WO2011039646A9 - Targeting of papilloma virus gene delivery particles - Google Patents

Targeting of papilloma virus gene delivery particles Download PDF

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WO2011039646A9
WO2011039646A9 PCT/IB2010/002654 IB2010002654W WO2011039646A9 WO 2011039646 A9 WO2011039646 A9 WO 2011039646A9 IB 2010002654 W IB2010002654 W IB 2010002654W WO 2011039646 A9 WO2011039646 A9 WO 2011039646A9
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vlp
cells
cancer
vlps
targeting
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PCT/IB2010/002654
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WO2011039646A2 (en
WO2011039646A3 (en
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Audrey Carpentier
Pierre L. Coursaget
Eva Alvarez
Antoine A. Touze
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Inserm (Institut National De La Sante Et De La Recherche Medicale)
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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20023—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/20045—Special targeting system for viral vectors

Definitions

  • the invention relates to papilloma virus particles (VLP) for delivering therapeutic or other agents.
  • VLP papilloma virus particles
  • chemotherapeutic agents are far from ideal because they have been selected for their activity against proliferating cells, and they do not discriminate between tumor cells and normal cells undergoing rapid division. Consequently, chemotherapy often causes specific organ toxicities, and produces a series of adverse side effects, including hair and nail loss, mouth ulcers, and sickness.
  • aspects of the invention relate to drug delivery methods and compositions. Aspects of the invention are based on novel papilloma virus particles that can encapsulate agents to be delivered to a subject (e.g. , a human patient). The particles can increase delivery efficiency and/or targeting. Aspects of the invention relate to the discovery that human papilloma virus (HPV)-based virus-like particles (VLPs) can be redirected to tissues and cells that are not normally infected by wild-type HPV. In certain embodiments, HPV-based VLP can be modified to specifically change or alter the tropism of the modified VLP to redirect the VLP to tissues and/or cells of interest.
  • HPV human papilloma virus
  • VLPs virus-based virus-like particles
  • aspects of the invention relate to HPV-based virus-like particles (VLPs) comprising LI or LI and L2 viral capsid proteins that have been modified to deliver an agent to a specific target site, tissue or cell in a subject (e.g. , a human subject, or an animal subject).
  • VLPs HPV-based virus-like particles
  • aspects of the invention provide VLPs that are adapted for delivering drugs (for example, cytotoxins or other drugs) and/or nucleic acids (for example siRNA, antisense R A and others) to one or more target areas in a subject.
  • viral capsid proteins comprise targeting peptides, such as heterologous protein fragments or peptide sequences that may be derived from mammals (e.g. , humans) or peptide sequences that may be derived from infectious agents, such as e.g. , viruses, bacteria, parasites.
  • the protein fragments or peptide sequences comprising targeting peptides provide means to specifically target VLPs in vivo to specific tissues or cells expressing antigens, such as tumor-associated antigens, tumor- specific antigens, tissue-specific antigens, cell type-specific antigens, other antigens, or express, for example, cellular receptors.
  • targeting peptides may be inserted into one or more surface-exposed or external loops of the papilloma viral capsid proteins, such as the BC loop, CD loop, DE loop, EF loop, FG loop, or HI loop of HPV LI viral capsid protein.
  • modified viral capsid proteins comprising targeting peptides in one or more of the surface-exposed loops maintain the ability to self- assemble into VLPs.
  • VLPs provided herein can be loaded with agents or drugs useful for therapeutic and/or diagnostic purposes.
  • aspects of the invention may be used to package and/or deliver a therapeutic agent that is useful to treat an infection, an inflammatory disorder, cancer and/or any other disease or disorder.
  • an agent may be a diagnostic agent, a reporter molecule (e.g., gene, protein, and/or RNA), and/or a therapeutic molecule.
  • a therapeutic molecule may be a small molecule, a polypeptide, a nucleic acid (e.g. , an RNA, a DNA, or other natural or synthetic nucleic acid molecule) a gene encoding a polypeptide, any other naturally occurring or synthetic therapeutic molecule, or any combination of two or more thereof.
  • VLPs may be used to target a diagnostic, prophylactic or therapeutic substance to an adversely affected area in a subject, such as, for example a tumor. Accordingly, aspects of the invention may be used to package and/or deliver a therapeutic agent that is an anti-cancer drug.
  • an anti-cancer drug may be 5- fluorouracil, leucovorin, capecitabine, cyclosphosphamide, docetaxel, placitaxel, or gemcitabine.
  • an anti-cancer drug may be a platin-based drug such as cisplatin or carboplatin.
  • aspects of the invention may be used to package and/or deliver a nucleic acid that may be used to silence the expression of one or more target genes.
  • methods and compositions of the invention may be used to deliver an siRNA, an antisense R A, or any combination thereof
  • VLPs possess the ability to carry cytotoxic drugs and display targeting molecules ⁇ e.g. , peptides) which are fused (in-frame) in one or more of the surface exposed loops of the LI protein.
  • HPV LI capsid protein comprises six loops, termed BC loop (amino acids 50-68), CD loop (amino acids 79-95), DE loop (amino acids 1 1 1-152), EF loop (amino acids 164-234), FG loop (amino acids 257-299), and HI loop (amino acids 349- 360), based on wild-type HPV- 16 LI capsid protein (amino acids 1-505), see, e.g. , FIG. 1.
  • the targeting molecules may direct VLPs to specific areas of the body of a subject, such as, for example, a site of a tumor, a site of inflammation, a site of a wound, a site of soft tissue, bone or cartilage damage, etc.
  • cellular peptides ⁇ e.g., receptor ligands or fragments thereof
  • other targeting peptides such as viral, bacterial, etc
  • Targeting specific areas of the body of a subject may reduce toxicity or may increase effective delivery of an agent to the site.
  • HPV-based VLPs are provided that comprise viral capsid proteins ⁇ e.g., LI or LI and L2) that have been modified.
  • the modification comprises mutating, deleting or inserting one or more amino acids in the viral capsid protein ⁇ e.g., LI or LI and L2).
  • the modification comprises insertion of one or more amino acids comprising a targeting motif that can be used to selectively target modified VLPs to specific sites, tissues or cells in vivo.
  • targeting involves interactions between cell surface receptors of cells at the target site and the modified viral capsid proteins.
  • HPV-based VLPs can be targeted to any site, tissue or cell in vivo.
  • modified VLPs comprising viral capsid proteins (e.g. , LI or LI and L2) comprising one or more targeting peptides can be targeted to cells or tissues other than the cells or tissue normally targeted by wild-type HPV (e.g., mucosal tissue, cervical epithelial cells).
  • modified VLPs described herein can be targeted to epithelial airway cells (lung), hepatocytes (liver), lymphocytes, and other tissues and/or cells.
  • the targeting peptide sequence is derived from infectious (exogenous) entities, such as viruses or bacteria (e.g. , Hepatitis B surface antigen pre-S2 region; human complement receptor 2 (CR2) binding domain of glycoprotein gp350/220 of the Epstein-Barr virus; HCV envelope glycoproteins (HCVpp) El and E2).
  • infectious (exogenous) entities such as viruses or bacteria (e.g. , Hepatitis B surface antigen pre-S2 region; human complement receptor 2 (CR2) binding domain of glycoprotein gp350/220 of the Epstein-Barr virus; HCV envelope glycoproteins (HCVpp) El and E2).
  • the targeting peptide sequence is derived from natural (endogenous) cellular receptor ligands (e.g., RGD motif for cells expressing integrins, such as ⁇ 3, ⁇ 5, and ⁇ integrins).
  • the targeting peptide sequence is derived from a nucleolin ligand.
  • the targeting peptide in some embodiments
  • F3 (AKVKDEPQPvRSARLSAKPAPPKPEPKPKKAPAKK, SEQ ID NO: 9).
  • F3 is a 34- amino acid fragment of a high mobility group protein, HMG2N (Christian et al. J Cell Biol. 163:871-878, 2003; Porkka et al , Proc. Natl. Acad. Sci. USA. 99:7444-7449 2002;
  • the F3 targeting peptide targets tumor vasculature, e.g. , tumor cells and angiogenic endothelial cells that express nucleolin in the cell surface.
  • the F3 peptide is internalized by its specific target cells and transported to the nucleus.
  • VLPs are provided comprising LI protein comprising targeting peptides that target the modified VLPs to airway cells.
  • LI proteins comprise small targeting peptides (5-15 amino acids) inserted within the major capsid protein.
  • the targeting peptides are airway cell ligands PI : THALWHT (SEQ ID NO: 1), P2: RFDSLKV (SEQ ID NO: 2) and P3: GHPRQMSHVY (SEQ ID NO: 3).
  • PI and/or P2 and/or P3 are inserted into one or more of the six HPV LI capsid protein loops (BC loop, CD loop, DE loop, EF loop, FG loop, and/or HI loop).
  • one or more other targeting peptides described herein may be inserted into one or more of the six HPV LI capsid protein loops (BC loop, CD loop, DE loop, EF loop, FG loop, and/or HI loop).
  • the targeting peptides for human lung cells are PHPNRAQ (SEQ ID NO: 7) and VDRLQQK (SEQ ID NO: 8).
  • the targeting peptides PHPNRAQ (SEQ ID NO: 7) and VDRLQQK (SEQ ID NO: 8) are inserted into one or more of the six HPV LI capsid protein loops (BC loop, CD loop, DE loop, EF loop, FG loop, and/or HI loop).
  • methods for treating a subject having an adverse condition comprising administering to the subject one or more compositions described herein in an amount effective to treat the conditioa
  • the HPV-based VLPs may be used to deliver therapeutic agents to treat any diseased cell, tissue, or subject, as aspects of the invention are not limited in this respect.
  • modified VLPs are provided comprising targeting peptides directing the modified VLPs to airway epithelial cells.
  • modified VLPs targeted to airway epithelial cells may be used to deliver agents (e.g. , nucleic acids, such as genes or inhibitory RNAs, e.g. siRNA and antisense RNA) as described herein to the airway epithelial cells.
  • agents e.g. , nucleic acids, such as genes or inhibitory RNAs, e.g. siRNA and antisense RNA
  • delivered nucleic acids may comprise a gene for gene transfer.
  • the gene for gene transfer may be used as part of a gene therapy to treat cystic fibrosis.
  • VLPs can be administered to a patient via any suitable route.
  • VLPs may be administered via one or more routes including, but not limited to, subcutaneous, intramuscular, intravenous, and transdermal routes, and through mucosal layers such as via oral, and intranasal routes.
  • VLPs may be used with a targeting molecule (for example, a targeting peptide inserted in one or more of the surface exposed loops of the LI capsid protein) for topical administration (for example, for treating melanoma).
  • a targeting molecule for example, a targeting peptide inserted in one or more of the surface exposed loops of the LI capsid protein
  • topical administration for example, for treating melanoma
  • VLPs may be used with a targeting peptide fused in one or more of the surface exposed loops of the LI and/or L2 capsid protein for intravenous (iv) administration (for example, to treat liver cancer, pancreatic cancer, breast cancer, or other organ cancer).
  • VLPs may be used with a targeting peptide fused in one or more of the surface exposed loops of the LI capsid protein for oral administration.
  • the invention also relates to a method of making a medicament for use in treating one or more diseases or conditions in a subject (e.g. , a human or other mammalian subject).
  • a subject e.g. , a human or other mammalian subject.
  • Such medicaments can be used for prophylactic treatment of a subject at risk for or suspected of having a disease or conditioa
  • one or more VLPs described herein may be used for the preparation of a medicament for use in any of the methods of treatment described herein.
  • Certain aspects of the invention provide for the use of one or more VLPs or compositions comprising VLPs for the manufacture of a medicament or pharmaceutical for treating a mammal (e.g. , a human) having one or more symptoms of, or at risk for, one or more diseases or conditions (e.g. , cancer, infection, and/or other diseases or conditions).
  • aspects of the invention relate to the use of one or more VLPs or compositions comprising VLPs for the preparation of a medicament for treating or preventing a disease or condition (e.g., cancer, infection, and/or other diseases or conditions) in a subject.
  • a disease or condition e.g., cancer, infection, and/or other diseases or conditions
  • aspects of the invention also relate to one or more compounds, VLPs or VLP compositions of the invention for use as a medicament
  • Certain aspects of the invention also relate to one or more of these compounds, VLPs or VLP compositions for use in methods described herein.
  • an HPV VLP may be modified to include a targeting peptide in one or more loops (e.g., 1 , 2, 3, 4, 5, or 6).
  • a targeting peptide may be inserted in each loop that is modified.
  • a different targeting peptide may be inserted into each modified loop (e.g., a first targeting peptide in a first loop and a second targeting peptide in a second loop, a third targeting peptide in a third loop, etc.).
  • a first targeting peptide is inserted in one or more loops and a second targeting peptide is inserted in one or more different loops, etc., wherein one or more of the peptides independently may be included in two or more loops.
  • one or more loops may include two or more targeting peptides (e.g., two or more copies of the same peptide, or one or more each of two or more different targeting peptides).
  • FIG. 1 Schematic representation of wild-type and chimeric LI proteins. PI, P2, P3 peptide ligands and the HBc fragment (DPASRE, SEQ ID NO:0) inserted into various loops (BG, CD, DE, EF, FG and HI) of the HPV-16 LI protein are depicted.
  • FIG. 2 Degrees (+/-) of binding and internalization of various peptide ligands in A-549 cells are indicated
  • FIG. 3 Electron micrographs of negatively stained, purified HPV-16 LI wild-type and mutant LI proteins self-assembled into VLPs are shown.
  • the HPV VLPs shown are composed of wild-type HPV-16 LI protein (LI wt), chimeric HBc/HPV-16 LI protein with insertion at position 140/141 and 266/267 (Ll-HBc 140/141 and Ll-HBc 266/267, respectively), with substitution at position 266-271 and 282-287 (Ll -HBc 266-271 and Ll- HBc 282-287, respectively), and point mutated HPV-16 LI protein at residue 270, 285 and both (L1(270A), L1(285A) and LI (270&85A), respectively). All electron micrographs were taken at a 50 000 x magnification (bar represent 200 nm).
  • FIG. 4 Photographs depicting internalization of wild-type and L1-P3179/180 VLP in 16HBel40- and Hela cells 8h after contact
  • A Fluorescent microscopy of CFDA-SE labeled VLP.
  • B FACS analysis of the number of fluorescent cells
  • C Internalization of CFDA-SE labeled wt and mutant VLP in 16HBel40- cells and Hela cells.
  • FIG. 5 Bar graphs depicting the targeting ratio for different mutants in comparison to HPV16 wt. The comparison was made between human cervical cells (Hela and CaSki) and human lung cells (S9, IB3 and Calu-3). A ratio of 100 means that the mutant targeted for example IB3 cells compared to Hela cells, 100 times better than do wild-type.
  • FIG. 6 Bar graphs depicting targeting efficiency of VLPs to lung cells, 16HBE (FIG. 6A) and A549 (FIG. 6B).
  • Lung cells were contacted with either liposomal transfection reagent (HIPERFECT, QIAgen) or VLPs (P2DE and VDR) to transfect fluorescently labeled siRNA.
  • Transfection efficiency was measured by counting fluorescently labeled cells (% positive cells) using flow cytometry.
  • FIG. 7A depicts the targeting efficiency of VLPs comprising lung targeting peptides (P2DE) to lung cells compared to non-modified VLPs (wt) as measured by fold luciferase reporter expression in the target cells.
  • FIG. 7B depicts a photograph showing immunofluorescence of 16HBe (left panel) and HeLa cells r(right panel) after contacting with P2DE VLP.
  • aspects of the invention relate to the discovery that human papilloma virus (HPV)- based virus-like particles (VLPs) can be redirected to tissues and cells that are not normally infected by wild-type HPV.
  • HPV-based VLP can be modified to specifically change or alter the tropism of the modified VLP to redirect the VLP to tissues and/or cells of interest.
  • modified papillomavirus VLPs that may be used as delivery vehicles, for the delivery of agents such as diagnostic or therapeutic agents (e.g., anti-cancer agents, anti-inflammatory agents, inhibitory nucleic acids, nucleic acids for gene delivery, etc) to specific target cells and/or tissues.
  • delivery vehicle or “viral vector” herein is meant a proteinaceous shell that self-assembles to form a structure with an interior cavity, for example a virus-like particle (VLP).
  • aspects of the invention are based on novel papilloma virus-like particles (VLPs) that can encapsulate agents to be delivered to a subject.
  • the particles can increase delivery efficiency and/or targeting.
  • aspects of the invention relate to human papilloma virus (HPV)-based virus-like particles comprising LI or LI and L2 viral capsid proteins that have been modified to deliver an agent to a specific target site, tissue or cell in a subject.
  • aspects of the invention provide VLP that are adapted for delivering drugs (for example, cytotoxins or other drugs) and/or nucleic acids (for example siRNA, antisense RNA and others) to one or more target areas in a subject
  • viral capsid proteins may comprise targeting peptides, such as heterologous protein fragments or peptide sequences that may be derived from mammals, e.g., humans, such as e.g. , cell receptor ligands, or peptide sequences that are derived from infectious agents, such as e.g. , viruses, bacteria, parasites, as described herein.
  • the protein fragments or peptide sequences comprising targeting peptides provide means to target the VLPs in vivo to specific tissues or cells expressing antigens, such as tumor-associated antigens, tumor-specific antigens, tissue-specific antigens, or cell type- specific antigens.
  • VLPs provided herein can be loaded with agents or drugs useful for therapeutic and/or diagnostic purposes.
  • Encapsidation of plasmids within HPV capsids has been used in models of DNA vaccine delivery systems (Huang et al , 2005; Shi et al, 2001 , Zhang et al, 2004) and gene delivery models (Fayad et al, 2004; Oh et al , 2004).
  • HPV Human Papillomaviruses
  • HPV Human Papillomaviruses
  • a subgroup of 15 HPVs including types 16, 18, 31 , 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and HPV-69 has been designated high risk.
  • mucosal-genital high-risk types such as HPV types 16 and 18 play a critical role in the development of cancer of the uterine cervix (Munoz et al , 2003;
  • the 8 kb double-stranded DNA genome is encapsidated in a structure consisting of 72 capsomers composed of LI and L2 proteins, the major and minor capsid proteins, respectively.
  • the major capsid protein LI in the presence or absence of the minor capsid protein L2, spontaneously assembles into empty virus-like particles (VLPs) when expressed in eukaryotic cells (for review see irnbauer 1996 ; Sapp et al , 1996 ; Schiller & Roden, 1995).
  • the initial step in transfection of viral-derived vectors is their interaction with cellular receptors that allow attachment and internalization.
  • Heparan sulfate and cell surface glycosaminoglycans were also identified as receptors for HPV6, 1 1 , 16, 18, 31 , 33, 39, 45, 58, 68 and bovine papillomavirus (BPV) (Combita et al , 2001 ; Drobni et al , 2003; Giroglou et al, 2001 ; Joyce et al , 1999; Shafti- Keramat et al , 2003: Christensen et al , 2001).
  • BPV bovine papillomavirus
  • VLPs virus-like particles
  • modified VLPs comprising viral capsid proteins comprising targeting peptides sufficient to target the VLPs to specific tissues and/or cells that are not normally targeted by non-modified, or wild- type VLPs, thereby redirecting the modified VLPs to novel sites of interest, in vivo (e.g., tissues/cells) or in vitro, that are not normally targeted.
  • the modified VLPs provided exhibit an altered (viral) tropism, that is the VLPs are redirected to specific tissues and/or cells that are not normally targeted by non-modified or wild-type VLPs that exhibit wild-type tropism for target tissues and/or cells.
  • viral tropism is principally related to the interaction of viruses at the cell surface
  • the most efficient modification to alter viral tropism and to redirect the virus to new cell receptors comprises modifying the viral capsid of non-enveloped viruses or the envelope of enveloped viruses.
  • a number of approaches have been used to generate cell-specific vector systems.
  • One approach is based on directly displaying cell-specific ligands (Cosset et al ,
  • Papillomavirus-like particles have not previously been modified to alter their tropism. It was unknown whether papillomavirus-based VLPs could be successfully redirected and if their tropism could be changed in such way that the modified VLPs could be used as a delivery vehicle (e.g., for delivery of therapeutic or diagnostic agents) to specifically target tissues and/or cells of interest.
  • a delivery vehicle e.g., for delivery of therapeutic or diagnostic agents
  • modified papillomavirus-based VLPs with novel and unexpected targeting characteristics, exhibiting altered tropism that allows targeting of the modified VLPs to tissues and/or cells of interest, that are not sufficiently targeted by non-modified or wild-type papilloma-virus particles.
  • the targeting ratio of the modified VLP relative to a non-modified (e.g. , wild-type) VLP of a particular tissue or cell of interest is increased.
  • the targeting ratio is increased by 2-fold, 5- fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, or more or less or an intermediate fold increase, when targeting efficiency for a target tissue and/or cell of interest of the modified, retargeted VLP, which comprises a targeting peptide of the target tissue and/or cell, is compared to a non- modified (e.g. , wild-type) VLP.
  • a non- modified VLP e.g. , wild-type
  • the targeting ratio can be calculated by comparing the targeting ability of the modified VLP comprising a targeting peptide of a cell of interest to the target cell of interest and the targeting ability of the modified VLP comprising a targeting peptide of a cell of interest to a target cell of wild-type tropism (e.g., for HPV-based VLP, human cervical cells) with the targeting ability to both cell (lines) of the non-modified or wild-type VLP.
  • wild-type tropism e.g., for HPV-based VLP, human cervical cells
  • modified HPV-based VLP comprising targeting peptides for lung cells are incubated with cells that would naturally be infected by HPV (wild-type tropism), e.g., human cervical cells, such as Hela and CaSki, and a rate of targeting is determined, for example in vitro.
  • HPV wild-type tropism
  • human cervical cells such as Hela and CaSki
  • a rate of targeting is determined, for example in vitro.
  • the same amount of modified HPV-based VLP comprising targeting peptides for lung cells are incubated with cells that are targeted by the targeting peptide, such as human lung cells (S9, IB3 and Calu-3), under the same conditions in a parallel experiment, and a rate of targeting is determined.
  • the rate of targeting is also determined for a non-modified (e.g.
  • wild-type VLP e.g., comprising HPV- 16 LI wt.
  • the ratio for targeting is determined.
  • a ratio of, for example, 100 means that the modified VLP targets the novel target cell (e.g., human lung cells) compared to cells of wild-type tropism (e.g., human cervical cells), 100-fold better than does a non-modified or wild-type VLP.
  • VLPs may consist entirely of LI or LI and L2 viral capsid proteins.
  • VLPs may further comprise one or more additional viral or heterologous protein fragments or peptide sequences.
  • heterologous protein fragments or peptide sequences may be derived from mammals, such as e.g., humans.
  • the protein fragments or peptide sequences may comprise targeting peptides, providing means to specifically target VLPs in vivo to specific tissues or cells expressing antigens, such as tumor-associated antigens, tumor-specific antigens, tissue-specific antigens, or cell type-specific antigens, and/or cells expressing other cell surface markers or surface receptors.
  • the protein fragments or peptide sequences may comprise one or more targeting peptides, providing means to specifically target VLPs in vivo to specific tissues or cells expressing cell surface receptors.
  • targeting peptides may direct VLPs in vivo to one or more of the following organs and/or tissues: heart, lung, liver, pancreas, gut, muscle, cervix, skin, tumoral tissue, lymphocytes, and other tissues, cells or organs.
  • a cell surface receptor may be selected from the group consisting of insulin receptor, insulin-like growth factor receptor (including both IGF-IR and IGF-2R), growth hormone receptor, transferrin receptor, epidermal growth factor receptor (EGF-R), low density lipoprotein receptor (LDLR), high density lipoprotein receptor (HDLR), leptin receptor; interleukin receptors including IL- 1 (CD 121 a/IL 1 Rl ,
  • CD121b/ILlR2 CD121b/ILlR2
  • IL-2 CD25/IL2RA, CD122/IL2RB, CD132/IL2RG
  • IL-3 CD123/IL3RA, CD131/IL3RB
  • IL-4 CD124/IL4R, CD132/IL2RG
  • IL-5 CD125/IL5RA, CD131/IL3RB
  • IL-6 CD126/IL6RA, CD130/IR6RB
  • IL-7 CD127/IL7RA, CD132/IL2RG
  • IL-8 CD121b/ILlR2
  • IL-2 CD25/IL2RA, CD122/IL2RB, CD132/IL2RG
  • IL-3 CD123/IL3RA, CD131/IL3RB
  • IL-4 CD124/IL4R, CD132/IL2RG
  • IL-5 CD125/IL5RA, CD131/IL3RB
  • IL-6 CD126/IL6RA, CD130/IR6RB
  • IL-7 CD127/IL7RA, CD
  • CDw218a/IL18Rl CDw218a/IL18Rl
  • IL21R IL21 receptors
  • human growth hormone receptor VEGF receptor
  • VEGF-R VEGF receptor
  • PDGF receptor PDGF receptor
  • transforming growth factor receptor transforming growth factor receptor
  • TGF-alpha and TGF-beta include TGF-alpha and TGF-beta), ciliary neurotrophic factor receptor, prolactin receptor, and T-cell receptors.
  • targeting peptides may comprise fragments of ligand peptides to one or more cell surface receptors. In some embodiments, targeting peptides comprising fragments of ligand peptides to one or more cell surface receptors may be inserted into one or more of the surface exposed loops of the LI capsid protein to generate modified VLP. It should be appreciated that targeting peptides comprising fragments of ligand peptides to one or more cell surface receptors may also be linked to the VLP surface by chemical means or by means of affinity. Chemical means may include the use of chemical linkers that covalently link the targeting peptide to the surface of the VLP comprising LI capsid protein.
  • Affinity means may comprise the use of affinity tags that may be inserted into one or more of the surface exposed loops of the VLP and the provision of a recognition sequence on the targeting peptide.
  • affinity tags include StrepTagTM technology (as described, for example, in PCT/US2009/004299, reference incorporated herein in its entirety).
  • the protein fragments or peptide sequences may comprise one or more targeting peptides derived from the proteins and peptides that interact with cellular receptors, such as insulin, insulin-like growth factor (IGF-1 or IGF-2), growth hormone (GH), transferrin, epidermal growth factor (EGF), low density lipoprotein (LDL), high density lipoprotein (HDL), leptin, interleukins IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and IL21 , VEGF, PDGF, TGF-alpha or TGF-beta, ciliary neurotrophic factor, and prolactin.
  • IGF-1 or IGF-2 insulin-like growth factor
  • GH growth hormone
  • transferrin epidermal growth factor
  • EGF epidermal growth factor
  • the heterologous protein fragments or peptide sequences may comprise one or more targeting peptides, providing means to specifically target VLPs in vivo to specific tissues or cells expressing integrins, such as ⁇ 3, ⁇ 5, and ⁇ integrins, wherein the targeting peptide comprises an RGD motif.
  • the targeting peptide sequence is derived from a nucleolin ligand.
  • the targeting peptide in some embodiments, is the F3 tumor-homing peptide, F3
  • F3 is a 34-amino acid fragment of a high mobility group protein, HMG2N (Christian et al. J Cell Biol.
  • the F3 targeting peptide targets tumor vasculature, e.g. , tumor cells and angiogenic endothelial cells that express nucleolin in the cell surface.
  • the F3 peptide is internalized by its specific target cells and transported to the nucleus.
  • the heterologous proteins or protein fragments may comprise one or more targeting peptides derived from viruses providing means to specifically target VLPs in vivo to specific tissues or cells.
  • targeting peptides derived from viruses that have been identified to be responsible for viral-mediated cell entry are provided.
  • peptides derived from the Hepatitis B surface antigen which identifies hepatocytes may be used for the treatment of liver disease and hepatocellular carcinoma Peptides derived from the Epstein Barr Virus, which identifies lymphocytes may be used for the treatment of lymphoma
  • the human complement receptor 2 (CR2) binding domain of glycoprotein gp350/220 of the Epstein-Barr virus is a virus-derived targeting peptide that may be used for targeting as described herein
  • the pre-S2 region of HB V is a virus-derived targeting peptide that may be used for targeting as described herein
  • HCV envelope glycoproteins (HCVpp) El and E2 may be used for targeting as described hereia
  • peptides are used to enhance oral delivery.
  • an integrin-adherent peptide motif, RGD can be used to improve oral deliver ofVLPs.
  • Patent Nos. 7,544,767 HMGN2 tumor blood vessels and tumor cells
  • 7,501,486 heart
  • 7,488,792 collagen/tumor vasculature
  • 7,192,921 tumor lymphatic vasculature
  • 6,743,892 selected organ
  • 6,610,651 variable organs and tissues
  • 6,576,239 angiogenic
  • 6,303,573 heart
  • 6,296,832 selected organs
  • 6,180,084 angiogenic
  • 6,177,542 integratedin
  • 5,981,478 integratedin
  • 5,912,234 integratedin
  • peptides can be identified by methods well known in the art, such as, for example, phage display of tumor peptide libraries, and other screening procedures using phage display cDNA library and combined ex vivo screening on cell suspensions and in vivo screening, e.g., for tumor homing. Accordingly, one or more of these peptides may be used in a VLP as described herein. In some embodiments, one or more protein fragments or peptide sequences used for targeting of VLPs are inserted into one or more external or surface-exposed loops.
  • the protein fragments or peptide sequences inserts can be 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200 or 250 amino acids long, or any length in between 4 amino acids and 250 amino acids.
  • the inserts are between 4 and 50, 5 and 25, or 5 and 15 amino acids long.
  • the inserts can be inserted anywhere in the loops. In some embodiments, the inserts are inserted approximately in the middle of a loop.
  • the inserts may comprise, for example, viral targeting domains. It will be appreciated that these domains are not limited to HPV. Viral targeting domains may be derived from any virus to target any cell that is desired.
  • the inserts may also comprise, for example, host- specific cellular recognition motifs, such as receptor recognition motifs.
  • wild-type amino acids comprising the loops are substituted or deleted, singly or in groups, to allow insertion of the targeting peptides while maintaining the ability of VLPs to self-assemble.
  • maintaining the ability to self-assemble it is meant that VLP maintain some ability to self-assemble. This ability may be reduced if compared to an unmodified VLP.
  • the ability to self-assemble may be measured by the ratio of LI (or LI and L2) protein expression and the number of self-assembled VLP observed by electron microscopy.
  • the ability to self-assemble is maintained resulting in VLPs that are well formed ⁇ e.g. , as judged by electron microscopy) and infectious (as assayed, e.g., in in vitro cell culture assays).
  • amino acids present in loops exposed on the virus surface can be deleted or mutated to reduce the immunogenicity of the VLP (as, for example, described in PCT/US2009/004299, reference incorporated herein in its entirety).
  • a tag polypeptide which provides an epitope to which an anti- tag antibody can selectively bind, is inserted into one or more external loops. Provision of the epitope tag enables VLPs to be readily detectable by contacting the VLPs with a labeled anti-tag antibody, or an anti-tag antibody that provides chemical groups that can be utilized for labeling.
  • tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (Field et ai , Mol. Cell.
  • tag polypeptides include the Flag-peptide (Hopp et al , BioTechnology, 6: 1204-1210 (1988)); the KT3 epitope peptide (Martin et al , Science, 255: 192-194 (1992)); tubulin epitope peptide (Skinner et al. , J. Biol. Chem., 266: 15163-15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al , Proc. Natl. Acad. Sci. USA, 87:6393- 6397 (1990)).
  • polypeptides encoding targeting sequences ⁇ e.g. , targeting peptides are inserted into one or more of the external loops of the viral capsid protein that allow VLPs to be targeted to specific organs, tissues or cells, without destroying the ability of the viral capsid protein (LI or LI and L2) to form ⁇ e.g. , to self-assemble into) VLPs.
  • VLPs comprising insertion of a targeting peptide into one or more of the BC loop (amino acids 50-68), CD loop (amino acids 79-95), DE loop (amino acids 1 1 1-152), EF loop (amino acids 164-234), FG loop (amino acids 257-299), and HI loop (amino acids 349-360), based on wild-type HPV-16 LI capsid protein (505 amino acids, e.g., Philippine strain as described in Touze et al , J Clin Microbiol 1998, incorporated herein by reference, or SEQ ID NO: 13) which still permits VLP assembly.
  • the resulting chimeric capsid protein is a fusion protein of the viral capsid protein and the targeting peptide sequence(s).
  • loops, BC loop, CD loop, DE loop, EF loop, FG loop, and HI loop may be positioned at slightly different positions in different strains. This may be the result for example of small deletions or insertions of one or more amino acids in the various papilloma virus strains. Thus, slightly different start and end positions may occur for the different loops, / ' . e.
  • the start and end position may shift one, two, three or more amino acids for one or more of the loops, e.g., the BC loop may start around amino acid positions 47, 48, 49, 50, 51, 52, 53 and may end around amino acid positions 65, 66, 67, 68, 69, 70, 71 depending on the papilloma virus strain.
  • the CD loop, DE loop, EF loop, FG loop, and HI loop may have variable start and end positions depending on the strain. It should be appreciated that a person of skill in the art will be able to identify loops corresponding to those described herein in the various strains.
  • targeting peptide herein is meant a peptide sequence that serves to target or direct the VLPs to a particular location, cell type, diseased tissue, or association.
  • the targeting peptide is directed against a target molecule and allows concentration of the VLPs loaded with an agent (e.g., a therapeutic agent) in a particular localization within a subject
  • VLPs comprising targeting peptides may enter mammalian cells as intact VLPs via active cellular transport, such as receptor-mediated endocytosis or other vesicular traffic mechanisms.
  • active cellular transport such as receptor-mediated endocytosis or other vesicular traffic mechanisms.
  • the VLPs may enter and leave animal cells via the binding interaction between ligand molecules (e.g. , the targeting peptides) expressed and displayed by the VLPs and their corresponding receptor molecules on the target cell membrane, which causes the membrane to wrap around and engulf the VLPs.
  • ligand molecules e.g. , the targeting peptides
  • VLPs may enter the target cell via receptor-mediated endocytosis as a consequence of interaction of the cellular receptor with a targeting peptide displayed on the surface of the VLPs.
  • the VLP membrane may fuse with the membrane of the endosome membrane, which leads to the release of the therapeutic agent trapped inside the VLP.
  • one or more external loops of the viral capsid protein may comprise a peptide sequence encoding e.g., antigen-specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof directed against specific receptor molecules such as e.g. , the integrin receptor family, the VEGF receptor family, the FGF receptor family, the IGF receptor family, the EGF receptor family and the hepatocyte receptor.
  • specific receptor molecules such as e.g. , the integrin receptor family, the VEGF receptor family, the FGF receptor family, the IGF receptor family, the EGF receptor family and the hepatocyte receptor.
  • antigen-specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof expressed in the external loops of VLPs are directed against one or more tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs), such as viral tumor antigens, cellular oncogene proteins, and/or tumor-associated differentiation antigens, including, but not limited to, CEA, TAG-72 (Yokata et al , 1992, Cancer Res. 52:3402-3408), C017-lA (Ragnhammar et a/. , 1993, Int J.
  • TAAs tumor-associated antigens
  • TSAs tumor-specific antigens
  • CEA C017-lA
  • GICA 19-9 (Herlyn et al , 1982, J. Clin. Immunol. 2: 135), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al, 1994, Blood 83: 1329-1336), human B-lymphoma antigen-CD20 (Relle al, 1994, Blood 83:435-445), CD33 (Sgouros et al , 1993, J. Nucl. Med. 34:422-430), melanoma specific antigens such as ganglioside GD2 (Saleh et al. , 1993, J.
  • ganglioside GD3 (Shitara et al, 1993, Cancer Immunol. Immunother. 36:373- 380), ganglioside GM2 (Livingston et al, 1994, J. Clin. Oncol. 12: 1036-1044), ganglioside GM3 (Hoon et al, 1993, Cancer Res.
  • tumor-specific transplantation type of cell-surface antigen such as virally-induced tumor antigens including T-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses, oncofetal antigen-alpha- fetoprotein such as CEA of colon, bladder tumor oncofetal antigen (Hellstrom et ah, 1985, Cancer. Res. 45:2210-2188), differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et ah , 1986, Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et ah, 1988, J. of
  • neoglycoprotein neoglycoprotein
  • sphingolipids breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (pl 85 HER2 ), polymorphic epithelial mucin (PEM) (Hilkens et ah , 1992, Trends in Bio. Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhard et ah , 1989, Science 245:301 -304),
  • differentiation antigen such as I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes, preimplantation embryos, I(Ma) found in gastric adenocarcinomas, Ml 8, M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D, 56-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Le y found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, Ei series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric
  • adenocarcinoma antigen CO-514 (blood group Le a ) found in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Le b ), G49 found in EGF receptor of A431 cells, MH2 (blood group ALe b /Le y ) found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, T 5A 7 found in myeloid cells, R 24 found in melanoma, 4.2, GD 3 , Dl .
  • acetylgalactosaminyltransferase GM2, GD2, GD3, JADE, MART, BAGE, GAGE, MAGE-1 , MAGE-2, MAGE-3, XAGE, MUC-1 , MUC-2, MUC-3, MUC-4, MUC-18, ICAM-1 , C-CAM, V-CAM, ELAM, NM23, EGFR, E-cadherin, N-CAM, LFA-3 (CD58), EpCAM, B7.1 , CEA, DCC, PSA, Her2-neu, UTAA, melanoma antigen p75, K19, HKer 8, pMel 17, TP10, tyrosinase related proteins 1 and 2, p97, p53, RB, APC, DCC, NF-1 , NF-2, WT-1 , MEN-I, MEN-II, BRCAl, VHL, FCC and MCC, ras, myc, neu, raf
  • antigen-specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof may also be linked to the VLP surface by chemical means or by means of affinity.
  • Chemical means may include the use of chemical linkers that covalently link the antigen-specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof to the surface of the VLP comprising LI or LI and L2 capsid protein.
  • Linker chemistry and the coupling of specific amino acids via linker chemistry are well known in the art.
  • Affinity means may comprise the use of affinity tags that may be inserted into one or more of the surface exposed loops of the VLP and the provision of a recognition sequence on the antigen- specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof.
  • affinity tags may be inserted into one or more of the surface exposed loops of the VLP and the provision of a recognition sequence on the antigen- specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof.
  • StrepTagTM technology as described, for example, in PCT/US2009/004299, reference incorporated herein in its entirety).
  • VLPs are provided comprising LI viral capsid protein comprising targeting peptides in one or more loops that facilitate targeting of the VLPs to airway epithelial cells.
  • modified LI proteins comprising one or more insertions of a targeting peptide maintain the ability to self-assemble into VLPs.
  • modifications in LI are insertions of targeting peptides within antigenic loops of the LI major protein of HPV. It should be appreciated that targeting peptides may be inserted at positions previously shown not to disturb the self-assembly of the LI protein into VLPs. (Sadeyen et al, 2003; Carpentier et al. ; 2005).
  • targeting peptides effective for targeting VLPs to specific tissues, organs, or cells may be identified by phage display technology or any other affinity-based technology known in the art.
  • targeting peptides effective for targeting VLPs to specific tissues, organs, or cells may be identified on the basis of greater internalization efficiency using specific cell lines in vitro.
  • the cell line(s) used to analyze greater internalization efficiency should be derived (or should correlate in vitro) to the tissue, organ or cell that is to be targeted in vivo by the modified VLPs comprising a targeting peptide.
  • targeting peptides effective to direct VLPs to human airway epithelial cells in vivo may be selected through binding or internalization studies using phage displaying peptide libraries on human airway epithelial cells (Drapkin et al , 2000; Gaden et al , 2005; Jost et al, 2001 ; Vaysse et al , 2000). Internalization can be monitored, for example, by fluorescence microscopy or any other method known in the art.
  • the targeting peptides can be inserted into one or more of the surface exposed loops of the viral capsid protein, e.g. , LI.
  • LI protein and optionally L2 protein to be modified can be derived from any known HPV type (e.g. , HPV 6, 1 1, 16, 18, 31 , 33, 34, 35, 39, 45, 51 , 52, 56, 58, 59, 68, 69, 73, and HPV91 , or any other known HPV type).
  • increase internalization of reassembled modified VLPs comprising targeting peptides may be measured by fluorescence microscopy.
  • VLPs may be labeled with a fluorescent dye or a substrate that becomes fluorescent upon internalization, e.g., carboxyfluorescein diacetate succinimidyl ester (CFDA-SE).
  • CFDA-SE carboxyfluorescein diacetate succinimidyl ester
  • VLPs comprising LI viral capsid protein comprising targeting peptides in one or more loops that facilitate targeting of the VLPs to airway epithelial cells, wherein the targeting peptides consist of or comprise the amino acid sequence as set forth in SEQ ID NO: 1 , THALWHT (PI , Jost et al , 2001); SEQ ID NO: 2, RFDSLKV (P2, Vaysse et al , 2000); SEQ ID NO: 3; GHPRQMSHVY (P3, Gaden et al , 2005); SEQ ID NO: 7, PHPNRAQ and SEQ ID NO: 8, VDRLQQK.
  • targeting peptides may be inserted into one or more external loops of the LI viral capsid protein, such as the BC loop (amino acids 50-68), CD loop (amino acids 79-95), DE loop (amino acids 1 1 1-152), EF loop (amino acids 164-234), FG loop (amino acids 257-299), and HI loop (amino acids 349-360), based on wild-type HPV-16 LI capsid protein (505 amino acids, e.g., Philippine strain as described in Touze et al , J Clin Microbiol 1998, incorporated herein by reference, or SEQ ID NO: 13).
  • the LI protein to be modified in the loops can be derived from any known HPV type and that the invention is not limited to modified LI capsid protein of HPV-16.
  • the targeting peptides may be 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200 or 250 amino acids long, or any length in between 4 amino acids and 250 amino acids.
  • the targeting peptide inserts are between 5 and 50, 5 and 25, 5 and 15, 5 and 10 or 7 and 10 amino acids long.
  • the targeting peptides are inserted at the N-terminal end of the loop.
  • a targeting peptide can be inserted between amino acids 49 and 50 of HPV-16 LI capsid protein, where amino acid 50 is the start (N-terminal end) of the BC loop, or it may be inserted between amino acids 50 and 51 , 51 and 52, 53 and 54, 55 and 56 and so on In certain embodiments, the targeting peptides are inserted at the C-terminal end of the loop.
  • a targeting peptide can be inserted between amino acids 68 and 69 of HPV-16 LI capsid protein, where amino acid 68 is the end (C-terminal end) of the BC loop, or it may be inserted between amino acids 67 and 68, 66 and 67, 65 and 66, 64 and 65 and so on.
  • the targeting peptides are inserted approximately in the middle of the loop.
  • a targeting peptide can be inserted between amino acids 59 and 60 of HPV-16 LI capsid protein, where amino acid 59 is approximately the middle (between N- terminal and C-terminal end) of the BC loop, or it may be inserted between amino acids 56 and 57, 57 and 58, 58 and 59, 60 and 61, 61 and 62, 62 and 63 and so on. It should be appreciated that insertions can be made into any of the loops of the viral capsid protein, following the procedure described herein.
  • target peptides are inserted into the DE loop. In some embodiments, target peptides are inserted into position 140/141 of the DE loop of HPV-16 LI protein, or an equivalent position within the DE loop of any other HPV serotype. In some embodiments, the target peptide is PI : THAL WHT (SEQ ID NO: 1). In some embodiments, the target cells are airway epithelial cells.
  • target peptides are inserted into the EF loop. In some embodiments, target peptides are inserted into position 179/180 of the EF loop of HPV-16 LI protein, or an equivalent position within the EF loop of any other HPV serotype. In some embodiments, the target peptide is P3: GHPRQMSHVY (SEQ ID NO: 3). In some embodiments, the target cells are airway epithelial cells.
  • the motif can be linear or structural, that is it may be based on the primary amino acid sequence or its secondary (or tertiary) structure.
  • the motif can be a cellular recognition motif, that may facilitate VLP uptake and/or target cell recognition, or it may be an epitope that is recognized by certain antibodies or that is known to be antigenic.
  • deletions, insertions, substitutions or mutations may be made in one or more of the amino acids of the one or more loops to be modified by insertion of targeting peptides. Such modifications may further be made outside the loops, e.g., N-terminal to the beginning of a loop or C-terminal to the end of a loop or at any place in the viral capsid proteia Any modifications described herein that maintain the ability of the viral capsid protein (e.g., LI) to self-assemble are contemplated herein.
  • LI viral capsid protein
  • mutations or insertions into the loops may be made according to those identified by Chen et al. (2000). In some embodiments, mutations or insertions into the loops may be made at the surface of the LI capsomers. In some embodiments, mutations or insertions into alpha-helices or beta-sheets of the LI capsomers are avoided (Sadeyen et al, 2003). In some embodiments, mutations certain mutations within loops that destabilize the structure are avoided (Kirnbauer et al, 1993).
  • Conservative amino acid changes may be made according to functional, chemical or structural considerations. For example, conservative amino acid changes may be made according to chemical similarity: acidic (D/E), aliphatic (A/G/I/L/V), amide (N/Q), aromatic (F/W/Y), basic (R/H/K), hydroxyl (S/T), imino (P), sulfur (CM); or functional similarity: acidic (D/E), basic (R/H/K), hydrophobic (A/I/L/M/F/P/W/V), polar (N/C/Q/G/S/T/Y); or similarity in charge: acidic, basic, neutral; or structural similarity: ambivalent
  • any amino acid of a group of amino acids in parentheses can be changed into another in that group and such change would be considered a conservative change according to the consideration applied, e.g. , structural, functional, or chemical.
  • one or more factors may be considered.
  • any changes in the wild-type amino acid sequence e.g., insertions of targeting peptides, deletions, and/or mutations/substitutions
  • the nucleic acid that encodes the modified viral capsid protein for example by genetic engineering (cloning) and other molecular biology techniques known in the art.
  • the chimeric peptides comprising e.g., fusions of targeting peptides and viral capsid protein may be generated by recombinant techniques generating chimeric genes inserted into expression vectors under the control of heterologous promoters driving expression of the chimeric gene in an expression system, e.g., bacterial, insect, yeast, plant or mammalian, producing recombinant chimeric fusion protein.
  • an expression system e.g., bacterial, insect, yeast, plant or mammalian, producing recombinant chimeric fusion protein.
  • L I protein and L I and L2 protein may be produced
  • recombinantly produced LI protein and L I and L2 protein may self-assemble to form virus-like particles (VLPs).
  • VLPs virus-like particles
  • Recombinant production may occur in a bacterial, insect, yeast or mammalian (heterologous) host system, or any other heterologous host system known in the art.
  • L I protein may be expressed or LI and L2 protein may be co-expressed in the host system.
  • VLPs are loaded with the one or more diagnostic and/or therapeutic agents.
  • VLPs may be loaded by dissembling and reassembling L I or LI and L2 viral particles, as described herein.
  • Salts that are useful in aiding dissembly/reassembly of viral capsid proteins into VLP, include Zn, Cu, Ni, Ru and Fe salts.
  • HPV nanoparticles may be loaded with one or more therapeutic agents.
  • HPV nanoparticles comprising L I protein, or L I and L2 protein further comprise one or more agents (e.g., therapeutic agents).
  • agents e.g., therapeutic agents.
  • the therapeutic agent comprises one or more siRNA molecules or one or more nucleic acids (e.g. , plasmid or other vector) that each is capable of expressing one or more siRNA molecules.
  • the therapeutic agent comprises one or more antisense nucleic acids or one or more nucleic acids (e.g. , plasmid or other vector) that each are capable of expressing one or more antisense nucleic acids.
  • the HPV nanoparticles comprise combinations of two or more therapeutic agents.
  • the therapeutic agent is an inducer of RNA interference (RNAi) or other inducer of gene silencing.
  • RNA interference may be a siRNA, a shRNA, a hybrid nucleic acid molecule comprising a first part that comprises a duplex ribonucleic acid (RNA) molecule and a second part that comprises a single stranded deoxyribonucleic acid (DNA) molecule, a longer double-stranded RNA or a DNA construct for expression of siRNA or longer RNA sequences.
  • RNA interference may be a siRNA, a shRNA, a hybrid nucleic acid molecule comprising a first part that comprises a duplex ribonucleic acid (RNA) molecule and a second part that comprises a single stranded deoxyribonucleic acid (DNA) molecule, a longer double-stranded RNA or a DNA construct for expression of siRNA or longer RNA sequences.
  • Other inducers of gene silencing include inducers of DNA methyl
  • siNA small nucleic acid molecules
  • antisense nucleic acids and short interfering nucleic acid include, for example: microRNA (miRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), and short hairpin RNA (shRNA) molecules to knockdown expression of target genes associated with a disease or disorder.
  • An siNA can be unmodified or chemically- modified.
  • An siNA can be chemically synthesized, expressed from a vector or enzymatically synthesized.
  • provided herein are chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating gene expression or activity in cells by RNA interference (RNAi).
  • RNAi RNA interference
  • the use of chemically- modified siNA may improve various properties of native siNA molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups,. for example, 2' amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31 , 163; Burgin et al , 1996, Biochemistry , 35, 14090).
  • Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al, International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565 568; Pieken et al Science, 1991, 253, 314317; Usman and
  • one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence identical to the nucleotide sequence or a portion thereof of the targeted RNA
  • one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is substantially complementary to a nucleotide sequence of a target RNA or a portion thereof
  • the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the target RNA
  • each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
  • an siNA is an shRNA, shRNA-mir, or microRNA molecule encoded by and expressed from a genomically integrated transgene or a plasmid-based expression vector.
  • a molecule capable of inhibiting mRNA expression, or microRNA activity is a transgene or plasmid-based expression vector that encodes a small-interfering nucleic acid.
  • Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siRNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue-specific expression systems.
  • transgenes and expression vectors are controlled by tissue-specific promoters.
  • transgenes and expression vectors are controlled by inducible promoters (e.g., such as tetracycline inducible expression systems).
  • the small interfering nucleic acid described herein are expressed in mammalian cells using a mammalian expression vector.
  • the recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type ⁇ e.g. , tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art
  • suitable tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid-specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters.
  • Developmentally- regulated promoters are also provided herein, for example the murine hox promoters and the alpha-fetoprotein promoter.
  • siRNA molecules are well know in the art and many siRNAs are known that target expression of tumor-specific proteins that may be mutated, overexpressed and/or
  • cyclin/cdk EGFR, brc/abl and the like.
  • modified VLP described herein can be used to deliver molecules that promote RNA interference using any of a variety of molecules known in the art, e.g. , short interfering RNA molecules (siRNA), which are double stranded RNA molecules.
  • siRNA short interfering RNA molecules
  • RNA interference is a phenomenon describing double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Synthetic duplexes of 21 nucleotide RNAs can mediate gene specific RNAi in mammalian cells, without invoking generic antiviral defense mechanisms (Elbashir et al. Nature 2001 , 41 1 :494-498; Caplen et al. Proc Natl Acad Sci 2001 , 98:9742-9747).
  • polynucleotides comprising an RNAi sequence that acts through an RNAi mechanism to attenuate or inhibit expression of a gene of interest, e.g., a gene that is overexpressed in cancer.
  • the siRNA sequence is between about 19 nucleotides and about 75 nucleotides in length, or between about 25 base pairs and about 35 base pairs in length.
  • An RNAi construct can contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript of a gene of interest.
  • the double-stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi.
  • the number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3 ' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition
  • sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters ⁇ e.g., University of Wisconsin Genetic Computing Group).
  • sequence identity between the inhibitory RNA and the portion of the target gene is greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is 100%.
  • polynucleotides comprising RNAi sequences can be produced by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase of the treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro.
  • the polynucleotides that modulate target gene activity by RNAi mechanisms may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g. , to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties.
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase.
  • the siRNA polynucleotides may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
  • RNAi constructs see, for example, Heidenreich et al. (1997) Nucleic Acids Res, 25:776- 780; Wilson et al. (1994) J Mol Recog 7:89-98; Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug Dev 7:55-61).
  • RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g. , 2'- substituted ribonucleosides).
  • the double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands.
  • RNA duplex formation may be initiated either inside or outside the cell.
  • the RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g. , at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications.
  • Inhibition generally is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
  • the subject RNAi constructs are "siRNAs.” These nucleic acids are between about 19-35 nucleotides in length, preferably between 25 and 30 or 21-23 nucleotides in length, e.g., with 21-23 nucleotides in length corresponding in length to the fragments generated by nuclease "dicing" of longer double-stranded RNAs. In certain embodiments, the length is about 27 nucleotides, e.g. 25, 26, 27, 28, 29, or 30. In certain embodiments, the length is exactly 27 nucleotides (Kim et al. 2005 Nat Biotechnol 23:222- 226).
  • the siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex or translation is inhibited.
  • the siRNA molecules comprise a 3' hydroxyl group.
  • the siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify such molecules.
  • non-denaturing methods such as non-denaturing column chromatography
  • chromatography e.g. , size exclusion chromatography
  • glycerol gradient centrifugation e.g., glycerol gradient centrifugation
  • affinity purification with antibody e.g., affinity purification with antibody
  • fluorescently labeled with a label such as FITC, Cy-3, Cy-5, etc.
  • At least one strand of the siRNA sequence has a 3' overhang from about 1 to about 6 nucleotides in length, or from 2 to 4 nucleotides in length. In other embodiments, the 3' overhangs are 1-3 nucleotides in length. In certain embodiments, one strand has a 3' overhang and the other strand is either blunt-ended or also has an overhang. In certain embodiments, the siRNA molecule is double stranded, double blunt-ended (e.g. U.S. patent 7,723,316). The length of the overhangs may be the same or different for each strand.
  • the 3' overhangs can be stabilized against degradation
  • the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • purine nucleotides such as adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g. , substitution of uridine nucleotide 3' overhangs by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi.
  • the absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium and may be beneficial in vivo.
  • siRNA that can be delivered by modified VLPs described herein include siRNA that mediate silencing of oncogenes, genes of infectious agents (including viruses), and other targets, including lung cancer specific target genes and cystic fibrosis-specific target genes.
  • siRNAs, shRNAs and/or miRNAs are known in the art.
  • Web-based online software systems for designing siRNA sequences and scrambled siRNA sequences are for example SIDIRECT, SISEARCH, SEQ2SVM, DEQOR, SIRNA WIZARD (InvivoGen, San Diego, CA).
  • the specificity can be predicted using for example SPECIFICITYSERVER, MIRACLE.
  • Target sequences can be researched for example at HuSiDa (Human siRNA Database), and siRNAdb (a database of siRNA sequences).
  • Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J. Cancer.
  • Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al , Embo J.
  • Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al, Nucleic Acids Symp Ser. (29): 121 -2, 1993).
  • Antisense nucleic acids include modified or unmodified RNA, DNA, or mixed polymer nucleic acids, and primarily function by specifically binding to matching sequences resulting in modulation of peptide synthesis (Wu-Pong, 1994, BioPharm, 20-33).
  • An antisense nucleic acid binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme.
  • Antisense molecules may also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit Rev. in Oncogenesis 7, 151 -190).
  • antisense nucleic acid describes a nucleic acid that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • the antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript.
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.
  • Triple helix approaches have also been investigated for sequence-specific gene suppression. Triple helix-forming oligonucleotides have been found in some cases to bind in a sequence- specific manner (Postel et al. , Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31 , 1991 ; Duval- Valentin et c/., Proc. Natl. Acad. Sci. U.S.A.
  • VLPs may be used to package and/or deliver small activating
  • RNAs saRNAs
  • uRNAs snRNA Ul
  • aspects of the invention relate to using VLPs to package and deliver any one or more of the different types of RNA or other nucleic acid molecules described herein.
  • VLPs are provided that are useful as delivery agents, such as for the in vivo delivery of one or more therapeutic agents, e.g., one or more anti-cancer agents, to a subject
  • therapeutic agents e.g., one or more anti-cancer agents
  • anti-cancer agents that may be delivered by the modified VLPs described herein include, but are not limited to, acivicin; aclarubicin; acodazole
  • hydrochloride acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium;
  • flurocitabine fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;
  • hydroxyurea idarubicin hydrochloride; ifosfamide; ilmofosine; interleulin II (including recombinant interleukin II; or rIL2); interferon alfa-2a; interferon alfa-2b; interferon alfa-nl ; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan
  • lometrexol sodium lomustine; losoxantrone hydrochloride; masoprocol; maytansine;
  • mechlorethamine mechlorethamine oxide hydrochloride rethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate;
  • methotrexate sodium metoprine; meturedepa; mitindomide; mitocarcin; mitocromin;
  • mitogillin mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;
  • mycophenolic acid nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;
  • pegaspargase peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;
  • porfiromycin prednimustine; procarbazine hydrochloride; puromycin; puromycin
  • vincristine sulfate vindesine
  • vindesine sulfate vinepidine sulfate; vinglycinate sulfate;
  • vinleurosine sulfate vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride; improsulfan; benzodepa; carboquone;
  • triethylenemelamrine triethylenephosphoramide; triethylenethiophosphoramide;
  • estermustine chlorozotocin; gemzar; nimustine; ranimustine; dacarbazine; mannomustine; mitobronitol; aclacinomycins; actinomycin F(l); azaserine; bleomycin; carubicin;
  • carzinophilin chromomycin; daunorubicin; daunomycin; 6-diazo-5-oxo-l-norleucine; doxorubicin; olivomycin; plicamyciri; porfiromycin; puromycin; tubercidin; zorubicin;
  • denopterin pteropterin; 6-mercaptopurine; ancitabine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; enocitabine; pulmozyme; aceglatone; aldophosphamide glycoside;
  • adecypenol adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;
  • amidox amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing mo hogenetic protein- 1 ; antiandrogen; prostatic carcinoma; antiestrogen;
  • antineoplaston antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; azatoxin;
  • azatyrosine baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists;
  • benzochlorins benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;
  • calcipotriol calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine;
  • carboxamide-amino-triazole carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4;
  • combretastatin analogue conagenin; crambescidin 816; crisnatol; cryptophycin 8;
  • cryptophycin A derivatives curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;
  • didemnin B didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifl uridine; droloxifene;
  • dronabinol duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; trasrabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin;
  • gallium nitrate gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol; iroplact;
  • irsogladine isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon;
  • leuprolide/estrogen/progesterone leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;
  • marimastat masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopraminde; MIF inhibitor;
  • mifepristone miltefosine; nirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody; human chorionic
  • gonadotrophin monophosphoryl lipid A+ myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N- substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine;
  • octreotide okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; taxel; taxel analogues; taxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate;
  • phosphatase inhibitors include picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin
  • placetin B plasminogen activator inhibitor
  • platinum complex platinum compounds
  • platinum-triamine complex porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase
  • oligonucleotides single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D;
  • spiromustine splenopentin
  • spongistatin 1 squalamine
  • stem cell inhibitor stem-cell division inhibitors
  • stipiamide stem-cell division inhibitors
  • stromelysin inhibitors sulfinosine
  • superactive vasoactive intestinal peptide antagonist suradista; suramin; swainsonine; synthetic glycosaminoglycans;
  • tallimustine tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide;
  • thrombopoietin mimetic thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine;
  • triciribine trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
  • tyrphostins UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system; erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer; 5-fluorouracil; leucovorin;
  • capsitabine cyclosphosphamide; and gemcitabine.
  • therapeutic agents or imaging agents that may also be delivered in vivo to a subject by the modified VLPs provided herein, are cellular components, genetically engineered or native, recombinant, soluble or any other type of proteins, peptides, cytokines or other signaling molecules, which can have pro- or anti-inflammatory effects, or pro- or anti- anti-apoptotic effects, polysaccharides, glycoproteins, heterogeneous mixtures of
  • macromolecules e.g. , a natural product extract
  • hybrid macromolecules e.g. ,
  • protein/nucleic acid hybrids albumin conjugated proteins, drugs, inorganic molecules, organic molecules, or combinations thereof), and/or other bioactive molecules, other nucleic acids (e.g., RNA, DNA, PNA, multiplexes of them (e.g. , triplex)), preferably siRNA and antisense RNA, and/or cytotoxic drugs.
  • nucleic acids e.g., RNA, DNA, PNA, multiplexes of them (e.g. , triplex)
  • siRNA and antisense RNA preferably siRNA and antisense RNA, and/or cytotoxic drugs.
  • a therapeutic substance may also be any of the following agents: adrenergic agent; adrenocortical steroid; adrenocortical suppressant; agents for treating cognition, antiplatelets, aldosterone antagonist; amino acid; anabolic; analeptic; analgesic; anesthetic; anorectic; antiacne agent; anti-adrenergic; anti-allergic; anti-Alzheimer's, anti-amebic; anti-anemic; anti- anginal; anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial; anticholinergic;
  • anticoagulant ; anticonvulsant; antidepressant; antidiabetic; antidiarrheal; antidiuretic; antiemetic; anti-epileptic; antifibrinolytic; antifungal; antihemorrhagic; antihistamine;
  • antihyperlipidemia antihypertensive; antihypo tensive; anti-infective; anti-inflammatory; antimicrobial; antimigraine; antimitotic; antimycotic, antinauseant, antineoplastic,
  • antineutropenic antiparasitic; antiproliferative; antipsychotic; antirheumatic; antiseborrheic; antisecretory; antispasmodic; antithrombotic; anti-ulcerative; antiviral; anxiolytics, appetite suppressant; blood glucose regulator; bone resorption inhibitor; bronchodilator; cardiovascular agent; cholinergic; COX1 inhibitors, COX2 inhibitors, direct thrombin inhibitors, depressant; diagnostic aid; diuretic; dopaminergic agent; estrogen receptor agonist; fibrinolytic;
  • fluorescent agent free oxygen radical scavenger
  • gastrointestinal motility effector
  • glucocorticoid hair growth stimulant; hemostatic; histamine H2 receptor antagonists; hormone; human growth hormone, hypocholesterolemic; hypoglycemic; hypolipidemic; hypnotics, hypotensive; imaging agent; immunological agents such as immunizing agents, immunomodulators, immunoregulators, immunostimulants, and immunosuppressants; keratolytic; LHRH agonist; mood regulator; mucolytic; mydriatic; nasal decongestant; neuromuscular blocking agent; neuroprotective; NMDA antagonist; non- hormonal sterol derivative; plasminogen activator; platelet activating factor antagonist;
  • platelet aggregation inhibitor platelet aggregation inhibitor; proton pump inhibitors, psychotropic; radioactive agent;
  • scabicide scabicide
  • sclerosing agent sedative; sedative-hypnotic; selective adenosine Al antagonist
  • serotonin antagonist serotonin inhibitor
  • serotonin receptor antagonist selective adenosine Al antagonist
  • statins steroid
  • thyroid hormone thyroid inhibitor
  • thyromimetic tranquilizer
  • amyotrophic lateral sclerosis agent cerebral ischemia agent
  • Paget's disease agent unstable angina agent
  • vasoconstrictor scabicide
  • vasodilator a vasodilator; wound healing agent; and/or xanthine oxidase inhibitor, but it is not so limited.
  • compositions and diagnostic, prophylactic or therapeutic substances or agents described herein may be used in combination with the modified VLPs described herein.
  • VLPs may be used to deliver one or more of the compositions and diagnostic, prophylactic or therapeutic substances or agents.
  • VLPs in some embodiments, may comprise one or more of the compositions and diagnostic, prophylactic or therapeutic substances or agents.
  • VLPs in some embodiments, may be formulated in one or more of the compositions described herein.
  • One or more of the compositions may comprise the modified VLPs described herein.
  • compositions and diagnostic, prophylactic or therapeutic substances or agents may be delivered by VLPs or may be administered in conjunction or combination with VLPs, for example as part of a treatment regimen.
  • the one or more of compositions and diagnostic, prophylactic or therapeutic substances or agents may be administered at substantially the same time as the VLPs, or may be administered at different times.
  • the one or more of compositions and diagnostic, prophylactic or therapeutic substances or agents may be administered by the same route of administration as the VLPs, or may be administered by different a different route.
  • compositions and diagnostic, prophylactic or therapeutic substances or agents may be administered in combination with the VLPs to augment or enhance the effects of the VLPs to treat the same disease or ailment, or, for example, to treat additional disease components, additional ailments and/or side effects that are not treated, targeted or alleviated by the VLP treatment.
  • the diagnostic, prophylactic or therapeutic substances used are preferably sterile.
  • solutions comprising diagnostic, prophylactic or therapeutic substances described herein are sterilized by mechanical filtration.
  • many sensitive pharmaceuticals or protein solutions may not be suitable for sterilization methods comprising exposure to heat, irradiation or chemical sterilization.
  • a filter with pore size 0.2 ⁇ may be used to effectively remove bacteria. If viruses must also be removed, a pore size around 20 nm may be needed.
  • the filtration equipment and the filters themselves may be purchased as pre- sterilized disposable units in sealed packaging, or may be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes.
  • the filtration system may be tested to ensure that the membranes have not been punctured prior to or during use.
  • _Pharmaceutical sterile filtration may be performed in a room with highly filtered air (HEPA filtration) or in a laminar flow cabinet.
  • Radiation sterilization may be used such as electron beams, X-rays, gamma rays, or subatomic particles.
  • Gamma rays may be used, for example, for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets.
  • Electron beam processing may be used, for example, for medical device sterilization.
  • X-rays may be used, for example, to irradiate large packages and pallet loads of medical devices.
  • Ultraviolet light irradiation may be used, for example, for sterilization of surfaces (e.g. , the interiors of biological safety cabinets) and some transparent objects.
  • Steam sterilization may be used.
  • autoclaves are commonly used for steam sterilization. Typical sterilization conditions comprise a holding time of at least 15 minutes at 121 °C or 3 minutes at 134 °C.
  • a “diagnostic substance” is any substance that has diagnostic capabilities, for example imaging agents, such as detectable markers, for example heavy metals, Gadolinium, Quantum dots, magnetic particle, radioactive particles, labeled antibodies, labeled nucleic acids (e.g. siRNA), luciferase and other chemoluminescent agents. These agents may be substances trapped inside the VLP, and/or on the surface (e.g. , outer-surface) of the membrane of the VLP. These agents may be used to detect an adverse condition by any medical detection device or method, such as for example Magnetic Resonance Imaging (MRI), Positron
  • Emission Tomography PET
  • CAT Computerized Axial Tomography
  • X-rays or other imaging modalities. These applications may provide for immediate monitoring and/or diagnosis of early metastasis.
  • imaging may be combined with therapy, for example, to allow for simultaneous treatment and monitoring (e.g., to confirm that treatment is appropriately localized to a target site such as a tumor or other diseased tissue).
  • the VLPs described herein may be used to deliver imaging agents and/or therapeutic agents.
  • the VLPs described herein that are used to deliver imaging agents and/or therapeutic agents may be administered in combination with one or more therapeutic and/or imaging agents. These agents may be different from the one or more agents delivered by VLPs or may be the same, but
  • Methods are provided for treating a subject in need of treatment administering the VLPs or VLP compositions described herein in an amount sufficient to treat the subject.
  • the terms “treat,” “treated,” or “treating” when used with respect to an adverse condition, such as a disorder or disease may refer to prophylaxis, amelioration, prevention and/or cure of the condition.
  • Treatment after a condition e.g., disease or disorder
  • Treatment of subjects before a condition has started aims to reduce the risk of developing the condition and/or lessen its severity if the condition does develop.
  • the term "prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the disorder, and/or to the inhibition of further development of an already established disorder. Desired outcomes may include a stabilization of the condition, a slowdown in progression of the disease or a full disease-free recovery of the subject.
  • Subjects in need of treatment are preferably subjects that have a disease or disorder.
  • the disease or disorder may be any disease or disorder, such as for example an infection (e.g., viral, bacterial, fungal), an allergy, a genetic disease, a cancer or lymphoma, a neurological or mental disease, an age-related disease, or any other disease or disorder.
  • Methods of treatment comprising administering modified VLPs comprising LI or LI and L2 comprising one or more targeting peptides, the VLPs further comprising one or more therapeutic agents and optionally diagnostic agents, to a subject in need of treatment in an amount effective to treat the disease or disorder.
  • the disease is a tumor, cancer or lymphoma
  • the modified VLPs described herein comprise targeting peptides that target the VLPs to specific tumor, cancer or lymphoma cells to deliver therapeutic or diagnostic agents to the tumor, cancer or lymphoma
  • cancer and “cancer” are being used interchangeably.
  • the cancer or tumor may be malignant or non-malignant. Cancers or tumors include but are not limited to biliary tract cancer; bladder cancer, brain cancer; breast cancer; cervical cancer;
  • choriocarcinoma colon cancer; endometrial cancer; esophageal cancer; gastric cancer;
  • intraepithelial neoplasms lymphomas; liver cancer; lung cancer (for example, small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.
  • the cancer is hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, or colon carcinoma
  • modified VLP may be used to deliver agents, e.g. diagnostic agents or therapeutic agents, such as small molecules, nucleic acids (DNA, RNA, e,g, siRNA, antisense etc.) or drugs (e.g. anti-cancer drugs) to cancerous cells, tissues, or organs.
  • modified VLPs that are targeted to airway epithelial cells as described herein can be used to delivery therapeutic or diagnostic agents to the lung.
  • modified VLPs that are targeted to airway epithelial cells can be used to deliver therapeutic agents and optionally diagnostic agents to the lung to treat cystic fibrosis.
  • modified VLPs that are targeted to airway epithelial cells can be used to deliver genes for lung cancer gene therapy. It should be appreciated that the modified VLPs can be modified in such a way to target any tissue, organ or cell in vivo, and can be used to deliver therapeutic or diagnostic agents to any tissue, organ or cell in vivo, for treatment of any disease or disorder that may be treated by the methods described herein.
  • treatment of a subject having a disease or disorder with modified VLPs comprising one or more therapeutic or diagnostic agents further comprises administration of one or more additional therapeutic or diagnostic agent(s).
  • additional therapeutic or diagnostic agent(s) may be co-administered at the same time or at a different time, for example before or after administration of modified VLPs comprising one or more therapeutic agents.
  • an effective amount of a composition refers to the amount necessary or sufficient for a composition alone, or together with further doses, to realize a desired biologic effect.
  • the desired response will depend on the particular condition being treated.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.
  • the effective amount for any particular application can vary depending on such factors as the disease or adverse condition being treated, the size of the subject, or the severity of the disease or adverse condition.
  • a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • One of ordinary skill in the art can empirically determine the effective amount without necessitating undue
  • VLPs for any VLPs, VLP composition and/or compound described herein the
  • therapeutically effective amount can be initially determined from animal models.
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered VLPs, VLP composition and/or compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well- known in the art is well within the capabilities of the ordinarily skilled artisan.
  • VLPs described herein and/or therapeutic agents may be administered per se (neat) or in the form of a pharmaceutically acceptable formulation.
  • VLPs may be in form of a VLP composition comprising the VLP and one or more additional substances, such as described herein.
  • VLPs and/or therapeutic agents are administered in pharmaceutically acceptable solutions, they may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • the solutions used are sterile.
  • the pharmaceutical compositions contain an effective amount of VLPs or therapeutic agents optionally included in a pharmaceutically-acceptable carrier.
  • VLPs and therapeutic agents described herein will vary depending upon the specific agents used and the disease being treated, as would either be known to those skilled in the art or can be established by routine experimentation using methods commonly employed in the art. In some embodiments, dependent upon these factors, the agents may be administered orally or parenterally.
  • oral formulations may include immediate release particle coatings, controlled release particle coatings or extended release particle coatings.
  • Parenteral modes of administration include intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intralesional, intrapleural, intrathecal, intra-arterial, and into lymphatic vessels or nodes and to bone or bone marrow.
  • VLPs and therapeutic agents may also be administered topically or
  • compositions described herein may be provided in the form of tablets, capsules, softgels, liquids, powders, or other forms for oral administration.
  • compositions described herein may be provided in the form of liquid or lyophilized preparations for injection.
  • VLPs, VLP compositions and/or compounds may be provided in the form of a metered dose, a dry powder, spray, e.g., for inhalation, foam or a nasal preparation for respiratory delivery.
  • these or other formulations may be used for ophthalmic, topical, and/or other forms of delivery.
  • the pharmaceutical compositions can be formulated readily by combining active VLPs and/or compound(s), e.g., VLPs described herein and optionally additional therapeutic agents, with pharmaceutically acceptable carriers well known in the art
  • Such carriers enable VLPs and optionally additional therapeutic agents to be formulated as tablets, pills, dragees, capsules, liquids, gels, hydrogels, pellets, granules, syrups, slurries, foams, suspensions and the like, for oral ingestion by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
  • polyvinylpyrrolidone PVP
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active VLP and/or compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • active VLPs and/or compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • VLPs, VLP compositions and/or compounds may be administered by inhalation to pulmonary tract, especially the bronchi and more particularly into the alveoli of the deep lung, using standard inhalation devices.
  • VLPs, VLP compositions and/or compounds may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • An inhalation apparatus may be used to deliver VLPs, VLP compositions and/or compounds to a subject.
  • An inhalation apparatus is any device for administering an aerosol, such as dry powdered form of VLPs, VLP compositions and/or compounds. This type of equipment is well known in the art and has been described in detail, such as that description found in Remington: The Science and Practice of Pharmacy, 19 th Edition, 1995, Mac Publishing Company, Easton, Pennsylvania, pages 1676-1692. Many U.S. patents also describe inhalation devices, such as U.S. Patent No. 6,1 16,237.
  • “Powder” as used herein refers to a composition that consists of finely dispersed solid particles.
  • VLPs, VLP compositions and/or compounds are relatively free flowing and capable of being dispersed in an inhalation device and subsequently inhaled by a subject so that the VLPs, VLP compositions and/or compounds reach the lungs to permit penetration into the alveoli.
  • a “dry powder” refers to a powder composition that has a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol. The moisture content is generally below about 10% by weight (% w) water, and in some embodiments is below about 5% w and preferably less than about 3% w.
  • the powder may be formulated with polymers or optionally may be formulated with other materials such as liposomes, albumin and/or other carriers.
  • Aerosol dosage and delivery systems may be selected for a particular therapeutic application by one of skill in the art, such as described, for example in Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract,” in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990), and in Moren, "Aerosol dosage forms and formulations,” in Aerosols in Medicine. Principles, Diagnosis and Therapy, Moren, et al , Eds., Esevier, Amsterdam, 1985.
  • compositions when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g. , by bolus injection or continuous infusion
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of active VLPs and/or compounds in water-soluble form. Additionally, suspensions of active VLPs and/or compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of VLPs and/or compounds to allow for the preparation of highly concentrated solutions.
  • the active compositions may be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use.
  • a suitable vehicle e.g. , sterile pyrogen-free water
  • compositions may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • compositions may also be formulated as a depot preparation
  • Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin
  • the pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active VLPs and/or compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 2
  • VLP preparations may be administered to a subject in combination (e.g. , simultaneously or separately but as part of the same therapeutic regimen) with a compound or other pharmaceutical preparation for treating one or more conditions or diseases described herein. Accordingly, aspects of the invention relate to combination preparations or kits that contain one or more VLPs along with one or more separate compounds (e.g., drugs) that are not packaged in VLPs.
  • the efficiency of HPV VLP-mediated gene transfer into human airway cells was improved. Indications and applications of these VLPs include, for example, delivery of therapeutic agents for the treatment of cystic fibrosis and lung cancer gene therapy.
  • VLPs HPV pseudovirions
  • chimeric VLPs were generated by insertion of 3 different peptide ligands within antigenic loops of the LI major protein of HPV- 16.
  • Peptides were inserted at positions previously shown not to disturb the self-assembly of the LI protein into capsid (Sadeyen et al , 2003; Carpentier et al ; 2005).
  • Three human airway cell ligands, which efficiently target human airway cells, have been identified by phage display technology. Two of these ligands, designated PI (THALWHT, SEQ ID NO: 1) (Jost et al , 2001) and P2 (RFDSLKV, SEQ ID NO: 2) (Vaysse et al , 2000), have been selected on the basis of phage binding to 16HBel4o " and CFT-2 cells, respectively.
  • Cos-7 African green monkey fibroblast-like cell line transformed by SV40, ATCC # CRL- 1651
  • Hela derived from an HPV-18 integrated human cervical adenocarcinoma
  • 16HBel4o human bronchial epithelial cells, Cozens et al, 1994
  • A549 adenocarcinoma of human airway epithelium, ATCC#CCL-185. All cell lines were cultivated in monolayer cultures in
  • Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin and 100 g/ml streptomycin and 1 mM Sodium pyruvate at the exception of 16HBel4o " cells for which the DMEM was replaced by MEM supplemented with 2mM L-glutamine. All cells were maintained in a humidified atmosphere with 5% C02 at 37°C.
  • HPV- 16 wild type (wt), HPV- 16 mutants Ll-HBc 140/141 and Ll-HBc 283/284, with insertion of a hepatitis B core (HBc) motif within the DE and FG loops of the LI protein respectively, have already been described [Sadeyen et al , 2003 ; Touze et al , 1998].
  • the other seven DNA-encoding chimeric LI proteins with insertion of the THALWHT motif (PI , SEQ ID NO: 1), (Jost et al , 2001) between HPV 16 LI positions 140/141 , 179/180 and 283/284, and RFDSL V motif (P2, SEQ ID NO: 2) (Vaysse et al , 2000) and GHPRQMSHVY motif (P3, SEQ ID NO:3) (Gaden et al, 2005) between HPV 16 LI positions 140/141 and 179/180, were obtained by mutagenesis using a two-step PCR protocol.
  • PI THALWHT motif
  • P2 RFDSL V motif
  • P3 GHPRQMSHVY motif
  • Recombinant plasmids were used to generate recombinant baculoviruses according to the Bac- to-Bac procedure (Invitrogen). Generation of recombinant baculoviruses and purification of Ll-VLPs. Sf21 cells, maintained in Grace's insect medium supplemented with 10% fetal calf serum (FCS,
  • Nonidet P40, pepstatin and leupeptin (lmg/ml). Cell lysates were then centrifuged at 14,000 g for 15 min at 4°C and nuclear pellets resuspended and sonicated. Nuclear cell extracts were then loaded on top of a preformed CsCl gradient and centrifuged to equilibrium in a Beckman SW28 rotor. CsCl gradient fractions were investigated for density by refractometry and for the presence of chimeric LI proteins by electrophoresis in 10% sodium dodecyl sulfate- polyacrylamide gel (SDS-PAGE) and Coomassie blue staining.
  • SDS-PAGE sodium dodecyl sulfate- polyacrylamide gel
  • Pseudovirions can be generated as previously described with some modifications (Touze et al , Nucleic Acids Res 1998; 26: 1317-23). For example, 1 ⁇ g of HPV VLPs is incubated in 50 mmol/L of Tris-HCl buffer (pH 7.5) containing 20 mmol/L of DTT and 1 mmol/L of EGTA for 30 min at room temperature. At this stage, expression plasmids encoding e.g. , shRNA or siRNA or a gene of interest, reporter plasmids, proteins or other cellular factors or small molecules (therapeutic or diagnostic agents) can be added to the disrupted VLPs.
  • Tris-HCl buffer pH 7.5
  • the preparation is then diluted with increasing concentrations of CaCl 2 up to a final concentration of 5 mmol/L, with or without ZnCl 2 (10 nmol/L).
  • ZnCl 2 is used because it has been reported that ZnCl 2 enhances the assembly of HPV capsomers into VLPs (Hanslip et al, Biotechnol Prog 2006; 22:554-60). Pseudovirions are then dialyzed against 1 x PBS overnight and stored at 4°C before use.
  • VLPs Assembly of chimeric LI proteins into VLPs was verified by electron microscopy.
  • purified VLP preparations were applied to carbon coated grids, negatively stained with 1.5 % uranyl acetate and observed at x 50,000 nominal magnification using a JEOL 1010 electron microscope.
  • the number of VLPs produced was semi-quantified by determination of the mean number of particles observed per field (calculated from 2 to 3 micrographs). Cell type specific targeting.
  • Purified HPV VLPs were labeled with carboxy-fluorescein diacetate succinimyl ester (CFDA-SE) (Vibrant CFDA-SE cell tracer kit, Molecular Probes).
  • CFDA-SE carboxy-fluorescein diacetate succinimyl ester
  • CFDA-SE acetate groups on the CFDA-SE are cleaved off by intracellular esterases and yields highly fluorescent particles.
  • CFDA-SE was added at a final concentration of 100 mM (Bergsdorf et al , 2003; Drobni et al , 2003) to VLPs at a concentration of 0.5 mg/ml in PBS (pH 8,5). After overnight incubation at room temperature, labeled VLPs were subjected to centrifugation in a sucrose density gradient (30 - 70%) to separate VLPs from free CFDA-SE. LI -positive fractions were pooled in PBS and sedimented by ultracentrifugation. The pellet was resuspended in 0.15 M NaCl.
  • VLPs To detect cell-entry of VLPs, cells were exposed to CFDA- SE-labeled VLPs in DMEM medium for 1 h at 37 °C. Unbound VLPs were removed by extensive washing with PBS. DMEM containing 10% FCS was then added and cells were incubated at 37°C for 7 h. Flow cytometry was used to measure the cell fluorescence resulting from the uptake of labeled VLPs. The relative fluorescent intensity was measured using FACsort and Cellquest software (Becton Dickinson). Typically, 20,000 events were collected for each experiment.
  • the internalization of each of the different VLPs was normalized by using a ratio corresponding to the percentage of fluorescent cells observed with 16HBe cells, divided by the percentage of fluorescent cells observed with Hela cells. The results are presented as relative internalization. This calculation was performed separately for each VLP. The data presented are the means of 2 to 3 determinations. Synthetic Peptides and analysis of their cell binding and internalization.
  • Results were scored + when OD values were lower than 3 times the value observed with wells coated with PBS-T containing 1% FCS (control), ++ when OD values were between 3 and 5 times the control well, and +++ when the OD values were greater than 5 times the control.
  • chimeric VLPs Cell specificity of chimeric VLPs. 3 different ligands were tested for their ability to specifically target human airway cells as compared to other cell lines. For this purpose, immunofluorescence microscopy was used to visualize the internalization of the different chimeric VLPs. The chimeric VLPs were initially tested for their ability to bind to -and to be internalized by- different cell lines. Targeting of airway cells by a given mutant VLP was determined comparing the percentage of 16HBe fluorescent cells with the percentage of Hela or Cos-7 fluorescent cells 8 hours after contact between these cells and CFDA-SE-labeled VLPs. The relative fluorescence intensity of the cells was measured by FACS. The data obtained (FIG.
  • Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer in these cells, one of the major problems confronting cystic fibrosis gene therapy.
  • Four peptide motifs, selected by binding or internalization studies using phage displaying peptide libraries on human airway epithelial cells were investigated for internalization within cells.
  • EF or FG might be associated with a decrease in the yield of VLP production.
  • the yield may also be dependent on the inserted sequence, since yields of L 1 production varied according to different amino acid sequences inserted at position 140/141. However, even with a decreased yield, modified VLP may be used as described herein.
  • 16HBel4o- cells was observed with the P3 sequence inserted at position 179/180 within the EF loop of the LI protein. All three ligands (PI , P2 and P3) were confirmed to target airway cells. In contrast, insertion of an unrelated sequence such as the HBc sequence did not induced a significant changes in the ability to target airway cells. The increased
  • 16HBel4o- cells and in Hela cells could also be related to the capacity of the VLPs to escape from the endosome.
  • FIG. 5 shows a comparison of targeting ratios for different modified VLP in comparison to HPV16 wt.
  • the comparisons were made between human cervical cells (Hela and CaSki) and human lung cells (S9, IB3 and Calu-3).
  • a ratio of 100 means that the modified VLP targeted, for example IB3 cells, compared to Hela cells, 100-fold better than wild-type VLP.
  • THALWHT SEQ ID NO: 1
  • ligand within the DE loop (P1DE) of the LI produced VLPs capable of targeting lung cancer cells, such as IB3-1 and S9 cells about 450-fold better than Llwt VLP when measured as compared to Hela cells (cervical) and about 2000-fold better when measured as compared to Ca Ski (cervical) cells.
  • P3EF the GHPRQMSHVY (SEQ ID NO: 3) ligand within the EF loop of the HPV-16 LI
  • P2DE the RFDSLKV (SEQ ID NO: 2) ligand within the DE loop of the HPV-16 LI
  • THALWHT SEQ ID NO: 1
  • FIG. 7A shows efficient targeting of the P2DE (DE) variant (RFDSLKV (SEQ ID NO: 2) ligand within the DE loop of the HPV-16 LI) to A549 and 16HBe lung cells compared to non-modified VLP (wt).
  • the P2DE variant has a 6 to 8-fold higher efficiency (measured by luciferase expression of the target cell) to deliver a nucleic acid (exemplified as a luciferase reporter plasmid) to the target cell compared to wild-type.
  • FIG. 7B shows internalization of the P2DE mutant in lung cells compared to HeLa cells by immunofluorescence, showing effective targeting of the lung cells by the modified VLP.
  • Table 2 Detection of virus-like particles by electron microscopy in the different LI proteins preparations ( a Mean number of VLPs per field when observed by electron microscopy) and LI antigenic titer.
  • GenBank AF043287.1 Human papillomavirus type 16 Phil late major capsid protein (LI) gene, complete cds:
  • variants of the HPV virus - like particles were produced to target them towards lung epithelia for drug or siRNA delivery.
  • Functional loops were inserted into the capsomer protein sequences and two variants were tested, P2DE and VDR.
  • the experiment was conducted to evaluate the ability of the targeted VLPs (VDR and P2DE) to deliver siRNA into lung cells as well as to compare the efficiency of siRNA delivery of the targeted VLPs with a standard transfection agent HIPERFECT (QIAgen).
  • VDR variable with VDRLQQK (SEQ ID NO: 8)
  • P2DE variant with RFDSLKV (SEQ ID NO: 2) ligand within the DE loop of the HPV-16 LI
  • HBE16e cells were seeded at 1.5 x 10 s cells/ well in one 12-well tissue culture plate.
  • VLPs VDR and P2DE were loaded with fluorescent siRNA using (200 ⁇ g VLP, 100 g siRNA per reaction).
  • A549 cells were seeded at 1 x 10 5 cells/ well in a 12-well plate.
  • encapsulated siRNA delivered for VDR is 25.6 ng and 6.4 ng for the high and low treatments, respectively.
  • siRNA encapsulation and delivery is estimated at 12.8 ng and 3.2 ng, respectively. These numbers are based on assumptions of 24 ng/10 ⁇ g (siRNA/VLP protein) encapsulation, based on volume, and 20% recovery of VLP during Disassembly/Reassembly procedure; based on prior data.
  • Additional controls included untreated cells, 140 ng siRNA + HIPERFECT, 35 ng siRNA + HIPERFECT, and HIPERFECT only.
  • FIG. 6 shows bar graphs for siRNA delivery into the two lung cell lines.
  • the most efficient transfer of fluorescent siRNA was observed using the P2DE particles (variant with RFDSLKV (SEQ ID NO: 2) ligand within the DE loop of the HPV-16 LI), followed by the VDR particles (variant with VDRLQQK (SEQ ID NO: 8)). Both VLPs were more efficient than the liposomal transfection reagent HIPERFECT.
  • P2DE particles were the most efficient in transferring fluorescent siRNA into cells followed by the VDR.
  • HIPERFECT transfection was able to reach a large number of cells, but only when much larger amounts of fluorescent siRNA were transfected.
  • the data demonstrate that lung targeted VLPs are very efficient in delivering fluorescently labeled siRNA into lung cells. Accordingly, HPV-based VLPs with lung targeting peptides may be used in methods of targeting lung cells or lung tissue.
  • Buck CB, Pastrana DV, Lowy DR, Schiller JT Efficient intracellular assembly of papillomaviral vectors. J Virol. 2004, 78:751 -757. Carpentier GS, Fleury MJ, Touze A, Sadeyen JR, Tourne S, Sumblet PY, Coursaget P. Mutations on the FG surface loop of human papillomavirus type 16 major capsid protein affect recognition by both type-specific neutralizing antibodies and cross-reactive antibodies. J Med Virol. 2005, 77:558-565. Chen XS, Garcea RL, Goldberg I, Casini G, Harrison SC. Structure of small virus-like particles assembled from the LI protein of human papillomavirus 16. Mol Cell. 2000, 5:557- 567.
  • Drapkin PT O'Riordan CR
  • Yi SM Chiorini JA
  • Cardella J Zabner J
  • Welsh MJ Targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia.
  • J Clin Invest. 2000 105:589-596.
  • Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry.
  • Gaden F Franqueville L, Magnusson MK, Hong SS, Merten MD, Lindholm L, Boulanger P. Gene transduction and cell entry pathway of fiber-modified adenovirus type 5 vectors carrying novel endocytic peptide ligands selected on human tracheal glandular cells. J Virol. 2004, 78:7227-47. Gillock ET, Rottinghaus S, Chang D, Cai X, Smiley SA, An K, Consigli RA. Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system. J Virol. 1997, 71 :2857-2865.
  • Laquerre S Anderson DB, Stolz DB, Glorioso JC. Recombinant herpes simplex virus type 1 engineered for targeted binding to erythropoietin receptor-bearing cells. J Virol. 1998, 72:9683-9697.
  • Lin AH Lin AH, Kasahara N, Wu W, Stripecke R, Empig CL, Anderson WF, Cannon PM.
  • Receptor- specific targeting mediated by the coexpression of a targeted murine leukemia virus envelope protein and a binding-defective influenza hemagglutinin protein.
  • Hum Gene Ther. 2001 12:323-332.
  • composition it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

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Abstract

Aspects of the invention provide modified virus-like particles for delivery of diagnostic or therapeutic agents. In particular, aspects of the invention provide modified viral capsid proteins that are modified to display targeting peptides when assembled in virus-like particles to direct the virus-like particles to specific sites in vivo. Aspects of the invention further provide methods for treating subjects with one or more modified virus-like particles.

Description

TARGETING OF PAPILLOMA VIRUS GENE DELIVERY PARTICLES
RELATED APPLICATIONS
The present application claims priority under 35 U.S.C. § 1 19(e) to U.S. provisional patent application USSN 61/247,504, filed September 30, 2009, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to papilloma virus particles (VLP) for delivering therapeutic or other agents.
BACKGROUND OF THE INVENTION
The efficacy of many pharmaceuticals and biologies is often limited by difficulties delivering these agents in vivo. Many drugs typically cannot be effectively delivered by conventional means, such as oral ingestion, injection, or inhalation. Not only are many drugs subjected to rapid degradation or metabolism, but they often are characterized by general low bioavailability, and systemic administration often causes many undesired side-effects.
For example, chemotherapeutic agents are far from ideal because they have been selected for their activity against proliferating cells, and they do not discriminate between tumor cells and normal cells undergoing rapid division. Consequently, chemotherapy often causes specific organ toxicities, and produces a series of adverse side effects, including hair and nail loss, mouth ulcers, and sickness.
Attempts have been made to develop delivery systems to improve both targeting and efficacy of drug delivery. However, there are considerable problems with many of the current in vivo delivery systems for therapeutic drugs, making it difficult to treat many major illnesses and diseases. Accordingly, there is an unmet need for delivery strategies that increase drug half-life, bioavailability, selectivity and targeted, sustained release of key drugs. SUMMARY OF THE INVENTION
Aspects of the invention relate to drug delivery methods and compositions. Aspects of the invention are based on novel papilloma virus particles that can encapsulate agents to be delivered to a subject (e.g. , a human patient). The particles can increase delivery efficiency and/or targeting. Aspects of the invention relate to the discovery that human papilloma virus (HPV)-based virus-like particles (VLPs) can be redirected to tissues and cells that are not normally infected by wild-type HPV. In certain embodiments, HPV-based VLP can be modified to specifically change or alter the tropism of the modified VLP to redirect the VLP to tissues and/or cells of interest. In particular, aspects of the invention relate to HPV-based virus-like particles (VLPs) comprising LI or LI and L2 viral capsid proteins that have been modified to deliver an agent to a specific target site, tissue or cell in a subject (e.g. , a human subject, or an animal subject). Aspects of the invention provide VLPs that are adapted for delivering drugs (for example, cytotoxins or other drugs) and/or nucleic acids (for example siRNA, antisense R A and others) to one or more target areas in a subject.
In certain embodiments, viral capsid proteins comprise targeting peptides, such as heterologous protein fragments or peptide sequences that may be derived from mammals (e.g. , humans) or peptide sequences that may be derived from infectious agents, such as e.g. , viruses, bacteria, parasites. In some embodiments, the protein fragments or peptide sequences comprising targeting peptides provide means to specifically target VLPs in vivo to specific tissues or cells expressing antigens, such as tumor-associated antigens, tumor- specific antigens, tissue-specific antigens, cell type-specific antigens, other antigens, or express, for example, cellular receptors. In some embodiments, targeting peptides may be inserted into one or more surface-exposed or external loops of the papilloma viral capsid proteins, such as the BC loop, CD loop, DE loop, EF loop, FG loop, or HI loop of HPV LI viral capsid protein. In some embodiments, modified viral capsid proteins comprising targeting peptides in one or more of the surface-exposed loops maintain the ability to self- assemble into VLPs.
In certain embodiments, VLPs provided herein can be loaded with agents or drugs useful for therapeutic and/or diagnostic purposes.
Aspects of the invention may be used to package and/or deliver a therapeutic agent that is useful to treat an infection, an inflammatory disorder, cancer and/or any other disease or disorder.
It should be appreciated that an agent may be a diagnostic agent, a reporter molecule (e.g., gene, protein, and/or RNA), and/or a therapeutic molecule. A therapeutic molecule may be a small molecule, a polypeptide, a nucleic acid (e.g. , an RNA, a DNA, or other natural or synthetic nucleic acid molecule) a gene encoding a polypeptide, any other naturally occurring or synthetic therapeutic molecule, or any combination of two or more thereof.
In some embodiments, VLPs may be used to target a diagnostic, prophylactic or therapeutic substance to an adversely affected area in a subject, such as, for example a tumor. Accordingly, aspects of the invention may be used to package and/or deliver a therapeutic agent that is an anti-cancer drug. In some embodiments, an anti-cancer drug may be 5- fluorouracil, leucovorin, capecitabine, cyclosphosphamide, docetaxel, placitaxel, or gemcitabine. In some embodiments, an anti-cancer drug may be a platin-based drug such as cisplatin or carboplatin.
Aspects of the invention may be used to package and/or deliver a nucleic acid that may be used to silence the expression of one or more target genes. For example, methods and compositions of the invention may be used to deliver an siRNA, an antisense R A, or any combination thereof
In some embodiments, VLPs possess the ability to carry cytotoxic drugs and display targeting molecules {e.g. , peptides) which are fused (in-frame) in one or more of the surface exposed loops of the LI protein. HPV LI capsid protein comprises six loops, termed BC loop (amino acids 50-68), CD loop (amino acids 79-95), DE loop (amino acids 1 1 1-152), EF loop (amino acids 164-234), FG loop (amino acids 257-299), and HI loop (amino acids 349- 360), based on wild-type HPV- 16 LI capsid protein (amino acids 1-505), see, e.g. , FIG. 1.
The targeting molecules (targeting peptides) may direct VLPs to specific areas of the body of a subject, such as, for example, a site of a tumor, a site of inflammation, a site of a wound, a site of soft tissue, bone or cartilage damage, etc. It should be appreciated that cellular peptides {e.g., receptor ligands or fragments thereof) or other targeting peptides (such as viral, bacterial, etc) that are recognized by the target tissue or target site {e.g. , by receptors expressed on the cells of target tissues or other target sites) may be fused (in-frame) in one or more of the surface exposed loops of the LI protein Targeting specific areas of the body of a subject may reduce toxicity or may increase effective delivery of an agent to the site.
In certain embodiments, HPV-based VLPs are provided that comprise viral capsid proteins {e.g., LI or LI and L2) that have been modified. In certain embodiments, the modification comprises mutating, deleting or inserting one or more amino acids in the viral capsid protein {e.g., LI or LI and L2). In certain embodiments, the modification comprises insertion of one or more amino acids comprising a targeting motif that can be used to selectively target modified VLPs to specific sites, tissues or cells in vivo. In certain embodiments, targeting involves interactions between cell surface receptors of cells at the target site and the modified viral capsid proteins.
In certain embodiments, HPV-based VLPs can be targeted to any site, tissue or cell in vivo. For example, modified VLPs comprising viral capsid proteins (e.g. , LI or LI and L2) comprising one or more targeting peptides can be targeted to cells or tissues other than the cells or tissue normally targeted by wild-type HPV (e.g., mucosal tissue, cervical epithelial cells). In certain embodiments, modified VLPs described herein can be targeted to epithelial airway cells (lung), hepatocytes (liver), lymphocytes, and other tissues and/or cells.
In certain embodiments, the targeting peptide sequence is derived from infectious (exogenous) entities, such as viruses or bacteria (e.g. , Hepatitis B surface antigen pre-S2 region; human complement receptor 2 (CR2) binding domain of glycoprotein gp350/220 of the Epstein-Barr virus; HCV envelope glycoproteins (HCVpp) El and E2). In other embodiments, the targeting peptide sequence is derived from natural (endogenous) cellular receptor ligands (e.g., RGD motif for cells expressing integrins, such as ανβ3, ανβ5, and ανβό integrins). In some embodiments, the targeting peptide sequence is derived from a nucleolin ligand. For example, the targeting peptide, in some embodiments, is the F3 tumor- homing peptide,
F3 (AKVKDEPQPvRSARLSAKPAPPKPEPKPKKAPAKK, SEQ ID NO: 9). F3 is a 34- amino acid fragment of a high mobility group protein, HMG2N (Christian et al. J Cell Biol. 163:871-878, 2003; Porkka et al , Proc. Natl. Acad. Sci. USA. 99:7444-7449 2002;
PCT US03/ 10240 Ruoslahti et al. ; incorporated by reference herein). In some embodiments, the F3 targeting peptide targets tumor vasculature, e.g. , tumor cells and angiogenic endothelial cells that express nucleolin in the cell surface. In some embodiments, the F3 peptide is internalized by its specific target cells and transported to the nucleus.
In certain embodiments, VLPs are provided comprising LI protein comprising targeting peptides that target the modified VLPs to airway cells. In certain embodiments, LI proteins comprise small targeting peptides (5-15 amino acids) inserted within the major capsid protein. In some embodiments, the targeting peptides are airway cell ligands PI : THALWHT (SEQ ID NO: 1), P2: RFDSLKV (SEQ ID NO: 2) and P3: GHPRQMSHVY (SEQ ID NO: 3). In certain embodiments, PI and/or P2 and/or P3 are inserted into one or more of the six HPV LI capsid protein loops (BC loop, CD loop, DE loop, EF loop, FG loop, and/or HI loop). In certain embodiments, one or more other targeting peptides described herein may be inserted into one or more of the six HPV LI capsid protein loops (BC loop, CD loop, DE loop, EF loop, FG loop, and/or HI loop). In some embodiments, the targeting peptides for human lung cells are PHPNRAQ (SEQ ID NO: 7) and VDRLQQK (SEQ ID NO: 8). In some embodiments, the targeting peptides PHPNRAQ (SEQ ID NO: 7) and VDRLQQK (SEQ ID NO: 8) are inserted into one or more of the six HPV LI capsid protein loops (BC loop, CD loop, DE loop, EF loop, FG loop, and/or HI loop). In certain embodiments, methods for treating a subject having an adverse condition are provided, comprising administering to the subject one or more compositions described herein in an amount effective to treat the conditioa In some embodiments, the HPV-based VLPs may be used to deliver therapeutic agents to treat any diseased cell, tissue, or subject, as aspects of the invention are not limited in this respect.
In certain embodiments, modified VLPs are provided comprising targeting peptides directing the modified VLPs to airway epithelial cells. In certain embodiments, modified VLPs targeted to airway epithelial cells may be used to deliver agents (e.g. , nucleic acids, such as genes or inhibitory RNAs, e.g. siRNA and antisense RNA) as described herein to the airway epithelial cells. In certain embodiments, delivered nucleic acids may comprise a gene for gene transfer. In certain embodiments, the gene for gene transfer may be used as part of a gene therapy to treat cystic fibrosis.
According to aspects of the invention, VLPs can be administered to a patient via any suitable route. For example, VLPs may be administered via one or more routes including, but not limited to, subcutaneous, intramuscular, intravenous, and transdermal routes, and through mucosal layers such as via oral, and intranasal routes.
In some embodiments, VLPs may be used with a targeting molecule (for example, a targeting peptide inserted in one or more of the surface exposed loops of the LI capsid protein) for topical administration (for example, for treating melanoma). In some
embodiments, VLPs may be used with a targeting peptide fused in one or more of the surface exposed loops of the LI and/or L2 capsid protein for intravenous (iv) administration (for example, to treat liver cancer, pancreatic cancer, breast cancer, or other organ cancer). In some embodiments, VLPs may be used with a targeting peptide fused in one or more of the surface exposed loops of the LI capsid protein for oral administration.
The invention also relates to a method of making a medicament for use in treating one or more diseases or conditions in a subject (e.g. , a human or other mammalian subject). Such medicaments can be used for prophylactic treatment of a subject at risk for or suspected of having a disease or conditioa Accordingly, one or more VLPs described herein may be used for the preparation of a medicament for use in any of the methods of treatment described herein. Certain aspects of the invention provide for the use of one or more VLPs or compositions comprising VLPs for the manufacture of a medicament or pharmaceutical for treating a mammal (e.g. , a human) having one or more symptoms of, or at risk for, one or more diseases or conditions (e.g. , cancer, infection, and/or other diseases or conditions).
Accordingly, aspects of the invention relate to the use of one or more VLPs or compositions comprising VLPs for the preparation of a medicament for treating or preventing a disease or condition (e.g., cancer, infection, and/or other diseases or conditions) in a subject.
Accordingly, aspects of the invention also relate to one or more compounds, VLPs or VLP compositions of the invention for use as a medicament Certain aspects of the invention also relate to one or more of these compounds, VLPs or VLP compositions for use in methods described herein.
In some aspects of any of the embodiments described herein, an HPV VLP may be modified to include a targeting peptide in one or more loops (e.g., 1 , 2, 3, 4, 5, or 6). In some embodiments, the same targeting peptide may be inserted in each loop that is modified. In some embodiments, a different targeting peptide may be inserted into each modified loop (e.g., a first targeting peptide in a first loop and a second targeting peptide in a second loop, a third targeting peptide in a third loop, etc.). In some embodiments, there may be a combination in that a first targeting peptide is inserted in one or more loops and a second targeting peptide is inserted in one or more different loops, etc., wherein one or more of the peptides independently may be included in two or more loops. In some embodiments, one or more loops may include two or more targeting peptides (e.g., two or more copies of the same peptide, or one or more each of two or more different targeting peptides).
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Schematic representation of wild-type and chimeric LI proteins. PI, P2, P3 peptide ligands and the HBc fragment (DPASRE, SEQ ID NO:0) inserted into various loops (BG, CD, DE, EF, FG and HI) of the HPV-16 LI protein are depicted.
FIG. 2. Degrees (+/-) of binding and internalization of various peptide ligands in A-549 cells are indicated
FIG. 3. Electron micrographs of negatively stained, purified HPV-16 LI wild-type and mutant LI proteins self-assembled into VLPs are shown. The HPV VLPs shown are composed of wild-type HPV-16 LI protein (LI wt), chimeric HBc/HPV-16 LI protein with insertion at position 140/141 and 266/267 (Ll-HBc 140/141 and Ll-HBc 266/267, respectively), with substitution at position 266-271 and 282-287 (Ll -HBc 266-271 and Ll- HBc 282-287, respectively), and point mutated HPV-16 LI protein at residue 270, 285 and both (L1(270A), L1(285A) and LI (270&85A), respectively). All electron micrographs were taken at a 50 000 x magnification (bar represent 200 nm).
FIG. 4. Photographs depicting internalization of wild-type and L1-P3179/180 VLP in 16HBel40- and Hela cells 8h after contact (A) Fluorescent microscopy of CFDA-SE labeled VLP. (B) FACS analysis of the number of fluorescent cells, (C) Internalization of CFDA-SE labeled wt and mutant VLP in 16HBel40- cells and Hela cells.
FIG. 5. Bar graphs depicting the targeting ratio for different mutants in comparison to HPV16 wt. The comparison was made between human cervical cells (Hela and CaSki) and human lung cells (S9, IB3 and Calu-3). A ratio of 100 means that the mutant targeted for example IB3 cells compared to Hela cells, 100 times better than do wild-type.
FIG. 6. Bar graphs depicting targeting efficiency of VLPs to lung cells, 16HBE (FIG. 6A) and A549 (FIG. 6B). Lung cells were contacted with either liposomal transfection reagent (HIPERFECT, QIAgen) or VLPs (P2DE and VDR) to transfect fluorescently labeled siRNA. Transfection efficiency was measured by counting fluorescently labeled cells (% positive cells) using flow cytometry.
FIG. 7. The bar graph of FIG. 7A depicts the targeting efficiency of VLPs comprising lung targeting peptides (P2DE) to lung cells compared to non-modified VLPs (wt) as measured by fold luciferase reporter expression in the target cells. FIG. 7B depicts a photograph showing immunofluorescence of 16HBe (left panel) and HeLa cells r(right panel) after contacting with P2DE VLP.
DETAILED DESCRIPTION OF THE INVENTION
Aspects of the invention relate to the discovery that human papilloma virus (HPV)- based virus-like particles (VLPs) can be redirected to tissues and cells that are not normally infected by wild-type HPV. In certain embodiments, HPV-based VLP can be modified to specifically change or alter the tropism of the modified VLP to redirect the VLP to tissues and/or cells of interest. Aspects of the invention relate to modified papillomavirus VLPs that may be used as delivery vehicles, for the delivery of agents such as diagnostic or therapeutic agents (e.g., anti-cancer agents, anti-inflammatory agents, inhibitory nucleic acids, nucleic acids for gene delivery, etc) to specific target cells and/or tissues. By "delivery vehicle" or "viral vector" herein is meant a proteinaceous shell that self-assembles to form a structure with an interior cavity, for example a virus-like particle (VLP).
Aspects of the invention are based on novel papilloma virus-like particles (VLPs) that can encapsulate agents to be delivered to a subject. The particles can increase delivery efficiency and/or targeting. In particular, aspects of the invention relate to human papilloma virus (HPV)-based virus-like particles comprising LI or LI and L2 viral capsid proteins that have been modified to deliver an agent to a specific target site, tissue or cell in a subject. Aspects of the invention provide VLP that are adapted for delivering drugs (for example, cytotoxins or other drugs) and/or nucleic acids (for example siRNA, antisense RNA and others) to one or more target areas in a subject
In certain embodiments, viral capsid proteins may comprise targeting peptides, such as heterologous protein fragments or peptide sequences that may be derived from mammals, e.g., humans, such as e.g. , cell receptor ligands, or peptide sequences that are derived from infectious agents, such as e.g. , viruses, bacteria, parasites, as described herein. In some embodiments, the protein fragments or peptide sequences comprising targeting peptides provide means to target the VLPs in vivo to specific tissues or cells expressing antigens, such as tumor-associated antigens, tumor-specific antigens, tissue-specific antigens, or cell type- specific antigens.
In certain embodiments, VLPs provided herein can be loaded with agents or drugs useful for therapeutic and/or diagnostic purposes.
Attempts to create papillomavirus and polyomavirus virus-like particles for DNA delivery applications have been under development for 20 years (Oppenheim et al, 1986). Approaches to produce viral-mimetic particles are based on the incorporation of plasmids or modified viral genomes into viral capsids to provide efficient cellular delivery of the transgene (Buck et al, 2004; Gillock et al , 1997; Pawlita et al , 1996; Touze and Coursaget 1998). Encapsidation of plasmids within HPV capsids has been used in models of DNA vaccine delivery systems (Huang et al , 2005; Shi et al, 2001 , Zhang et al, 2004) and gene delivery models (Fayad et al, 2004; Oh et al , 2004). However, successful targeting of a
papillomavirus-based VLPs to cells and/or tissues that are not natural target sites for the virus has not previously been shown.
Human Papillomaviruses (HPV), a group of more than 100 genotypes, are small double-stranded non-enveloped DNA viruses that infect mucosal or cutaneous epithelium and induce a variety of proliferative lesions. A subgroup of 15 HPVs including types 16, 18, 31 , 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and HPV-69 has been designated high risk. A subset of them (referred to as mucosal-genital high-risk types), such as HPV types 16 and 18 play a critical role in the development of cancer of the uterine cervix (Munoz et al , 2003;
Walboomers et al , 1999). The 8 kb double-stranded DNA genome is encapsidated in a structure consisting of 72 capsomers composed of LI and L2 proteins, the major and minor capsid proteins, respectively. The major capsid protein LI , in the presence or absence of the minor capsid protein L2, spontaneously assembles into empty virus-like particles (VLPs) when expressed in eukaryotic cells (for review see irnbauer 1996 ; Sapp et al , 1996 ; Schiller & Roden, 1995). The initial step in transfection of viral-derived vectors is their interaction with cellular receptors that allow attachment and internalization. The binding and internalization of papillomavirus VLPs to a wide range of cell types (El Mehdaoui et al. , 2000; Muller et al. , 1995; Roden et al. , 1994; Volpers et al. , 1995), suggested that they have potential as vehicles for gene delivery Alpha-6 integrin was proposed as an attachment receptor for HPV6b VLP (Evander et al. , 1997; McMillan et al, 1999), but alpha-6 integrin was shown to be
dispensable for VLP binding of all other genotypes investigated (Joyce et al , 1999; Sibbet et al. , 2000). Heparan sulfate and cell surface glycosaminoglycans were also identified as receptors for HPV6, 1 1 , 16, 18, 31 , 33, 39, 45, 58, 68 and bovine papillomavirus (BPV) (Combita et al , 2001 ; Drobni et al , 2003; Giroglou et al, 2001 ; Joyce et al , 1999; Shafti- Keramat et al , 2003: Christensen et al , 2001).
Some aspects of the invention relate to the targeting of virus-like particles (VLPs) to specific tissues and/or cells in vitro and/or in vivo. In certain embodiments, modified VLPs are provided, comprising viral capsid proteins comprising targeting peptides sufficient to target the VLPs to specific tissues and/or cells that are not normally targeted by non-modified, or wild- type VLPs, thereby redirecting the modified VLPs to novel sites of interest, in vivo (e.g., tissues/cells) or in vitro, that are not normally targeted. In certain embodiments, the modified VLPs provided exhibit an altered (viral) tropism, that is the VLPs are redirected to specific tissues and/or cells that are not normally targeted by non-modified or wild-type VLPs that exhibit wild-type tropism for target tissues and/or cells.
Since viral tropism is principally related to the interaction of viruses at the cell surface, the most efficient modification to alter viral tropism and to redirect the virus to new cell receptors comprises modifying the viral capsid of non-enveloped viruses or the envelope of enveloped viruses. A number of approaches have been used to generate cell-specific vector systems. One approach is based on directly displaying cell-specific ligands (Cosset et al ,
1995; Laquerre et al , 1998, Kasahara N et al , 1994; Shin & Folk, 2003 ; Wang et al, 2005) or cell envelope fusion proteins (Lin et al , 2001) on the surface of the viral vectors. Another strategy is the insertion of docking modules in viral vectors such as the immunoglobulin binding domain of S. aureus (Gleiter & Lilie, 2003; Tai et al. 2003). Binding to a cell-specific antibody forms a bridge between the vector and the target cell receptors.
Papillomavirus-like particles have not previously been modified to alter their tropism. It was unknown whether papillomavirus-based VLPs could be successfully redirected and if their tropism could be changed in such way that the modified VLPs could be used as a delivery vehicle (e.g., for delivery of therapeutic or diagnostic agents) to specifically target tissues and/or cells of interest.
In some embodiments, provided herein are modified papillomavirus-based VLPs with novel and unexpected targeting characteristics, exhibiting altered tropism that allows targeting of the modified VLPs to tissues and/or cells of interest, that are not sufficiently targeted by non-modified or wild-type papilloma-virus particles. In some embodiments, the targeting ratio of the modified VLP relative to a non-modified (e.g. , wild-type) VLP of a particular tissue or cell of interest is increased. In some embodiments, the targeting ratio is increased by 2-fold, 5- fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, or more or less or an intermediate fold increase, when targeting efficiency for a target tissue and/or cell of interest of the modified, retargeted VLP, which comprises a targeting peptide of the target tissue and/or cell, is compared to a non- modified (e.g. , wild-type) VLP. For example, the targeting ratio can be calculated by comparing the targeting ability of the modified VLP comprising a targeting peptide of a cell of interest to the target cell of interest and the targeting ability of the modified VLP comprising a targeting peptide of a cell of interest to a target cell of wild-type tropism (e.g., for HPV-based VLP, human cervical cells) with the targeting ability to both cell (lines) of the non-modified or wild-type VLP.
As a non-limiting example, modified HPV-based VLP comprising targeting peptides for lung cells are incubated with cells that would naturally be infected by HPV (wild-type tropism), e.g., human cervical cells, such as Hela and CaSki, and a rate of targeting is determined, for example in vitro. The same amount of modified HPV-based VLP comprising targeting peptides for lung cells are incubated with cells that are targeted by the targeting peptide, such as human lung cells (S9, IB3 and Calu-3), under the same conditions in a parallel experiment, and a rate of targeting is determined. The rate of targeting is also determined for a non-modified (e.g. , wild-type) VLP (e.g., comprising HPV- 16 LI wt). The ratio for targeting is determined. A ratio of, for example, 100 means that the modified VLP targets the novel target cell (e.g., human lung cells) compared to cells of wild-type tropism (e.g., human cervical cells), 100-fold better than does a non-modified or wild-type VLP.
In some embodiments, VLPs may consist entirely of LI or LI and L2 viral capsid proteins. In other embodiments, VLPs may further comprise one or more additional viral or heterologous protein fragments or peptide sequences. In certain embodiments, such heterologous protein fragments or peptide sequences may be derived from mammals, such as e.g., humans. In some embodiments, the protein fragments or peptide sequences may comprise targeting peptides, providing means to specifically target VLPs in vivo to specific tissues or cells expressing antigens, such as tumor-associated antigens, tumor-specific antigens, tissue-specific antigens, or cell type-specific antigens, and/or cells expressing other cell surface markers or surface receptors.
In some embodiments, the protein fragments or peptide sequences may comprise one or more targeting peptides, providing means to specifically target VLPs in vivo to specific tissues or cells expressing cell surface receptors. For example, targeting peptides may direct VLPs in vivo to one or more of the following organs and/or tissues: heart, lung, liver, pancreas, gut, muscle, cervix, skin, tumoral tissue, lymphocytes, and other tissues, cells or organs.
In some embodiments, a cell surface receptor may be selected from the group consisting of insulin receptor, insulin-like growth factor receptor (including both IGF-IR and IGF-2R), growth hormone receptor, transferrin receptor, epidermal growth factor receptor (EGF-R), low density lipoprotein receptor (LDLR), high density lipoprotein receptor (HDLR), leptin receptor; interleukin receptors including IL- 1 (CD 121 a/IL 1 Rl ,
CD121b/ILlR2), IL-2 (CD25/IL2RA, CD122/IL2RB, CD132/IL2RG), IL-3 (CD123/IL3RA, CD131/IL3RB), IL-4 (CD124/IL4R, CD132/IL2RG), IL-5 (CD125/IL5RA, CD131/IL3RB), IL-6 (CD126/IL6RA, CD130/IR6RB), IL-7 (CD127/IL7RA, CD132/IL2RG), IL-8
(CXCR1/IL8RA, CXCR2/IL8RB/CD 128), IL-9 (CD129/IL9R), IL-10 (CD210/IL10RA, CD W21 OB/IL 1 ORB), IL- 1 1 (IL 1 1 RA), IL- 12 (CD212/IL 12RB 1 , IR 12RB2), IL- 13 (IL 13R), IL-14, IL-15 (IL15RA), IL-16 (CD4), IL-17 (CDw217/IL17RA, IL17RB), IL-18
(CDw218a/IL18Rl), and IL21 (IL21R) receptors, human growth hormone receptor, VEGF receptor (VEGF-R), PDGF receptor (PDGF-R), transforming growth factor receptor
(including TGF-alpha and TGF-beta), ciliary neurotrophic factor receptor, prolactin receptor, and T-cell receptors.
In some embodiments, targeting peptides may comprise fragments of ligand peptides to one or more cell surface receptors. In some embodiments, targeting peptides comprising fragments of ligand peptides to one or more cell surface receptors may be inserted into one or more of the surface exposed loops of the LI capsid protein to generate modified VLP. It should be appreciated that targeting peptides comprising fragments of ligand peptides to one or more cell surface receptors may also be linked to the VLP surface by chemical means or by means of affinity. Chemical means may include the use of chemical linkers that covalently link the targeting peptide to the surface of the VLP comprising LI capsid protein. Linker chemistry and the coupling of specific amino acids via linker chemistry are well known in the art. Affinity means may comprise the use of affinity tags that may be inserted into one or more of the surface exposed loops of the VLP and the provision of a recognition sequence on the targeting peptide. Examples include StrepTag™ technology (as described, for example, in PCT/US2009/004299, reference incorporated herein in its entirety).
In some embodiments, the protein fragments or peptide sequences may comprise one or more targeting peptides derived from the proteins and peptides that interact with cellular receptors, such as insulin, insulin-like growth factor (IGF-1 or IGF-2), growth hormone (GH), transferrin, epidermal growth factor (EGF), low density lipoprotein (LDL), high density lipoprotein (HDL), leptin, interleukins IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and IL21 , VEGF, PDGF, TGF-alpha or TGF-beta, ciliary neurotrophic factor, and prolactin.
In some embodiments, the heterologous protein fragments or peptide sequences may comprise one or more targeting peptides, providing means to specifically target VLPs in vivo to specific tissues or cells expressing integrins, such as ανβ3, ανβ5, and ανβό integrins, wherein the targeting peptide comprises an RGD motif. In some embodiments, the targeting peptide sequence is derived from a nucleolin ligand. For example, the targeting peptide, in some embodiments, is the F3 tumor-homing peptide, F3
(AKVKDEPQRRSARLSAKPAPPKPEPKPKKAPA K, SEQ ID NO: 9). F3 is a 34-amino acid fragment of a high mobility group protein, HMG2N (Christian et al. J Cell Biol.
163:871-878, 2003; Porkka et al. , Proc. Natl. Acad. Sci. USA. 99:7444-7449 2002;
PCT US03/10240 Ruoslahti et al. ; incorporated by reference herein). In some embodiments, the F3 targeting peptide targets tumor vasculature, e.g. , tumor cells and angiogenic endothelial cells that express nucleolin in the cell surface. In some embodiments, the F3 peptide is internalized by its specific target cells and transported to the nucleus.
In some embodiments, the heterologous proteins or protein fragments may comprise one or more targeting peptides derived from viruses providing means to specifically target VLPs in vivo to specific tissues or cells. In certain embodiments targeting peptides derived from viruses that have been identified to be responsible for viral-mediated cell entry are provided.
For example peptides derived from the Hepatitis B surface antigen which identifies hepatocytes may be used for the treatment of liver disease and hepatocellular carcinoma Peptides derived from the Epstein Barr Virus, which identifies lymphocytes may be used for the treatment of lymphoma In certain embodiments, the human complement receptor 2 (CR2) binding domain of glycoprotein gp350/220 of the Epstein-Barr virus is a virus-derived targeting peptide that may be used for targeting as described herein
In certain embodiments, the pre-S2 region of HB V is a virus-derived targeting peptide that may be used for targeting as described herein
In certain embodiments, the HCV envelope glycoproteins (HCVpp) El and E2 may be used for targeting as described hereia
In some embodiments, peptides are used to enhance oral delivery. In certain embodiments, an integrin-adherent peptide motif, RGD, can be used to improve oral deliver ofVLPs.
It should be appreciated that many targeting peptides to particular tissues and/or cells and many tumor homing peptides are known in the art. For example, the following U.S. patent applications and U.S. patents describe targeting peptides and the cells and/or tissues they target:
U.S. Publication Nos. 2009/0214429 (tumors and wounds); 2009/0098633 (HMGN2 tumor blood vessels and tumor cells); 2008/0139479 (bladder tumor); 2007/0219134 (lymphatic tumors and premalignant lesions); 2007/0149444 (lymphatic tumors and premalignant lesions); 2006/0160743 (heart); 2006/0002854 (pancreas malignancies and premalignancies); 2005/0074812 (selected organs and tissues); 2004/0186056 (HMGN2 tumor blood vessels and tumor cells); 2004/0131623 (tumors); 2004/0087499 (tumor lymphatic vasculature); 2003/0232762 (breast); 2003/0152578 (tumors); 2003/01 13320 (angiogenic vasculature); U.S. Patent Nos. 7,544,767 (HMGN2 tumor blood vessels and tumor cells); 7,501,486 (heart); 7,488,792 (collagen/tumor vasculature); 7,192,921 (tumor lymphatic vasculature); 6,743,892 (selected organ); 6,610,651 (various organs and tissues); 6,576,239 (angiogenic); 6,303,573 (heart); 6,296,832 (selected organs); 6,180,084 (angiogenic); 6,177,542 (integrin); 5,981,478 (integrin); 5,912,234 (integrin). Accordingly, one or more of these peptides may be used in a VLP as described herein.
It should further be appreciated that additional peptides can be identified by methods well known in the art, such as, for example, phage display of tumor peptide libraries, and other screening procedures using phage display cDNA library and combined ex vivo screening on cell suspensions and in vivo screening, e.g., for tumor homing. Accordingly, one or more of these peptides may be used in a VLP as described herein. In some embodiments, one or more protein fragments or peptide sequences used for targeting of VLPs are inserted into one or more external or surface-exposed loops. The protein fragments or peptide sequences inserts can be 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200 or 250 amino acids long, or any length in between 4 amino acids and 250 amino acids. In certain embodiments, the inserts are between 4 and 50, 5 and 25, or 5 and 15 amino acids long. The inserts can be inserted anywhere in the loops. In some embodiments, the inserts are inserted approximately in the middle of a loop.
In some embodiments, where insertions are used to promote targeting or cellular uptake, the inserts may comprise, for example, viral targeting domains. It will be appreciated that these domains are not limited to HPV. Viral targeting domains may be derived from any virus to target any cell that is desired. The inserts may also comprise, for example, host- specific cellular recognition motifs, such as receptor recognition motifs.
In some embodiments wild-type amino acids comprising the loops are substituted or deleted, singly or in groups, to allow insertion of the targeting peptides while maintaining the ability of VLPs to self-assemble. By "maintaining the ability to self-assemble" it is meant that VLP maintain some ability to self-assemble. This ability may be reduced if compared to an unmodified VLP. For example, the ability to self-assemble may be measured by the ratio of LI (or LI and L2) protein expression and the number of self-assembled VLP observed by electron microscopy. In some embodiments, the ability to self-assemble is maintained resulting in VLPs that are well formed {e.g. , as judged by electron microscopy) and infectious (as assayed, e.g., in in vitro cell culture assays).
In some embodiments, amino acids present in loops exposed on the virus surface can be deleted or mutated to reduce the immunogenicity of the VLP (as, for example, described in PCT/US2009/004299, reference incorporated herein in its entirety).
In some embodiments, a tag polypeptide, which provides an epitope to which an anti- tag antibody can selectively bind, is inserted into one or more external loops. Provision of the epitope tag enables VLPs to be readily detectable by contacting the VLPs with a labeled anti-tag antibody, or an anti-tag antibody that provides chemical groups that can be utilized for labeling.
Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (Field et ai , Mol. Cell. Biol., 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al , Molecular and Cellular Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al , Protein Engineering, 3(6):547-553 (1990)). Other tag polypeptides include the Flag-peptide (Hopp et al , BioTechnology, 6: 1204-1210 (1988)); the KT3 epitope peptide (Martin et al , Science, 255: 192-194 (1992)); tubulin epitope peptide (Skinner et al. , J. Biol. Chem., 266: 15163-15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al , Proc. Natl. Acad. Sci. USA, 87:6393- 6397 (1990)).
In some embodiments, polypeptides encoding targeting sequences {e.g. , targeting peptides) are inserted into one or more of the external loops of the viral capsid protein that allow VLPs to be targeted to specific organs, tissues or cells, without destroying the ability of the viral capsid protein (LI or LI and L2) to form {e.g. , to self-assemble into) VLPs. In certain embodiments, VLPs are provided comprising insertion of a targeting peptide into one or more of the BC loop (amino acids 50-68), CD loop (amino acids 79-95), DE loop (amino acids 1 1 1-152), EF loop (amino acids 164-234), FG loop (amino acids 257-299), and HI loop (amino acids 349-360), based on wild-type HPV-16 LI capsid protein (505 amino acids, e.g., Philippine strain as described in Touze et al , J Clin Microbiol 1998, incorporated herein by reference, or SEQ ID NO: 13) which still permits VLP assembly. The resulting chimeric capsid protein is a fusion protein of the viral capsid protein and the targeting peptide sequence(s).
It should be appreciated that the loops, BC loop, CD loop, DE loop, EF loop, FG loop, and HI loop may be positioned at slightly different positions in different strains. This may be the result for example of small deletions or insertions of one or more amino acids in the various papilloma virus strains. Thus, slightly different start and end positions may occur for the different loops, /'. e. BC loop, CD loop, DE loop, EF loop, FG loop, and HI loop, in the various strains, e.g., the start and end position may shift one, two, three or more amino acids for one or more of the loops, e.g., the BC loop may start around amino acid positions 47, 48, 49, 50, 51, 52, 53 and may end around amino acid positions 65, 66, 67, 68, 69, 70, 71 depending on the papilloma virus strain. Likewise, the CD loop, DE loop, EF loop, FG loop, and HI loop may have variable start and end positions depending on the strain. It should be appreciated that a person of skill in the art will be able to identify loops corresponding to those described herein in the various strains.
By "targeting peptide" herein is meant a peptide sequence that serves to target or direct the VLPs to a particular location, cell type, diseased tissue, or association. In general, the targeting peptide is directed against a target molecule and allows concentration of the VLPs loaded with an agent (e.g., a therapeutic agent) in a particular localization within a subject
In certain embodiments, VLPs comprising targeting peptides may enter mammalian cells as intact VLPs via active cellular transport, such as receptor-mediated endocytosis or other vesicular traffic mechanisms. In embodiments, wherein VLPs enter the target cell mediated by receptor mediated endocytosis, the VLPs may enter and leave animal cells via the binding interaction between ligand molecules (e.g. , the targeting peptides) expressed and displayed by the VLPs and their corresponding receptor molecules on the target cell membrane, which causes the membrane to wrap around and engulf the VLPs.
In certain embodiments, VLPs may enter the target cell via receptor-mediated endocytosis as a consequence of interaction of the cellular receptor with a targeting peptide displayed on the surface of the VLPs. In certain embodiments, the VLP membrane may fuse with the membrane of the endosome membrane, which leads to the release of the therapeutic agent trapped inside the VLP.
In some embodiments, one or more external loops of the viral capsid protein may comprise a peptide sequence encoding e.g., antigen-specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof directed against specific receptor molecules such as e.g. , the integrin receptor family, the VEGF receptor family, the FGF receptor family, the IGF receptor family, the EGF receptor family and the hepatocyte receptor. In other embodiments, antigen-specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof expressed in the external loops of VLPs are directed against one or more tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs), such as viral tumor antigens, cellular oncogene proteins, and/or tumor-associated differentiation antigens, including, but not limited to, CEA, TAG-72 (Yokata et al , 1992, Cancer Res. 52:3402-3408), C017-lA (Ragnhammar et a/. , 1993, Int J. Cancer 53:751-758); GICA 19-9 (Herlyn et al , 1982, J. Clin. Immunol. 2: 135), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al, 1994, Blood 83: 1329-1336), human B-lymphoma antigen-CD20 (Reffet al, 1994, Blood 83:435-445), CD33 (Sgouros et al , 1993, J. Nucl. Med. 34:422-430), melanoma specific antigens such as ganglioside GD2 (Saleh et al. , 1993, J. Immunol., 151 , 3390-3398), ganglioside GD3 (Shitara et al, 1993, Cancer Immunol. Immunother. 36:373- 380), ganglioside GM2 (Livingston et al, 1994, J. Clin. Oncol. 12: 1036-1044), ganglioside GM3 (Hoon et al, 1993, Cancer Res. 53:5244-5250), tumor-specific transplantation type of cell-surface antigen (TSTA) such as virally-induced tumor antigens including T-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses, oncofetal antigen-alpha- fetoprotein such as CEA of colon, bladder tumor oncofetal antigen (Hellstrom et ah, 1985, Cancer. Res. 45:2210-2188), differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et ah , 1986, Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et ah, 1988, J. of
Immunospecifically. 141 : 1398-1403), neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (pl 85HER2), polymorphic epithelial mucin (PEM) (Hilkens et ah , 1992, Trends in Bio. Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhard et ah , 1989, Science 245:301 -304),
differentiation antigen (Feizi, 1985, Nature 314:53-57) such as I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes, preimplantation embryos, I(Ma) found in gastric adenocarcinomas, Ml 8, M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D,56-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, Ei series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric
adenocarcinoma antigen, CO-514 (blood group Lea) found in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Leb), G49 found in EGF receptor of A431 cells, MH2 (blood group ALeb/Ley) found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, T5A7 found in myeloid cells, R24 found in melanoma, 4.2, GD3, Dl . l , OFA-1 , G 2, OFA-2, GD2, and Ml :22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos T cell receptor derived peptides from
Cutaneous T cell Lymphoma (Edelson, 1998, The Cancer Journal 4:62), IL-1 , IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IFN-a, IFN-β, IFN-β 17 mutants, IFN-65, CD2, CD3, CD4, CD5, CD8, CD1 la, CD1 lb, CD1 lc, CD 16, CD 18, CD21 , CD28, CD32, CD34, CD35, CD40, CD44, CD45, CD54, CD56, OX40L, 4-1 BBL, K2, Kl , Ρβ, Oct, Ma, Μβ2, Μβΐ , Hepsin, Pim-1 , LMP1 , TAP2, LMP7, TAP1 , TRP, Οβ, ΙΑβ, IAa, ΙΕβ, ΙΕβ2, IEa, CYP21, C4B, CYP21P, C4A, Bf, C2, HSP, G7a/b, TNF-a, TNF-β, D, L, Qa, Tla, COLl 1A2, ϋΡβ2, DPa2, ϋΡβΙ , DPal, DNa, DMa, ϋΜβ, LMP2, TAPil, LMP7, ϋθβ, DQ 2, DQa2, DQJ3, DQJ31 , DQal , DRβ, DRa, G250, HSP-70, HLA-B, HLA-C, HLA-X, HLA-E, HLA-J, HLA-A, HLA-H, HLA-G, HLA-F, nerve growth factor, somatotropin, somatomedins, parathormone, FSH, LH, EGF, TSH, THS-releasing factor, HGH, GRHR, PDGF, IGF-I, IGF- II, TGF-β, GM-CSF, M-CSF, G-CSF1 , erythropoietin, β-HCG, 4-N
acetylgalactosaminyltransferase, GM2, GD2, GD3, JADE, MART, BAGE, GAGE, MAGE-1 , MAGE-2, MAGE-3, XAGE, MUC-1 , MUC-2, MUC-3, MUC-4, MUC-18, ICAM-1 , C-CAM, V-CAM, ELAM, NM23, EGFR, E-cadherin, N-CAM, LFA-3 (CD58), EpCAM, B7.1 , CEA, DCC, PSA, Her2-neu, UTAA, melanoma antigen p75, K19, HKer 8, pMel 17, TP10, tyrosinase related proteins 1 and 2, p97, p53, RB, APC, DCC, NF-1 , NF-2, WT-1 , MEN-I, MEN-II, BRCAl, VHL, FCC and MCC, ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl, Clq, Clr, Cl s, C4, C2, Factor D, Factor B, properdin, C3, C5, C6, C7, C8, C9, Cllnh, Factor H, C4b-binding protein, DAF, membrane cofactor protein, anaphylatoxin inactivator S protein, HRF, MIRL, CR1 , CR2, CR3, CR4, C3a/C4a receptor, C5a receptor, Epstein-Barr Virus antigens (EBNA), BZLF-1 , BXLF-1 , and Nuclear Matrix Proteins, modified TAAs or TSAs, splice variants of TAAs or TS As, functional epitopes, epitope agonists, and degenerate variations thereof.
It should be appreciated that antigen-specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof may also be linked to the VLP surface by chemical means or by means of affinity. Chemical means may include the use of chemical linkers that covalently link the antigen-specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof to the surface of the VLP comprising LI or LI and L2 capsid protein. Linker chemistry and the coupling of specific amino acids via linker chemistry are well known in the art. Affinity means may comprise the use of affinity tags that may be inserted into one or more of the surface exposed loops of the VLP and the provision of a recognition sequence on the antigen- specific fragments, single chain antibodies, nanobodies (VHH antibodies, single domain antibodies) and/or camel/llama (heavy-chain) nanobodies or fragments thereof. Examples include StrepTag™ technology (as described, for example, in PCT/US2009/004299, reference incorporated herein in its entirety).
In some embodiments, VLPs are provided comprising LI viral capsid protein comprising targeting peptides in one or more loops that facilitate targeting of the VLPs to airway epithelial cells. In some embodiments, modified LI proteins comprising one or more insertions of a targeting peptide maintain the ability to self-assemble into VLPs.
In some embodiments, modifications in LI are insertions of targeting peptides within antigenic loops of the LI major protein of HPV. It should be appreciated that targeting peptides may be inserted at positions previously shown not to disturb the self-assembly of the LI protein into VLPs. (Sadeyen et al, 2003; Carpentier et al. ; 2005). In some embodiments, targeting peptides effective for targeting VLPs to specific tissues, organs, or cells may be identified by phage display technology or any other affinity-based technology known in the art. In some embodiments, targeting peptides effective for targeting VLPs to specific tissues, organs, or cells may be identified on the basis of greater internalization efficiency using specific cell lines in vitro. It should be appreciated that the cell line(s) used to analyze greater internalization efficiency should be derived (or should correlate in vitro) to the tissue, organ or cell that is to be targeted in vivo by the modified VLPs comprising a targeting peptide. For example, targeting peptides effective to direct VLPs to human airway epithelial cells in vivo may be selected through binding or internalization studies using phage displaying peptide libraries on human airway epithelial cells (Drapkin et al , 2000; Gaden et al , 2005; Jost et al, 2001 ; Vaysse et al , 2000). Internalization can be monitored, for example, by fluorescence microscopy or any other method known in the art. In some embodiments, increased localization of targeting peptides may be observed in the cytoplasm, nucleus or both. After identification of effective targeting peptides the targeting peptides can be inserted into one or more of the surface exposed loops of the viral capsid protein, e.g. , LI. It should be appreciated that the LI protein and optionally L2 protein to be modified can be derived from any known HPV type (e.g. , HPV 6, 1 1, 16, 18, 31 , 33, 34, 35, 39, 45, 51 , 52, 56, 58, 59, 68, 69, 73, and HPV91 , or any other known HPV type).
In some embodiments, increase internalization of reassembled modified VLPs comprising targeting peptides may be measured by fluorescence microscopy. In some embodiments, VLPs may be labeled with a fluorescent dye or a substrate that becomes fluorescent upon internalization, e.g., carboxyfluorescein diacetate succinimidyl ester (CFDA-SE).
In some embodiments, VLPs are provided comprising LI viral capsid protein comprising targeting peptides in one or more loops that facilitate targeting of the VLPs to airway epithelial cells, wherein the targeting peptides consist of or comprise the amino acid sequence as set forth in SEQ ID NO: 1 , THALWHT (PI , Jost et al , 2001); SEQ ID NO: 2, RFDSLKV (P2, Vaysse et al , 2000); SEQ ID NO: 3; GHPRQMSHVY (P3, Gaden et al , 2005); SEQ ID NO: 7, PHPNRAQ and SEQ ID NO: 8, VDRLQQK.
In some embodiments, targeting peptides may be inserted into one or more external loops of the LI viral capsid protein, such as the BC loop (amino acids 50-68), CD loop (amino acids 79-95), DE loop (amino acids 1 1 1-152), EF loop (amino acids 164-234), FG loop (amino acids 257-299), and HI loop (amino acids 349-360), based on wild-type HPV-16 LI capsid protein (505 amino acids, e.g., Philippine strain as described in Touze et al , J Clin Microbiol 1998, incorporated herein by reference, or SEQ ID NO: 13). It should be appreciated that the LI protein to be modified in the loops can be derived from any known HPV type and that the invention is not limited to modified LI capsid protein of HPV-16. The targeting peptides may be 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200 or 250 amino acids long, or any length in between 4 amino acids and 250 amino acids. In certain embodiments, the targeting peptide inserts are between 5 and 50, 5 and 25, 5 and 15, 5 and 10 or 7 and 10 amino acids long. In certain embodiments, the targeting peptides are inserted at the N-terminal end of the loop. For example, a targeting peptide can be inserted between amino acids 49 and 50 of HPV-16 LI capsid protein, where amino acid 50 is the start (N-terminal end) of the BC loop, or it may be inserted between amino acids 50 and 51 , 51 and 52, 53 and 54, 55 and 56 and so on In certain embodiments, the targeting peptides are inserted at the C-terminal end of the loop. For example, a targeting peptide can be inserted between amino acids 68 and 69 of HPV-16 LI capsid protein, where amino acid 68 is the end (C-terminal end) of the BC loop, or it may be inserted between amino acids 67 and 68, 66 and 67, 65 and 66, 64 and 65 and so on. In certain embodiments, the targeting peptides are inserted approximately in the middle of the loop. For example, a targeting peptide can be inserted between amino acids 59 and 60 of HPV-16 LI capsid protein, where amino acid 59 is approximately the middle (between N- terminal and C-terminal end) of the BC loop, or it may be inserted between amino acids 56 and 57, 57 and 58, 58 and 59, 60 and 61, 61 and 62, 62 and 63 and so on. It should be appreciated that insertions can be made into any of the loops of the viral capsid protein, following the procedure described herein.
In some embodiments, target peptides are inserted into the DE loop. In some embodiments, target peptides are inserted into position 140/141 of the DE loop of HPV-16 LI protein, or an equivalent position within the DE loop of any other HPV serotype. In some embodiments, the target peptide is PI : THAL WHT (SEQ ID NO: 1). In some embodiments, the target cells are airway epithelial cells.
In some embodiments, target peptides are inserted into the EF loop. In some embodiments, target peptides are inserted into position 179/180 of the EF loop of HPV-16 LI protein, or an equivalent position within the EF loop of any other HPV serotype. In some embodiments, the target peptide is P3: GHPRQMSHVY (SEQ ID NO: 3). In some embodiments, the target cells are airway epithelial cells. It will be appreciated that if certain motifs are known to be presented by a certain loop and it is desired to maintain the motif, the insertion will be made outside the motif, either N- terminal or C-terminal of the motif On the other hand, if it is desired to disrupt a certain motif presented in a certain loop then the insertion will be made within the motif. The motif can be linear or structural, that is it may be based on the primary amino acid sequence or its secondary (or tertiary) structure. The motif can be a cellular recognition motif, that may facilitate VLP uptake and/or target cell recognition, or it may be an epitope that is recognized by certain antibodies or that is known to be antigenic.
It will further be appreciated that additional deletions, insertions, substitutions or mutations may be made in one or more of the amino acids of the one or more loops to be modified by insertion of targeting peptides. Such modifications may further be made outside the loops, e.g., N-terminal to the beginning of a loop or C-terminal to the end of a loop or at any place in the viral capsid proteia Any modifications described herein that maintain the ability of the viral capsid protein (e.g., LI) to self-assemble are contemplated herein.
In some embodiments, mutations or insertions into the loops may be made according to those identified by Chen et al. (2000). In some embodiments, mutations or insertions into the loops may be made at the surface of the LI capsomers. In some embodiments, mutations or insertions into alpha-helices or beta-sheets of the LI capsomers are avoided (Sadeyen et al, 2003). In some embodiments, mutations certain mutations within loops that destabilize the structure are avoided (Kirnbauer et al, 1993).
It should be appreciated that the modifications described herein, e.g. , insertions of targeting peptides into external loops of viral capsid proteins {e.g., LI or L2), are carried out in such a manner that the ability of the protein to self-assemble into VLPs is maintained. It should be appreciated that the ability of modified LI or LI and L2 to self-assemble into VLPs may be enhanced or reduced when compared to wild-type LI and L2, respectively.
It should further be appreciated that additions, deletions, mutations and substitutions of amino acids in one or more of the external loops of the viral capsid proteins outside and/or in addition to the insertion of targeting peptides into these one or more loops are also contemplated. These modifications may be made, for example, to improve functionality of the loop, e.g. , improve presentation of the targeting peptide or domain, or to improve stability of the motif of the targeting peptide, or to additionally suppress immunogenicity of the one or more loops. Such modifications also include conservative amino acid changes in one or more of the amino acids of the one or more exposed loops and/or the remainder of the amino acid sequence of the viral capsid protein(s), LI or L2. Conservative amino acid changes may be made according to functional, chemical or structural considerations. For example, conservative amino acid changes may be made according to chemical similarity: acidic (D/E), aliphatic (A/G/I/L/V), amide (N/Q), aromatic (F/W/Y), basic (R/H/K), hydroxyl (S/T), imino (P), sulfur (CM); or functional similarity: acidic (D/E), basic (R/H/K), hydrophobic (A/I/L/M/F/P/W/V), polar (N/C/Q/G/S/T/Y); or similarity in charge: acidic, basic, neutral; or structural similarity: ambivalent
(A/C/G P/S/T/W/Y), external (R/N/D/Q/E/H/K), internal (I/L/M/F/V), wherein any amino acid of a group of amino acids in parentheses can be changed into another in that group and such change would be considered a conservative change according to the consideration applied, e.g. , structural, functional, or chemical. In some embodiments, one or more factors may be considered.
It should be appreciated that any changes in the wild-type amino acid sequence (e.g., insertions of targeting peptides, deletions, and/or mutations/substitutions) taught herein are introduced in the nucleic acid that encodes the modified viral capsid protein, for example by genetic engineering (cloning) and other molecular biology techniques known in the art. For example, the chimeric peptides comprising e.g., fusions of targeting peptides and viral capsid protein may be generated by recombinant techniques generating chimeric genes inserted into expression vectors under the control of heterologous promoters driving expression of the chimeric gene in an expression system, e.g., bacterial, insect, yeast, plant or mammalian, producing recombinant chimeric fusion protein. These techniques are well known in the art, e.g., Sambrook et l. Molecular Cloning: A laboratory Manual (3rd edition, CSHL Press, 2001)
In certain embodiments, L I protein and L I and L2 protein may be produced
recombinantly. In certain embodiments, recombinantly produced LI protein and L I and L2 protein may self-assemble to form virus-like particles (VLPs). Recombinant production may occur in a bacterial, insect, yeast or mammalian (heterologous) host system, or any other heterologous host system known in the art. L I protein may be expressed or LI and L2 protein may be co-expressed in the host system.
Methods for expressing and purifying LI and L2 recombinant viral proteins in host systems, methods for disassembly and re-assembly of HPV nanoparticles or VLPs, and examples of modifications to the amino acid sequences of L I and L2, administration of VLPs to subjects, and pharmaceutical compositions comprising VLPs are well known in the art and are taught herein. For example, U.S. Patent Nos: 6,416,945; 6,991 ,795; and 7,205, 126, incorporated herein by reference. It should be appreciated however, that the methods and modes provided herein are not limited to those described in the aforementioned U.S. patents. Other methods and modes known to those in the art may also be employed.
In certain embodiments, VLPs are loaded with the one or more diagnostic and/or therapeutic agents. VLPs may be loaded by dissembling and reassembling L I or LI and L2 viral particles, as described herein. Salts that are useful in aiding dissembly/reassembly of viral capsid proteins into VLP, include Zn, Cu, Ni, Ru and Fe salts.
Other methods of loading may be used as the invention is not limited in this respect. In some embodiments, HPV nanoparticles may be loaded with one or more therapeutic agents.
In some embodiments, HPV nanoparticles comprising L I protein, or L I and L2 protein, further comprise one or more agents (e.g., therapeutic agents). In certain
embodiments the therapeutic agent comprises one or more siRNA molecules or one or more nucleic acids (e.g. , plasmid or other vector) that each is capable of expressing one or more siRNA molecules. In some embodiments, the therapeutic agent comprises one or more antisense nucleic acids or one or more nucleic acids (e.g. , plasmid or other vector) that each are capable of expressing one or more antisense nucleic acids. In some embodiments, the HPV nanoparticles comprise combinations of two or more therapeutic agents.
In some embodiments, the therapeutic agent is an inducer of RNA interference (RNAi) or other inducer of gene silencing. An inducer of RNA interference may be a siRNA, a shRNA, a hybrid nucleic acid molecule comprising a first part that comprises a duplex ribonucleic acid (RNA) molecule and a second part that comprises a single stranded deoxyribonucleic acid (DNA) molecule, a longer double-stranded RNA or a DNA construct for expression of siRNA or longer RNA sequences. Other inducers of gene silencing include inducers of DNA methylation, or ribozymes, or aptamers. In other embodiments, the therapeutic agent can be a modulator of gene expression such as a PNA (Peptide Nucleic Acid).
Some aspects of the invention relate to the use of small nucleic acid molecules, including antisense nucleic acids and short interfering nucleic acid (siNA), the latter include, for example: microRNA (miRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), and short hairpin RNA (shRNA) molecules to knockdown expression of target genes associated with a disease or disorder. An siNA can be unmodified or chemically- modified. An siNA can be chemically synthesized, expressed from a vector or enzymatically synthesized. In some embodiments, provided herein are chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating gene expression or activity in cells by RNA interference (RNAi). In certain embodiments, the use of chemically- modified siNA may improve various properties of native siNA molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al, International Publication No. WO 92/07065; Perrault et al, 1990 Nature 344, 565; Pieken et al , 1991 , Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al, International Publication No. WO
93/15187; and Rossi et al , International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,71 1 ; and Burgin et al, supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein). In some embodiments, modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are provided.
There are several examples in the art describing sugar, base and phosphate
modifications that can be introduced into nucleic acid molecules with significant
enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups,. for example, 2' amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31 , 163; Burgin et al , 1996, Biochemistry , 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al, International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565 568; Pieken et al Science, 1991, 253, 314317; Usman and
Cedergren, Trends in Biochem. Sci., 1992, 17, 334 339; Usman et al. International
Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,71 1 and Beigelman et al , 1995, J. Biol. Chem., 270, 25702; Beigelman et al , International PCT publication No. WO 97/26270; Beigelman et al , U.S. Pat No. 5,716,824; Usman et al , molecule comprises one or more chemical modifications.
In some embodiments, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence identical to the nucleotide sequence or a portion thereof of the targeted RNA In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is substantially complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the target RNA In another embodiment, each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
In some embodiments an siNA is an shRNA, shRNA-mir, or microRNA molecule encoded by and expressed from a genomically integrated transgene or a plasmid-based expression vector. Thus, in some embodiments a molecule capable of inhibiting mRNA expression, or microRNA activity, is a transgene or plasmid-based expression vector that encodes a small-interfering nucleic acid. Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siRNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue-specific expression systems. In some embodiments, transgenes and expression vectors are controlled by tissue-specific promoters. In certain embodiments transgenes and expression vectors are controlled by inducible promoters (e.g., such as tetracycline inducible expression systems).
In some embodiments, the small interfering nucleic acid described herein are expressed in mammalian cells using a mammalian expression vector. The recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type {e.g. , tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art Non- limiting examples of suitable tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid-specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters. Developmentally- regulated promoters are also provided herein, for example the murine hox promoters and the alpha-fetoprotein promoter.
siRNA molecules are well know in the art and many siRNAs are known that target expression of tumor-specific proteins that may be mutated, overexpressed and/or
deregulated, such as for example cyclin/cdk, EGFR, brc/abl and the like.
Accordingly, modified VLP described herein can be used to deliver molecules that promote RNA interference using any of a variety of molecules known in the art, e.g. , short interfering RNA molecules (siRNA), which are double stranded RNA molecules. As described herein, RNA interference (RNAi) is a phenomenon describing double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Synthetic duplexes of 21 nucleotide RNAs can mediate gene specific RNAi in mammalian cells, without invoking generic antiviral defense mechanisms (Elbashir et al. Nature 2001 , 41 1 :494-498; Caplen et al. Proc Natl Acad Sci 2001 , 98:9742-9747).
In some embodiments, polynucleotides are provided comprising an RNAi sequence that acts through an RNAi mechanism to attenuate or inhibit expression of a gene of interest, e.g., a gene that is overexpressed in cancer. In some embodiments, the siRNA sequence is between about 19 nucleotides and about 75 nucleotides in length, or between about 25 base pairs and about 35 base pairs in length. An RNAi construct can contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript of a gene of interest. In certain embodiments, the double-stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi. In certain embodiments, the number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3 ' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition
In certain embodiments, sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters {e.g., University of Wisconsin Genetic Computing Group). In certain embodiments, the sequence identity between the inhibitory RNA and the portion of the target gene is greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is 100%.
Production of polynucleotides comprising RNAi sequences is well known in the art. For example, polynucleotides comprising RNAi sequences can be produced by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase of the treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro. In certain embodiments, the polynucleotides that modulate target gene activity by RNAi mechanisms, may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g. , to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase. In certain embodiments, the siRNA polynucleotides may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
Methods of chemically modifying RNA molecules can be adapted for modifying RNAi constructs (see, for example, Heidenreich et al. (1997) Nucleic Acids Res, 25:776- 780; Wilson et al. (1994) J Mol Recog 7:89-98; Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate, the backbone of an RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g. , 2'- substituted ribonucleosides).
The double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g. , at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition generally is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
In certain embodiments, the subject RNAi constructs are "siRNAs." These nucleic acids are between about 19-35 nucleotides in length, preferably between 25 and 30 or 21-23 nucleotides in length, e.g., with 21-23 nucleotides in length corresponding in length to the fragments generated by nuclease "dicing" of longer double-stranded RNAs. In certain embodiments, the length is about 27 nucleotides, e.g. 25, 26, 27, 28, 29, or 30. In certain embodiments, the length is exactly 27 nucleotides (Kim et al. 2005 Nat Biotechnol 23:222- 226). The siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex or translation is inhibited. In a particular embodiment, the siRNA molecules comprise a 3' hydroxyl group. The siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify such molecules.
Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA molecules. In addition, chromatography (e.g. , size exclusion chromatography), glycerol gradient centrifugation, and affinity purification with antibody can be used to purify siRNAs. In certain embodiments, siRNA molecules are labeled, e.g.
fluorescently labeled with a label such as FITC, Cy-3, Cy-5, etc.
In certain embodiments, at least one strand of the siRNA sequence has a 3' overhang from about 1 to about 6 nucleotides in length, or from 2 to 4 nucleotides in length. In other embodiments, the 3' overhangs are 1-3 nucleotides in length. In certain embodiments, one strand has a 3' overhang and the other strand is either blunt-ended or also has an overhang. In certain embodiments, the siRNA molecule is double stranded, double blunt-ended (e.g. U.S. patent 7,723,316). The length of the overhangs may be the same or different for each strand. In order to further enhance the stability of the siRNA sequence, the 3' overhangs can be stabilized against degradation In one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g. , substitution of uridine nucleotide 3' overhangs by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi. The absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium and may be beneficial in vivo.
Examples of siRNA that can be delivered by modified VLPs described herein include siRNA that mediate silencing of oncogenes, genes of infectious agents (including viruses), and other targets, including lung cancer specific target genes and cystic fibrosis-specific target genes.
Tools for design and quality of siRNAs, shRNAs and/or miRNAs are known in the art. Web-based online software systems for designing siRNA sequences and scrambled siRNA sequences are for example SIDIRECT, SISEARCH, SEQ2SVM, DEQOR, SIRNA WIZARD (InvivoGen, San Diego, CA). The specificity can be predicted using for example SPECIFICITYSERVER, MIRACLE. Target sequences can be researched for example at HuSiDa (Human siRNA Database), and siRNAdb (a database of siRNA sequences).
Other inhibitor molecules that can be used (e.g. , packaged in VLPs) include sense and antisense nucleic acids (single or double stranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, antibodies, aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins. Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al , Leukemia 6(1 1): 1786-94, 1993; Valera et a/. , J. Biol. Chem. 269(46):28543-6, 1994; Dosaka-Akita et al, Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng et al, Cancer Res. 55(10):2024-8, 1995; Quattrone et al, Cancer Res. 55(l):90-5, 1995; Lewin et al , Nat Med. 4(8):967-71 , 1998). For example, neoplastic reversion was obtained using a ribozyme targeted to an H- Ras mutation in bladder carcinoma cells (Feng et al , Cancer Res. 55(10):2024-8, 1995). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al , Nat. Med. 2(6):643-8, 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al , Embo J. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9, 1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al, Nucleic Acids Symp Ser. (29): 121 -2, 1993).
Antisense nucleic acids include modified or unmodified RNA, DNA, or mixed polymer nucleic acids, and primarily function by specifically binding to matching sequences resulting in modulation of peptide synthesis (Wu-Pong, 1994, BioPharm, 20-33). An antisense nucleic acid binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules may also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit Rev. in Oncogenesis 7, 151 -190).
As used herein, the term "antisense nucleic acid" describes a nucleic acid that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. Triple helix approaches have also been investigated for sequence-specific gene suppression. Triple helix-forming oligonucleotides have been found in some cases to bind in a sequence- specific manner (Postel et al. , Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31 , 1991 ; Duval- Valentin et c/., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A. 93(7):281 1-6, 1996; Porumb et al, Cancer Res. 56(3):515-22, 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Hanvey et al, Antisense Res. Dev. 1(4):307-17, 1991 ; Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor et al, Arch. Surg. 132(1 1): 1 177-83, 1997). Minor-groove binding polyamides can bind in a sequence-specific manner to DNA targets and hence may represent useful small molecules for suppression at the DNA level
(Trauger et al. , Chem. Biol. 3(5):369-77, 1996). In addition, suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz Nature 329(6136):219-22, 1987; Rimsky et al , Nature 341(6241):453-6, 1989; Wright et al , Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989). In some cases suppression strategies have led to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein.
In some embodiments, VLPs may be used to package and/or deliver small activating
RNAs (saRNAs) and/or snRNA Ul (uRNAs).
Aspects of the invention relate to using VLPs to package and deliver any one or more of the different types of RNA or other nucleic acid molecules described herein.
In certain embodiments, VLPs are provided that are useful as delivery agents, such as for the in vivo delivery of one or more therapeutic agents, e.g., one or more anti-cancer agents, to a subject Anti-cancer agents that may be delivered by the modified VLPs described herein include, but are not limited to, acivicin; aclarubicin; acodazole
hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium;
bropirimine; busulfan; cactinomycin; calusterone; capsitabine; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;
dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin;
dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil;
flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;
hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleulin II (including recombinant interleukin II; or rIL2); interferon alfa-2a; interferon alfa-2b; interferon alfa-nl ; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan
hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride;
lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine;
mechlorethamine; mechlorethamine oxide hydrochloride rethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate;
methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin;
mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;
mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;
pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;
porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin
hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;
semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;
vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;
vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride; improsulfan; benzodepa; carboquone;
triethylenemelamrine; triethylenephosphoramide; triethylenethiophosphoramide;
trimethylolomelainine; chlomaphazine; novembichin; phenesterine; trofosfamide;
estermustine; chlorozotocin; gemzar; nimustine; ranimustine; dacarbazine; mannomustine; mitobronitol; aclacinomycins; actinomycin F(l); azaserine; bleomycin; carubicin;
carzinophilin; chromomycin; daunorubicin; daunomycin; 6-diazo-5-oxo-l-norleucine; doxorubicin; olivomycin; plicamyciri; porfiromycin; puromycin; tubercidin; zorubicin;
denopterin; pteropterin; 6-mercaptopurine; ancitabine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; enocitabine; pulmozyme; aceglatone; aldophosphamide glycoside;
bestrabucil; defofamide; demecolcine; elfornithine; elliptinium acetate; etoglucid; flutamide; hydroxyurea; lentinan; phenamet; podophyllinic acid; 2-ethylhydrazide; razoxane;
spirogermanium; tamoxifen; taxotere; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; urethan; vinblastine; vincristine; vindesine and related agents; 20-epi-
1 , 25; dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene;
adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;
amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing mo hogenetic protein- 1 ; antiandrogen; prostatic carcinoma; antiestrogen;
antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; azatoxin;
azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;
calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4;
combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;
didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifl uridine; droloxifene;
dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin;
gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol; iroplact;
irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon;
leuprolide/estrogen/progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;
lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;
marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopraminde; MIF inhibitor;
mifepristone; miltefosine; nirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody; human chorionic
gonadotrophin; monophosphoryl lipid A+ myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N- substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; taxel; taxel analogues; taxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate;
phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin
A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase
C inhibitor; protein kinase C inhibitors; microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rheniuim Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B l ; ruboxyl; safingol; saintopin; SarCNU; sarcophytpl A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense
oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D;
spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans;
tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin;
thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system; erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer; 5-fluorouracil; leucovorin;
capsitabine; cyclosphosphamide; and gemcitabine.
Examples of therapeutic agents or imaging agents that may also be delivered in vivo to a subject by the modified VLPs provided herein, are cellular components, genetically engineered or native, recombinant, soluble or any other type of proteins, peptides, cytokines or other signaling molecules, which can have pro- or anti-inflammatory effects, or pro- or anti- anti-apoptotic effects, polysaccharides, glycoproteins, heterogeneous mixtures of
macromolecules (e.g. , a natural product extract) and hybrid macromolecules (e.g. ,
protein/nucleic acid hybrids, albumin conjugated proteins, drugs, inorganic molecules, organic molecules, or combinations thereof), and/or other bioactive molecules, other nucleic acids (e.g., RNA, DNA, PNA, multiplexes of them (e.g. , triplex)), preferably siRNA and antisense RNA, and/or cytotoxic drugs. A therapeutic substance may also be any of the following agents: adrenergic agent; adrenocortical steroid; adrenocortical suppressant; agents for treating cognition, antiplatelets, aldosterone antagonist; amino acid; anabolic; analeptic; analgesic; anesthetic; anorectic; antiacne agent; anti-adrenergic; anti-allergic; anti-Alzheimer's, anti-amebic; anti-anemic; anti- anginal; anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial; anticholinergic;
anticoagulant; anticonvulsant; antidepressant; antidiabetic; antidiarrheal; antidiuretic; antiemetic; anti-epileptic; antifibrinolytic; antifungal; antihemorrhagic; antihistamine;
antihyperlipidemia; antihypertensive; antihypo tensive; anti-infective; anti-inflammatory; antimicrobial; antimigraine; antimitotic; antimycotic, antinauseant, antineoplastic,
antineutropenic, antiparasitic; antiproliferative; antipsychotic; antirheumatic; antiseborrheic; antisecretory; antispasmodic; antithrombotic; anti-ulcerative; antiviral; anxiolytics, appetite suppressant; blood glucose regulator; bone resorption inhibitor; bronchodilator; cardiovascular agent; cholinergic; COX1 inhibitors, COX2 inhibitors, direct thrombin inhibitors, depressant; diagnostic aid; diuretic; dopaminergic agent; estrogen receptor agonist; fibrinolytic;
fluorescent agent; free oxygen radical scavenger; gastrointestinal motility effector;
glucocorticoid; GPIIbllla antagonists, hair growth stimulant; hemostatic; histamine H2 receptor antagonists; hormone; human growth hormone, hypocholesterolemic; hypoglycemic; hypolipidemic; hypnotics, hypotensive; imaging agent; immunological agents such as immunizing agents, immunomodulators, immunoregulators, immunostimulants, and immunosuppressants; keratolytic; LHRH agonist; mood regulator; mucolytic; mydriatic; nasal decongestant; neuromuscular blocking agent; neuroprotective; NMDA antagonist; non- hormonal sterol derivative; plasminogen activator; platelet activating factor antagonist;
platelet aggregation inhibitor; proton pump inhibitors, psychotropic; radioactive agent;
scabicide; sclerosing agent; sedative; sedative-hypnotic; selective adenosine Al antagonist; serotonin antagonist; serotonin inhibitor; serotonin receptor antagonist; statins, steroid; thyroid hormone; thyroid inhibitor; thyromimetic; tranquilizer; amyotrophic lateral sclerosis agent; cerebral ischemia agent; Paget's disease agent; unstable angina agent; vasoconstrictor;
vasodilator; wound healing agent; and/or xanthine oxidase inhibitor, but it is not so limited.
It should be appreciated by one of ordinary skill, that the compositions and diagnostic, prophylactic or therapeutic substances or agents described herein may be used in combination with the modified VLPs described herein. For example, in some embodiments, VLPs may be used to deliver one or more of the compositions and diagnostic, prophylactic or therapeutic substances or agents. VLPs, in some embodiments, may comprise one or more of the compositions and diagnostic, prophylactic or therapeutic substances or agents. VLPs, in some embodiments, may be formulated in one or more of the compositions described herein. One or more of the compositions may comprise the modified VLPs described herein. The one or more of compositions and diagnostic, prophylactic or therapeutic substances or agents may be delivered by VLPs or may be administered in conjunction or combination with VLPs, for example as part of a treatment regimen. The one or more of compositions and diagnostic, prophylactic or therapeutic substances or agents may be administered at substantially the same time as the VLPs, or may be administered at different times. The one or more of compositions and diagnostic, prophylactic or therapeutic substances or agents may be administered by the same route of administration as the VLPs, or may be administered by different a different route. The one or more of compositions and diagnostic, prophylactic or therapeutic substances or agents may be administered in combination with the VLPs to augment or enhance the effects of the VLPs to treat the same disease or ailment, or, for example, to treat additional disease components, additional ailments and/or side effects that are not treated, targeted or alleviated by the VLP treatment.
The diagnostic, prophylactic or therapeutic substances used are preferably sterile. In some embodiments solutions comprising diagnostic, prophylactic or therapeutic substances described herein are sterilized by mechanical filtration. For example, many sensitive pharmaceuticals or protein solutions may not be suitable for sterilization methods comprising exposure to heat, irradiation or chemical sterilization. A filter with pore size 0.2 μιτι may be used to effectively remove bacteria. If viruses must also be removed, a pore size around 20 nm may be needed. The filtration equipment and the filters themselves may be purchased as pre- sterilized disposable units in sealed packaging, or may be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure sterility, the filtration system may be tested to ensure that the membranes have not been punctured prior to or during use._Pharmaceutical sterile filtration may be performed in a room with highly filtered air (HEPA filtration) or in a laminar flow cabinet.
Radiation sterilization may be used such as electron beams, X-rays, gamma rays, or subatomic particles. Gamma rays may be used, for example, for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets. Electron beam processing may be used, for example, for medical device sterilization. X-rays may be used, for example, to irradiate large packages and pallet loads of medical devices. Ultraviolet light irradiation may be used, for example, for sterilization of surfaces (e.g. , the interiors of biological safety cabinets) and some transparent objects. Steam sterilization may be used. For example, autoclaves are commonly used for steam sterilization. Typical sterilization conditions comprise a holding time of at least 15 minutes at 121 °C or 3 minutes at 134 °C.
A "diagnostic substance" is any substance that has diagnostic capabilities, for example imaging agents, such as detectable markers, for example heavy metals, Gadolinium, Quantum dots, magnetic particle, radioactive particles, labeled antibodies, labeled nucleic acids (e.g. siRNA), luciferase and other chemoluminescent agents. These agents may be substances trapped inside the VLP, and/or on the surface (e.g. , outer-surface) of the membrane of the VLP. These agents may be used to detect an adverse condition by any medical detection device or method, such as for example Magnetic Resonance Imaging (MRI), Positron
Emission Tomography (PET), Computerized Axial Tomography (CAT), X-rays, or other imaging modalities. These applications may provide for immediate monitoring and/or diagnosis of early metastasis. It should be appreciated that imaging may be combined with therapy, for example, to allow for simultaneous treatment and monitoring (e.g., to confirm that treatment is appropriately localized to a target site such as a tumor or other diseased tissue). It should be appreciated that the VLPs described herein may be used to deliver imaging agents and/or therapeutic agents. Further, it should be appreciated that the VLPs described herein that are used to deliver imaging agents and/or therapeutic agents may be administered in combination with one or more therapeutic and/or imaging agents. These agents may be different from the one or more agents delivered by VLPs or may be the same, but
administered via a different route and/or at different doses and/or at different times.
Methods are provided for treating a subject in need of treatment administering the VLPs or VLP compositions described herein in an amount sufficient to treat the subject.
As used herein, the terms "treat," "treated," or "treating" when used with respect to an adverse condition, such as a disorder or disease (e.g., cancer, infection, neurodegenerative disorder, or any other disease or disorder) may refer to prophylaxis, amelioration, prevention and/or cure of the condition. Treatment after a condition (e.g., disease or disorder) has started aims to reduce, ameliorate or altogether eliminate the condition, and/or its associated symptoms, or prevent it from becoming worse. Treatment of subjects before a condition has started (e.g., prophylactic treatment) aims to reduce the risk of developing the condition and/or lessen its severity if the condition does develop. As used herein, the term "prevent" refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the disorder, and/or to the inhibition of further development of an already established disorder. Desired outcomes may include a stabilization of the condition, a slowdown in progression of the disease or a full disease-free recovery of the subject.
Subjects in need of treatment are preferably subjects that have a disease or disorder. The disease or disorder may be any disease or disorder, such as for example an infection (e.g., viral, bacterial, fungal), an allergy, a genetic disease, a cancer or lymphoma, a neurological or mental disease, an age-related disease, or any other disease or disorder.
Methods of treatment are provided, comprising administering modified VLPs comprising LI or LI and L2 comprising one or more targeting peptides, the VLPs further comprising one or more therapeutic agents and optionally diagnostic agents, to a subject in need of treatment in an amount effective to treat the disease or disorder. In some
embodiments, the disease is a tumor, cancer or lymphoma
In some embodiments, the modified VLPs described herein comprise targeting peptides that target the VLPs to specific tumor, cancer or lymphoma cells to deliver therapeutic or diagnostic agents to the tumor, cancer or lymphoma
As used herein, "tumor" and "cancer" are being used interchangeably. The cancer or tumor may be malignant or non-malignant. Cancers or tumors include but are not limited to biliary tract cancer; bladder cancer, brain cancer; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer;
intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (for example, small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas. In one embodiment the cancer is hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, or colon carcinoma In certain embodiments, modified VLP may be used to deliver agents, e.g. diagnostic agents or therapeutic agents, such as small molecules, nucleic acids (DNA, RNA, e,g, siRNA, antisense etc.) or drugs (e.g. anti-cancer drugs) to cancerous cells, tissues, or organs.
In some embodiments, modified VLPs that are targeted to airway epithelial cells as described herein can be used to delivery therapeutic or diagnostic agents to the lung. In some embodiments, modified VLPs that are targeted to airway epithelial cells can be used to deliver therapeutic agents and optionally diagnostic agents to the lung to treat cystic fibrosis. In some embodiments, modified VLPs that are targeted to airway epithelial cells can be used to deliver genes for lung cancer gene therapy. It should be appreciated that the modified VLPs can be modified in such a way to target any tissue, organ or cell in vivo, and can be used to deliver therapeutic or diagnostic agents to any tissue, organ or cell in vivo, for treatment of any disease or disorder that may be treated by the methods described herein.
In certain embodiments, treatment of a subject having a disease or disorder with modified VLPs comprising one or more therapeutic or diagnostic agents further comprises administration of one or more additional therapeutic or diagnostic agent(s). These agents may be co-administered at the same time or at a different time, for example before or after administration of modified VLPs comprising one or more therapeutic agents.
The term "effective amount" of a composition refers to the amount necessary or sufficient for a composition alone, or together with further doses, to realize a desired biologic effect. The desired response, of course, will depend on the particular condition being treated. Combined with the teachings provided herein, by choosing among the various active VLPs, VLP compositions and/or compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or adverse condition being treated, the size of the subject, or the severity of the disease or adverse condition. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. One of ordinary skill in the art can empirically determine the effective amount without necessitating undue
experimentation.
For any VLPs, VLP composition and/or compound described herein the
therapeutically effective amount can be initially determined from animal models. The applied dose can be adjusted based on the relative bioavailability and potency of the administered VLPs, VLP composition and/or compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well- known in the art is well within the capabilities of the ordinarily skilled artisan.
VLPs described herein and/or therapeutic agents may be administered per se (neat) or in the form of a pharmaceutically acceptable formulation. For example, VLPs may be in form of a VLP composition comprising the VLP and one or more additional substances, such as described herein. If VLPs and/or therapeutic agents are administered in pharmaceutically acceptable solutions, they may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. In some embodiments, the solutions used are sterile. In some embodiments, the pharmaceutical compositions contain an effective amount of VLPs or therapeutic agents optionally included in a pharmaceutically-acceptable carrier.
Modes of administering VLPs and therapeutic agents described herein will vary depending upon the specific agents used and the disease being treated, as would either be known to those skilled in the art or can be established by routine experimentation using methods commonly employed in the art. In some embodiments, dependent upon these factors, the agents may be administered orally or parenterally. In some embodiments, oral formulations may include immediate release particle coatings, controlled release particle coatings or extended release particle coatings. Parenteral modes of administration include intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intralesional, intrapleural, intrathecal, intra-arterial, and into lymphatic vessels or nodes and to bone or bone marrow. VLPs and therapeutic agents may also be administered topically or
transdermally, buccally or sublingually, or by a nasal, pulmonary, vaginal, or anal route. Accordingly, in some embodiments, compositions described herein may be provided in the form of tablets, capsules, softgels, liquids, powders, or other forms for oral administration. In some embodiments, compositions described herein may be provided in the form of liquid or lyophilized preparations for injection. In some embodiments, VLPs, VLP compositions and/or compounds may be provided in the form of a metered dose, a dry powder, spray, e.g., for inhalation, foam or a nasal preparation for respiratory delivery. In some embodiments, these or other formulations may be used for ophthalmic, topical, and/or other forms of delivery.
For oral administration, the pharmaceutical compositions can be formulated readily by combining active VLPs and/or compound(s), e.g., VLPs described herein and optionally additional therapeutic agents, with pharmaceutically acceptable carriers well known in the art Such carriers enable VLPs and optionally additional therapeutic agents to be formulated as tablets, pills, dragees, capsules, liquids, gels, hydrogels, pellets, granules, syrups, slurries, foams, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active VLP and/or compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, active VLPs and/or compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
In some embodiments, VLPs, VLP compositions and/or compounds may be administered by inhalation to pulmonary tract, especially the bronchi and more particularly into the alveoli of the deep lung, using standard inhalation devices. In some embodiments, VLPs, VLP compositions and/or compounds may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. An inhalation apparatus may be used to deliver VLPs, VLP compositions and/or compounds to a subject. An inhalation apparatus, as used herein, is any device for administering an aerosol, such as dry powdered form of VLPs, VLP compositions and/or compounds. This type of equipment is well known in the art and has been described in detail, such as that description found in Remington: The Science and Practice of Pharmacy, 19th Edition, 1995, Mac Publishing Company, Easton, Pennsylvania, pages 1676-1692. Many U.S. patents also describe inhalation devices, such as U.S. Patent No. 6,1 16,237.
"Powder" as used herein refers to a composition that consists of finely dispersed solid particles. In certain embodiments, VLPs, VLP compositions and/or compounds are relatively free flowing and capable of being dispersed in an inhalation device and subsequently inhaled by a subject so that the VLPs, VLP compositions and/or compounds reach the lungs to permit penetration into the alveoli. A "dry powder" refers to a powder composition that has a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol. The moisture content is generally below about 10% by weight (% w) water, and in some embodiments is below about 5% w and preferably less than about 3% w. The powder may be formulated with polymers or optionally may be formulated with other materials such as liposomes, albumin and/or other carriers.
Aerosol dosage and delivery systems may be selected for a particular therapeutic application by one of skill in the art, such as described, for example in Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990), and in Moren, "Aerosol dosage forms and formulations," in Aerosols in Medicine. Principles, Diagnosis and Therapy, Moren, et al , Eds., Esevier, Amsterdam, 1985.
The compositions, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g. , by bolus injection or continuous infusion Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of active VLPs and/or compounds in water-soluble form. Additionally, suspensions of active VLPs and/or compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of VLPs and/or compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active compositions may be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use.
The compositions may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compositions may also be formulated as a depot preparation Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active VLPs and/or compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249: 1527-1533, 1990, which is incorporated herein by reference.
In some embodiments, VLP preparations may be administered to a subject in combination (e.g. , simultaneously or separately but as part of the same therapeutic regimen) with a compound or other pharmaceutical preparation for treating one or more conditions or diseases described herein. Accordingly, aspects of the invention relate to combination preparations or kits that contain one or more VLPs along with one or more separate compounds (e.g., drugs) that are not packaged in VLPs.
Aspects of the invention are not limited in its application to the details of construction and the arrangement of components set forth in the preceding description or illustrated in the examples or in the drawings. Aspects of the invention are capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
EXAMPLES
The present invention will be more specifically illustrated by the following examples. However, it should be understood that the present invention is not limited by these examples in any manner.
Example 1
In a non-limiting example, the efficiency of HPV VLP-mediated gene transfer into human airway cells was improved. Indications and applications of these VLPs include, for example, delivery of therapeutic agents for the treatment of cystic fibrosis and lung cancer gene therapy. Experiments with HPV pseudovirions (VLPs) indicated that airway epithelial cells were poorly permissive to HPV pseudovirions. In attempts to achieve greater selectivity of HPV pseudoinfection for airway cells, chimeric VLPs were generated by insertion of 3 different peptide ligands within antigenic loops of the LI major protein of HPV- 16. Peptides were inserted at positions previously shown not to disturb the self-assembly of the LI protein into capsid (Sadeyen et al , 2003; Carpentier et al ; 2005). Three human airway cell ligands, which efficiently target human airway cells, have been identified by phage display technology. Two of these ligands, designated PI (THALWHT, SEQ ID NO: 1) (Jost et al , 2001) and P2 (RFDSLKV, SEQ ID NO: 2) (Vaysse et al , 2000), have been selected on the basis of phage binding to 16HBel4o" and CFT-2 cells, respectively. The third ligand, designated P3
(GHPRQMSHVY, SEQ ID NO: 3) (Gaden et al, 2005), was selected on the basis of greater internalization efficiency in CF-KM4 cells.
Seven chimeric VLPs were produced and investigated for their capacity to be internalized into human airway cells and compared to other cell types. Materials and methods
Cell lines and cell culture experiments. The following cell lines were used : Cos-7 (African green monkey fibroblast-like cell line transformed by SV40, ATCC # CRL- 1651), Hela (derived from an HPV-18 integrated human cervical adenocarcinoma), 16HBel4o" (human bronchial epithelial cells, Cozens et al, 1994) and A549 (adenocarcinoma of human airway epithelium, ATCC#CCL-185). All cell lines were cultivated in monolayer cultures in
Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin and 100 g/ml streptomycin and 1 mM Sodium pyruvate at the exception of 16HBel4o" cells for which the DMEM was replaced by MEM supplemented with 2mM L-glutamine. All cells were maintained in a humidified atmosphere with 5% C02 at 37°C.
Production of wild-type and chimeric HPV-16 VLPs. HPV- 16 wild type (wt), HPV- 16 mutants Ll-HBc 140/141 and Ll-HBc 283/284, with insertion of a hepatitis B core (HBc) motif within the DE and FG loops of the LI protein respectively, have already been described [Sadeyen et al , 2003 ; Touze et al , 1998]. The other seven DNA-encoding chimeric LI proteins, with insertion of the THALWHT motif (PI , SEQ ID NO: 1), (Jost et al , 2001) between HPV 16 LI positions 140/141 , 179/180 and 283/284, and RFDSL V motif (P2, SEQ ID NO: 2) (Vaysse et al , 2000) and GHPRQMSHVY motif (P3, SEQ ID NO:3) (Gaden et al, 2005) between HPV 16 LI positions 140/141 and 179/180, were obtained by mutagenesis using a two-step PCR protocol. In the first step, two fragments corresponding to the 5' and 3' part of the LI chimeric gene were obtained using the wild-type HPV-16 LI DNA sequence as template, with primers HPV 16 L 1 For and P Rev and P For and HPV 16 L 1 Rev, respectively (Table I). All these fragments overlapped in their 3' and 5' sequences and were used in the second PCR step as DNA templates using HPV 16 LI For and HPV 16 LI Rev primers. The resulting PCR fragments were cloned into the pCR2.1 by TOPO cloning (Invitrogen, Cergy Pontoise, France). The chimeric genes were then sequenced with an ABI PRISM 3100 automated sequencing system (Perkin Elmer/ Applied Biosystem, Courtaboeuf, France). The different genes were cloned into the BamHI restriction site of pFastBacl (Invitrogen).
Recombinant plasmids were used to generate recombinant baculoviruses according to the Bac- to-Bac procedure (Invitrogen). Generation of recombinant baculoviruses and purification of Ll-VLPs. Sf21 cells, maintained in Grace's insect medium supplemented with 10% fetal calf serum (FCS,
Invitrogen), were infected with the different recombinant baculoviruses and incubated for 72 h at 27°C. Chimeric proteins were purified as previously described [Touze et al , 1998].
Briefly, cells were harvested, resuspended in phosphate-buffered saline (PBS) containing
Nonidet P40, pepstatin and leupeptin (lmg/ml). Cell lysates were then centrifuged at 14,000 g for 15 min at 4°C and nuclear pellets resuspended and sonicated. Nuclear cell extracts were then loaded on top of a preformed CsCl gradient and centrifuged to equilibrium in a Beckman SW28 rotor. CsCl gradient fractions were investigated for density by refractometry and for the presence of chimeric LI proteins by electrophoresis in 10% sodium dodecyl sulfate- polyacrylamide gel (SDS-PAGE) and Coomassie blue staining. Positive fractions were pooled, diluted in PBS and pelleted in a Beckman SW 28 rotor. After centrifugation, VLPs were resuspended in 0.15 M NaCl and sonicated by a one 5-s burst at 60% maximum power.
Production of the mutant proteins was analyzed by Western blotting. For this purpose, a sample of the different CsCl fractions for each mutant was boiled in Laemmli sample buffer and the lysates were subjected to SDS-PAGE in 10% gels. LI proteins were detected using CamVir-1 MAb as primary antibody and with anti-mouse IgG antibodies coupled with alkaline-phosphatase (Sigma) as secondary antibody. To control the protein loading, the titer of each LI preparation was assessed by ELISA using two-fold dilution starting at 1/10 using CamVir-1 as primary antibody and a peroxidase-labeled anti-mouse IgG as secondary antibody (Carpentier et al , 2005). The data presented are the means of 2 to 3 determinations.
Pseudovirions can be generated as previously described with some modifications (Touze et al , Nucleic Acids Res 1998; 26: 1317-23). For example, 1 μg of HPV VLPs is incubated in 50 mmol/L of Tris-HCl buffer (pH 7.5) containing 20 mmol/L of DTT and 1 mmol/L of EGTA for 30 min at room temperature. At this stage, expression plasmids encoding e.g. , shRNA or siRNA or a gene of interest, reporter plasmids, proteins or other cellular factors or small molecules (therapeutic or diagnostic agents) can be added to the disrupted VLPs. The preparation is then diluted with increasing concentrations of CaCl2 up to a final concentration of 5 mmol/L, with or without ZnCl2 (10 nmol/L). ZnCl2 is used because it has been reported that ZnCl2 enhances the assembly of HPV capsomers into VLPs (Hanslip et al, Biotechnol Prog 2006; 22:554-60). Pseudovirions are then dialyzed against 1 x PBS overnight and stored at 4°C before use.
Assembly of chimeric LI proteins into VLPs was verified by electron microscopy. For this purpose, purified VLP preparations were applied to carbon coated grids, negatively stained with 1.5 % uranyl acetate and observed at x 50,000 nominal magnification using a JEOL 1010 electron microscope. The number of VLPs produced was semi-quantified by determination of the mean number of particles observed per field (calculated from 2 to 3 micrographs). Cell type specific targeting. Purified HPV VLPs were labeled with carboxy-fluorescein diacetate succinimyl ester (CFDA-SE) (Vibrant CFDA-SE cell tracer kit, Molecular Probes). Inside cells, acetate groups on the CFDA-SE are cleaved off by intracellular esterases and yields highly fluorescent particles. CFDA-SE was added at a final concentration of 100 mM (Bergsdorf et al , 2003; Drobni et al , 2003) to VLPs at a concentration of 0.5 mg/ml in PBS (pH 8,5). After overnight incubation at room temperature, labeled VLPs were subjected to centrifugation in a sucrose density gradient (30 - 70%) to separate VLPs from free CFDA-SE. LI -positive fractions were pooled in PBS and sedimented by ultracentrifugation. The pellet was resuspended in 0.15 M NaCl. To detect cell-entry of VLPs, cells were exposed to CFDA- SE-labeled VLPs in DMEM medium for 1 h at 37 °C. Unbound VLPs were removed by extensive washing with PBS. DMEM containing 10% FCS was then added and cells were incubated at 37°C for 7 h. Flow cytometry was used to measure the cell fluorescence resulting from the uptake of labeled VLPs. The relative fluorescent intensity was measured using FACsort and Cellquest software (Becton Dickinson). Typically, 20,000 events were collected for each experiment. For data analysis, the internalization of each of the different VLPs was normalized by using a ratio corresponding to the percentage of fluorescent cells observed with 16HBe cells, divided by the percentage of fluorescent cells observed with Hela cells. The results are presented as relative internalization. This calculation was performed separately for each VLP. The data presented are the means of 2 to 3 determinations. Synthetic Peptides and analysis of their cell binding and internalization. Six peptides representing the amino acid sequences of the PI , P2 and P3 ligands, P2 and P3 ligands with the addition at their C-terminus of the AKRKKKRKL (SEQ ID NO: 4) sequence corresponding to the nuclear localization signal identified at the C-terminus of the LI protein (El Mehdaoui et al. ,2000 ; Zhou et al, 1995), and P4 (CDCLNGGTCVSNKY, SEQ ID NO: 5) a ligand of the urokinase plasminogen activator receptor present on the airway epithelia described by Drapkin et al. (2000) were synthesized (N -terminal biotinylated with an SGSG spacer (SEQ ID NO: 6); Pepsets, Mimotopes, Clayton Victoria, Australia).
Cell internalization of these peptides was investigated on A549 and Hela cells. Cells grown at confluence in 96- well plates (TPP, ATGC, France) were washed with PBS IX, saturated with PBS IX containing 1% fetal calf serum CS. One hundred μΐ of peptides diluted at 20 μg/ml in PBS 5X containing 0.5% BSA were then added to the wells and incubated for 1 h at 37°C. After two washes with PBS IX, cells were fixed with PBS IX containing 4% paraformaldehyde and 0.1 % glutaraldehyde for 15 min at room temperature and then washed with PBS IX Cells were permeabilized using PBS containing 0.2% Triton X-100 for 5 min at room temperature and then washed twice with PBS. Alexa Fluor 430 streptavidin conjugate (Molecular Probe, Invitrogen, France) diluted 1/200 in PBS IX was then added. Cells were washed twice with PBS IX and observed using an inverted fluorescence microscope (1X51 , Olympus, France).
Results were scored + when OD values were lower than 3 times the value observed with wells coated with PBS-T containing 1% FCS (control), ++ when OD values were between 3 and 5 times the control well, and +++ when the OD values were greater than 5 times the control. Results
Selection of peptide ligand insertions within LI VLP to redirect HPV-16 VLP cell tropism/cell targeting to human airway epithelial cells. Cell internalization of four different peptides (PI to P4) that have been identified for their capability to bind and to be internalized by A549 cells was analyzed; two peptides (P2 and P3) with the addition of the LI C-terminal sequence containing a nuclear localisation signal (NLS). Localization of the peptides into the cells was analyzed by binding of the peptides to a fluorescent probe. The P4 peptide was poorly internalized into A-549 cells and the PI peptide (THALWHT, SEQ ID NO: 1) showed the highest degree of internalization into cells (Fig 2). The addition of the LI NLS to P2 and P3 peptides increased their internalization. Without the addition of LI NLS the peptides were mainly present in the cytoplasm. Nuclear fluorescence was not detected for PI and P4 peptides and fluorescence was weak for P2 and P3 peptides, but a much stronger signal was observed when the LI NLS sequence was fused to these peptides. From these data, PI , P2 and P3 sequences were selected for insertion within the LI sequence to redirect the cell tropism of HPV-16 VLP.
Production and characterization of chimeric HPV 16 VLPs. Immunoblotting of purified VLPs indicated that the peptide sequences were incorporated into the LI protein in all cases tested. Electron-microscopy analysis indicated that all chimeric LI proteins retained the capacity to self-assemble into VLPs (FIG. 3). The antigenicity of the chimeric VLPs was also investigated by testing their reactivity against Camvir-1 , a monoclonal antibody directed against a LI linear epitope. The results obtained (Table II) indicated that the production yield and folding into VLP was retained. A lower yield of both LI and VLP was observed for the mutant corresponding to the insertion of the P3 peptide sequence at position 140/141. This reduction was not observed for other sequences inserted at the same position, or for chimeras having the P3 peptide sequence inserted at position 179/180. A low number of VLP was observed for the LI -PI 283/284 mutant compared with its LI antigenic titre.
Cell specificity of chimeric VLPs. 3 different ligands were tested for their ability to specifically target human airway cells as compared to other cell lines. For this purpose, immunofluorescence microscopy was used to visualize the internalization of the different chimeric VLPs. The chimeric VLPs were initially tested for their ability to bind to -and to be internalized by- different cell lines. Targeting of airway cells by a given mutant VLP was determined comparing the percentage of 16HBe fluorescent cells with the percentage of Hela or Cos-7 fluorescent cells 8 hours after contact between these cells and CFDA-SE-labeled VLPs. The relative fluorescence intensity of the cells was measured by FACS. The data obtained (FIG. 4b) indicate that the highest targeting (a ratio of 7.7) was observed when the P3 sequence was inserted at position 179/180 within the LI protein. No targeting was observed for VLPs containing the P2 sequence at position 140/141. Insertion of the HBc sequence within LI at positions 179/180 and 283/284 led to both a lower level of
internalization and a lower capacity to be internalized by 16HBe cells as compared to Hela cells.
Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer in these cells, one of the major problems confronting cystic fibrosis gene therapy. Four peptide motifs, selected by binding or internalization studies using phage displaying peptide libraries on human airway epithelial cells (Drapkin et al , 2000; Gaden et al , 2005; Jost et al, 2001 ; Vaysse et al , 2000) were investigated for internalization within cells. Evidence that 3 of these ligands (PI , P2 and P3) promoted the interaction and/or internalization was provided by a fluorescence microscopy experiment in which increased localization of the peptides was observed in the cytoplasm or nucleus of A549 cells. These 3 peptide ligands were inserted in 3 different external loops of the HPV- 16 LI protein to redirect the cellular tropism of HPV-16 pseudovirions. None of the three ligands, 7 to 10 amino acids long, affected the capacity of the chimeric LI protein to self- assemble into VLP, suggesting that VLPs can accommodate the insertion of ligands of at least 10 amino acids, or more.
Previous results have indicated that mutations or insertions into the loops identified by Chen et al. (2000) at the surface of the LI capsomers have lower chances of affecting the three-dimensional protein structure compared to mutations or insertions into alpha-helices or beta-sheets (Sadeyen et al, 2003). Although it has been reported that other mutations within loops destabilize the structure (Kirnbauer et al, 1993). These findings confirmed previous observations (Sadeyen et al , 2003; Carpentier et al, 2005) that the yield of VLP production of mutants with insertions within the DE loop of the HPV-16 LI protein appeared generally similar to that observed with the wild-type LI proteia Insertion in other loops {e.g. , EF or FG) might be associated with a decrease in the yield of VLP production. The yield may also be dependent on the inserted sequence, since yields of L 1 production varied according to different amino acid sequences inserted at position 140/141. However, even with a decreased yield, modified VLP may be used as described herein.
The presentation of peptide ligands on the surface of LI VLPs improved the cellular internalization process in human airway cells. The highest efficacy of targeting to
16HBel4o- cells was observed with the P3 sequence inserted at position 179/180 within the EF loop of the LI protein. All three ligands (PI , P2 and P3) were confirmed to target airway cells. In contrast, insertion of an unrelated sequence such as the HBc sequence did not induced a significant changes in the ability to target airway cells. The increased
internalization of the VLPs into cells, also suggested that the peptide ligands are presented correctly at the surface of the VLPs and that no misfolding of the LI occurs. Differences in targeting efficiency between the three ligands may be due to different presentation or levels of expression of receptors present on the surface of different cell lines, or may be due to receptor/ligand affinity, may reflect different cell membrane receptor regulation in
16HBel4o- cells and in Hela cells, and/or could also be related to the capacity of the VLPs to escape from the endosome.
The insertion of the GHPRQMSHVY (SEQ ID NO: 3) ligand within the EF loop of the HPV-16 LI proteins or the insertion of THALWHT (SEQ ID NO: 1) ligand within the DE loop of the LI produced VLPs capable of becoming specifically internalized in 16HBel4o- cells, demonstrating that these pseudovirions can target airway epithelial cells in vitro.
FIG. 5 shows a comparison of targeting ratios for different modified VLP in comparison to HPV16 wt. The comparisons were made between human cervical cells (Hela and CaSki) and human lung cells (S9, IB3 and Calu-3). A ratio of 100 means that the modified VLP targeted, for example IB3 cells, compared to Hela cells, 100-fold better than wild-type VLP. It can be seen that, for example, the insertion of THALWHT (SEQ ID NO: 1) ligand within the DE loop (P1DE) of the LI produced VLPs capable of targeting lung cancer cells, such as IB3-1 and S9 cells about 450-fold better than Llwt VLP when measured as compared to Hela cells (cervical) and about 2000-fold better when measured as compared to Ca Ski (cervical) cells. P3EF (the GHPRQMSHVY (SEQ ID NO: 3) ligand within the EF loop of the HPV-16 LI) and P2DE (the RFDSLKV (SEQ ID NO: 2) ligand within the DE loop of the HPV-16 LI) show targeting ratios of about 100 to about 500, particularly for S9 lung cells). The insertion of THALWHT (SEQ ID NO: 1) ligand within the FG loop (P1FG) of the LI produced VLPs capable of targeting Calu-3 lung cancer cells. This data demonstrate that HPV-based VLP comprising airway cell-specific targeting peptides can target airway epithelial cells in vitro to a high degree of specificity.
FIG. 7A shows efficient targeting of the P2DE (DE) variant (RFDSLKV (SEQ ID NO: 2) ligand within the DE loop of the HPV-16 LI) to A549 and 16HBe lung cells compared to non-modified VLP (wt). The P2DE variant has a 6 to 8-fold higher efficiency (measured by luciferase expression of the target cell) to deliver a nucleic acid (exemplified as a luciferase reporter plasmid) to the target cell compared to wild-type. FIG. 7B shows internalization of the P2DE mutant in lung cells compared to HeLa cells by immunofluorescence, showing effective targeting of the lung cells by the modified VLP.
Table 1 : Primers used for the generation of HPV-16 LI mutants.
Figure imgf000053_0001
P3 179/180 For GGTCATCCTCGACAAATGTCTCATGTATATGTAAATCCAGGTGATTGTCC ( SEQ ID
NO : 30 )
Table 2: Detection of virus-like particles by electron microscopy in the different LI proteins preparations (a Mean number of VLPs per field when observed by electron microscopy) and LI antigenic titer.
Figure imgf000054_0001
Nucleotide Sequence of 16Llh (humanized version): SEQ ID NO: 14
GGGCGAATTGGGTACCGCTAGCCACCATGAGCCTGTGGCTGCCCAGCGAGGCCACCGTGTAC CTGCCCCCCGTGCCCGTGAGCAAGGTGGTGTCCACCGACGAGTACGTGGCCCGGACCAACAT CTACTACCACGCCGGCACCAGCAGGCTGCTGGCCGTGGGCCACCCCTACTTCCCCATCAAGA AGCCCAACAACAACAAGATCCTGGTGCCCAAGGTGTCCGGCCTGCAGTACCGGGTGTTCCGG ATTCACCTGCCCGACCCCAACAAGTTCGGCTTCCCCGACACCAGCTTCTACAACCCCGACAC CCAGCGGCTGGTGTGGGCCTGCGTGGGCGTGGAAGTGGGCAGAGGCCAGCCCCTGGGCGTGG GCATCAGCGGCCACCCCCTGCTGAACAAGCTGGACGACACCGAGAACGCCAGCGCCTACGCC GCCAACGCCGGCGTGGACAACCGGGAGTGCATCAGCATGGACTACAAGCAGACCCAGCTGTG CCTGATCGGCTGCAAGCCCCCCATCGGCGAGCACTGGGGCAAGGGCTCCCCCTGCACCAACG TGGCCGTGAACCCCGGCGACTGCCCCCCTCTGGAACTGATCAACACCGTGATCCAGGACGGC GACATGGTGGACACCGGCTTCGGCGCCATGGACTTCACCACCCTGCAGGCCAACAAGAGCGA GGTGCCCCTGGACATCTGCACCAGCATCTGCAAGTACCCCGACTACATCAAGATGGTGTCCG AGCCCTACGGCGACAGCCTGTTCTTCTACCTGCGGCGGGAGCAGATGTTCGTGCGGCACCTG TTCAACAGGGCCGGCACCGTGGGCGAGAACGTGCCCGACGACCTGTACATCAAGGGCAGCGG CAGCACCGCCAACCTGGCCAGCAGCAACTACTTCCCTACCCCCAGCGGCTCCATGGTGACCA GCGACGCCCAGATCTTCAACAAGCCCTACTGGCTGCAGCGGGCCCAGGGCCACAACAACGGC ATCTGCTGGGGCAACCAGCTGTTCGTGACCGTGGTGGATACCACCCGGTCCACCAACATGTC CCTGTGCGCCGCCATCAGCACCAGCGAGACCACCTACAAGAACACCAACTTCAAAGAGTACC TGCGGCACGGCGAGGAATACGACCTGCAGTTCATCTTCCAGCTGTGCAAGATCACCCTGACC GCCGACGTGATGACCTACATCCACAGCATGAACAGCACCATCCTGGAAGATTGGAACTTCGG CCTGCAGCCCCCTCCTGGCGGCACCCTGGAAGATACCTACCGGTTCGTGACCAGCCAGGCCA TCGCCTGCCAGAAGCACACCCCCCCTGCCCCCAAAGAGGACCCCCTGAAGAAGTACACCTTC TGGGAGGTGAACCTGAAAGAGAAGTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCCGGAA GTTCCTGCTGCAGGCCGGGCTGAAGGCCAAGCCCAAGTTCACCCTGGGCAAGCGGAAGGCCA CCCCCACCACCTCCAGCACCAGCACCACCGCCAAGCGGAAAAAAAGGAAGCTGTGATGAATT CGAGCTCCAGCTTTTGTTCCC (SEQ ID NO: 14)
LI human papilloma virus 16 amino acid sequence (505 amino acids):
1 mslwlpseat vylppvpvsk wstdeyvar tniyyhagts rllavghpyf pikkpnnnki
61 lvpkvsglqy rvfrihlpdp nkfgfpdtsf ynpdtqrlvw acvgvevgrg qplgvgisgh
121 pllnklddte nasayaanag vdnrecismd ykqtqlclig ckppigehwg kgspctnvav
181 npgdcpplel intviqdgdm vdtgfgamdf ttlqanksev pldictsick ypdyikmvse
241 pygdslffyl rreqtnfvrhl fnragavgen vpddlyikgs gstanlassn yfptpsgsmv 301 tsdaqifnkp ywlqraqghn ngicwgnqlf vtwdttrst nmslcaaist settykntnf
361 keylrhgeey dlqfifqlck itltadvmty ihsmnstile dwnfglqppp ggtledtyrf
421 vtsqaiacqk htppapkedp lkkytfwevn lkekfsadld qfplgrkfll qaglkakpkf
481 tlgkrkatpt tsststtakr kkrkl (SEQ ID NO: 13)
(Leder C. et al, J. Virol. 75: 9201-9209, 2001)
Phil HPV-16 LI DNA sequence:
GenBank: AF043287.1 Human papillomavirus type 16 Phil late major capsid protein (LI) gene, complete cds:
ATGTCTCTTTGGCTGCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCTAAGGT TGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTATCATGCAGGAACATCCAGAC TACTTGCAGTTGGACATCCCTATTTTCCTATTAAAAAACCTAACAATAACAAAATATTAGTT CCTAAAGTATCAGGATTACAATACAGGGTATTTAGAATACATTTACGTGACCCCAATAAGTT TGGTTTTCCTGACACCTCATTTTACAATCCAGATACACAGCGGCTGGTTTGGGCCTGTGTAG GTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTAGGCATTAGTGGCCATCCTTTATTAAAT AAATTGGATGACACAGAAAATGCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGAGA ATGTATATCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCACCTATAG GGGAACACTGGGGCAAAGGATCCCCATGTAACAATGTTGCAGTAAATCCAGGTGATTGTCCA CCATTAGAGTTAATAAACACAGTTATTCAGGATGGTGATATGGTTGATACCGGCTTTGGTGC TATGGACTTTACTACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCTA TTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATATGGCGACAGCTTATTTTTT TATTTACGAAGGGAACAAATGTTTGTTAGACATTTATTTAATAGGGCTGGTGCTGTTGGTGA AAATGTACCAGACGATTTATACATTAAAGGCTCTGGGTCTACTGCAAATTTAGCCAGTTCAA ATTATTTTCCTACACCTAGTGGTTCTATGGTTACCTCTGATGCCCAAATATTTAATAAACCT TATTGGTTACAACGAGCACAGGGCCACAATAATGGCATTTGTTGGGGTAACCAACTGTTTGT TACTGTTGTTGATACTACACGCAGTACAAATATGTCATTATGTGCTGCCATATCTACTTCAG AACCTACATATAAAAATACTAACTTTAAAGAGTACCTACGACATGGGGAGGAATATGATTTA CAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACGTTATGTCATACATACATTC TATGAATTCCACTATTTTGGAGGACTGGAATTTTGGTTTACAACCTCCTCCAGGAGGCACAC TAGAAGATACTTATAGGTTTGTAACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCA GCACCTAAAGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTAAAGGAAAAGTT TTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAAATTTTTACTACAAGCAGGATTGAAGG CCAAACCCAAATTTACATTAGGAAAACGAAAAGCTACACCCACCACCTCATCTACCTCTACA ACTGCTAAACGCAAAAAACGTAAGCTGTAA ( SEQ ID NO : 31 ) Example 2
In a non-limiting example, variants of the HPV virus - like particles (VLPs) were produced to target them towards lung epithelia for drug or siRNA delivery. Functional loops were inserted into the capsomer protein sequences and two variants were tested, P2DE and VDR. The experiment was conducted to evaluate the ability of the targeted VLPs (VDR and P2DE) to deliver siRNA into lung cells as well as to compare the efficiency of siRNA delivery of the targeted VLPs with a standard transfection agent HIPERFECT (QIAgen).
Materials and methods - VLPs: VDR (variant with VDRLQQK (SEQ ID NO: 8)) and P2DE (variant with RFDSLKV (SEQ ID NO: 2) ligand within the DE loop of the HPV-16 LI)
- Cy3 -labelled fluorescent siRNA (10 mg/ mL) targeting human GAPDH
- HBE16 and A549 cells
- 1 Ox PBS (ISC Bioexpress, J373-4L)
- DMEM (ISC Bioexpress, 1 19651 18), incomplete and complete with FBS (ATCC, 30-2021) and penicillin/ streptomycin (Sigma, P4333-100ML)
- Trypsin
- Guava EASYCYTE flow cytometer (Millipore)
1. On Day 1, HBE16e cells were seeded at 1.5 x 10s cells/ well in one 12-well tissue culture plate.
2. On the same day, the VLPs (VDR and P2DE) were loaded with fluorescent siRNA using (200 μg VLP, 100 g siRNA per reaction).
3. The samples were dialyzed using SPECTRAPOR dialysis tubing (3 kDa) in lx PBS overnight at 4° C with stirring in the dark.
4. On Day 2, the samples were concentrated with PEG 20000 while still in dialysis tubing. After concentration to <200 μΐ, the samples were stored at 4° C overnight.
5. On the same day, A549 cells were seeded at 1 x 105 cells/ well in a 12-well plate.
6. On Day 3, appropriate confluency was determined for each of the cell lines (A549- 80%, HBE16e- 80%) prior to invasion.
7. Cells were washed with lx PBS. Recovery volumes were evenly distributed among the two cell lines, each including a high (80%) and low (20%) treatment group.
8. Approximate encapsulated siRNA delivered for VDR is 25.6 ng and 6.4 ng for the high and low treatments, respectively. For the P2DE VLP siRNA encapsulation and delivery is estimated at 12.8 ng and 3.2 ng, respectively. These numbers are based on assumptions of 24 ng/10μg (siRNA/VLP protein) encapsulation, based on volume, and 20% recovery of VLP during Disassembly/Reassembly procedure; based on prior data.
9. Additional controls included untreated cells, 140 ng siRNA + HIPERFECT, 35 ng siRNA + HIPERFECT, and HIPERFECT only.
10. Invasion proceeded for two hours in incubator before harvest.
1 1. Samples were harvested in 100 μΐ trypsin, followed by addition of 400 μΐ complete DMEM once cells detached from plates. Samples were spun to pellet cells, which were then resuspended in 350 μΐ of 0.5% BSA in PBS.
12. Readout was completed on Millipore GUAVA Flow Cytometry System. Results
FIG. 6 shows bar graphs for siRNA delivery into the two lung cell lines. For 16HBE cells (FIG. 6A), the most efficient transfer of fluorescent siRNA was observed using the P2DE particles (variant with RFDSLKV (SEQ ID NO: 2) ligand within the DE loop of the HPV-16 LI), followed by the VDR particles (variant with VDRLQQK (SEQ ID NO: 8)). Both VLPs were more efficient than the liposomal transfection reagent HIPERFECT. For A549 cells (FIG 6B), P2DE particles were the most efficient in transferring fluorescent siRNA into cells followed by the VDR. HIPERFECT transfection was able to reach a large number of cells, but only when much larger amounts of fluorescent siRNA were transfected. The data demonstrate that lung targeted VLPs are very efficient in delivering fluorescently labeled siRNA into lung cells. Accordingly, HPV-based VLPs with lung targeting peptides may be used in methods of targeting lung cells or lung tissue.
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Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. , from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a
composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc. , certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
OTHER EMBODIMENTS
The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.
What is claimed is:

Claims

1. An isolated viral-like particle (VLP) comprising a modified viral capsid protein, wherein the modified viral capsid protein comprises a targeting peptide, wherein the modified viral capsid protein is capable of self-assembly into the VLP and the targeting peptide is capable of directing the self-assembled VLP to one or more specific sites in vivo.
2. The VLP of claim 1 , wherein the targeting peptide is inserted into one or more surface-exposed loops of the modified viral capsid protein.
3. The VLP of claims 1 or 2, wherein the modified viral capsid protein is an
papilloma virus capsid protein.
4. The VLP of any one of claims 1 to 3, wherein the papilloma virus capsid protein is LI , L2 or LI and L2.
5. The VLP of claim 4, wherein the papilloma virus capsid protein is LI .
6. The VLP of claim 5, wherein the targeting peptide is capable of directing the self- assembled VLP to airway epithelial cells in vivo.
7. The VLP of claim 6, wherein the targeting peptide is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 8.
8. The VLP of claim 7, wherein the targeting peptide is inserted into the DE loop or EF loop.
9. The VLP of claim 8, wherein the papilloma virus capsid protein is LI is LI of human papilloma virus HPV-16.
10. The VLP of claim 9, wherein the targeting peptide PI (SEQ ID NO.T) is inserted into amino acid position 140 and 141 of wild-type LI.
1 1. The VLP of claim 9, wherein the targeting peptide P3 (SEQ ID NO:3) is inserted into amino acid position 179 and 180 of wild-type LI.
12. The VLP of any one of claims 1 to 4, wherein the targeting peptide is between 5 and 50, 5 and 25, 5 and 15, 5 and 10 or between 7 and 10 amino acids long.
13. The VLP of claim 12, wherein the targeting peptide is inserted approximately in the middle of the one or more surface exposed loops.
14. The VLP of claims 12 or 13, wherein the targeting peptide is capable of directing the self-assembled VLP to liver cells, lung cells, heart cells, kidney cells, blood cells, brain cells, skin cells, or gut cells in vivo.
15. The VLP of claims 12 or 13, wherein the targeting peptide is capable of directing the self-assembled VLP to stem cells in vivo.
16. The VLP of claims 12 or 13, wherein the targeting peptide is capable of directing the self-assembled VLP to cancer cells in vivo.
17. The VLP of claim 16, wherein the cancer cell is a call of biliary tract cancer,
bladder cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasms, lymphoma, liver cancer, lung cancer, melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and renal cancer, hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, or colon carcinoma
18. The VLP of claims 12 or 13, wherein the targeting peptide is capable of directing the self-assembled VLP to infected cells in vivo.
19. The VLP of claim 18, wherein the infected cell is a cell infected with a virus, a bacteria, a fungus, or a parasite.
20. The VLP of any one of claims 1-19, further comprising an agent.
21. The VLP of claim 20, wherein the agent is a diagnostic agent.
22. The VLP of claim 21 , wherein the agent is an imaging agent.
23. The VLP of claim 20, wherein the agent is a therapeutic agent.
24. The VLP of claim 23, wherein the agent is a small molecule or a nucleic acid.
25. The VLP of claim 24, wherein the small molecule is a cytotoxic drug.
26. The VLP of claim 24, wherein the nucleic acid is a gene for gene therapy.
27. The VLP of claim 24, wherein the nucleic acid is a RNAi nucleic acid, a
microRNA or an antisense nucleic acid.
28. The VLP of claim 23, wherein the agent is a recombinant protein or cytokine.
29. A method for treating or diagnosing a disease or disorder, comprising:
administering to a subject having or suspected of having a disease or disorder the VLP of any of claims 20 to 28 in an amount sufficient to treat or diagnose the disease or disorder.
30. The method of claim 29, wherein the disease or disorder is infection, cancer, age- related disease, genetic disorder, allergy, or auto-immune disease.
31. The method of claims 29 or 30, further comprising administering one or more additional therapeutic or diagnostic agents.
32. The method of claim 31, wherein the one or more additional therapeutic or
diagnostic agents are administered separately from the VLP.
33. The method of claim 32, wherein separate administration comprises
administration of the one or more additional therapeutic or diagnostic agents at the same time or different time, at the same site or different site, via the same route or different route, by the same means or different means as administration of the VLP.
34. Use of the VLP of any of claims 20 to 28 in the manufacture of a medicament for treatment or diagnosis of a disease or disorder.
35. The use of claim 34, wherein the disease or disorder is infection, cancer, age- related disease, genetic disorder, allergy, or auto-immune disease.
36. A method of redirecting the tropism a papilloma virus-like particle comprising viral capsid protein to target a specific tissue and/or cell in vivo, the method comprising:
inserting one or more targeting peptides, sufficient to target a self-assembled virus-like particle to a specific tissue and/or cell, into one or more surface exposed loops of the viral capsid protein, thereby producing a modified viral capsid protein;
expressing the modified viral capsid protein in a heterologous expression system; at least partially purifying the modified viral capsid protein;
allowing the modified viral capsid protein to self-assemble into a papilloma viruslike particle (VLP) in vitro, thereby producing a modified VLP capable of sufficiently targeting specific tissues and/or cells in vivo that are not sufficiently targeted by non-modified or wild-type papilloma virus-like particles, thereby redirecting the VLP tropism.
37. The method of claim 36, wherein the modified papilloma virus-like particle is the VLP of any of claims 4-28.
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