DEX, 750 kD for PEI, 4 kD-6 kD for polybrene, 5.1 kD for protamine sulfate. The inventors believe that the lower the molecular weight of the cationic polymer, the more polymer is required in the complex in order to convert the negative charge of the PDP such that it is positive at physiological pH.
[76] The cationic polymer may comprise DEAE, more preferably DEAE.DEX. Preferably, the polymer comprises PDL. In some embodiments, the cationic polymer may comprise a combination of any of the polymers described herein, such as DEAE.DEX and PDL.
[77] The complex may comprise a weight weight ratio of about 50 ng-500 ng polymer: 1 pg PDP, more preferably about 100 ng-400 ng polymer: 1 pg PDP, and even more preferably about 130 ng-320 ng polymer: 1 pg PDP. In embodiments where the polymer is PDL, the complex preferably comprises a weightweight ratio of about 50 ng- 300 ng polymer: 1 pg PDP, more preferably about 100 ng-200 ng polymer: 1 pg PDP, and even more preferably about 120 ng-150 ng polymer: 1 pg PDP. In embodiments where the polymer is DEAE, the complex preferably comprises a weight: weight ratio of about 100 ng-500 ng polymer: 1 pg PDP, more preferably about 200 ng-400 ng polymer: 1 pg PDP, and even more preferably about 250 ng-350 ng polymer: 1 pg PDP.
Modified Phage Derived Particles
[78] In certain embodiments, the PDPs provided herein comprise modified coat proteins (e.g., modified 10A and/or 10B coat proteins) that impart certain beneficial properties upon the PDP. For example, in certain embodiments, the PDPs comprise modified coat proteins that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived.
[79] In some embodiments, the PDPs provided herein comprise a modified 10A coat protein. In certain embodiments, the modified 10A coat protein comprises an amino acid sequence that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to a sequence of a wildtype 10A coat protein. In some embodiments, the PDP comprises a modified 10A coat protein that comprises an amino acid sequence that has no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type 10A coat protein sequence. In some embodiments, the PDP comprises a modified 10A coat protein that comprises an amino acid sequence that has at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type 10A coat protein sequence. In some embodiments, the PDP comprises a modified 10A coat protein that comprises an amino acid sequence that includes deletions of at least 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids compared to a wild-type 10A coat protein sequence. In some embodiments, the PDP comprises a modified 10A coat protein that comprises an amino acid sequence that includes additions of at least 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids compared to a wild-type 10A coat protein sequence. In certain embodiment, a PDP comprising the modified 10A coat protein has improved improve targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared an identical PDP but comprising a wild-type 10A coat protein instead of the modified 10A coat protein.
[80] In some embodiments, the PDPs provided herein comprise a modified 10B coat protein. In certain embodiments, the modified 10B coat protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to a sequence of a wild-type 10B coat protein. In some embodiments, the PDP comprises a modified 10B coat protein that comprises an amino acid sequence that has no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type 10B coat protein sequence. In some embodiments, the PDP comprises a modified 10B coat protein that comprises an amino acid sequence that has at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type 10B coat protein sequence. In some embodiments, the PDP comprises a modified 10B coat protein that comprises an amino acid sequence that includes deletions of at least 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids compared to a wild-type 10B coat protein sequence. In some embodiments, the PDP comprises a modified 10B coat protein that comprises an amino acid sequence that includes additions of at least 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids compared to a wild-type 10B coat protein sequence. In certain embodiment, a PDP comprising the modified 10B coat protein has improved improve targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared an identical PDP but comprising a wild-type 10B coat protein instead of the modified 10B coat protein.
[81] In certain embodiments, the PDPs provided herein comprise moieties that impart certain beneficial properties upon the PDP. For example, in certain embodiments, the PDPs comprise moieties that improve PDP targeting, endosomal escape, nuclear shuttling, immune evasion, pharmacokinetics and/or pharmacodynamics as compared to a phage of the type from which the PDP was derived. In certain embodiments, such moieties are displayed on one or more of the phage coat proteins of the PDP (e.g., a 10A and/or 10B coat protein). In some embodiments, such moieties are covalently attached to the phage coat protein. In certain embodiments, such moieties are non-covalently attached to the phage coat protein. In some embodiments, the phage coat protein displaying the moiety is a fusion protein comprising the phage coat protein and the moiety, linkers and conjugation methods
[82] In certain embodiments, functional moieties provided herein are displayed on a coat protein PDPs disclosed herein. In some embodiments, the coat protein is 10A and/or 10B coat protein (e.g., 10A and/or 10B coat protein configured to display a moiety provided herein). In certain embodiments, the moiety is a domain of a coat protein fusion protein (e.g., a 10A and/or 10B coat protein fusion protein).
[83] Without being bound by theory, constructing a targeted bacteriophage with more than one 10A/10B major coat protein (i) provides the choice to use one 10A/10B major coat protein for the display of large foreign peptide or proteins while keeping the wild type 10A/10B intact, which may be important to achieve efficient phage assembly and subsequently high titers of the phage vector, (ii) allows the display of a considerable copy number of the peptide in order to yield a phage that displays hundreds or even thousands of functional foreign peptides, and (iii) offers the possibility of the simultaneous display of two different functional peptides on the capsid of a single bacteriophage particle. It will be appreciated that a foreign peptide or protein is one that is not normally or naturally expressed by the phage, i.e., it can be heterologous.
[84] In some embodiments, the coat proteins of the PDP may be conjugated to a moiety using heterobifunctional crosslinking reagents. Other methods for chemical conjugation are available in the art, for example by using heterobifunctional linkers including, but not limited to N-succinimidyl 3-(2-pyridyl dithio) propionate (SPDP), m- maleimidobenzoyl-N-hydroxysulfosuccinimida ester, and N-succinimidyl-(4-iodoacetyl) amino-benzoate. Such heterobifunctional crosslinking reagents can also be used to link the drug to the coat proteins of the bacteriophage via a linker such as a peptide, a polypeptide, a peptide derivative, an oligonucleotide, a lipid, a glycolipid, an oligosaccharide and the like.
[85] The linkers disclosed herein may also comprise non-covalent bonds either within the linker, or between the linker and the coat protein and/or the moiety to be linked. Thus, for example, such linkers may be conjugated to the coat protein by means of an avidin/biotin complex. The PDP may be modified to express on its surface avidin or a portion thereof that selectively binds to biotin with the requisite binding affinity. Modification of the PDP to express avidin is easily accomplished by inserting the nucleic acid encoding avidin or a functionally active portion thereof into a helper plasmid used in the making of the PDP such that the avidin or avidin portion is expressed on the PDP surface (e.g., on a coat protein). In this manner, an avidin-expressing PDP is produced which serves as an intermediate for attachment of a biotinylated moiety to the PDP surface. Alternatively, avidin or a functionally active portion thereof can be chemically coupled to the PDP surface using standard cross-linking chemistries, such as those described above. The avidin-labeled PDP permits non-covalent, yet high affinity, attachment of pre-selected biotinylated moieties to the bacteriophage surface. Alternatively, the PDP can be biotinylated and an avidin-labeled moiety provided herein, can be used to form the PDPs described herein. The term "conjugated" thus explicitly includes both covalent and non-covalent links between the PDP and the moieties.
[86] The PDP provided herein may be conjugated to multiple different moieties modulating different PDP properties (e.g., PDP targeting, immunogenicity, endosomal escape, nuclear localization, pharmacokinetics, and/or pharmacodynamics). As a non- limitative example, the PDP may be pegylated (i.e., conjugated to polyethylene glycol), resulting in reduced immunogenicity. Such conjugations may be done prior or following the conjugation of other moieties, and typically involve a different conjugation method than that used for conjugating the first moiety to the PDP. For example, a first moiety may be conjugated to an amino group of a coat protein while a second moiety may be conjugated to a thiol group engineered into the coat protein. Such dual conjugation chemistries may also be used for conjugating a plurality of moieties to the PDP. Alternatively, different moieties can be displayed on different moiety/coat protein fusion proteins.
[87] In some embodiments, the linker is a branched linker or a dendrimer. The term "dendrimer" refers to a three-dimensionally branched, multi-branched compound, and generally refers to all of hyper-branched polymer having a low regularity and dendrimers having a high regularity. A branched linker suitable for linking moieties to the PDP coat proteins by means of chemical conjugation contains at least two reactive residues that may be used for conjugation. The residues may be selected from amine, carboxyl, hydroxyl and sulfhydryl residues. In certain embodiments, the branched linker has high water solubility, and is thus useful for conjugating hydrophobic drugs.
[88] In one particular embodiment, the linker is an aminoglycoside. The aminoglycoside antibiotics are highly hydrophilic substances, which are naturally produced by the actinomycetes. Most of the molecules in the group comprise multiple amino sugars. The aminoglycosides vary in the form and quanta of amine residues within the molecules, which range between 1-7 amine residues per molecule. The chemical structure of the aminoglycoside antibiotic drug kanamycin, which comprises three amine sugars and four amine residues.
[89] Amplification of the carrying capacity of the PDP may be facilitated by chemical conjugation of a single amine from an aminoglycoside molecule to a carboxyl residue of the carrier, thereby converting the single carboxyl residue to an amine branched linker.
[90] In some embodiments, suitable aminoglycoside molecules have two or more reactive residues. Exemplary aminoglycosides include, but are not limited to, hygromycin, kanamycin, gentamycin, amikacin, neomycin, pardomycin, tobramycin and viomycin.
[91] By means of a non-limitative example, amine conjugation methods well-known in the art may be used to conjugate aminoglycosides to the PDP carrier and to the drug molecules, including, but not limited to, NHS chemistry, paranitophenyl phosphate (PNP) chemistry, isothiocyanate chemistry and N-(3-dimethylaminopropyl)-N'- ethycarbodiimide (EDC) chemistry.
[92] The linker, moiety, and/or a component thereof, may be displayed on the PDP as a result of genetic modification. As explained above, a peptide linker and/or moiety may be fused to a coat protein of a PDP disclosed herein, to which other moieties may be linked by means of chemical conjugation or genetic modification. By means of a non-limitative example, a peptide comprising a moiety (e.g, a moiety disclosed herein) can be fused to protein of the phage. In some embodiments, the peptide comprising a moiety (e.g, a moiety disclosed herein) can be fused to the N’ of a coat protein of the phage (e.g., 10A, 10B), such that the moiety is displayed by the PDP.
[93] The PDP surface can be modified to include a poly-glycine (polyG) motif (e.g., a GGGGG docking site, where G(n) can vary from 1-10). A sortase enzyme can catalyze the formation of a peptide bond between the C-terminal LPXTG amino acid motif, where X is any amino acid, of a desired moiety and the N-terminal of a poly-glycine motif, to functionalize the PDP surface with the desired moiety. In some embodiments, the PDP surface can be modified to include an LPXTG amino acid motif and the desired moiety can contain the G(n) sequence for the sortase conjugation reaction.
PDP Nucleic Acid Payloads
[94] In certain aspects, provided herein are icosahedral (e.g., T7) phage derived particles (PDPs) with improved functional properties, as well as methods of making and using such PDPs. Generally, the PDPs provided herein are useful for the delivery of nucleic acid payloads to mammalian cells.
[95] Typically, the nucleic acid payload of the PDPs provided herein is a linear double stranded DNA (dsDNA) construct comprising a non-phage sequence encoding a therapeutic agent to be delivered to a cell of interest. Typically, the nucleic acid payload of a PDP substantially lacks the genome of the phage from which the PDP is derived (e.g., the nucleic acid payload does not encode the phage-derived coat proteins in which it is encapsulated). In some embodiments, the PDP does not comprise nucleic acid sequences encoding at least 50% (e.g., 60%, 70%, 80%, 90%, 95%) of the phage genome of the phage from which the PDP is derived. In some embodiments, the PDP does not comprise a nucleic acid sequence encoding a phage coat protein. In certain embodiments, the PDP comprises no more that 300 bases of the genome of the phage from which the PDP was derived. [96] Where a nucleic acid sequence delivered by a PDP provided herein comprises a sequence to be expressed, the application envisages the use of codon-optimized sequences. An example of a codon optimized sequence may be a sequence optimized for expression in a eukaryote, e.g., humans (i.e., being optimized for expression in humans), or for another eukaryote, animal or mammal. Codon optimization for a host species other than human, or for codon optimization for specific organs is known. In some embodiments, the coding sequence encoding a protein may be codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell may generally reflect the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database" available on the internet at www.kazusa.orjp/codon/ and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
[97] In certain embodiments, provided herein are improved PDPs for gene therapy. In some embodiments, the nucleic acid payloads of the PDPs are modified to (1) increase the cargo-carrying capacity by using minimal sequence requirements for packaging and (2) improve the levels, kinetics, and durability of transgene expression following cellular transduction. [98] Accordingly, in certain embodiments the nucleic acid payload of the PDPs provided herein is a linear double stranded DNA (dsDNA) construct.
[99] In certain embodiments, payload dsDNA construct comprises a DNA sequence element that enhance expression and/or specificity in mammalian cells. In some embodiments, the DNA sequence element is selected from a promoter, an enhancer, a silencer, an insulator, an untranslated region, and a microRNA binding site. In certain embodiments, the payload dsDNA construct comprises a coding region that is codon optimized for mammalian expression. In some embodiments, the dsDNA construct comprises a terminator sequence. In some embodiments, the episomal expression of the dsDNA construct is durable in slow and/or non-dividing cells (e.g., lasting at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days).
[100] In certain embodiments, the dsDNA construct encodes a transgene (e.g., a transgene encoding a therapeutic agent provided herein). The term "transgene" refers to any nucleic acid molecule that is introduced into a cell, that may be intermittently termed herein as a recipient cell. The resultant cell after receiving a transgene may be referred to a transgenic cell. A transgene may include a gene that is partly or entirely heterologous (z.e., foreign) to the transgenic organism or cell, or may represent a gene homologous to an endogenous gene of the organism or cell. In some cases, transgenes include any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacks an expression control element, such as a promoter that drives transcription). Transcripts and encoded polypeptides may be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[101] In certain embodiments, the dsDNA construct comprises promoters, e.g., to drive expression of a therapeutic agent described herein. The term "promoter," as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the disclosure include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter may be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. A "tissue-specific promoter" initiates transcription only in one or a few particular tissue types, whereas a "non-specific promoter" is one that is capable of initiating transcription in nearly all tissue types. A “constitutive promoter” is a promoter that is capable of driving expression of a coding sequence in most or all environmental conditions. For example, the cytomegalovirus (CMV) promoter is a constitutive promoter that is often used when continual expression is desired. In contrast to a constitutive promoter, an "inducible promoter" is one that initiates transcription only under particular environmental conditions, developmental conditions, or drug or chemical conditions. Exemplary inducible promoter may be a doxycycline or a tetracycline inducible promoter. Tetracycline regulated promoters may be both tetracycline inducible or tetracycline repressible, called the tet-on and tet-off systems. The tet regulated systems rely on two components, i.e., a tetracycline-controlled regulator (also referred to as transactivator) (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of a downstream cDNA, in a tetracycline-dependent manner. tTA is a fusion protein containing the repressor of the TnlO tetracycline-resistance operon of Escherichia coli and a carboxyl-terminal portion of protein 16 of herpes simplex virus (VP 16). The tTA- dependent promoter consists of a minimal RNA polymerase II promoter fused to tet operator (tetO) sequences (an array of seven cognate operator sequences). This fusion converts the tet repressor into a strong transcriptional activator in eukaryotic cells. In the absence of tetracycline or its derivatives (such as doxycycline), tTA binds to the tetO sequences, allowing transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target and transcription does not occur. The tet system that uses tTA is termed /e -OFF, because tetracycline or doxycycline allows transcriptional down-regulation. In contrast, in the tet-ON system, a mutant form of tTA, termed rtTA, has been isolated using random mutagenesis. In contrast to tTA, rtTA is not functional in the absence of doxycycline but requires the presence of the ligand for transactivation.
[102] The term "termination sequence" refers to a nucleic acid sequence which is recognized by the polymerase of a host cell and results in the termination of transcription. The termination sequence is a sequence of DNA that, at the 3' end of a natural or synthetic gene, provides for termination of mRNA transcription or both mRNA transcription and ribosomal translation of an upstream open reading frame. Prokaryotic termination sequences commonly comprise a GC-rich region that has a two-fold symmetry followed by an AT-rich sequence. A commonly used termination sequence is the T7 termination sequence. A variety of termination sequences are known in the art and may be employed in the nucleic acid constructs of the present invention, including the TINT3, TL13, TL2, TRI, TR2, and T6S termination signals derived from the bacteriophage lambda, and termination signals derived from bacterial genes, such as the trp gene of E. coli.
[103] The terms "polyadenylation sequence" (also referred to as a "poly A site" or "poly A sequence") refers to a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, as transcripts lacking a poly A tail are typically unstable and rapidly degraded. The poly A signal utilized in an expression vector disclosed herein may be "heterologous" or "endogenous". An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene, e.g., coding sequence for a protein. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on a 237 bp BamFE/BclI restriction fragment and directs both termination and polyadenylation. Another commonly used heterologous poly A signal is derived from the bovine growth hormone (BGH) gene; the BGH poly A signal is also available on a number of commercially available vectors. The poly A signal from the Herpes simplex virus thymidine kinase (HSV tk) gene is also used as a poly A signal on a number of commercial expression vectors. The polyadenylation signal facilitates the transportation of the RNA from within the cell nucleus into the cytosol as well as increases cellular halflife of such an RNA. The polyadenylation signal is present at the 3 ’-end of an mRNA.
[104] In some embodiment, ccsDNA construct comprises a the DNA sequence element is selected from the DNA sequence elements listed in Table 1.
Table 1: Exemplary Sequence Elements for Mammalian Expression
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
[105] In certain embodiments, the PDPs provided herein are designed to inhibit the expression of a gene (e.g., a disease-associated gene) in a cell, for example, wherein reduction of such a gene in a cell would have a therapeutic effect. In certain embodiments, such PDPs would comprise a nucleic acid payload that encodes an inhibitory RNA disclosed herein and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that targets the gene.
[106] In certain embodiments, the PDPs provided herein are designed to enhance the expression of a peptide (e.g., a therapeutic peptide) in a cell (e.g., a peptide whose expression treats and/or prevents a disease). In certain embodiments, such PDPs would comprise a nucleic acid payload that encodes the peptide and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate insertion of a sequence encoding the peptide into the genome of the cell. In some embodiments, the sequence encoding a peptide is inserted into safe harbor locus in the cell. Exemplary safe harbor loci are listed in Table 2.
Table 2: Safe Harbor Loci
Figure imgf000039_0002
[107] In certain embodiments, the PDPs provided herein are designed to inhibit the expression of a gene (e.g., a disease-associated gene) in a cell and enhance expression of a peptide (e.g., a therapeutic peptide) in a cell. For example, in some embodiments, the gene for which expression is inhibited is a mutant gene associated with a disease, while the peptide is the peptide encoded by the wild-type version of that gene. In certain embodiments, such PDPs would comprise a nucleic acid payload that comprises a sequence that encodes an inhibitory RNA and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that targets the gene; and (2) a sequence that encodes the peptide and/or one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate insertion of a sequence encoding the peptide into the genome of the cell (e.g., into the mutant gene locus and/or a safe harbor locus in the cell, such as a safe harbor locus listed in Table 2).
[108] In certain embodiments, the PDPs provided herein are designed to modify the sequence of a gene in a cell. In certain embodiments, such PDPs would comprise a nucleic acid payload that encodes one or more components of a gene editing system disclosed herein (e.g., a CRISPR/Cas system) that could facilitate modification of the gene in the genome of the cell (e.g., converting mutant version of a gene into a wild-type version of a gene).
Cell Targeting
[109] In certain aspects, provided herein are PDPs able to be targeted to specific cells and/or tissues. For example, in certain embodiments provided herein are PDPs that display a ligand, antigen-binding, or other targeting moiety endowing them with specificity towards target molecules, cells, tissues and/or other biological structures. These PDPs can be used to deliver internal nucleic acid payloads and/or external conjugated moieties (e.g., conjugated through a labile/non labile linker or directly) to specific cells and/or tissues and are thus useful as targeted delivery vessels for the treatment of a disease. In certain embodiments, the PDPs comprise a phage coat protein displaying a cell-targeting moiety specific for a cell type selected from erythrocytes, granulocytes, agranulocytes, platelets, neurons, neuroglial cells, skeletal muscle cells, cardiac muscle cells, smooth muscle cells, chondrocytes, lymphocytes, osteoblasts, osteoclasts, osteocytes, lining cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, epithelial cells (e.g. hepatocytes), endothelial cells, white adipocytes, brown adipocytes, spermatozoa, ova, exocrine and endocrine secretory cells (e.g. pancreatic islet cells), embryonic stem cells, adults stem cells, extracellular matrix cells (e.g. fibroblasts). [HO] In certain aspects, PDPs disclosed herein may display a targeting moiety that selectively binds a target molecule on a target cell and/or in a target tissue. The term displaying a “targeting moiety” as used herein encompasses targeting moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both. The targeting moiety and its manner of expression and/or linkage is designed to facilitate the PDP to selectively bind a target cell. This term further includes a targeting moiety comprising a moiety conjugated to the PDP that binds non-covalently to a second target or molecule capable of binding the target cell.
[Hl] The targeting moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs. Targeting moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
[112] An isolated nucleic acid sequence encoding a targeting moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof. A nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional targeting moiety of the present invention. A nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
[113] A targeting moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
[114] The targeting moiety may be any biological or synthetic substance endowed with specific binding properties towards a selected target cell and/or tissue. For example and without limitation, targeting moieties may be antibody -based moieties, including, but not limited to: monoclonal antibodies, polyclonal antibodies, and antibody fragments such as recombinant antibody fragments, single-chain antibodies (scFv), single antibody variable domains, and the like (Borrebaeck, 1995; Lo, 2003). Single-chain antibodies are small recognition units consisting of the variable regions of the immunoglobulin heavy (VH) and light (VL) chains which are connected by a synthetic linker sequence. Single antibody domain proteins (dAbs) are minimized antibody fragments comprising either an individual VL domain or an individual VH domain.
[115] Methods of generating monoclonal and polyclonal antibodies are well known in the art. Antibodies may be generated via any one of several known methods, which may employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries, or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique. Antibody fragments may be obtained using methods well known in the art, including, but not limited to by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary (CHO) cell culture or other protein expression systems) of DNA encoding the fragment (Fab')2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments retain the ability to bind to the antigen that is recognized by the intact antibody. An Fv is composed of paired heavy chain variable and light chain variable domains. This association may be non- covalent. Alternatively, as described hereinabove, the variable domains may be linked to generate a single-chain Fv by an intermolecular disulfide bond, or alternately such chains may be cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv is a singlechain Fv. Single-chain Fvs are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains. In addition, techniques may be utilized to change a “murine” antibody to a “human” antibody, without altering the binding specificity of the antibody. [116] The targeting moiety may be a peptide endowed with binding specificity towards the target cell (linear, circularly constrained or cyclic) or a short peptide selected from a library of short peptide sequences that is endowed with binding specificity towards the target cell (Kay et al., 1996). Methods for constructing libraries and using them for screening for ligands having an affinity to a selected target molecule or cell are known in the art.
[117] The targeting moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to selectively bind a target molecule on a target cell. For instance, the ligand may include known ligands of cell surface receptors, or any natural or synthetic derivative thereof..
[118] The targeting moiety is chosen according to the target cell and/or tissue that is to be targeted. For certain applications, targeting moieties are chosen such that they are internalized by the target cell upon binding the target molecule, thereby enabling the internalization of the PDP. Methods of constructing and selecting for internalizing phages are known in the art (see, for example, Becerril et al., 1999, Kassner et al., 1999, Poul and Marks, 1999, Larocca and Baird, 2001, Larocca et al., 2001, Urbanelli et al., 2001).
[119] The targeting moieties used in the compositions and/or methods disclosed herein do not necessarily retain any of their in vivo biological activities, other than binding a target molecule on a target cell and/or tissue. However, it may be desirable in certain contexts that a ligand exerts certain of its biological activities. For example, the targeting moiety may act as an agonist, or alternatively as an antagonist, upon binding a cell surface receptor.
[120] In some embodiments, the targeting moiety displayed by the PDP disclosed herein is selected so as to facilitate selective binding of the conjugate to a target cell and/or tissue involved in a disease or disorder in a subject in need thereof (e.g., a disease or disorder disclosed herein).
[121] Aptamers are another class of binding agent that can be used as a targeting moiety. Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g., membrane lipid rafts).
Aptamers that specifically bind a marker of the cell (e.g., a cell surface moiety or receptor) are useful in the methods of the invention. Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No. 5,660,985; No. 5,567,588; No. 5,683,867; No. 5,637,459; and No. 6,011,020. In some embodiments, the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics. In some embodiments, aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups (e.g., 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
Immune Cells
[122] In certain embodiments, the PDPs provided herein are designed to target immune cells. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of the function of the targeted immune cell would have a therapeutic effect.
[123] In some embodiments, the targeted immune cell is a neutrophil. In certain embodiments, the PDPs provided herein comprise a neutrophil-targeting moiety specific for a neutrophil surface antigen. In some embodiments, the neutrophil-targeting moiety can be specific for any protein expressed on the surface of a neutrophil. Exemplary neutrophil surface antigens are listed in Table 3.
Table 3: Exemplary Neutrophil Surface Markers
Figure imgf000044_0001
Figure imgf000045_0001
[124] In certain embodiments, the neutrophil-targeting moiety comprises an antibody or antibody fragment specific for a neutrophil surface antigen (e.g., an antigen listed in Table 3). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the neutrophil-targeting moiety comprises an aptamer specific for a neutrophil surface antigen (e.g., an antigen listed in Table 3).
[125] In some embodiments, the neutrophil-targeting moiety comprises a ligand that binds to a neutrophil surface antigen. [126] In some embodiments, the targeted immune cell is an eosinophil. In certain embodiments, the PDPs provided herein comprise an eosinophil-targeting moiety specific for a eosinophil surface antigen. In some embodiments, the eosinophil-targeting moiety can be specific for any protein expressed on the surface of an eosinophil. Exemplary eosinophil surface antigens are listed in Table 4.
Table 4: Exemplary Eosinophil Surface Markers
Figure imgf000046_0001
Figure imgf000047_0001
[127] In certain embodiments, the eosinophil-targeting moiety comprises an antibody or antibody fragment specific for an eosinophil surface antigen (e.g., an antigen listed in Table 4). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the eosinophil-targeting moiety comprises an aptamer specific for an eosinophil surface antigen (e.g., an antigen listed in Table 4).
[128] In some embodiments, the eosinophil-targeting moiety comprises a ligand that binds to an eosinophil surface antigen.
[129] In some embodiments, the targeted immune cell is a basophil. In certain embodiments, the PDPs provided herein comprise a basophil-targeting moiety specific for a basophil surface antigen. In some embodiments, the basophil-targeting moiety can be specific for any protein expressed on the surface of a basophil. Exemplary basophil surface antigens are listed in Table 5.
Table 5: Exemplary Basophil Surface Markers
Figure imgf000047_0002
Figure imgf000048_0001
[130] In certain embodiments, the basophil-targeting moiety comprises an antibody or antibody fragment specific for a basophil surface antigen (e.g., an antigen listed in Table 5 or a biologically active fragment thereof). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the basophil-targeting moiety comprises an aptamer specific for a basophil surface antigen (e.g., an antigen listed in Table 5 or a biologically active fragment thereof).
[131] In some embodiments, the basophil-targeting moiety comprises a ligand that binds to a basophil surface antigen.
[132] In some embodiments, the targeted immune cell is a lymphocyte (e.g., a T cell, a B cell, a natural killer (NK) cell)).
[133] In some embodiments, the targeted lymphocyte is a T cell. In some embodiments, the targeted T cell is a CD4 helper T cell (e.g., a Thl cell, a Th2 cell, a Thl7 cell, a Th9 cell, a Tfh cell, a Th22 cell). In certain embodiments, the targeted T cell is a cytotoxic CD8 T cell (e.g., a Tel cell, a Tc2 cell, a Tc9 cell, a Tcl7 cell). In some embodiments, the targeted T cell is a naive T cell (Tn). In certain embodiments, the targeted T cell is an effector T cell (Teff). In some embodiments, the targeted T cell is a memory T cell (e.g., a central memory T cell (Tern), an effector memory T cell (Tern), a tissue-resident memory T cell (Trm), a stem cell memory T cell (Tscm)). In some embodiments, the targeted T cell is a regulatory T cell (Treg). In certain embodiments, the targeted T cell is a natural killer T cell (NKT). In some embodiments, a combination of the above T cell sub types are targeted.
[134] In some embodiments, the lymphocyte is a B cell. In some embodiments, the targeted B cell is a naive B cell. In some embodiments, the targeted B cell is a memory B cell. In certain embodiments, the targeted B cell is a plasmablast. In some embodiments, the targeted B cell is a plasma cell. In some embodiments, the targeted B cell is a lymphoplasmacytoid cell. In certain embodiments, the targeted B cell is a B-2 cell. In some embodiments, the targeted B cell is a B-l cell. In certain embodiments, the targeted B cell is a regulatory B cell (Breg). In some embodiments, a combination of the above B cell sub types are targeted.
[135] In certain embodiments, the lymphocyte is a natural killer (NK) cell.
[136] In certain embodiments, the PDPs provided herein comprise a lymphocytetargeting moiety specific for a lymphocyte surface antigen. In some embodiments, the lymphocyte-targeting moiety can be specific for any protein expressed on the surface of a lymphocyte. Exemplary lymphocyte surface antigens are listed in Table 6. Table 6: Exemplary Lymphocyte Surface Markers
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
[137] In certain embodiments, the lymphocyte-targeting moiety comprises an antibody or antibody fragment specific for a lymphocyte surface antigen (e.g., an antigen listed in Table 6). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the lymphocyte-targeting moiety comprises an aptamer specific for a lymphocyte surface antigen (e.g., an antigen listed in Table 6).
[138] In some embodiments, the lymphocyte-targeting moiety comprises a ligand that binds to a lymphocyte surface antigen.
[139] In some embodiments, the targeted immune cell is a monocyte. In certain embodiments, the PDPs provided herein comprise a monocyte-targeting moiety specific for a monocyte surface antigen. In some embodiments, the monocyte-targeting moiety can be specific for any protein expressed on the surface of a monocyte. Exemplary monocyte surface antigens are listed in Table 7.
Table 7: Exemplary Monocyte Surface Markers
Figure imgf000053_0001
Figure imgf000054_0001
[140] In certain embodiments, the monocyte-targeting moiety comprises an antibody or antibody fragment specific for a monocyte surface antigen (e.g., an antigen listed in Table 7). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the monocyte-targeting moiety comprises an aptamer specific for a monocyte surface antigen (e.g., an antigen listed in Table 7).
[141] In some embodiments, the monocyte-targeting moiety comprises a ligand that binds to a monocyte surface antigen.
[142] In some embodiments, the targeted immune cell is a macrophage. In certain embodiments, the PDPs provided herein comprise a macrophage-targeting moiety specific for a macrophage surface antigen. In some embodiments, the macrophagetargeting moiety can be specific for any protein expressed on the surface of a macrophage. Exemplary macrophage surface antigens are listed in Table 8.
Table 8: Exemplary Macrophage Surface Markers
Figure imgf000054_0002
Figure imgf000055_0001
[143] In certain embodiments, the macrophage-targeting moiety comprises an antibody or antibody fragment specific for a macrophage surface antigen (e.g., an antigen listed in Table 8). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the macrophage-targeting moiety comprises an aptamer specific for a macrophage surface antigen (e.g., an antigen listed in Table 8).
[144] In some embodiments, the macrophage-targeting moiety comprises a ligand that binds to a macrophage surface antigen.
[145] In some embodiments, the targeted immune cell is a dendritic cell. In certain embodiments, the PDPs provided herein comprise a dendritic cell-targeting moiety specific for a dendritic cell surface antigen. In some embodiments, the dendritic cell-targeting moiety can be specific for any protein expressed on the surface of a dendritic cell. Exemplary dendritic cell surface antigens are listed in Table 9.
Table 9: Exemplary Dendritic cell Surface Markers
Figure imgf000056_0001
Figure imgf000057_0001
[146] In certain embodiments, the dendritic cell-targeting moiety comprises an antibody or antibody fragment specific for a dendritic cell surface antigen (e.g., an antigen listed in Table 9). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the dendritic cell-targeting moiety comprises an aptamer specific for a dendritic cell surface antigen (e.g., an antigen listed in Table 9).
[147] In some embodiments, the dendritic cell-targeting moiety comprises a ligand that binds to a dendritic cell surface antigen.
Neuronal Cells
[148] In certain embodiments, the PDPs provided herein comprise a neuronal cell-targeting moiety specific for a neuronal cell surface antigen. In some embodiments, the targeted neuronal cell is a neuroepithelial cell, a radial glia, an intermediate progenitor, an immature neuron, an oligodendrocyte precursor cell, a mature oligodendrocyte, a schwann cell, an astrocyte, a microglia cell,, an Ml microglia cell, a M2 microglia cell, a mature neuron, a glutamatergic neuron, a GABAergic neuron, a domaminergic neuron, a serotonergic neuron, and/or a cholinergic neuron. In some embodiments, a combination of the above neuronal cell sub types are targeted. In some embodiments, the neuronal cell-targeting moiety can be specific for any protein expressed on the surface of a neuronal cell. Exemplary neuronal cell surface antigens are listed in Table 10.
Table 10: Exemplary Neuronal Cell Surface Markers
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
[149] In certain embodiments, the neuronal cell-targeting moiety comprises an antibody or antibody fragment specific for a neuronal cell surface antigen (e.g., an antigen listed in Table 10). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the neuronal cell-targeting moiety comprises an aptamer specific for a neuronal cell surface antigen (e.g., an antigen listed in Table 10).
[150] In some embodiments, the neuronal cell-targeting moiety comprises a ligand that binds to a neuronal cell surface antigen.
Epithelial Cells
[151] In certain embodiments, the PDPs provided herein comprise an epithelial cell-targeting moiety specific for a epithelial cell surface antigen. In some embodiments, the epithelial cell-targeting moiety can be specific for any protein expressed on the surface of an epithelial cell. Exemplary epithelial cell surface antigens are listed in Table 11.
Table 11: Exemplary Epithelial Cell Surface Markers
Figure imgf000060_0002
Figure imgf000061_0001
[152] In certain embodiments, the epithelial cell-targeting moiety comprises an antibody or antibody fragment specific for an epithelial cell surface antigen (e.g., an antigen listed in Table 11). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the epithelial cell-targeting moiety comprises an aptamer specific for an epithelial cell surface antigen (e.g., an antigen listed in Table 11).
[153] In some embodiments, the epithelial cell-targeting moiety comprises a ligand that binds to an epithelial cell surface antigen.
Adipocytes
[154] In certain embodiments, the PDPs provided herein comprise an adipocytetargeting moiety specific for a adipocyte surface antigen. In some embodiments, the adipocyte-targeting moiety can be specific for any protein expressed on the surface of an adipocyte. Exemplary adipocyte surface antigens are listed in Table 12.
Table 12: Exemplary Adipocyte Surface Markers
Figure imgf000062_0001
[155] In certain embodiments, the adipocyte-targeting moiety comprises an antibody or antibody fragment specific for an adipocyte surface antigen (e.g., an antigen listed in Table 12). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the adipocyte-targeting moiety comprises an aptamer specific for an adipocyte surface antigen (e.g., an antigen listed in Table 12).
[156] In some embodiments, the adipocyte -targeting moiety comprises a ligand that binds to an adipocyte surface antigen.
Hepatocytes
[157] In certain embodiments, the PDPs provided herein comprise an hepatocyte-targeting moiety specific for a hepatocyte surface antigen. In some embodiments, the hepatocyte-targeting moiety can be specific for any protein expressed on the surface of an hepatocyte. Exemplary hepatocyte surface antigens are listed in Table 13.
Table 13: Exemplary Hepatocyte Surface Markers
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
[158] In certain embodiments, the hepatocyte-targeting moiety comprises an antibody or antibody fragment specific for an hepatocyte surface antigen (e.g., an antigen listed in Table 13). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the hepatocyte-targeting moiety comprises an aptamer specific for an hepatocyte surface antigen (e.g., an antigen listed in Table 13).
[159] In some embodiments, the hepatocyte-targeting moiety comprises a ligand that binds to an hepatocyte surface antigen.
Fibroblasts
[160] In certain embodiments, the PDPs provided herein comprise a fibroblasttargeting moiety specific for a fibroblast surface antigen. In some embodiments, the fibroblast-targeting moiety can be specific for any protein expressed on the surface of a fibroblast. Exemplary fibroblast surface antigens are listed in Table 14.
Table 14: Exemplary Fibroblast Surface Markers
Figure imgf000070_0002
Figure imgf000071_0001
[161] In certain embodiments, the fibroblast-targeting moiety comprises an antibody or antibody fragment specific for a fibroblast surface antigen (e.g., an antigen listed in Table 14). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the fibroblast-targeting moiety comprises an aptamer specific for a fibroblast surface antigen (e.g., an antigen listed in Table 14).
[162] In some embodiments, the fibroblast-targeting moiety comprises a ligand that binds to a fibroblast surface antigen.
Pancreatic Cells
[163] In certain embodiments, the PDPs provided herein comprise a pancreatic cell-targeting moiety specific for a pancreatic cell surface antigen. In some embodiments, the pancreatic cell-targeting moiety can be specific for any protein expressed on the surface of a pancreatic cell. Exemplary pancreatic cell surface antigens are listed in Table 15.
Table 15: Exemplary Pancreatic Cell Surface Markers
Figure imgf000071_0002
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
[164] In certain embodiments, the pancreatic cell-targeting moiety comprises an antibody or antibody fragment specific for a pancreatic cell surface antigen (e.g., an antigen listed in Table 15). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the pancreatic cell- targeting moiety comprises an aptamer specific for a pancreatic cell surface antigen (e.g., an antigen listed in Table 15).
[165] In some embodiments, the pancreatic cell-targeting moiety comprises a ligand that binds to a pancreatic cell surface antigen.
Osteoblasts
[166] In certain embodiments, the PDPs provided herein comprise an osteoblasttargeting moiety specific for a osteoblast surface antigen. In some embodiments, the osteoblast-targeting moiety can be specific for any protein expressed on the surface of an osteoblast. Exemplary osteoblast surface antigens are listed in Table 16.
Table 16: Exemplary Osteoblast Surface Markers
Figure imgf000081_0001
[167] In certain embodiments, the osteoblast-targeting moiety comprises an antibody or antibody fragment specific for an osteoblast surface antigen (e.g., an antigen listed in Table 16). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the osteoblast-targeting moiety comprises an aptamer specific for an osteoblast surface antigen (e.g., an antigen listed in Table 16).
[168] In some embodiments, the osteoblast-targeting moiety comprises a ligand that binds to an osteoblast surface antigen. Stem Cells
[169] In certain embodiments, the PDPs provided herein comprise a stem celltargeting moiety specific for a stem cell surface antigen. In some embodiments, the stem cell-targeting moiety can be specific for any protein expressed on the surface of a stem cell. Exemplary stem cell surface antigens are listed in Table 17.
Table 17: Exemplary Stem Cell Surface Markers
Figure imgf000082_0001
[170] In certain embodiments, the stem cell-targeting moiety comprises an antibody or antibody fragment specific for a stem cell surface antigen (e.g., an antigen listed in Table 17). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the stem cell-targeting moiety comprises an aptamer specific for a stem cell surface antigen (e.g., an antigen listed in Table 17).
[171] In some embodiments, the stem cell-targeting moiety comprises a ligand that binds to a stem cell surface antigen.
Skeletal Muscle Cells
[172] In certain embodiments, the PDPs provided herein comprise a skeletal muscle cell-targeting moiety specific for a skeletal muscle cell surface antigen. In some embodiments, the skeletal muscle cell-targeting moiety can be specific for any protein expressed on the surface of a skeletal muscle cell. Exemplary skeletal muscle cell surface antigens are listed in Table 18.
Table 18: Exemplary Skeletal Muscle Cell Surface Markers
Figure imgf000083_0001
Figure imgf000084_0002
[173] In certain embodiments, the skeletal muscle cell-targeting moiety comprises an antibody or antibody fragment specific for a skeletal muscle cell surface antigen (e.g., an antigen listed in Table 18). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the skeletal muscle celltargeting moiety comprises an aptamer specific for a skeletal muscle cell surface antigen (e.g., an antigen listed in Table 18).
[174] In some embodiments, the skeletal muscle cell -targeting moiety comprises a ligand that binds to a skeletal muscle cell surface antigen.
Endothelial Cells
[175] In certain embodiments, the PDPs provided herein are designed to target endothelial cells (e.g., vascular endothelial cell). Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of endothelial cells (e.g., vascular endothelial cell) function would have a therapeutic effect.
[176] In certain embodiments, the PDPs provided herein comprise an endothelial cell-targeting moiety specific for an endothelial cell surface antigen. In some embodiments, the endothelial cell-targeting moiety can be specific for any protein expressed on the surface of an endothelial cell (e.g., a vascular endothelial cell.
Exemplary endothelial cell surface antigens are listed in Table 19.
Table 19: Exemplary Endothelial Cell Surface Markers
Figure imgf000084_0001
CD117/c-Kit
Figure imgf000085_0001
Figure imgf000086_0001
[177] In certain embodiments, the endothelial cell-targeting moiety comprises an antibody or antibody fragment specific for an endothelial cell surface antigen (e.g., an antigen listed in Table 19). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the endothelial celltargeting moiety comprises an aptamer specific for an endothelial cell surface antigen (e.g., an antigen listed in Table 19).
[178] In some embodiments, the endothelial cell-targeting moiety comprises a ligand that binds to an endothelial cell surface antigen. In certain embodiments, the ligand is P-selectin glycoprotein ligand 1, CD44, or E-selectin ligand-1.
Cardiomyocytes
[179] In certain embodiments, the PDPs provided herein are designed to target cardiomyocytes. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of cardiomyocyte function would have a therapeutic effect.
[180] In certain embodiments, the PDPs provided herein comprise a cardiomyocyte-targeting moiety specific for a cardiomyocyte surface antigen. In some embodiments, the cardiomyocyte-targeting moiety can be specific for any protein expressed on the surface of a cardiomyocyte. Exemplary cardiomyocyte surface antigens are listed in Table 20.
Table 20: Exemplary Cardiomyocyte Surface Markers
Figure imgf000086_0002
Figure imgf000087_0001
Figure imgf000088_0001
[181] In certain embodiments, the cardiomyocyte-targeting moiety comprises an antibody or antibody fragment specific for a cardiomyocyte surface antigen (e.g., an antigen listed in Table 20). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the cardiomyocyte- targeting moiety comprises an aptamer specific for a cardiomyocyte surface antigen (e.g., an antigen listed in Table 20).
[182] In some embodiments, the cardiomyocyte-targeting moiety comprises a ligand that binds to a cardiomyocyte surface antigen.
Retinal Pigment Epithelium (RPE) Cells
[183] In certain embodiments, the PDPs provided herein are designed to target retinal pigment epithelium (RPE) cells. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of RPE cell function would have a therapeutic effect.
[184] In certain embodiments, the PDPs provided herein comprise an RPE celltargeting moiety specific for a RPE cell surface antigen. In some embodiments, the RPE cell-targeting moiety can be specific for any protein expressed on the surface of an RPE cell. Exemplary RPE cell surface antigens are listed in Table 21.
Table 21 Exemplary RPE Cell Surface Markers
Figure imgf000089_0001
[185] In certain embodiments, the RPE cell-targeting moiety comprises an antibody or antibody fragment specific for an RPE cell surface antigen (e.g., an antigen listed in Table 21). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the RPE cell-targeting moiety comprises an aptamer specific for an RPE cell surface antigen (e.g., an antigen listed in Table 21).
[186] In some embodiments, the RPE cell-targeting moiety comprises a ligand that binds to an RPE cell surface antigen. Chondrocytes
[187] In certain embodiments, the PDPs provided herein are designed to target chondrocytes. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of chondrocyte function would have a therapeutic effect.
[188] In certain embodiments, the PDPs provided herein comprise a chondrocyte-targeting moiety specific for a chondrocyte surface antigen. In some embodiments, the chondrocyte-targeting moiety can be specific for any protein expressed on the surface of an chondrocyte. Exemplary chondrocyte surface antigens are listed in Table 22.
Table 22 Exemplary Chondrocyte Surface Markers
Figure imgf000090_0001
[189] In certain embodiments, the chondrocyte-targeting moiety comprises an antibody or antibody fragment specific for a chondrocyte surface antigen (e.g., an antigen listed in Table 22). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the chondrocyte-targeting moiety comprises an aptamer specific for a chondrocyte surface antigen (e.g., an antigen listed in Table 22).
[190] In some embodiments, the chondrocyte-targeting moiety comprises a ligand that binds to a chondrocyte surface antigen.
Keratinocytes [191] In certain embodiments, the PDPs provided herein are designed to target keratinocytes. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of keratinocyte function would have a therapeutic effect.
[192] In certain embodiments, the PDPs provided herein comprise a keratinocyte-targeting moiety specific for a keratinocyte surface antigen. In some embodiments, the keratinocyte-targeting moiety can be specific for any protein expressed on the surface of an keratinocyte. Exemplary keratinocyte surface antigens are listed in Table 23.
Table 23 Exemplary Keratinocyte Surface Markers
Figure imgf000091_0001
[193] In certain embodiments, the keratinocyte-targeting moiety comprises an antibody or antibody fragment specific for a keratinocyte surface antigen (e.g., an antigen listed in Table 23). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the keratinocyte-targeting moiety comprises an aptamer specific for a keratinocyte surface antigen (e.g., an antigen listed in Table 23). [194] In some embodiments, the keratinocyte-targeting moiety comprises a ligand that binds to a keratinocyte surface antigen.
Neuroglia cell
[195] In certain embodiments, the PDPs provided herein are designed to target neuroglia cells. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of neuroglia cell function would have a therapeutic effect.
[196] In certain embodiments, the PDPs provided herein comprise a neuroglia cell-targeting moiety specific for a neuroglia cell surface antigen. In some embodiments, the neuroglia cell-targeting moiety can be specific for any protein expressed on the surface of a neuroglia cell. Exemplary neuroglia cell surface antigens are listed in Table 24.
Table 24 Exemplary Neuroglia Cell Surface Markers
Figure imgf000092_0001
[197] In certain embodiments, the neuroglia cell-targeting moiety comprises an antibody or antibody fragment specific for a neuroglia cell surface antigen (e.g., an antigen listed in Table 24). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the neuroglia celltargeting moiety comprises an aptamer specific for a neuroglia cell surface antigen (e.g., an antigen listed in Table 24).
[198] In some embodiments, the neuroglia cell-targeting moiety comprises a ligand that binds to a neuroglia cell surface antigen.
Melanocytes
[199] In certain embodiments, the PDPs provided herein are designed to target melanocytes. Such PDPs can be used to treat and/or prevent diseases and/or disorders for which modification of melanocyte function would have a therapeutic effect.
[200] In certain embodiments, the PDPs provided herein comprise a melanocytetargeting moiety specific for a melanocyte surface antigen. In some embodiments, the melanocyte-targeting moiety can be specific for any protein expressed on the surface of an melanocyte. Exemplary melanocyte surface antigens are listed in Table 25.
Table 25 Exemplary Melanocyte Surface Markers
Figure imgf000093_0001
[201] In certain embodiments, the melanocyte-targeting moiety comprises an antibody or antibody fragment specific for a melanocyte surface antigen (e.g., an antigen listed in Table 25). In some embodiments, the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3. In some embodiments, the melanocyte-targeting moiety comprises an aptamer specific for a melanocyte surface antigen (e.g., an antigen listed in Table 25).
[202] In some embodiments, the melanocyte-targeting moiety comprises a ligand that binds to a melanocyte surface antigen.
Improved Cellular Uptake
[203] In certain aspects, provided herein are PDPs that have improved cellular uptake compared to the phage from which they are derived. For example, in certain embodiments provided herein are PDPs that display an internalization moiety endowing them improved cellular uptake. These PDPs can be used to deliver internal nucleic acid payloads and/or external conjugated moieties (e.g., conjugated through a labile/non labile linker or directly) into cells and are thus useful as delivery vessels for the treatment and/or prevention of a disease. In certain embodiments, the PDPs comprise a phage coat protein displaying a internalization moiety.
[204] In certain aspects, PDPs disclosed herein may display a internalization moiety that facilitates internalization of the PDP into a mammalian cell. The term displaying a “internalization moiety” as used herein encompasses internalization moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both. The internalization moiety and its manner of expression and/or linkage is designed to facilitate internalization of the PDP into a cell. This term further includes a internalization moiety comprising a moiety conjugated to the PDP that binds non-covalently to a second target or molecule capable of binding the target cell.
[205] The internalization moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs. Internalization moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
[206] An isolated nucleic acid sequence encoding a internalization moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof. A nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional internalization moiety of the present invention. A nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
[207] An internalization moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
[208] The internalization moiety may be any biological or synthetic substance that facilitates internalization of the PDP. For example and without limitation, internalization moieties may be antibody-based moieties, including, but not limited to: monoclonal antibodies, polyclonal antibodies, and antibody fragments such as recombinant antibody fragments, single-chain antibodies (scFv), single antibody variable domains, and the like (Borrebaeck, 1995; Lo, 2003). Single-chain antibodies are small recognition units consisting of the variable regions of the immunoglobulin heavy (VH) and light (VL) chains which are connected by a synthetic linker sequence. Single antibody domain proteins (dAbs) are minimized antibody fragments comprising either an individual VL domain or an individual VH domain.
[209] Methods of generating monoclonal and polyclonal antibodies are well known in the art. Antibodies may be generated via any one of several known methods, which may employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries, or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique. Antibody fragments may be obtained using methods well known in the art, including, but not limited to by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary (CHO) cell culture or other protein expression systems) of DNA encoding the fragment (Fab')2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments retain the ability to bind to the antigen that is recognized by the intact antibody. An Fv is composed of paired heavy chain variable and light chain variable domains. This association may be non- covalent. Alternatively, as described hereinabove, the variable domains may be linked to generate a single-chain Fv by an intermolecular disulfide bond, or alternately such chains may be cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv is a singlechain Fv. Single-chain Fvs are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains. In addition, techniques may be utilized to change a “murine” antibody to a “human” antibody, without altering the binding specificity of the antibody.
[210] The internalization moiety may be a peptide that facilitates PDP internalization into a mammalian cells. Methods for constructing libraries and using them for screening for ligands having a particular functional property are known in the art.
[211] The internalization moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to facilitate internalization of a PDP into a mammalian cell. For instance, the ligand may include known ligands of cell surface receptors, or any natural or synthetic derivative thereof..
[212] Aptamers are another class of binding agent that can be used as an internalization moiety. Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g, membrane lipid rafts). Aptamers that specifically bind a marker of the cell (e.g., a cell surface moiety or receptor) are useful in the methods of the invention. Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No. 5,660,985; No. 5,567,588; No. 5,683,867; No. 5,637,459; and No. 6,011,020. In some embodiments, the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics. In some embodiments, aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups (e.g, 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
[213] Accordingly, in certain embodiments the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an internalization moiety. In some embodiments, the internalization moiety is covalently attached to the phage coat protein displaying the internalization moiety. In some embodiments, the internalization moiety is non-covalently attached to the phage coat protein displaying the internalization moiety. In some embodiments, the internalization moiety is a fusion protein comprising the phage coat protein and the internalization moiety. In some embodiments, the internalization moiety acts via membrane penetration. In certain embodiments, the internalization moiety is selected from a cell-penetrating peptide (CPP), transbody, cationic polymer, sugar, lipid, inorganic small molecule, and a mammalian viral particle (e.g. AAV).
[214] In some embodiments, the internalization moiety acts via membrane endocytosis. In certain embodiments, the internalization moiety is selected from a high- affinity antibody (e.g. mAb, scFv, VHH), peptide sequence, endogenous ligands (e.g. glucose; growth hormones), sugars (e.g. beta-galNAc), lipids, nucleotide sequence (e.g. aptamers).
[215] In some embodiments, the PDP can be internalized by any mechanism. In certain embodiments the PDP is internalized by a mechanism selected from macropinocytosis, phagocytosis, clathrin-mediated, caveolin-mediated, interaction of hydrophilic lipid membrane and fusogenic moieties, interaction with hydrophobic portion of lipid membrane, and hydrophobic cloaking.
[216] In certain embodiments, the internalization of the PDP is improved compared to the internalization of the phage type from which the PDP is derived. In some embodiments, internalization of the PDP is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more efficient that internalization of the phage type from which the PDP is derived. In some embodiments, internalization can be measured by any means known in the art. For example, in some embodiments internalization is measured via flow cytometry, western blotting, immunofluore scent staining, density gradient separation of cellular compartments, ELISA, or other published assay.
[217] In certain embodiments, internalization moiety is selected from the moieties listed in Table 26.
Table 26. Exemplary Internalization Moieties.
Figure imgf000097_0001
Figure imgf000098_0001
Endosomal Escape
[218] In certain aspects, provided herein are PDPs that have improved endosomal escape compared to the phage from which they are derived. For example, in certain embodiments provided herein are PDPs that display an endosomal escape moiety endowing them improved endosomal escape. These PDPs can be used to deliver internal nucleic acid payloads and/or external conjugated moieties (e.g., conjugated through a labile/non labile linker or directly) into cells and are thus useful as delivery vessels for the treatment and/or prevention of a disease. In certain embodiments, the PDPs comprise a phage coat protein displaying an endosomal escape moiety.
[219] In certain aspects, PDPs disclosed herein may display an endosomal escape moiety that facilitates endosomal escape of the PDP in a mammalian cell. The term displaying a “endosomal escape moiety” as used herein encompasses endosomal escape moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both. The endosomal escape moiety and its manner of expression and/or linkage is designed to facilitate endosomal escape of the PDP in a cell..
[220] In some embodiments, PDP is generated using a helper plasmid encoding a coat protein (e.g., a 10A and/or 10B coat protein) modified to display an endosomal escape moiety. The genetic modification may result in the display of an endosomal escape moiety on the PDP coat. In some embodiments, the genetic modification is in the form of an endosomal escape moiety-coding DNA sequence fused to a gene coding for a coat protein of the PDP (e.g., a 10A and/or 10B coat protein).
[221] The endosomal escape moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs. Endosomal escape moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
[222] An isolated nucleic acid sequence encoding an endosomal escape moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof. A nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional endosomal escape moiety of the present invention. A nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
[223] An endosomal escape moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
[224] The endosomal escape moiety may be any biological or synthetic substance that facilitates endosomal escape of the PDP. For example and without limitation, endosomal escape moieties may be antibody -based moieties, including, but not limited to: monoclonal antibodies, polyclonal antibodies, and antibody fragments such as recombinant antibody fragments, single-chain antibodies (scFv), single antibody variable domains, and the like (Borrebaeck, 1995; Lo, 2003). Single-chain antibodies are small recognition units consisting of the variable regions of the immunoglobulin heavy (VH) and light (VL) chains which are connected by a synthetic linker sequence. Single antibody domain proteins (dAbs) are minimized antibody fragments comprising either an individual VL domain or an individual VH domain.
[225] Methods of generating monoclonal and polyclonal antibodies are well known in the art. Antibodies may be generated via any one of several known methods, which may employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries, or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique. Antibody fragments may be obtained using methods well known in the art, including, but not limited to by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary (CHO) cell culture or other protein expression systems) of DNA encoding the fragment (Fab')2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments retain the ability to bind to the antigen that is recognized by the intact antibody. An Fv is composed of paired heavy chain variable and light chain variable domains. This association may be non- covalent. Alternatively, as described hereinabove, the variable domains may be linked to generate a single-chain Fv by an intermolecular disulfide bond, or alternately such chains may be cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv is a singlechain Fv. Single-chain Fvs are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains. In addition, techniques may be utilized to change a “murine” antibody to a “human” antibody, without altering the binding specificity of the antibody.
[226] The endosomal escape moiety may be a peptide that facilitates PDP endosomal escape in a mammalian cells. Methods for constructing libraries and using them for screening for ligands having a particular functional property are known in the art.
[227] The endosomal escape moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to facilitate endosomal escape of a PDP in a mammalian cell.
[228] Aptamers are another class of binding agent that can be used as an endosomal escape moiety. Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g., membrane lipid rafts). Aptamers that specifically bind a marker of the cell (e.g., a cell surface moiety or receptor) are useful in the methods of the invention. Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No. 5,660,985; No. 5,567,588; No. 5,683,867; No. 5,637,459; and No. 6,011,020. In some embodiments, the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics. In some embodiments, aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups e.g., 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
[229] Accordingly, in certain embodiments the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an endosomal escape moiety. In some embodiments, the endosomal escape moiety is covalently attached to the phage coat protein displaying the endosomal escape moiety. In some embodiments, the endosomal escape moiety is non-covalently attached to the phage coat protein displaying the endosomal escape moiety. In some embodiments, the endosomal escape moiety is a fusion protein comprising the phage coat protein and the endosomal escape moiety.
[230] In certain embodiments, the endosomal escape moiety is an endosomal escape peptide. Non-limiting examples of endosomal escape peptides include, H5WYG peptides, INF7 peptides, and PCI peptides. In certain embodiments the endosomal escape moiety is selected from an endosomal-escape peptide (EEP), transbody, cationic polymer, sugar, lipid, inorganic small molecule, mammalian viral particle (e.g. AAV), and nucleotides.
[231] In certain embodiments, the endosomal escape moiety can act via any mechanism. In some embodiments, the endosomal escape moiety acts via proton sponge and/or osmotic disruption. In certain embodiments, the endosomal escape moiety acts via compartment membrane disruption. In some embodiments, the endosomal escape moiety acts via membrane pore formation.
[232] In certain embodiments, the endosomal escape moiety is an endosomal escape peptide (EEP). EEPs are known for their potential to promote escape of vectors from endosomes by inducing disruption of endosomes (endosmolytic peptides) or by fusion with the endosomal membranes (fusogenic peptides). Accordingly, in one embodiment, the EEP is an endosmolytic peptide. In another embodiment, the EEP is a fusogenic peptide. For example and without limitation, EEPs may include the H5WYG peptide, the INF7 peptide, and the PCI peptide, or functional fragments or variants thereof.
[233] In certain embodiments, the endosomal escape of the PDP is improved compared to the endosomal escape of the phage from which the PDP was derived. In some embodiments, endosomal escape of the PDP is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more efficient than endosomal escape of the phage type from which the PDP is derived. In some embodiments, endosomal escape can be measured by any means known in the art. In some embodiments, endosomal escape is measured via flow cytometry, western blotting, immunofluore scent staining, density gradient separation of cellular compartments, or ELISA.
[234] In some embodiments the endosomal escape moiety is selected from a moiety listed in Table 27.
Table 27. Exemplary Endosomal Escape Moieties
Figure imgf000103_0001
Nuclear Shuttling
[235] The rate of transport of the bacteriophage of the invention to the nucleus may also represent another rate-limiting step to transgene expression. In certain aspects, provided herein are PDPs that have improved nuclear shuttling compared to the phage from which they are derived. For example, in certain embodiments provided herein are PDPs that display a nuclear localization moiety endowing them improved nuclear shuttling. These PDPs can be used to deliver internal nucleic acid payloads into the nuclei of cells and are thus useful as delivery vessels for the treatment of a disease. In certain embodiments, the PDPs comprise a phage coat protein displaying a nuclear localization moiety.
[236] In certain aspects, PDPs disclosed herein may display a nuclear localization moiety that facilitates nuclear shuttling of the PDP in a mammalian cell. The term displaying a “nuclear localization moiety” as used herein encompasses nuclear localization moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both. The nuclear localization moiety and its manner of expression and/or linkage is designed to facilitate nuclear shuttling of the PDP into the nucleus of a cell.
[237] In some embodiments, PDP is generated using a helper plasmid encoding a coat protein (e.g., a 10A and/or 10B coat protein) modified to display an nuclear localization moiety. The genetic modification may result in the display of a nuclear localization moiety on the PDP coat. In some embodiments, the genetic modification is in the form of an nuclear localization moiety-coding DNA sequence fused to a gene coding for a coat protein of the PDP (e.g., a 10A and/or 10B coat protein).
[238] The nuclear localization moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs. Nuclear localization moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
[239] An isolated nucleic acid sequence encoding a nuclear localization moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof. A nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional nuclear localization moiety of the present invention. A nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
[240] A nuclear localization moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
[241] The nuclear localization moiety may be a peptide that facilitates PDP nuclear shuttling in a mammalian cells. Methods for constructing libraries and using them for screening for ligands having a particular functional property are known in the art.
[242] The nuclear localization moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to facilitate nuclear shuttling of a PDP in a mammalian cell.
[243] Aptamers are another class of binding agent that can be used as an nuclear localization moiety. Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g., membrane lipid rafts). Aptamers that specifically bind a marker of the cell (e.g., a cell surface moiety or receptor) are useful in the methods of the invention. Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No. 5,660,985; No. 5,567,588; No. 5,683,867; No. 5,637,459; and No. 6,011,020. In some embodiments, the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics. In some embodiments, aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups (e.g., 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
[244] Accordingly, in certain embodiments the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an nuclear localization moiety. In some embodiments, the nuclear localization moiety is covalently attached to the phage coat protein displaying the nuclear localization moiety. In some embodiments, the nuclear localization moiety is non-covalently attached to the phage coat protein displaying the nuclear localization moiety. In some embodiments, the nuclear localization moiety is a fusion protein comprising the phage coat protein and the nuclear localization moiety.
[245] In certain embodiments the nuclear localization moiety is a nuclear localization signal (NLS). Examples of NLS include, but are not limited to, SV40 T antigen, an optimized SV40 NLS, an optimized short M9 (osM9), a c-Myc NLS, a nucleoplasmin NLS, or a heptamer NLS peptide. In some embodiments, the nuclear localization moiety is selected from a nuclear localization signal peptide (NLS), transbody, cationic polymer, sugar, lipid, inorganic small molecule, mammalian viral particle (e.g. AAV), and nucleotides.
[246] In certain embodiments the nuclear localization moiety can act via any mechanism. In certain embodiments, the nuclear localization moiety acts via direct transport (nuclear pore entry) of the PDP into the nucleus. In some embodiments, the nuclear localization moiety acts via indirect transport (nuclear membrane translocation) of the PDP into the nucleus.
[247] In certain embodiments, the PDP has improved nuclear localization in mammalian cells compared to the nuclear localization of the phage from which the PDP was derived. In some embodiments, nuclear localization of the PDP is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more more efficient than nuclear localization of the phage type from which the PDP is derived. In some embodiments, nuclear localization can be measured by any means known in the art. In certain embodiments, nuclear localization is measured via flow cytometry, western blotting, immunofluorescent staining, density gradient separation of cellular compartments, or ELISA.
[248] In some embodiments, the nuclear localization moiety is selected from a moiety listed in Table 28. Table 28. Exemplary Nuclear Localization Moieties.
Figure imgf000107_0001
Immune Evasion
[249] In certain aspects, provided herein are PDPs that have immune evasion compared to the phage from which they are derived. These PDPs can be used to deliver internal nucleic acid payloads to cells in a subject while eliciting a reduced immune response (or without eliciting an immune response) in the subject and are thus useful as delivery vessels for the treatment and/or prevention of a disease.
[250] In certain embodiments, the PDP comprises a phage coat protein that is modified to enhance immune evasion of the PDP (e.g., a modified 10A and/or 10B coat protein). In some embodiments, the phage coat protein is modified such that the PDP avoids neutralizing antibodies and/or immune cell uptake. In some embodiments, the modified phage coat protein is modified to reduce antibody epitope recognition, to reduce T cell epitope recognition, and/or to reduce surface charge.
[251] In some embodiments, the modified phage coat protein is modified to display an immune evasion moiety. The term displaying a “immune evasion moiety” as used herein encompasses immune evasion moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both. The immune evasion moiety and its manner of expression and/or linkage is designed to reduce or eliminate the immune response elicited by the PDP when it is administered to a subject.
[252] In some embodiments, PDP is generated using a helper plasmid encoding a coat protein (e.g., a 10A and/or 10B coat protein) modified to display an immune evasion moiety. The genetic modification may result in the display of a immune evasion moiety on the PDP coat. In some embodiments, the genetic modification is in the form of an immune evasion moiety-coding DNA sequence fused to a gene coding for a coat protein of the PDP (e.g., a 10A and/or 10B coat protein).
[253] The immune evasion moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs. Immune evasion moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
[254] An isolated nucleic acid sequence encoding a immune evasion moiety can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof. A nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional immune evasion moiety of the present invention. A nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). Methods for inserting foreign coding sequences into a phage gene are well known (see e.g., Sambrook et al., 1989; Brent et al., 2003).
[255] An immune evasion moiety provided herein may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g., Boc or f-Moc chemistry) and solution phase synthesis methods.
[256] The immune evasion moiety may be any biological or synthetic substance that reduces the immune response generated by the PDP when it is administered to a subject. The endosomal escape moiety may be a peptide that inhibits an immune response by a mammalian subject. Methods for constructing libraries and using them for screening for ligands having a particular functional property are known in the art.
[257] The immune evasion moiety may be a polypeptide, a carbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid and the like, which is able to inhibit the immune response elicited by a PDP when administered to a subject.
[258] Aptamers are another class of binding agent that can be used as an immune evasion moiety. Aptamers are nucleic acid-based molecules that bind specific ligands, for example, proteins, peptides, carbohydrates, or lipids (e.g., membrane lipid rafts).
Aptamers that specifically bind a marker of the cell (e.g., a cell surface moiety or receptor) are useful in the methods of the invention. Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patents No. 5,475,096; No. 5,670,637; No. 5,696,249; No. 5,270,163; No. 5,707,796; No. 5,595,877; No. 5,660,985; No. 5,567,588; No. 5,683,867; No. 5,637,459; and No. 6,011,020. In some embodiments, the aptamer comprises a naturally occurring nucleosides (e.g., cytidine, uridine, adenosine, guanine, thymidine, and inosine) and has a sugar-phosphate backbone; however, aptamers may comprise nucleoside analogs to modulate binding kinetics. In some embodiments, aptamers comprise a backbone having at least one modification (e.g., a phosphorothioate, phosphodiester, or phosphorothioate backbone). Additionally, aptamers may be further modified to include additional groups (e.g., 2’ methyl or methoxy ethyl) that may increase aptamer stability and/or otherwise improve aptamer function.
[259] Accordingly, in certain embodiments the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an immune evasion moiety. In some embodiments, the immune evasion moiety is covalently attached to the phage coat protein displaying the immune evasion moiety. In some embodiments, the immune evasion moiety is non-covalently attached to the phage coat protein displaying the immune evasion moiety. In some embodiments, the immune evasion moiety is a fusion protein comprising the phage coat protein and the immune evasion moiety.
[260] In certain embodiments, the immune evasion moiety is an antiinflammatory signal. Examples of anti-inflammatory signals include, but are not limited to IL 10 and other “anti-inflammatory” cytokines
[261] In certain embodiments, the immune evasion moiety is a mammalian selfprotein signal. Examples of mammalian self-protein signals include, but are not limited to, CD47, NKR-P1B- R, PD-1, FcyRIIB, CD22, and Siglec-G.
[262] In certain embodiments, the immune evasion moiety is a capsid masking moiety. Examples of capsid masking moieties include, but are not limited to, polymers (e.g. PEG), sugars (e.g. glycoproteins), lipids, and protein corona (e.g. serum proteins).
[263] In certain embodiments, the PDP provided herein elicits a reduced immune response when administered to a subject as compared to the immune response that occurs when a phage from which the PDP was derived is administered to a subject. In some embodiments, the reduced immune response includes reduced production of anti-PDP antibodies, reduced B cell activation, reduced B cell proliferation, reduced T cell activation, reduced T cell proliferation, reduced expression of inflammatory cytokines (e.g., IFNy, TNFa, IL12, etc.). In some embodiments, the immune response is measured via flow cytometry, western blotting, immunofluorescent staining, density gradient separation of cellular compartments, ELISA, multiplexed cytokine measurement (e.g. Luminex), or other published assay.
[264] In certain embodiments, the phage coat protein modification to enhance immune evasion is selected from the modifications listed in Table 29.
Figure imgf000110_0001
Figure imgf000111_0001
Pharmacokinetics
[265] In certain embodiments, the PDPs provided herein contain phage coat proteins that are modified to improve pharmacokinetic and/or pharmacodynamics properties of the PDP. In some embodiments the one or more pharmacokinetic parameters that are improved include, but are not limited to, area under the plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), agent half-life (t’ ), and volume of distribution at steady state (Vss). In some embodiments, the PDP comprises a phage coat protein that is modified to extend circulation half-life of the PDP. In some embodiments, the PDP comprises a phage coat protein that is modified to increase stability of the PDP in circulation. In some embodiments, the PDP comprises a phage coat protein that is modified to reduce degradation of the PDP. In some embodiments, the PDP comprises a phage coat protein that is modified to reduce clearance of the PDP. In some embodiments, the PDP comprises a phage coat protein that is modified to kidney localization of the PDP. In some embodiments, the PDP comprises a phage coat protein that is modified to reduce off-target binding of the PDP. In some embodiments, the PDP comprises the modified phage coat protein is a 10A and/or 10B coat protein and/or a derivative or fragment thereof.
[266] In certain embodiments, the modified phage coat protein is modified to display pharmacokinetics or pharmacodynamics enhancing moiety. The term displaying a “pharmacokinetics or pharmacodynamics moiety” as used herein pharmacokinetics or pharmacodynamics moieties that are not naturally expressed or displayed on a bacteriophage coat, i.e., the PDP coat, which are either expressed as a part of a fusion coat protein or linked to the PDP by means of genetic modification, chemical (covalent or non-covalent) conjugation, or both. The pharmacokinetics or pharmacodynamics moiety and its manner of expression and/or linkage is designed to facilitate improved pharmacokinetics or pharmacodynamics of the PDP when administered to a subject.
[267] In some embodiments, PDP is generated using a helper plasmid encoding a coat protein (e.g., a 10A and/or 10B coat protein) modified to display an pharmacokinetics or pharmacodynamics moiety. The genetic modification may result in the display of a pharmacokinetics or pharmacodynamics moiety on the PDP coat. In some embodiments, the genetic modification is in the form of an pharmacokinetics or pharmacodynamics moiety-coding DNA sequence fused to a gene coding for a coat protein of the PDP (e.g., a 10A and/or 10B coat protein).
[268] The pharmacokinetics or pharmacodynamics moiety may be linked to the PDP by chemical conjugation described above for chemically conjugating moieties to PDPs. Pharmacokinetics or pharmacodynamics moieties to be chemically conjugated may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis, by methods well known to the skilled artisan.
[269] Accordingly, in certain embodiments the PDPs provided herein comprise a phage coat protein (e.g., a 10A and/or 10B coat protein) displaying an pharmacokinetics or pharmacodynamics enhancing moiety. In some embodiments, the pharmacokinetics or pharmacodynamics enhancing moiety is covalently attached to the phage coat protein displaying pharmacokinetics or pharmacodynamics enhancing moiety. In some
I l l embodiments, the pharmacokinetics or pharmacodynamics enhancing moiety is non- covalently attached to the phage coat protein displaying the pharmacokinetics or pharmacodynamics enhancing moiety. In some embodiments, the pharmacokinetics or pharmacodynamics enhancing moiety is a fusion protein comprising the phage coat protein and the pharmacokinetics or pharmacodynamics enhancing moiety. In certain embodiments, the pharmacokinetics or pharmacodynamics enhancing moiety is selected from a peptide sequence, polymer, sugar, lipid, inorganic small molecule, and nucleotides.
[270] In certain embodiments, the phage coat protein modification that enhances the pharmacokinetics or pharmacodynamics of the PDP is selected from the modifications listed in Table 30.
Table 30. Exemplary Pharmacokinetics Enhancing Modifications.
Figure imgf000113_0001
Figure imgf000114_0001
Expression Systems
[271] Applications of the present disclosure encompass but are not limited to methods and compositions related to expression of an exogenous nucleic acid in a cell that is delivered by a PDP described herein (i.e., as part of the PDP’s nucleic acid payload). In some embodiments, the exogenous nucleic acid delivered by the PDP is configured for stable integration in the genome of a cell. In some embodiments, the stable integration of the exogenous nucleic acid may be at specific targets within the genome of the cell. In some embodiments, the exogenous nucleic acid comprises one or more coding sequences. An exogenous nucleic acid can refer to a nucleic acid that was not originally in a cell and is added from outside the cell, irrespective of whether it comprises a sequence that may already be present in the cell endogenously. An exogenous nucleic acid may be a DNA or an RNA molecule or a hybrid thereof. An exogenous nucleic acid may comprise a sequence encoding a transgene. An exogenous nucleic acid may encode a recombinant protein.
[272] In one aspect, provided herein are methods and compositions for delivery inside a cell, including stable incorporation of one or more nucleic acids, comprising nucleic acid sequences encoding one or more proteins. In some embodiments, provided herein is a method of delivering a composition inside a cell, the composition comprising one or more nucleic acid sequences encoding one or more proteins (e.g., one or more therapeutic proteins described herein).
[273] In certain embodiments, the nucleic acid payloads of the PDPs provided herein comprise nucleic acid sequences encoding one or more proteins for expression in a cell (e.g., a therapeutic protein disclosed herein). In one embodiment, the nucleic acid sequence is designed for stable expression of the one or more proteins or polypeptides encoded by the recombinant nucleic acid. In some embodiments, the stable expression is achieved by incorporation of the nucleic acid sequence within the genome of the cell.
Gene Editing Systems
[274] In certain embodiments, the nucleic acid payload of a PDP provided herein encodes one or more components of a gene editing system. Gene editing systems allow for the targeted modification of a cell’s genome by inserting, deleting and/or modifying sequences within the genome. In certain embodiments, the gene editing system is a CRISPR/Cas system. Typically, CRISPR/Sy stems include at least a Cas nuclease (or nucleic acid encoding a Cas nuclease) and a guide RNA (gRNA) that targets the Cas nuclease to a particular sequence in a cell genome. In certain embodiments, the Cas nuclease is a Cas9 nuclease. A CRISPR/Cas system can also include a template sequence to be inserted into the cell genome at the position targeted by the gRNA.
[275] CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3 -aided processing of pre- crRNA. Subsequently, the Cas9/crRNA/tracrRNA complex endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species (see, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference). Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself. Cas9 nuclease sequences and structures are well known to those of ordinary skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al.. J. J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471 :602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes S. thermophiles, Geobaciullus stearothermophilus, Corynebacterium ulcerous, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquisl, Listeria innocua, Campylobacter jejuni, or Neisseria meningitidis. In some embodiments, the Cas9 ortholog is an saCas9 domain, an spCas9 domain comprising one or more mutations to alter the PAM specificity, or a Cpfl domain. Additional suitable Cas9 nucleases and sequences will be apparent to those of ordinary skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that inactive the DNA cleavage domain, that is, the Cas9 is a nickase and/or a nuclease-inactivated Cas9 protein.
[276] In some embodiments, proteins comprising fragments of Cas9 are encoded by the PDP nucleic acid payload. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA- cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is 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%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.
[277] In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
[278] In some embodiments, Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1. In some embodiments, Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2, (nucleotide); and Uniprot Reference Sequence: Q99ZW2. In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC 017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP 820832.1), Geobacillus stearothermophilus (NCBI Ref: NZ_CP008934.1); Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100.1).
[279] In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of ordinary skill in the art. In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC 017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP 820832.1); Geobacillus stearothermophilus (NCBI Ref: NZ_CP008934.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria, meningitidis (NCBI Ref: YP_002342100.1).
[280] The term “nucleic acid programmable DNA binding protein” or “napDNAbp” refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid (e.g, gRNA), that guides the napDNAbp to a specific nucleic acid sequence, for example, by hybridizing to the target nucleic acid sequence. For example, a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence is has complementary to the guide RNA. In some embodiments, the napDNAbp is a class 2 microbial CRISPR-Cas effector. In some embodiments, the napDNAbp is a Cas9 domain, for example, a nuclease active Cas9, a Cas9 nickase (Cas9n), or a nuclease inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g, dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, and Argonaute. It should be appreciated, however, that nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind RNA. For example, the napDNAbp may be associated with a nucleic acid that guides the napDNAbp to an RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they may not be specifically described in this disclosure.
[281] The term “nucleic acid programmable DNA binding protein” or “napDNAbp” refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence, for example, by hybridizing to the target nucleic acid sequence. For example, a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence is has complementary to the guide RNA. In some embodiments, the napDNAbp is a class 2 microbial CRISPR-Cas effector. In some embodiments, the napDNAbp is a Cas9 domain, for example, a nuclease active Cas9, a Cas9 nickase (Cas9n), or a nuclease inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, and Argonaute. It should be appreciated, however, that nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind RNA. For example, the napDNAbp may be associated with a nucleic acid that guides the napDNAbp to an RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they may not be specifically described in this disclosure.
[282] In some embodiments, the napDNAbp is an “RNA-programmable nuclease” or “RNA-guided nuclease.” The terms are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA(s) that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). Guide RNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. Guide RNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is also used to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as a single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (z.e., directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA and comprises a stem-loop structure. In some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al.. Science 337:816-821 (2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in International Patent Application PCT/US2014/054252, filed September 5, 2014, published as Patent Publication No. WO2015/035139, published March 12, 2015, entitled “Switchable Cas9 Nucleases And Uses Thereof,” and International Patent Application PCT/US2014/054247, filed September 5, 2014, published as Patent Publication No. WO2015/035136, published March 12, 2015, entitled “Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” For example, an extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. In some embodiments, the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (also known as Csnl) from Streptococcus pyogenes (see, c.g, “Complete genome sequence of an Ml strain of Streptococcus pyogenes"' Ferretti J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); “CRISPR RNA maturation by transencoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471 : 602-607 (2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference).
[283] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to target, in principle, any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong et al., Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W.Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227- 229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J.E. et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Research (2013); Jiang, W. et al., RNA- guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).
Base Editing
[284] In various embodiments, the PDPs disclosed herein comprise a nucleic acid payload encoding a gene editing system comprising one or more base editors comprising one or more nucleic acid effector domains.
[285] The term “base editor (BE),” or “nucleobase editor (NBE),” as used herein, refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating a cytosine (C) in DNA. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein fused to a nucleic acid editing domain. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a Cas9 domain (e.g., dCas9 or Cas9n), CasX, CasY, Cpfl, C2cl, C2c2, C2c3, or Argonaute protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a Cas9 nickase (Cas9n) fused to an cytidine deaminase domain. In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to a cytidine deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain, or a dISN domain. In some embodiments, the base editor comprises a CasX protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a CasY protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a Cpfl protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a C2cl protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a C2c2 protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a C2c3 protein fused to a cytidine deaminase domain. In some embodiments, the base editor comprises an Argonaute protein fused to a cytidine deaminase domain. Base editors have been described, e.g., in Patent Publication No. W02017/070632, published April 27, 2017, entitled “Nucleobase Editors and Uses Thereof’, in Patent Publication No. W02018/027078, published February 8, 2018, entitled “Adenosine Base Editors and Uses Thereof’, in Patent Publication No. WO2018/165629, published September 13, 2018, entitled “Cytosine to Guanine Base Editor”, and in Patent Publication No. W02018/176009, published September 27, 2018, entitled “Nucleobase Editors Comprising Nucleic Acid Progrrammable DNA Binding Proteins”; the entire contents of each of which are incorporated by reference herein.
[286] The term “deaminase” or “deaminase domain,” as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism, that does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism.
[287] In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, the cytidine deaminase catalyzes the hydrolytic deamination of cytidine or cytosine in deoxyribonucleic acid (DNA). In some embodiments, the cytidine deaminase or cytidine deaminase domain is a naturally- occurring cytidine deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the cytidine deaminase or cytidine deaminase domain is a variant of a naturally-occurring cytidine deaminase from an organism that does not occur in nature. For example, in some embodiments, the cytidine deaminase or cytidine deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring cytidine deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.
[288] In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, catalyzing the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g. , engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism, such as a bacterium. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium, such as E.coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may be missing one or more N-terminal amino acids relative to a full-length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine. [289] It should be appreciated that, in some embodiments, effector domains may be used in place of any of the deaminases or deaminase domains provided herein. As used herein, an “effector domain” refers to a molecule (e.g., a protein) that regulates a biological activity and/or is capable of modifying a biological molecule (e.g., a protein, or a nucleic acid such as DNA or RNA). In some embodiments, the effector domain is a protein. In some embodiments, the effector domain is capable of modifying a protein (e.g., a histone). In some embodiments, the effector domain is capable of modifying DNA (e.g., genomic DNA). In some embodiments, the effector domain is capable of modifying RNA (e.g., mRNA). In some embodiments, the effector molecule is a nucleic acid editing domain. In some embodiments, the effector molecule is capable of regulating an activity of a nucleic acid (e.g., transcription, and/or translation). Exemplary effector domains include, without limitation, a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.
[290] In some embodiments, the base editor is capable of deaminating an adenosine (A) in DNA. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein fused to a nucleic acid editing domain. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase domain. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to one or more adenosine deaminase domains. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to two adenosine deaminase domains. In some embodiments, the base editor comprises a Cas9 (e.g., dCas9 and Cas9n), CasX, CasY, Cpfl, C2cl, C2c2, C2c3, or Argonaute protein fused to an adenosine deaminase domain. In some embodiments, the base editor comprises a Cas9 nickase (Cas9n) fused to an adenosine deaminase domain. In some embodiments, the base editor comprises a Cas9 nickase (Cas9n) fused to two adenosine deaminase domains. In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase domain. In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to two adenosine deaminase domains. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain, or a dISN domain. In some embodiments, the base editor comprises a CasX protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a CasY protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a Cpfl protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a C2cl protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a C2c2 protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises a C2c3 protein fused to one or more adenosine deaminase domains. In some embodiments, the base editor comprises an Argonaute protein fused to one or more adenosine deaminase domains.
[291] In various embodiments, the nucleic acid effector domain may be any protein, enzyme, or polypeptide (or functional fragment thereof) which is capable of modifying a DNA or RNA molecule. Nucleobase modification moieties can be naturally occurring, or can be recombinant. For example, a nucleobase modification moiety can include one or more DNA repair enzymes, for example, and an enzyme or protein involved in base excision repair (BER), nucleotide excision repair (NER), homology-dependnent recombinational repair (HR), non-homologous end-joining repair (NHEJ), microhomology end-joining repair (MMEJ), mismatch repair (MMR), direct reversal repair, or other known DNA repair pathway. A nucleobase modification moiety can have one or more types of enzymatic activities, including, but not limited to endonuclease activity, polymerase activity, ligase activity, replication activity, proofreading activity. Nucleobase modification moieties can also include DNA or RNA-modifying enzymes and/or mutagenic enzymes, such as, DNA methylases and deaminating enzymes (i.e., deaminases, including cytidine deaminases and adenosine deaminases, all defined above), which deaminate nucleobases leading in some cases to mutagenic corrections by way of normal cellular DNA repair and replication processes. The “nucleic acid effector domain” (c.g, a DNA effector domain or an RNA effector domain) as used herein may also refer to a protein or enzyme capable of making one or more modifications (e.g., deamination of a cytidine residue) to a nucleic acid (e.g., DNA or RNA). Exemplary nucleic acid editing domains include, but are not limited to a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments the nucleic acid editing domain is a deaminase (e.g., an adenosie deaminase and/or a cytidine deaminase). [292] In some embodiments, the PDP comprises a nucleic acid sequence encoding a fusion protein comprising a nucleic acid binding domain and a deaminase domain. For example, an adenosine deaminase (e.g, an adenosine deaminase that deaminates deoxyadenosine in DNA as described herein) and a domain (e.g., a Cas9 or a Cpfl protein) capable of binding to a specific nucleotide sequence. The deamination of an adenosine by an adenosine deaminase can lead to a point mutation, thereby editing the nucleic acid. For example, the adenosine may be converted to an inosine residue, which typically base pairs with a cytosine residue. Such fusion proteins are useful for targeted editing of nucleic acid sequences. Such fusion proteins may be used for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re-introduced into the same or another subject; and for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a subject.
[293] In some embodiments, the nucleobase editors provided herein can be made by fusing together one or more protein domains, thereby generating a fusion protein. In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity (e.g, efficiency, selectivity, and specificity) of the fusion proteins. For example, the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic residue (e.g., H840) maintains the activity of the Cas9 to cleave the nonedited (e.g., non-deaminated) strand containing a T opposite the targeted A. Mutation of the catalytic residue (e.g., DIO to A10) of Cas9 prevents cleavage of the edited strand containing the targeted A residue. Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a T to C change on the non-edited strand.
[294] Some aspects of the disclosure provide adenosine deaminases. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA. The adenosine deaminase may be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of skill in the art will be able to identify the corresponding residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally- occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.
[295] In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase, e.g., a cytidine deaminase (rAPOBECl) which converts a DNA base cytosine to uracil. One such base editor is referred to as “BE1” in the literature. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 fused to a deaminase and further fused to a UGI domain (uracil DNA glycosylase inhibitor, which prevents the subsequent U:G mismatch from being repaired back to a C:G base pair). One such base editor is referred to as “BE2” in the literature. In other embodiments, to improve base editing efficiency, the catalytic His residue at position 840 in the Cas9 HNH domain of BE2 can be restore (resulting in “BE3” as described in the literature), which nicks only the non-edited strand, simulating newly synthesized DNA and leading to the desired U:A product. In other embodiments, the dCas9 is any dCas9 disclosed or described in PCT/US2017/045381 (published as WO 2018/027078), which is incorporated herein by reference in its entirety. The terms “nucleobase editors (NBEs)” and “base editors (BEs)” may be used interchangeably. The term “base editors” encompasses any base editor known or described in the art at the time of this filing, but also the improved base editors described herein. The base editors known in the state of the art which may be modified by the methods and strategies described herein to improve editing efficiency include, for example, BE1, BE2, BE3, or BE4.
[296] In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase. In other embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC 1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a Lamprey CDA1 (pmCDAl) deaminase. In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is from a human. In some embodiments the deaminase is from a rat.
[297] Some exemplary suitable nucleic-acid editing domains, e.g., deaminases and deaminase domains, that can be fused to Cas9 domains according to aspects of this disclosure are provided below. It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).
[298] Some aspects of the disclosure provide cytidine deaminases. In some embodiments, second protein comprises a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain can catalyze a C to U base change. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC 1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3 A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBECl.
[299] In some aspects, a nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting, exemplary Cas9 domains are provided herein. The Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase. In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain may be a Cas9 domain that cuts both strands of a duplexed nucleic acid (e.g., both strands of a duplexed DNA molecule).
[300] In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain may bind to a duplexed nucleic acid molecule e.g., via a gRNA molecule) without cleaving either strand of the duplexed nucleic acid molecule Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure.
[301] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase cleaves the target strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired to (complementary to) a gRNA e.g., an sgRNA) that is bound to the Cas9.
[302] Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region, where the “N” in “NGG” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the G is guanine. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein need to be positioned at a precise location, for example, where a target base is within a 4 base region (e.g, a “deamination window”), which is approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base region. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g, NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.
[303] Some aspects of the disclosure provide fusion proteins comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an adenosine deaminase. In some embodiments, any of the fusion proteins provided herein are base editors. In some embodiments, the napDNAbp is a Cas9 domain, a Cpfl domain, a CasX domain, a CasY domain, a C2cl domain, a C2c2 domain, aC2c3 domain, or an Argonaute domain. In some embodiments, the napDNAbp is any napDNAbp provided herein. Some aspects of the disclosure provide fusion proteins comprising a Cas9 domain and an adenosine deaminase. The Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the adenosine deaminases provided herein.
[304] Some aspects of the disclosure provide fusion proteins that comprise a nucleic acid programmable DNA binding protein (napDNAbp) and at least two adenosine deaminase domains. Without wishing to be bound by any particular theory, dimerization of adenosine deaminases (e.g., in cis or in trans) may improve the ability (e.g., efficiency) of the fusion protein to modify a nucleic acid base, for example to deaminate adenine. In some embodiments, any of the fusion proteins may comprise 2, 3, 4 or 5 adenosine deaminase domains. In some embodiments, any of the fusion proteins provided herein comprise two adenosine deaminases. In some embodiments, any of the fusion proteins provided herein contain only two adenosine deaminases. In some embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminases are any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminases are different. In some embodiments, the first adenosine deaminase is any of the adenosine deaminases provided herein, and the second adenosine is any of the adenosine deaminases provided herein, but is not identical to the first adenosine deaminase.
[305] Some aspects of the disclosure provide methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to correct a point mutation in a target gene. In some embodiments, the disclosure provides methods of using base editors (e.g., any of the fusion proteins provided herein) and gRNAs to generate an A to G and/or T to C mutation in a target gene. In some embodiments, the disclosure provides method for deaminating an adenosine nucleobase (A) in a target gene, the method comprising contacting the target gene with a base editor and a guide RNA bound to the base editor, where the guide RNA comprises a guide sequence that is complementary to a target nucleic acid sequence in the target gene. In some embodiments, the target gene comprises a C to T or G to A mutation. In some embodiments, the C to T or G to A mutation in the target gene impairs function of the target protein encoded by the target gene. In some embodiments, the C to T or G to A mutation in the target gene impairs function of the target protein encoded by the target gene by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%.
[306] In some embodiments, deaminating an adenosine (A) nucleobase complementary to the T corrects the C to T or G to A mutation in the target gene. In some embodiments, the C to T or G to A mutation in the target gene leads to a Cys (C) to Tyr (Y) mutation in the target protein encoded by the target gene. In some embodiments, deaminating the adenosine nucleobase complementary to the T corrects the Cys to Tyr mutation in the target protein. [307] In some embodiments, the guide sequence of the gRNA comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleic acids that are 100% complementary to a target nucleic acid sequence of the target gene. In some embodiments, the base editor nicks the target sequence that is complementary to the guide sequence.
TALE Nucleases (TALENs)
[308] In some aspects, a PDP is provided comprising a nucleic acid sequence encoding a transcription activator- like effector nuclease (TALEN).
[309] The term “Transcriptional Activator-Like Effector,” (TALE) as used herein, refers to proteins comprising a DNA binding domain, which contains a highly conserved 33-34 amino acid sequence comprising a highly variable two-amino acid motif (Repeat Variable Diresidue, RVD). The RVD motif determines binding specificity to a nucleic acid sequence and can be engineered according to methods well known to those of skill in the art to specifically bind a desired DNA sequence. The simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
[310] The term “Transcriptional Activator-Like Element Nuclease,” (TALEN) as used herein, refers to an artificial nuclease comprising a transcriptional activator like effector DNA binding domain fused to a DNA nuclease or cleavage domain, for example, a FokI domain.
[311] TALEN-induced double-strand breaks can result in targeted mammalian gene knockout through non-homologous end joining (NHEJ) or targeted genomic sequence replacement through homology-directed repair (HDR) using an exogenous DNA template.
[312] In some embodiments, the nuclease domain, also sometimes referred to as a nucleic acid cleavage domain is a non-specific cleavage domain, e.g., a FokI nuclease domain. In some embodiments, the nuclease domain is monomeric and must dimerize or multimerize in order to cleave a nucleic acid. Homo- or heterodimerization or multimerization of TALEN monomers typically occurs via binding of the monomers to binding sequences that are in sufficiently close proximity to allow dimerization, e.g., to sequences that are proximal to each other on the same nucleic acid molecule (e.g., the same double-stranded nucleic acid molecule).
[313] In some embodiments, a TALEN is provided herein that comprises a canonical N-terminal domain, a TALE repeat array, a modified C-terminal domain, and a nuclease domain. In some embodiments, a TALEN is provided herein that comprises a modified N-terminal domain, a TALE repeat array, a canonical C-terminal domain, and a nuclease domain. In some embodiments, a TALEN is provided herein that comprises a modified N-terminal domain, a TALE repeat array, a modified C-terminal domain, and a nuclease domain. In some embodiments, the nuclease domain is a FokI nuclease domain. In some embodiments, the FokI nuclease domain is a homodimeric FokI domain, or a Fokl-EL, Fokl-KK, Fokl-ELD, or Fokl-KKR domain.
[314] In some embodiments, the TALEN cleaves the target sequence upon dimerization. In some embodiments, a TALEN provided herein cleaves a target site within an allele that is associated with a disease or disorder. In some embodiments, the TALEN cleaves a target site the cleavage of which results in the treatment or prevention of a disease or disorder.
[315] In some embodiments, the canonical N-terminal domain and/or the canonical C-terminal domain is modified to replace an amino acid residue that is positively charged at physiological pH with an amino acid residue that is not charged or is negatively charged to arrive at the isolated N-terminal and/or C-terminal domain provided herein. In some embodiments, the modification includes the replacement of a positively charged residue with a negatively charged residue. In some embodiments, the modification includes the replacement of a positively charged residue with a neutral (uncharged) residue. In some embodiments, the modification includes the replacement of a positively charged residue with a residue having no charge or a negative charge. In some embodiments, the net charge of the isolated N-terminal domain and/or of the isolated C- terminal domain provided herein is less than or equal to +10, less than or equal to +9, less than or equal to +8, less than or equal to +7, less than or equal to +6, less than or equal to +5, less than or equal to +4, less than or equal to +3, less than or equal to +2, less than or equal to +1, less than or equal to 0, less than or equal to -1, less than or equal to -2, less than or equal to -3, less than or equal to -4, or less than or equal to -5, or less than or equal to -10 at physiological pH. In some embodiments, the net charge of the isolated N- terminal domain and/or of the isolated C-terminal domain is between +5 and -5, between +2 and -7, between 0 and -5, between 0 and -10, between -1 and -10, or between -2 and -15 at physiological pH. In some embodiments, the net charge of the isolated N- terminal TALE domain and/or of the isolated C-terminal TALE domain is negative. In some embodiments, an isolated N-terminal TALE domain and an isolated C-terminal TALE domain are provided and the net charge of the isolated N-terminal TALE domain and of the isolated C-terminal TALE domain, together, is negative. In some embodiments, the net charge of the isolated N-terminal TALE domain and/or of the isolated C-terminal TALE domain is neutral or slightly positive (e.g., less than +2 or less than +1 at physiological pH). In some embodiments, an isolated N-terminal TALE domain and an isolated C-terminal TALE domain are provided, and the net charge of the isolated N-terminal TALE domain and of the isolated C-terminal TALE domain, together, is neutral or slightly positive (e.g., less than +2 or less than +1 at physiological pH).
[316] In some embodiments, the isolated N-terminal domain and/or the isolated C- terminal domain provided herein comprise(s) an amino acid sequence that differs from the respective canonical domain sequence in that at least one cationic amino acid residue of the canonical domain sequence is replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 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 cationic amino acid(s) is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH in the isolated N-terminal domain and/or in the isolated C-terminal domain provided. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 cationic amino acid(s) is/are replaced with an amino acid residue that exhibits no charge or a negative charge at physiological pH in the isolated N-terminal domain and/or in the isolated C-terminal domain.
[317] In some embodiments, the cationic amino acid residue is arginine (R), lysine (K), or histidine (H). In some embodiments, the cationic amino acid residue is R or H. In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is glutamine (Q), glycine (G), asparagine (N), threonine (T), serine (S), aspartic acid (D), or glutamic acid (E). In some embodiments, the amino acid residue that exhibits no charge or a negative charge at physiological pH is Q. In some embodiments, at least one lysine or arginine residue is replaced with a glutamine residue in the isolated N-terminal domain and/or in the isolated C-terminal domain. [318] In some embodiments, an isolated N-terminal TALE domain is provided that is a truncated version of the canonical N-terminal domain. In some embodiments, an isolated C-terminal TALE domain is provided that is a truncated version of the canonical C-terminal domain. In some embodiments, the truncated N-terminal domain and/or the truncated C-terminal domain comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% of the residues of the canonical domain. In some embodiments, the truncated C-terminal domain comprises less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, or less than 20 amino acid residues. In some embodiments, the truncated C-terminal domain comprises 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 residues. In some embodiments, an isolated N-terminal TALE domain and/or an isolated C-terminal domain is provided herein that is/are truncated and comprise(s) one or more amino acid replacement(s).
Inhibitory Nucleic Acids
[319] In certain embodiments, the PDPs provided herein comprise a payload encoding interfering nucleic acid molecules that selectively target a mRNA encoded by a gene whose expression is to be suppressed. Interfering nucleic acids generally include a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence. Interfering RNA molecules include, but are not limited to, antisense molecules, siRNA molecules, single-stranded siRNA molecules, miRNA molecules and shRNA molecules.
[320] Typically at least 17, 18, 19, 20, 21, 22 or 23 nucleotides of the complement of the target mRNA sequence are sufficient to mediate inhibition of a target transcript. Perfect complementarity is not necessary. In some embodiments, the interfering nucleic acid molecule is double-stranded RNA. The double-stranded RNA molecule may have a
2 nucleotide 3’ overhang. In some embodiments, the two RNA strands are connected via a hairpin structure, forming a shRNA molecule. shRNA molecules can contain hairpins derived from microRNA molecules. For example, an RNAi vector can be constructed by cloning the interfering RNA sequence into a pCAG-miR30 construct containing the hairpin from the miR30 miRNA. RNA interference molecules may include DNA residues, as well as RNA residues.
[321] In some embodiments, the interfering nucleic acid molecule is a siRNA molecule. Such siRNA molecules should include a region of sufficient homology to the target region, and be of sufficient length in terms of nucleotides, such that the siRNA molecule down-regulate target RNA. The term “ribonucleotide” or “nucleotide” can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions. It is not necessary that there be perfect complementarity between the siRNA molecule and the target, but the correspondence must be sufficient to enable the siRNA molecule to direct sequencespecific silencing, such as by RNAi cleavage of the target RNA. In some embodiments, the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.
[322] Each strand of an siRNA molecule can be equal to or less than 35, 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the strand is at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. In some embodiments, siRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, such as one or two 3' overhangs, of 2-3 nucleotides.
[323] A “small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
[324] In some embodiments, shRNAs are about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, or are about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded shRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, or about 18-22, 19-20, or 19-21 base pairs in length). shRNA duplexes may comprise 3’ overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides on the antisense strand and/or 5 ’-phosphate termini on the sense strand. In some embodiments, the shRNA comprises a sense strand and/or antisense strand sequence of from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), or from about 19 to about 40 nucleotides in length (e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length), or from about 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).
[325] Non-limiting examples of shRNA include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a doublestranded polynucleotide molecule with a hairpin secondary structure having self- complementary sense and antisense regions. In some embodiments, the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.
[326] Additional embodiments related to the shRNAs, as well as methods of designing and synthesizing such shRNAs, are described in U.S. patent application publication number 2011/0071208, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
[327] In some embodiments, provided herein are micro RNAs (miRNAs). miRNAs represent a large group of small RNAs produced naturally in organisms, some of which regulate the expression of target genes. miRNAs are formed from an approximately 70 nucleotide single-stranded hairpin precursor transcript by Dicer. miRNAs are not translated into proteins, but instead bind to specific messenger RNAs, thereby blocking translation. In some instances, miRNAs base-pair imprecisely with their targets to inhibit translation.
[328] In some embodiments, antisense oligonucleotide compounds are provided herein. In certain embodiments, the degree of complementarity between the target sequence and antisense targeting sequence is sufficient to form a stable duplex. The region of complementarity of the antisense oligonucleotides with the target RNA sequence may be as short as 8-11 bases, but can be 12-15 bases or more, e.g., 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers in between these ranges. An antisense oligonucleotide of about 14-15 bases is generally long enough to have a unique complementary sequence.
[329] In certain embodiments, antisense oligonucleotides may be 100% complementary to the target sequence, or may include mismatches, e.g., to improve selective targeting of allele containing the disease-associated mutation, as long as a heteroduplex formed between the oligonucleotide and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo. Hence, certain oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 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%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence. Oligonucleotide backbones that are less susceptible to cleavage by nucleases are discussed herein. Mismatches, if present, are typically less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
Manufacturing Phage Derived Particles
[330] In some aspects, methods of manufacturing a PDP are provided. The assembly of the T7 is now well enough understood to allow for manipulations that have had significant impact on the ease of dsDNA isolation and E. coli infection/transformation of foreign sequences carried by phage. Phagemid systems and helper phage systems have borrowed or altered T7 origins of replication that allow for the packaging of foreign sequences within a phage body.
[331] Generally, a template phagemid with a T7 origin insert is co-infected with a helper phage that will express all the necessary phage assembly proteins to yield PDP carrying the sequence from the template phagemid. These PDP can be produced in bacterial culture at high yields and purified for acquisition of desired dsDNA sequences.
[332] In some embodiments, the dsDNA sequences of interest may be directly integrated into different areas of the T7 phage genome and packaged into T7 phage particles. The dsDNA sequences of interest may be placed at any site of the T7 genome that does not disrupt any protein coding genes, sequence elements related to genome replication, and/or sequence elements related to T7 packaging.
[333] In some embodiments, packageable dsDNA are generated with chosen sizes (i.e. 100s to 1000s base-pairs), which are much shorter than the ~40 kb observed in wildtype T7 phage. These packagable genomes, known as phagemids, can be of varying lengths and contain the T7 phage packaging signal, T7 origin of replication, and terminal repeats required for replication, but none of the T7 phage protein genes. In this case, the T7 phage particles may preferentially package the phagemid over the wild-type T7 phage genome by sequence manipulation of the T7 packaging signal in the wild-type T7 phage genome.
Pharmaceutical Compositions
[334] Pharmaceutical compositions comprising a PDP of the present disclosure are provided herein. Such compositions further comprise a pharmaceutically acceptable carrier and can be supplied a sterile pharmaceutical composition. This composition can be in any suitable form (depending upon the desired method of administering it to a patient).
[335] Administration of the pharmaceutical composition may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. The most suitable route for administration in any given case will depend on the particular PDP, the subject, and the nature and severity of the disease and the physical condition of the subject.
[336] Pharmaceutical compositions can be conveniently presented in unit dose forms containing a predetermined amount of a PDP of the disclosure per dose. Such a unit can contain for example but without limitation 5 mg to 5 g, for example 10 mg to 1 g, or 20 to 50 mg. Pharmaceutically acceptable carriers for use in the disclosure can take a wide variety of forms depending, e.g., on the condition to be treated or route of administration.
[337] Therapeutic formulations of the PDPs of the disclosure can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the PDPs having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are referred to herein as “carriers”), i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be nontoxic to the recipients at the dosages and concentrations employed.
[338] Buffering agents help to maintain the pharmaceutical composition’s pH in the range that approximates physiological conditions. They can be present at concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof such as citrate buffers (e.g, monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g, succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-di sodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid- sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used.
[339] Preservatives can be added to retard microbial growth, and can be added in amounts ranging from 0.2%4% (w/v). Suitable preservatives for use with the present disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3 -pentanol. Isotonicifiers sometimes known as “stabilizers” can be added to ensure isotonicity of liquid compositions of the present disclosure and include polyhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or fewer); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccacharides such as raffinose; and polysaccharides such as dextran. Stabilizers can be present in the range from 0.1 to 10,000 weights per part of weight active protein.
[340] Non-ionic surfactants or detergents (also known as “wetting agents”) can be added to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.). Non- ionic surfactants can be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, for example about 0.07 mg/ml to about 0.2 mg/ml.
[341] Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.
[342] The formulation herein can also contain a second therapeutic agent in addition to the PDP of the disclosure.
[343] The dosing schedule for subcutaneous administration can vary from once a month to daily depending on a number of clinical factors, including the type of disease, severity of disease, and the patient's sensitivity to the PDP. [344] The dosage of a PDP of the disclosure to be administered will vary according to the particular PDP, the type of disease, the subject, and the nature and severity of the disease, the physical condition of the subject, the therapeutic regimen (e.g., whether a second therapeutic agent is used), and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art.
INCORPORATION BY REFERENCE
[345] All publications and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. Equivalents
EQUIVALENTS
[346] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:
1. A phage derived particle (PDP) comprising phage coat proteins encapsulating a nucleic acid payload, wherein the nucleic acid payload comprises a sequence encoding an therapeutic agent, wherein the PDP comprises a modification that conveys improved cell or tissue specificity, improved endosomal escape, improved nuclear shuttling, improved immune evasion, improved pharmacokinetics, and/or improved mammalian expression compared to the wild-type phage from which the PDP is derived; wherein the PDP is derived from an icosahedral phage.
2. The PDP of claim 1, wherein the PDP is derived from family Podoviridae .
3. The PDP of claim 1 or 2, wherein the PDP is derived from phages T7, T3, 029, or
P22.
4. The PDP of any one of claims 1-3, wherein the PDP is derived from phage T7.
5. The PDP of any one of claims 1-4, wherein the therapeutic agent is a therapeutic peptide.
6. The PDP of any one of claims 1-4, wherein the therapeutic agent is an inhibitory RNA.
7. A PDP of any one of claims 1-4, wherein the therapeutic agent comprises one or more components of a CRISPR/Cas system.
8. The PDP of claim 7, wherein the one or more components of a CRISPR/Cas system comprises a Cas nuclease.
9. The PDP of claim 8, wherein the Cas nuclease is a Cas9 nuclease.
10. The PDP of any one of claims 7-9, wherein the one or more components of the
CRISPR/Cas system comprises a guide RNA (gRNA).
11. The PDP of claim 10, wherein the gRNA targets a safe harbor locus.
12. The PDP of claim 11, wherein the safe harbor locus is selected from the safe harbor loci listed in Table 2.
13. The PDP of any one of claims 7-12, wherein the one or more components of the CRISPR/Cas system comprises a template nucleic acid sequence.
14. The PDP of any one of claims 1-13, further comprising a phage coat protein displaying a cell-targeting moiety.
15. The PDP of claim 13, wherein the cell-targeting moiety targets a cell type selected from erythrocytes, granulocytes, agranulocytes, platelets, neurons, neuroglial cells, skeletal muscle cells, cardiac muscle cells, smooth muscle cells, chondrocytes, osteoblasts, osteoclasts, osteocytes, lining cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, epithelial cells (e.g. hepatocytes), endothelial cells, white adipocytes, brown adipocytes, spermatozoa, ova, exocrine and endocrine secretory cells (e.g. pancreatic islet cells), embryonic stem cells, adults stem cells, extracellular matrix cells (e.g. fibroblasts).
16. The PDP of claim 14 or 15, wherein the phage coat protein displaying a celltargeting moiety is a 10A and/or 10B coat protein and/or a derivative or fragment thereof.
17. The PDP of any one of claims 14-16, wherein the cell -targeting moiety comprises an antibody or antibody fragment specific for a cell surface antigen.
18. The PDP of claim 17, wherein the antibody or antibody fragment is an Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabody, single-chain antibody, NANOBODIES®, or isolated CDRH3.
19. The PDP of any one of claims 14-18, wherein the cell -targeting moiety is covalently attached to the phage coat protein displaying the cell-targeting moiety.
20. The PDP of any one of claims 14-18, wherein the cell-targeting moiety is non- covalently attached to the phage coat protein displaying the cell-targeting moiety.
21. The PDP of any one of claims 14-18, wherein the phage coat protein displaying the cell-targeting moiety is a fusion protein comprising the phage coat protein and the cell-targeting moiety.
22. The PDP of any one of claims 1-21, wherein the PDP comprises at least 300 bases on non-phage DNA.
23. The PDP of any one of claims 1-22, wherein at least one PDP coat proteins comprises a chemical or structural modification as compared the coat proteins of the phage from which the PDP is derived.
24. The PDP of claim 23, wherein one or more modified PDP coat proteins comprises a 10A and/or 10B coat protein.
25. The PDP of any one of claims 1-24, further comprising a phage coat protein displaying an internalization moiety.
26. The PDP of claim 25, wherein the phage coat protein displaying an internalization moiety is a 10A and/or 10B coat protein and/or a derivative or fragment thereof.
27. The PDP of claim 25 or 26, wherein the internalization moiety is covalently attached to the phage coat protein displaying the internalization moiety.
28. The PDP of claim 25 or 26, wherein the internalization moiety is non-covalently attached to the phage coat protein displaying the internalization moiety.
29. The PDP of claim 25 or 26, wherein the coat protein displaying the internalization moiety is a fusion protein comprising the phage coat protein and the internalization moiety.
30. The PDP of any one of claims 25-29, wherein the internalization moiety acts via membrane penetration.
31. The PDP of claim 30, wherein the internalization moiety is selected from a cellpenetrating peptide (CPP), transbody, cationic polymer, sugar, lipid, inorganic small molecule, and a mammalian viral particle (e.g. AAV).
32. The PDP of any one of claims 25-29, wherein the internalization moiety acts via membrane endocytosis.
33. The PDP of claim 32, wherein the internalization moiety is selected from a high- affinity antibody (e.g. mAb, scFv, VHH), peptide sequence, endogenous ligands (e.g. glucose; growth hormones), sugars (e.g. beta-galNAc), lipids, nucleotide sequence (e.g. aptamers).
34. The PDP of any one of claims 25-33, wherein the internalization moiety is selected from the moieties listed in Table 26.
35. The PDP of any one of claims 25-34, wherein the internalization of the PDP is improved compared to the internalization of the phage type from which the PDP is derived.
36. The PDP of claim 35, wherein internalization is measured via flow cytometry, western blotting, immunofluorescent staining, density gradient separation of cellular compartments, ELISA, or other published assay.
37. The PDP of any one of claims 25-36, wherein the PDP is internalized by a mechanism selected from macropinocytosis, phagocytosis, clathrin-mediated, caveolin- mediated, interaction of hydrophilic lipid membrane and fusogenic moieties, interaction with hydrophobic portion of lipid membrane, and hydrophobic cloaking.
38. The PDP of any one of claims 1-37, further comprising a phage coat protein displaying an endosomal escape moiety.
39. The PDP of claim 38, wherein the phage coat protein displaying an endosomal escape moiety is a 10A and/or 10B coat protein and/or a derivative or fragment thereof.
40. The PDP of claim 38 or 39, wherein the endosomal escape moiety is covalently attached to the phage coat protein displaying the endosomal escape moiety.
41. The PDP of claim 38 or 39, wherein the endosomal escape moiety is non- covalently attached to the phage coat protein displaying the endosomal escape moiety.
42. The PDP of claim 38 or 39, wherein the coat protein displaying the endosomal escape moiety is a fusion protein comprising the phage coat protein and the endosomal escape moiety.
43. The PDP of any one of claims 38-42, wherein the endosomal escape moiety is an endosomal escape peptide.
44. The PDP of claim 43, wherein the endosomal escape peptide is a H5WYG peptide, INF7 peptide, or PCI peptide.
45. The PDP of any one of claims 38-42, wherein the endosomal escape moiety acts via proton sponge and/or osmotic disruption.
46. The PDP of any one of claims 38-42, wherein the endosomal escape moiety acts via compartment membrane disruption.
47. The PDP of any one of claims 38-42, wherein the endosomal escape moiety acts via membrane pore formation.
48. The PDP of any one of claims 38-47, the endosomal escape moiety is selected from an endosomal-escape peptide (EEP), transbody, cationic polymer, sugar, lipid, inorganic small molecule, mammalian viral particle (e.g. AAV), and nucleotides.
49. The PDP of any one of claims 38-48, wherein the endosomal escape of the PDP is improved compared to the endosomal escape of the phage from which the PDP was derived.
50. The PDP of claim 49, wherein endosomal escape is measured via flow cytometry, western blotting, immunofluorescent staining, density gradient separation of cellular compartments, or ELISA.
51. The PDP of any one of claims 38-50, wherein the endosomal escape moiety is selected from a moiety listed in Table 27.
52. The PDP of any one of claims 1-51, further comprising a phage coat protein displaying a nuclear localization moiety.
53. The PDP of claim 52, wherein the phage coat protein displaying a nuclear localization moiety is a 10A and/or 10B coat protein and/or a derivative or fragment thereof.
54. The PDP of claim 52 or 53, wherein the nuclear localization moiety is covalently attached to the phage coat protein displaying the nuclear localization moiety.
55. The PDP of claim 52 or 53, wherein the nuclear localization moiety is non- covalently attached to the phage coat protein displaying the nuclear localization moiety.
56. The PDP of claim 52 or 53, wherein the coat protein displaying the nuclear localization moiety is a fusion protein comprising the phage coat protein and the nuclear localization moiety.
57. The PDP of any one of claims 52-54, wherein the nuclear localization moiety is a nuclear localization signal (NLS).
58. The PDP of claim 57, wherein the NLS is an NLS peptide from SV40 T antigen, an optimized SV40 NLS, an optimized short M9 (osM9), a c-Myc NLS, a nucleoplasmin NLS, or a heptamer NLS peptide.
59. The PDP of any one of claims 52-58, wherein the nuclear localization moiety acts via direct transport (nuclear pore entry) of the PDP into the nucleus.
60. The PDP of any one of claims 52-58, wherein the nuclear localization moiety acts via indirect transport (nuclear membrane translocation) of the PDP into the nucleus.
61. The PDP of any one of claims 52-58, wherein the nuclear localization moiety is selected from a nuclear localization signal peptide (NLS), transbody, cationic polymer, sugar, lipid, inorganic small molecule, mammalian viral particle (e.g. AAV), and nucleotides.
62. The PDP of any one of claims 52-61, wherein the PDP has improved nuclear localization in mammalian cells compared to the nuclear localization of the phage from which the PDP was derived.
63. The PDP of claim 62, wherein nuclear localization is measured via flow cytometry, western blotting, immunofluorescent staining, density gradient separation of cellular compartments, or ELISA.
64. The PDP of any one of claims 52-63, wherein the nuclear localization moiety is selected from a moiety listed in Table 28.
65. The PDP of any one of claims 1-64, further comprising a phage coat protein that is modified to enhance immune evasion of the PDP.
66. The PDP of claim 65, wherein the modified phage coat protein is a 10A and/or 10B coat protein and/or a derivative or fragment thereof.
67. The PDP of claims 65 or 66, wherein the phage coat protein is modified such that the PDP avoids neutralizing antibodies and/or immune cell uptake.
68. The PDP of any one of claims 65-67, wherein the modified phage coat protein is modified to reduce antibody epitope recognition, to reduce T cell epitope recognition, and/or to reduce surface charge.
69. The PDP of any one of claims 65 to 69, wherein the modified phage coat protein is modified to display an immune evasion moiety.
70. The PDP of claim 69, wherein the immune evasion moiety is covalently attached to the phage coat protein displaying the immune evasion moiety.
71. The PDP of claim 69, wherein the immune evasion moiety is non-covalently attached to the phage coat protein displaying the immune evasion moiety.
72. The PDP of claim 69, wherein the coat protein displaying the immune evasion moiety is a fusion protein comprising the phage coat protein and immune evasion moiety.
73. The PDP of any one of claims 69-72, wherein the immune evasion moiety is selected from an anti-inflammatory signal, a mammalian self-protein signal, and a capsid masking moiety.
74. The PDP of claim 73, wherein the mammalian self-protein signal is CD47.
75. The PDP of claim 73, wherein the capsid masking moiety is selected from a polymer (e.g. PEG), sugars (e.g. glycoproteins), lipids, and protein corona (e.g. serum proteins).
76. The PDP of any one of claims 69-75, wherein the PDP elicits a reduced immune response when administered to a subject as compared to the immune response that occurs when a phage from which the PDP was derived is administered to a subject.
77. The PDP of claim 76, wherein the immune response is measured via flow cytometry, western blotting, immunofluorescent staining, density gradient separation of cellular compartments, ELISA, multiplexed cytokine measurement (e.g. Luminex), or other published assay and via in vivo animal studies e.g. in mice and/or non-human primates.
78. The PDP of any one of claims 69-77, wherein the phage coat protein modification to enhance immune evasion is selected from the modifications listed in Table 29.
79. The PDP of any one of claims 1-78, further comprising a phage coat protein that is modified to extend or reduce circulation half-life of the PDP.
80. The PDP of any one of claims 1-79, further comprising a phage coat protein that is modified to increase or reduce stability of the PDP in circulation.
81. The PDP of any one of claims 1-80, further comprising a phage coat protein that is modified to reduce or increase degradation of the PDP.
82. The PDP of any one of claims 1-87, further comprising a phage coat protein that is modified to reduce or increase clearance of the PDP.
83. The PDP of any one of claims 1-82, further comprising a phage coat protein that is modified to reduce or increase kidney localization of the PDP.
84. The PDP of any one of claims 1-83, further comprising a phage coat protein that is modified to reduce or increase off-target binding of the PDP.
85. The PDP of any one of claims 79-84, wherein the modified phage coat protein is a 10A and/or 10B coat protein and/or a derivative or fragment thereof.
86. The PDP of any one of claims 79 to 85, wherein the modified phage coat protein is modified to display pharmacokinetics or pharmacodynamics enhancing moiety.
87. The PDP of claim 86, wherein the pharmacokinetics or pharmacodynamics enhancing moiety is covalently attached to the phage coat protein displaying the pharmacokinetics or pharmacodynamics enhancing moiety.
88. The PDP of claim 86, wherein the pharmacokinetics or pharmacodynamics or pharmacodynamics enhancing is non-covalently attached to the phage coat protein displaying the pharmacokinetics or pharmacodynamics enhancing moiety.
89. The PDP of claim 86, wherein the coat protein displaying the pharmacokinetics or pharmacodynamics enhancing moiety is a fusion protein comprising the phage coat protein and pharmacokinetics or pharmacodynamics enhancing moiety.
90. The PDP of any one of claims 86 to 89, wherein the pharmacokinetics or pharmacodynamics enhancing moiety is selected from a peptide sequence, polymer, sugar, lipid, inorganic small molecule, and nucleotides.
91. The PDP of any one of claims 79-90, wherein the phage coat protein modification is selected from the modifications listed in Table 30.
92. The PDP of any one of claims 1-91, wherein the nucleic acid payload is a linear double stranded DNA (dsDNA) construct comprising DNA secondary structures that enhance expression in a mammalian system.
93. The PDP of any one of claims 1-92, wherein the nucleic acid payload is a linear double stranded DNA (dsDNA) construct.
94. The PDP of claim 93, wherein the dsDNA construct comprises a DNA sequence element that enhances expression and/or specificity in mammalian cells.
95. The PDP of claim 93 or 94, wherein the DNA sequence element is selected from a promoter, an enhancer, a silencer, an insulator, an untranslated region, and a microRNA binding site.
96. The PDP of any one of claims 93-95, wherein the dsDNA construct comprises a coding region that is codon optimized for mammalian expression.
97. The PDP of any one of claims 93-96, wherein the dsDNA construct comprises a terminator sequence.
98. The PDP of any one of claims 93-97, wherein the episomal expression of the dsDNA construct is durable in slow and/or non-dividing cells (e.g., lasting at least 7 days).
99. The PDP of any one of claims 94-98, wherein the DNA sequence element is selected from the DNA sequence elements listed in Table 1.
100. A method of treating a disease in a subject in need thereof comprising administering to the subject a PDP of any one of claims 1-99.
101. A method of modifying the genome of a cell in a subject, the method comprising administering to the subject a composition comprising a PDP of any one of claims 1-99.
102. A method of inducing expression of a therapeutic peptide in a cell in a subject, the method comprising administering to the subject a composition comprising a PDP of any one of claims 1-99.
103. A method of inhibiting expression of a gene in a cell in a subject, the method comprising administering to the subject a composition comprising a PDP of any one of claims 1-99.
104. A system for producing a PDP of any one of claims 1-99 from a prokaryotic host comprising:
(i) a phage vector comprising a packaging signal for replication of the vector into the nucleic acid payload of the PDP, wherein the nucleic acid payload is a linear double stranded DNA (dsDNA); and
(ii) a second vector comprising nucleic acid sequences encoding the phage coat proteins of the PDP.
105. A method of making a PDP, the method comprising delivering into a prokaryotic cell the system of claim 104 and culturing the prokaryotic cell under conditions such that it produces the PDP.
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