WO2013009717A1 - Nanoparticules de protéine virionique pour l'administration d'agents diagnostiques ou thérapeutiques pour le traitement de maladies cutanées - Google Patents

Nanoparticules de protéine virionique pour l'administration d'agents diagnostiques ou thérapeutiques pour le traitement de maladies cutanées Download PDF

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WO2013009717A1
WO2013009717A1 PCT/US2012/045989 US2012045989W WO2013009717A1 WO 2013009717 A1 WO2013009717 A1 WO 2013009717A1 US 2012045989 W US2012045989 W US 2012045989W WO 2013009717 A1 WO2013009717 A1 WO 2013009717A1
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protein
composition
therapeutic agent
proteins
hpv
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Elisabet De Los Pinos
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Elisabet De Los Pinos
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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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • 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
    • 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
    • C12N2710/20042—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule

Definitions

  • Sequence Listing provides exemplary polynucleotide sequences of the invention. The traits associated with the used of the sequences are included in the Examples.
  • the invention relates to methods for loading protein nanoparticles with therapeutic, diagnostic or other agents, wherein the protein nanoparticles are based on viral proteins. More particularly, the present invention relates to a method for using papilloma-derived protein nanoparticles to deliver drugs to the keratinocytes and basal membrane cells for the treatment of skin-related diseases such as alopecia or patterned hair loss, non-melanoma skin cancer, psoriasis, and treatment of skin related genetic diseases (e.g. Pachyonychia Congenita and Xeroderma pigmentosum).
  • skin-related diseases such as alopecia or patterned hair loss, non-melanoma skin cancer, psoriasis
  • skin related genetic diseases e.g. Pachyonychia Congenita and Xeroderma pigmentosum.
  • the first three systems reviewed are the phagemid particles and Herpes Simplex Virus- 1 (HSV) amplicons, in which the RNAi is derived from DNA, and the SV40 in vitro- packaged vectors. These three systems are examples of "trans-packaging systems in which viral machinery and proteins have been repurposed to package genetic material other than the original viral genome" (Lund et al. 401).
  • HSV Herpes Simplex Virus- 1
  • influenza Virosome and the HVJ-E a derivative of the Hemagglutinating Virus of Japan
  • viral envelope systems in which native enveloped virus particles are inactivated and used to package other cargos
  • the object of the present invention is to overcome the shortcomings disclosed in the prior art.
  • the products of the current invention are pseudo-viruses derived from
  • papillomaviruses in particular from the genus betapapillomaviridae to deliver drugs to the keratinocytes and basal membrane cells for the treatment of skin related diseases.
  • the present in vention wil l allow for impro ved methods for administering treatment of skin diseases capable of targeting epithelial cells.
  • pseudovirions and in particular type 5 betaHPV viral shells as an ideal delivery vehicle for the delivery of drugs to the skin for the treatment of skin related diseases such as alopecia or patterned hair loss, non-melanoma skin cancer, psoriasis, and treatment of skin related genetic diseases (e.g. Pachyonychia Congenita and Xeroderma pigmentosum).
  • aspects of the invention relate to methods and compositions for producing papilloma-derived (e.g., human papilloma virus (HPV)-derived) protein nanoparticles containing one or more therapeutic or diagnostic agents.
  • aspects of the invention relate to methods and compositions for encapsulating an agent within a virus like particle (VLP) requiring an initial isolation of capsid proteins produced in a host cell system (e.g. yeast, mammalian cell, insect cell, E.coli) and subsequent reassembly in vitro.
  • a host cell system e.g. yeast, mammalian cell, insect cell, E.coli
  • FIG. 1 depicts shuttle vector information.
  • FIG. 3 shows results from in vitro reassembly of capsid protein produced in insect cell culture. DLS demonstrates presence of capsid protein in form of monomers and oligomers after harvest from nuclear fraction (left) and appearance of well formed loaded VLPs after the reassembly procedure (right).
  • FIG. 4 is a graph showing the amount of luminescence/luciferase signal measured 48 hrs after treatment of HeLa cells with loaded VLP, where luminescence is reported on a scale of 0 to 30,000 units along the y-axis.
  • FIG. 5 is a graph the same data in FIG.4, showing the amount of
  • luminescence/luciferase signal measured 48 hrs after treatment of HeLa cells with loaded VLP, where luminescence is reported on a scale of 0 to 20 units along the y-axis.
  • FIG. 6 shows a fl ow chart diagram of a preferred embodiment of the present invention for the treatment of alopecia.
  • FIG. 7 shows a flow chart diagram of a preferred embodiment of the present in vention for the treatment of non-melanoma skin cancer.
  • FIG. 8 shows a flo w chart diagram of a preferred embodiment of the present invention for the treatment of skin related genetic diseases.
  • FIG. 9 shows a flow chart diagram of a preferred embodiment of the present invention for the treatment of psoriasis.
  • SEQ ID NO: 1 shows DNA sequence for baculovirus L! X plasmid encoding HP V 16/31 L 1 (pFastBacTM).
  • SEQ ID NO: 2 shows DNA sequence for baculovirus L2 plasmid encoding
  • HPV16L2 (pFastBacTM).
  • SEQ ID NO: 3 shows forward primer DNA sequence used for generation of shE7- 1 RNA construct.
  • SEQ ID NO: 4 shows reverse primer DNA sequence used for generation of shE7- 1 RNA construct.
  • SEQ ID NO: 5 shows plasmid pl 6Ll *L2 DNA sequence encoding 16/31 L I (LI *) and L.2 human codon -optimized,
  • [0041 , 1 ] shows a sequence for the complete genome for Human
  • SEQ ID NO: 8 shows a sequence for P5 SHELL circular plasmid DS-DNA. [0037], DETAILED DESCRIPTION OF THE INVENTION [0038], Assembly Of Particles
  • the components can be associated with the nanoparticles through a linkage.
  • used as a carrier associated with it is meant that the component is carried by the nanoparticles.
  • the component can be dissolved and incorporated in the nanoparticles non-covalently.
  • aspects of the invention relate to methods and compositions for producing protein nanoparticles that contain therapeutic and/or diagnostic agents for delivery to a subject.
  • Methods and compositions have been developed for effectively encapsulating therapeutic and/or diagnostic agents within papilloma virus proteins (e.g., HPV proteins) that can be used for delivery to a subject (e.g. , a human subject).
  • papilloma virus proteins e.g., HPV proteins
  • LI and L2 capsid proteins are useful to isolate LI and L2 capsid proteins directly from host cells as opposed to disassembling VLPs that were isolated from host cells.
  • LI and L2 capsid proteins that are isolated directly from cells can be used in in vitro assembly reactions to encapsulate a therapeutic or diagnostic agent. This avoids the additional steps of isolating and disassembling VLPs. This also results in a cleaner preparation of LI and L2 proteins, because there is a lower risk of contamination with host cell material (e.g., nucleic acid, antigens or other material) that can be contained in VLPs that are isolated from cells.
  • host cell material e.g., nucleic acid, antigens or other material
  • LI and L2 capsid proteins it is useful to independently produce LI and L2 capsid proteins.
  • they can be produced from two independent nucleic acids (e.g., different vectors).
  • they can be produced in the same cell (e.g., using two different vectors within the same cell).
  • they can be produced in different cells (e.g., different host cells of the same type or different types of host cell).
  • This approach allows the ratio of LI and L2 proteins to be varied for either in vitro or intracellular assembly.
  • VLPs to be assembled (e.g., in vitro or intracellularly) with higher or lower LI to L2 ratios than in a wild type VLP.
  • H PV nanoparticles as deliver ⁇ ' vehicles for therapeutic agents.
  • a higher ratio of L,2 in the assembled structure may allow the resultant VLP to have a higher nucleic acid binding affinity and a better efficiency in delivering these intracellularly.
  • LI and L2 proteins are expressed in a host cell system (e.g., both in the same host cell or independently in different host cells). LI and/or L2 are isolated from nuclei of the host cells. In some embodiments, certain LI and/or L2 structures that are formed during cellular growth (e.g., during the fermentation process) are disrupted. Any suitable method may be used. In some embodiments, sonication may be used (e.g., nuclei may be isolated and then sonicated). Capsid proteins then may be purified using any suitable process. For example, in some embodiments, capsid proteins may be purified using chromatography.
  • Isolated capsid proteins can then be used as described herein in a cell free system to assemble together with different payloads to create superstructures that contain a drug or diagnostic agent in its interior.
  • directly isolating capsid proteins provides several benefits, In some embodiments, there is a reduced risk of encapsulating and transferring genetic information (DNA, RNA) from the host cell to the treated subject.
  • de-novo assembly of VL Ps during the assembly procedure ensures formation of a larger percentage of loaded VLPs as opposed to using already-formed VLPs for loading where a certain fraction can remain unloaded.
  • one or more therapeutic or diagnostic agents may be loaded intracellularly by expressing LI and/or L2 in the presence of intracellular levels of one or more agents of interest,
  • this method is used for encapsulating a silencing plasmid which will encode for expression of short hairpin RN A (shRNA),
  • this plasmid will have a size of 2kB-6kB.
  • any suitable size may be used.
  • a plasmid is designed to be functional within the cells of the patient or subject to be treated (to which the loaded V LP is administered). Accordingly, the plasmid will be active within the target cells resulting in knockdown of the targeted gene(s).
  • this method may be used to encapsulate short interfering RNA (siRNA) or antisense nucleic acids (DN A or RNA) transfected into the host cells (e.g., 293 cel ls or other mammalian or insect host cells) during the production of the V LPs.
  • siRNA short interfering RNA
  • DN A or RNA antisense nucleic acids
  • loaded VLPs may be produced intracellularly to provide gene silencing functions when delivered to a subject.
  • RNA interference RNA interference
  • LI and L2 proteins are expressed in a host ceil system (e.g. mammalian cells or insect cells) from independent expression nucleic acids (e.g., vectors, for example, plasmids) as opposed to both being expressed from the same nucleic acid.
  • independent expression nucleic acids e.g., vectors, for example, plasmids
  • LI and L2 proteins are expressed in a host ceil system (e.g. mammalian cells or insect cells) from independent expression nucleic acids (e.g., vectors, for example, plasmids) as opposed to both being expressed from the same nucleic acid.
  • VLP structures can be produced to fit the needs of the different classes of payloads (e.g., DNA, R A, small molecule, large molecule) both in terms of charge and other functions (e.g. DNA binding domains, VLP inner volume, endosomal release function).
  • VLPs with a higher content of L2 protein will be better to bind nucleic acids (L2 contains a DNA binding domain) whereas VLPs with a smaller content of L2 protein will be better for other small molecules.
  • VLPs with different ratios of LI :L2 protein will have different inner volumes that w r ill allow a higher concentration of drug to be encapsulated.
  • the release of pay load into the cell will also be modulated.
  • structures containing more L2 protein may have a higher ability to transfer nucleic acids intracellularly. It should be appreciated that different ratios of L1/L2 may be used, in some embodiments, ratios may be 1 :1 , 1 :2, 1 :4, 1:5, 1 :20 or 1 :100. However, other ratios may be used as aspects of the invention are not limited in this respect.
  • each separate expression nucleic acid encodes an LI (but not an L,2) or an L2 (but not an LI ) sequence operably linked to a promoter.
  • the separate expression nucleic acids may use the same or different promoters and/or other regulatory sequences and/or replication origins, and/or selectable markers.
  • the separate nucleic acids may be vectors (e.g., plasmids, or other independently replicating nucleic acids).
  • separate nucleic acids may be independently integrated into the genome of a host cell (e.g., a first nucleic acid integrated and a second nucleic acid on a vector, two different nucleic acids integrated at different positions, etc.).
  • the relative expression levels of LI and L2 may be different in different cells, different using different expression sequences, independently regulated, or a combination thereof.
  • an expression vector is used to produce a mutant LI or L2 protein.
  • a mutant HPV16L1 protein (called LI *) is expressed along with L2 in a host system (e.g., a 293 cell system). These can then be isolated and assembled as described herein to encapsulate a therapeutic or diagnostic payload (e.g. therapeutic plasmid, siRNA, small molecule drugs, etc.).
  • a therapeutic or diagnostic payload e.g. therapeutic plasmid, siRNA, small molecule drugs, etc.
  • loaded VLPs are produced using certain LI and/or L2 variant sequences that are not recognized by existing antibodies against HPV (e.g.,
  • loaded VLPs can be produced using LI and/or L2 proteins that are modified to reduce antigenicity against other HPV serotype antibodies and/or to target the loaded VLP to particular organs or tissues (e.g., lung) or cells or subcellular locations.
  • the papilloma virus particles are HPV-VLP.
  • the methods described herein utilize HPV- VLP that contain one or more naturally occurring HPV capsid proteins (e.g., LI and/or L2 capsid proteins).
  • HPV-VLP may be comprised of capsid protein oligomers or monomers.
  • VLP refers to the capsid-like structures which result upon assembly of a HPV L I capsid protein alone or in combination with a HPV L2 capsid protein.
  • VLPs are morphologically and antigenically similar to authentic virions. VLPs lack viral genetic material (e.g., viral nuclei acid), rendering the VLP non-infectious.
  • VLP may be produced in vivo, in suitable host cells, e.g., mammalian, yeast, bacterial and insect host cells.
  • a “capsomere” refers to an oligomeric configuration of LI capsid protein.
  • Capsomeres may comprise at least one L I (e.g., a pentamer of LI).
  • capsid protein refers to LI or L2 proteins that are involved in building the viral capsid structure. Capsid proteins can form oligomeric structures i.e. pentamers, trimers or be in single units as monomers.
  • a VLP can be loaded with one or more medical, diagnostic and/or therapeutic agents, or a combination of two or more thereof.
  • the methods described herein utilize HPV-VLP that contain one or more variant capsid proteins (e.g., variant LI and/or L2 capsid proteins) that have reduced or modified immunogenicity in a subject. Examples of varian t capsid proteins are described in WO 2010/120266.
  • the modification may be an amino acid sequence change that reduces or avoids neutralization by the immune system of the subject.
  • a modified HPV-VLP contains a recombinant HPV protein (e.g., a recombinant L I and/or L2 protein) that includes one or more amino acid changes that alter the immunogenicity of the protein in a subject ( . e.g., in a huma subject).
  • a modified HPV-VLP has an altered immunogenicity but retains the ability to package and deliver molecules to a subject.
  • amino acids of the viral wild-type capsid proteins such as LI and/or LI +1,2, assembling into the HPV-VLP, are mutated and/or substituted and/or deleted.
  • these amino acids are modified to enhance the positive charge of the VLP interior.
  • modifications are introduced to allow a stronger electrostatic interaction of nucleic acid molecules with one or more of the amino acids facing the interior of the VLP and/or to avoid leakage of nucleic acid molecules out of the VLP. Examples of modifications are described in WO 2010/120266. It should be appreciated that any modified HPV-VLP may be loaded with one or more agents. Such particles may be delivered to a subject without inducing an immune response that would be induced by a naturally-occurring HPV,
  • HPV- VLP loaded according to the methods described herein, are useful for delivering one or more therapeutic agents to diseased tissue (e.g., diseased mucosal tissue).
  • a diseased tissue e.g. , mucosal tissue, epithelial tissue, or endothelial tissue
  • a infected tissue e.g., infected with a virus such as HP V or HSV.
  • the mucosal tissue is cervical tissue and the disease is dysplasia or cancer (e.g., cervical dysplasia, cervical cancer, for example associated with persistent HPV infection).
  • HPV-VLP loaded according to the methods described herein, may be used to deliver compositions to other tissues (e.g. , epidermis), In some embodiments, HPV-VLP may be used to treat HPV related diseases, In some embodiments, HPV-VLP, loaded according to the methods described herein, may be used to deliver therapeutic agents to treat a disease or condition, and/or may be used to deliver diagnostic agents to diagnose other diseases or conditions. For example, quantum dots, metals, and/or other imaging agents may be loaded into VLP according to the methods described herein and delivered. In some embodiments, agents may be used to track early stage diseases (e.g. , early stage metastasis).
  • early stage diseases e.g. , early stage metastasis
  • any suitable therapeutic, diagnostic and/or other medical agent may be loaded into VLP according to the methods described herein and delivered to a subject.
  • Examples of administration of VLP to subjects are described for example in U.S. Patent No. 7,205,126, incorporated herein by reference.
  • HPV-VLPs that are modified to display epitopes from two or more different naturally-occurring HPV variants are used in the agent loading methods described herein. Such modified VLPs may be used to provide immunization against infection by any of two or more naturally occurring HPV variants.
  • HPV-VLPs comprise viral LI capsid proteins.
  • HPV-VLPs comprise viral LI capsid proteins and viral L2 capsid proteins.
  • the LI and/or L2 proteins may, in some embodiments, be wild-type viral proteins.
  • L i and/or L2 capsid proteins may be altered by mutation and/or deletion and/or insertion so that the resulting LI and/or L2 proteins comprise only 'minimal' domains essential for assembly of a VLP.
  • LI and/or L2 proteins may also be fused to other proteins and/or peptides that provide additional functionality. Examples of modifications are described for example in U.S. Patent No.
  • VLPs may be based on particles containing one or more recombinant proteins or fragments thereof (e.g. , one or more HPV membrane and/or surface proteins or fragments thereof). In some embodiments, VLPs may be based on naturally-occurring particles that are processed to incorporate one or more agents as described herein, as aspects of the invention are not limited in this respect, In certain embodiments, particles comprising one or more targeting peptides may be used. Other combinations of HPV proteins (e.g., capsid proteins) or peptides may be used as aspects of the invention are not limited in this respect.
  • viral wild-type capsid proteins are altered by mutations, insertions and deletions. All conformation-dependent type-specific epitopes identified to date are found on the HPV-VLP surface within hyper-variable loops where the amino acid sequence is highly divergent between H PV types, which are designated BC, DE, EF, FG and HI loops. Most neutralizing antibodies are generated against epitopes in these variable loops and are type-specific, with limited cross-reactivity, cross-neutralization and cross-protection. Different HPV serotypes induce antibodies directed to different type-specific epitopes and/or to different loops. Examples of variant capsid proteins are described in WO 2010/120266.
  • viral capsid proteins HPV L I and/or L2 are mutated at one or more amino acid positions located in one or more hyper-variable and/or surface- exposed loops.
  • the mutations are made at amino acid positions within the loops that are not conserved between HPV serotypes. These positions can be completely non-conserved, that is that any amino acid can be at this position, or the position can be conserved in that only conservative amino acid changes can be made.
  • LI protein and L1 ⁇ L2 protein may be produced
  • reeombraantly produced LI protein and L1+L2 protein may self-assemble to form vims-like particles (VLP).
  • VLP vims-like particles
  • Recombinant production may occur in a bacterial, insect, yeast or mammalian host system.
  • LI protein may be expressed or L1 ⁇ L2 protein may be co-expressed in the host system.
  • HPV LI and/or L2 proteins may be expressed in Spodoptera frugiperla (8f21) cells.
  • Baculoviruses encoding the LI and/or L2 gene of any HPV or recombinant versions thereof from different serotypes e.g., HPV 16, HPV18, HPV31, and HPV58
  • HPV LI and/or L,2 genes may be cloned into a plasmid, such as pFastBacl (Invitrogen).
  • Sf21 cells may be maintained in Grace's insect medium (Invitrogen) supplemented with 10% fetal calf serum (PCS, invitrogen) and infected with recombinant baculoviruses and incubated at 27°C. Three days post infection, cells can be harvested and VLP can be purified.
  • cells may be resuspended in PBS containing Nonidet P40 (0.5%), pepstatin A, and leupeptin (1 ⁇ each, Sigma Aldrich), and allowed to stand for 30 min at 4°C.
  • Nuclear lysates may then be centrifuged and pellets can be resuspended in ice cold PBS containing pepstatin A and leupeptin and then sonicated. Samples may then be loaded on a CsCl gradient and centrifuged to equilibrium (e.g., 22 h, 27,000 rpm in a SW28 rotor, 4°C).
  • CsCl gradient fractions may be investigated for density by refractonietry and for the presence of L1/L2 protein by electrophoresis in 10% sodium dodecyl sulfate-polyacryiamide gel (SDS-PAGE) and Coomassie blue staining. Positive fractions can be pooled, diluted in PBS and pelleted e.g., in a Beckman SW 28 rotor (3 h, 28,000 rpm, 4°C), After centrifugation, VLP can be resuspended in 0.15 mol/ ' L NaCl and sonicated, e.g., by one 5 second burst at 60% maximum power. Total protein content may be determined.
  • Viral capsid proteins may also be expressed using galactose-inducible Saccharomyces cerevisiae expression system, Leucine-free selective culture medium used for the propagation of yeast cultures, yeast can be induced with medium containing glucose and galactose. Cells can be harvested using filtration, After resuspension, cells may be treated with Benzonase and subsequently mechanically disrupted (e.g., using a homogenizer). Ceil lysate may be clarified using filtration.
  • An exemplary protocol can be found in Cook et al. Protein Expression and Purification 17, 477-484 (1999).
  • Transiently transfected cells e.g., 293 cells, for example 293T or 293TT cells
  • 293 cells for example 293T or 293TT cells
  • Benzonase and exonuclease V can be lysed by adding Brij58 or similar nonionic polyoxyethylene surfactant detergent, followed by benzonase and exonuclease V and incubating at 37°C for 24 h to remove unpackaged cellular and viral DNA and to allow capsid maturation.
  • the lysate can be incubated on ice with 5 M NaCl and cleared by centrifugation.
  • VLP can be collected by high-speed centrifugation.
  • Capsid proteins may also be expressed in E. coli.
  • E.coli one important potential contaminant of protein solutions is endotoxin, a lipopolysaccharide (LPS) that is a major component of the outer membrane of Gram-negative bacteria (Schadlich et /. Vaccine 27, 1 11-1522 (2009)).
  • LPS lipopolysaccharide
  • transformed BL21 bacteria may be grown in LB medium containing ImM ampiciliin and incubated with shaking at 200 rpm at 37 ' C. At an optical density (ODgoo nm) of 0.3-0.5, bacteria can be cooled down and IPTG may be added to induce protein expression. After 16-18 h bacteria may be harvested by centrifugation.
  • VLPs are loaded with the one or more therapeutic agents. After isolation of LI and L2 capsid proteins which may be in the form of monomers or oligomers, VLPs may be assembled and loaded by disassembling and reassembling LI or LI and L2 viral capsid proteins, as described herein. Salts that are useful in aiding
  • VLPs include Zn, Cu and Ni, Ru and Fe salts.
  • VLPs may be loaded with one or more therapeutic agents.
  • VLPs Loading of VLPs with agents utilizing a disassembly-reassembly method has been described previously, for example in U.S. Patent Nos: 6,416,945 and WO 2010/120266, incorporated herein by reference. Generally, these methods involve incubation of the VLP in a buffer comprising EGTA and DTT, Under these conditions, VLP completely disaggregated into structures resembling capsid proteins in monomeric or oligomeric form, A therapeutic or diagnostic agent, as described herein, may then be added and the preparation diluted in a buffer containing DMSO and CaCl 2 with or without ZnCl 2 in order to reassemble the VLP.
  • ZnCl 2 increases the reassembly of capsid proteins into VLP.
  • these reassembly methods may be used to assemble capsid proteins to form VLPs that encapsulate one or more agents without requiring an initial VLP disassembly procedure, as described herein.
  • VLP are loaded with the one or more therapeutic agents.
  • these may mixed directly after purification from the host cell with the therapeutic agent and reassembled into loaded VLPs as described herein, the preparation diluted in a buffer containing DMSO and CaCl 2 with or without ZnCl 2 in order to reassemble the VLP.
  • the presence of ZnCl 2 increases the reassembly of capsid proteins into VLP.
  • VLP loaded with agents using the methods described herein are able to deliver agent to 65%, 75%, 85%, 95%, 96%», 97%, 98%, or 99% of target cells.
  • One non imiting example of the improved method is exemplified in the Examples.
  • VLP may be loaded with a nucleic acid using a method comprising: a) contacting a preparation of capsid proteins with the nucleic acid in a reaction volume, wherein i) the ratio of capsid protein to reaction volume ranges from 0.
  • the ratio of nucleic acid to capsid protein ranges from 0.1 ⁇ g nucleic acid per 1 p.g capsid protein to 10 p.g nucleic acid per 1 ⁇ g capsid protein; and/or iii) the ratio of nucleic acid to reaction volume ranges from 0.01 ⁇ g nucleic acid per 1 ⁇ reaction volume to 10 g nucleic acid per 1 ⁇ reaction volume, and b) reassembling the capsid proteins to form a VLP, thereby
  • the ratio of HPV- capsid protein to reaction volume ranges from 0.2 ⁇ g HPV-capsid protein per 1 ⁇ reaction volume to 0.6 ⁇ g HPV-capsid protein per 1 ⁇ reaction volume.
  • the ratio of nucleic acid to HP V-capsid protein ranges from 0.5 ⁇ g nucleic acid per 1 ug HPV-capsid protein to 3.5 ⁇ nucleic acid per 1 ⁇ 3 ⁇ 4 HPV-capsid protein.
  • the ratio of nucleic acid to reaction volume ranges from 0.2 ⁇ 3 ⁇ 4 nucleic acid per 1 ⁇ reaction volume to 3 ⁇ g nucleic acid per I ⁇ reaction volume.
  • the step of dissociating the VLP or capsid protein oligomers can be carried out in a solution comprising ethylene glycol tetraacetic acid (EGTA) and dithiothreitol (DTT), wherein the concentration of EGTA ranges from 0,3 mM to 30 mM and the concentration of DTT ranges from 2 mM to 200 mM. in certain embodiments, the concentration of EGTA ranges from 1 mM to 5 mM. In certain embodiments, the concentration of DTT ranges from 5 mM to 0 mM.
  • EGTA ethylene glycol tetraacetic acid
  • DTT dithiothreitol
  • the step of reassembling of capsid proteins into a VLP can be carried out in a solution comprising dimethyl sulfoxide (DMSO), ⁇ aCL and ZnC , wherein the
  • concentration of DMSO ranges from 0.03% to 3% volume/volume
  • concentration of CaCl 2 ranges from 0.2 mM to 20 mM
  • concentration of ZnCL ranges from 0.5 ⁇ , ⁇ to 50 ⁇ .
  • concentration of DMSO ranges from 0.1% to 1% volume/volume.
  • concentration of ZnCL ranges from 1 ⁇ , ⁇ to 20 ⁇ .
  • concentration of CaCl 2 ranges from 1 mM to 10 mM.
  • the loading method is further modified to stabilize the VLP, in thai the loading reaction is dialyzed against hypertonic NaCl solution (e.g., using a NaCl concentration of about 500 mM) instead of phosphate-buffered saline (PBS), as was previously described. Surprisingly, this reduces the tendency of the loaded VLP to form larger agglomerates and precipitate.
  • the concentration ofNaCl ranges between 5 mM and 5 M. In certain embodiments, the concentration of NaCl ranges between 20 mM and 1 M.
  • VLPs can be loaded with one or more therapeutic agents according to the methods described herein.
  • the therapeutic agent is a nucleic acid molecule capable of inducing RNA interference or a nucleic acid (e.g. , plasmid or other vector) that is capable of expressing a nucleic acid molecule capable of inducing RNA interference.
  • VLP can be loaded with combinations of two or more therapeutic or diagnostic agents,
  • the therapeutic agent is an inducer of RNA interference 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.
  • RNA duplex ribonucleic acid
  • DNA deoxyribonucleic acid
  • Other inducers of gene silencing include inducers of DNA methylation, or rihozymes, or aptamers.
  • the therapeutic agent can be a modulator of gene expression such as a PNA (Peptide Nucleic Acid).
  • RNA interference is a process whereby the introduction of double stranded RNA (dsRNA) into a cell inhibits gene expression post-transcriptionally, in a sequence dependent fashion, RNAi can be mediated by short (for example 19-25 nucleotides) dsRNAs or small interfering R ' NAs (siRN A).
  • dsRNA is cleaved in the cell to create siRNAs that are incorporated into an RNA-induced silencing complex (RISC), guiding the complex to a homologous endogenous mRNA, cleaving the mRNA transcript, and resulting in the destruction of the mRNA.
  • RISC RNA-induced silencing complex
  • dsRNA may be introduced into the cell as an isolated nucleic acid fragment or via a transgene, plasmid, or virus.
  • VLP are used to deliver dsRNA to the target cells,
  • a short hairpin RNA molecule is expressed in the cell
  • a shRNA comprises short inverted repeats separated by a small loop sequence, One inverted repeat is complimentary to the gene target.
  • the shRN A is then processed into an siRNA which degrades the target gene mRNA.
  • siiRNAs can produced within a cell with a DNA construct encoding the shRNA sequence under control of a RN A polymerase III promoter, such as the human HI, U6 or 7SK promoter.
  • the shRNA may be synthesized exogenous! ⁇ ' and introduced directly into the cell, for example through VLP deliver ⁇ '.
  • the shRNA sequence is between 40 and 100 bases in length or between 40 and 70 bases in length.
  • the stem of the hairpin are, for example, between 19 and 30 base pairs in length.
  • the stem may contain G-U pairings to stabilize the hairpin striscture.
  • siRNA sequences are selected on the basis of their homology to the target gene. Homology between two nucleotide sequences may be determined using a variety of programs including the BLAST program (Altschul et al (1990) J. Mol. Biol. 215: 403-10), or BestFit (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA., Wisconsin 53711). Sequence comparisons may be made using FAST A and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology 183: 63-98). Tools for design and quality of siRNAs, shRNAs and/or miRNAs are known in the art.
  • siRNA sequences and scrambled siRNA sequences are for example siDirect, siSearch, SEQ2SVM, Deqor, siRNA Wizard (invivoGen).
  • 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). Sequence comparison may be made over the full length of the relevant sequence, or may more preferably be over a contiguous sequence of about or 10,15, 20,25 or 30 bases.
  • the degree of homology between the siRNA and the target gene is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, or 100% ⁇ .
  • the siRN A may be between lObp and 30bp in length, or between 20bp and 25bp, or the siRNA is 20, 21 or 22bp in length.
  • RNAi can be detected by transfecting cultured cells with the siRNA, followed by RT-PCR of the mRNA of interest. Where R NAi is induced by the siRNA, levels of the mRNA of interest will be reduced in traiisiected ceils as compared to control cells. A reduction in protein production can be confirmed by Western blotting of cell lysates followed by probing with an antibody reactive to the protein of interest,
  • siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques which are known in the art. [0093], In some embodiments, the siRNA has an overhang at one or both ends of one or more deoxythymidme bases to increase the stability of the siRN A within cells by reducing its susceptibility to degradation by nucleases,
  • the siRNA is 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.
  • RNA duplex ribonucleic acid
  • DNA deoxyribonucleic acid
  • Preferred targets for the RNA interference would include disease causing genes e.g.
  • the target genes would be E6 & E7 HPV viral oncogenes (e.g., E7 siRNA), In other embodiments of the invention the target genes would be p53, Sirt-1, survivin, EGFR, VEGFR, VEGF, CTNNB1 or other oncogenes.
  • Linkages between nucleotides may be phosphodiester bonds or alternatives, for example, linking groups of the formula P (O) 8, (thioate) ; P (S) 8, (dithioate); P (O) NR'2 ; P (O) R' ; P (O) OR6; CO ; or CONR'2 wherein R is H (or a salt) or alkyl ( 1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S-.
  • Modified nucleotide bases can be used in addition to the naturally occurring bases, For example, modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for silencing,
  • the term modified nucleotide base encompasses nucleotides with a covIERly modified base and/or sugar.
  • modified nucleotides include nucleotides having sugars which are covIERly attached to low molecular weight organic groups other than a hydroxy! group at the 3'position and other than a phosphate group at the 5 'position.
  • modified nucleotides may also include ⁇ substituted sugars such as 2'- 0-methyl- ; 2-0- alkyl : 2-0-allyl; 2'-S-alkyl; 2'-S-allyl ; 2'-fiuoro- ; 2'-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
  • ⁇ substituted sugars such as 2'- 0-methyl- ; 2-0- alkyl : 2-0-allyl; 2'-S-alkyl; 2'-S-allyl ; 2'-fiuoro- ; 2'-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugar
  • Modified nucleotides include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4, N4- ethanocytosine, 8-hydroxy-N6-methylademne, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylammomethyl uracil, dihydrouracil, inosine, N6 ⁇ isopentyl-adenine, 1- methyladenine, l-methylpseudouracil, 1- methylguanine, 2,2-dimethylguanine, 2methylad nine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
  • siRNA molecules or longer dsRNA molecules may be made recombinantly by transcription of a nucleic acid sequence, for example contained within a vector as described herein.
  • the vector may be any RNA or DNA vector,
  • the vector can be an expression vector, wherein the nucleotide sequence is operably linked to a promoter compatible with the cell.
  • promoters suitable for use in various vertebrate systems are well known in the art.
  • suitable promoters include viral promoters such as mammalian retrovirus or DN A virus promoters, e.g., MLV, CMV, RSV, SV40 IEP (immediate early promoter) and adenovirus promoters and metallothionein promoter. Strong mammalian promoters may also be used. It will be appreciated that variants of such promoters retaining substantially similar transcriptional activities may also be used.
  • the vector may have at least two promoters, one to direct expression of the sense strand and one to direct expression of the antisense strand of the dsRNA. In other embodiments, two vectors may be used, one for the sense strand and one for the antisense strand. Alternatively the vector may encode RNAs which form stem-loop structures which are subsequently cleaved by the cell to produce dsRNA,
  • the nucleic acid construct may contain a specific cellular, viral or other promoter or repressor of gene expression.
  • the promoter or repressor may be designed to reflect the context of the cell into which the constract is introduced.
  • the construct may contain a viral promoter so expression from the construct is dependent upon the presence of a viral protein, so that the construct is expressed only in viral-infected cells.
  • the construct may have a promoter or repressor specific to certain cell types or to certain developmental stages.
  • a viral promoter whic matches the disease-causing vims should be used, e.g., a HPV promoter (such as the promoter causing expression of HPV E6/E7) for HPV- infected ceils.
  • a HPV promoter such as the promoter causing expression of HPV E6/E7 for HPV- infected ceils.
  • the vector will only be expressed in the virally- infected cells.
  • nucleic acids are highly charged and do not cross ceil membranes by free diffusion.
  • the hydrophilic character and anionic backbone of nucleic acids reduces their uptake by the cells, in certain embodiments, nucleic acids (e.g., siRNA) may be loaded into VLP (e.g., HPV-VLP) to efficiently deliver them to a subject through VLP (e.g., HPV-VLP) to efficiently deliver them to a subject through
  • V LP administration of V LP
  • encapsulating the nucleic acid into a V LP increases cellular uptake, allows traversal of biological membrane barriers in vivo, and/or increases the bioavailability of the nucleic acid (e.g., siRNA).
  • the agent loaded into the VLP is an anti-viral agent, in certain embodiments, the agent is an anticancer agent, In a preferred embodiment, the anticancer agent is a taxane,
  • the therapeutic agent that may be loaded into a VLP using the methods described herein is a chemotherapeutic agent, for instance, methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5- fiuorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, vairubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase inhibitor, MiV!P, MTA/LY231514,
  • chemotherapeutic agent for instance, methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5- fiuorouracil, mitomycin C,
  • LY264618 Lometexol. Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH- 4556, CS-682, 9-AC, AG3340, AG3433, Incel VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative, Temodal/Temozolomide, Evacet liposomal doxorubicin, Y ewtaxan Paclitaxel, Taxol/Paclitaxel, Xeload/
  • Camptosar/Irinotecan Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine,
  • Aminogiutethimide Asparaginase, Busulfan, Carboplatin, Chlorambucil, Cytarabine HCI, Dactinomycin, Daunorubicin HCI, Estramustine phosphate sodium, Etoposide (VP 16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),
  • Lomustine (CCNU), Mechlorethamine HCI (nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o.p ' -DDD), Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m ⁇ AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2,
  • Mitoguazone methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG
  • Pentostatin (2 ' deox coform cin
  • Semustine methyl-CCNU
  • Teniposide VM-26
  • Viiidesine sulfate but it is not so limited.
  • the therapeutic agent that may be loaded into a VLP using the methods described herein is an immunotherapeutic agent, for instance, Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncoiym, SMART Ml 95, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-1 1, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE- 1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Giiomab-H, GNI-250, EMD-72000,
  • an immunotherapeutic agent for instance, Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncoiym, SMART Ml 95, ATRAGEN, Ovarex, Bexxar, LDP-03
  • LymphoCide CMA 676, Monopharm-C, 4B5, ior egf r3, ior c5, BABS, anti-FLK-2, MDX- 260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab or IrrunuRAIT-CEA, but it is not so limited.
  • the therapeutic agent that may be loaded into a VLP using the methods described herein is an antiviral agent.
  • anti-viral agents are:
  • Polysulfates PVAS
  • Polysulfonates PVS
  • Polycarboxylates Polyoxometalates
  • Chicoric acid zintevir, cosalane derivatives
  • Bicyclams i.e., AMD3100
  • T-22, T-134, ALX-40-4C CGP-64222, TAK-779
  • AZT azidothymidine
  • ddl, ddC, d4T didehydrodideoxythymidine
  • 3TC 3'-thiadideoxycytidine
  • ABC and other ddN (2',3'-dideoxynucleoside) analogs
  • Nevirapine delavirdine, efavirenz, emivirine (MKC-442), capravirine, thiocarboxanilide UC- 781 , acyclovir, valaciclovir, penciclovir, famciclovir, bromovinyldeoxyuridine
  • Bacmid/cellfectin complex and plate Incubate plates for four days in a humidified 27C tissue culture incubator. Transfer conditioned media to 30ml SF 8f9 culture. Grow culture 3-5 days. Monitor for cell viability and cell diameter using Vi-Cell. Harvest conditioned media and cell pellet when viability is less than 75%. Perform titer (BacPA RapidTiter Kit) and Western Plot analysis. Expand recombinant virus by infecting a 1 L, culture of Sf9 ceils at an MOI of 0.1 with the best expressing Baculovirus clone. Harvest conditioned media by centrifugation once viability has dropped less than 75%. Perform titer analysis using
  • the cell pellet was obtained by tangential- flow filtration, washed in PBS, re-pelleted by centrifugation, and stored at -80°C. Western blot using protein-specific antibodies for LI and L2 proteins were then used to verify the presence of the recombinant protein.
  • Bac-to-Bac selective plates Incubate plates at 37C for two. days.
  • bacmid Cellfectin complex by mixing 1 ug of bacmid that has been diluted with 100 ul of Grace's media with 6ul of cellfectin transfection reagent that has been diluted with 100 ul of Grace's media. Let complexes form for 30 minutes at room temperature. Remove media from the cells in upper left comer well, dilute bacmid cellfectin complex with SOOul of Grace's media, add transfection solution to the upper left corner well. Place plates into a humidified incubator at 27C. After five hours, remove transfection solution from the cells in the upper left corner well and add 2ml of growth media (i.e. Sf-900 II).
  • baculo viral stock For each baculo viral stock, add I ml of the PO viral stock to a 30ml culture in a 125ml shake flask of Sf9 cell at a cell density of le6 cells/ml. An additional SF is utilized as a negative control and lml of growth media added, Shaking incubator parameters are 120 rpm and 27.5C. Cultures are monitored daily with the Vi-Cell for cell density, cell viability, and diameter. In a proper infection, within 48 hours the insect ceil culture should have significantly lower cell density and cell viability and increased cell diameter. Cultures are maintained for three to five days and harvested by centrifugation (2500 x g, 10 minutes) once viability has dropped belo 75%. Transfer the conditioned media (PI) viral stock to a fresh tube and store at 4C. Reserve cell pellet for Western analysis. Determine titer for the pi viral stock using the Clontech BacPAK Rapid Titer Ket according to manufacturer's protocol.
  • baculoviral stock For the best expressing baculoviral stock (i.e. Western Analysis), add 1.5e8 pfu of PI viral stock to a II. cultureof Sf9 ceils in a3L Shake Flask at 1.5e6 ceils per ml (i.e. MOI of 0.1 ). Shaking incubator parameters are 120 rpm and 27.5C. Cultures are monitored daily with Vi- Cell for cell density, cell viability, and cell diameter. Cultures are maintained for two to five days and harvested by centrifugation (2500 x g, 10 minutes) once viability has dropped below 75%. Transfer the conditioned media (PI) viral stock to a fresh sterile bottle and store at 4C. Determine titer for the P2 viral stock using the Clontech BacPAK Rapid Titer Kit according to manufacturer's protocol.
  • Solution 3 (2 mM ( aCk 5 ⁇ CaCL, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 434 iL dH 2 0) was then added to the above mixture and incubated at 37°C for 30 min.
  • Solution 4 (4 mM CaCi 2 , 10 ⁇ CaCL, 50 mM Tris-HCl pH 7.5, 150 mM NaCL 1224 ⁇ dH 2 0) was then added to the above mixture and incubated at 37°C for 2 hrs.
  • Capsid proteins isolated from insect cells were assembled into VLPs as described. Dynamic light scattering (DLS) demonstrates presence of capsid proteins in moiiomeric and oligomeric forms ( ⁇ 1 Onm) after harvest and prior to the loading procedure. After the reassembly in presence of the nucleic acid payload, VLPs are seen by DLS (50-70nm diameter) (FIG. 3).
  • DLS 50-70nm diameter
  • Results show functional transfer of luciferase expression.
  • VLPs were generated using different production methods to compare efficacy. Transfection of luciferase plasmid (pClucF) using standard iipofectamine transfection at various plasmid amounts (O.lng well, 1 tig/well, lOng/weil) was used to create a range of positive controls. lOng of pClucF plasmid was used without transfection reagent as a reagent/background control.
  • AB1-2 refers to HPV16L1L2 VLP generated using the methods described above, where a single plasmid like p!6sheLL (SEQ ID NO: 6) was used to co-express wildtype HPV L I and 1.2 proteins.
  • Capsid proteins were purified, as described above, from 293 cells transfected with the co-expression plasmid for LI and L2. Capsid proteins were then subjected to the following loading protocol, thereby forming loaded VLP.
  • Solution 3 (2 mM CaCl 2 , 5 ⁇ CaCl 2 , 50 mM Tris-HCl pH 7.5, 150 mM NaCL 434 iL d3 ⁇ 40) was then added to the above mixture and incubated at 37°C for 30 min.
  • Solution 4 (4 mM CaCi 2 , 10 ⁇ CaCi 2 , 50 mM Tris-HCl pH 7.5, 150 mM Nad, 1224 ⁇ dH 2 0) was then added to the above mixture and incubated at 37°C for 2 hrs.
  • Loaded VLP were then used to treat Hela cells in 96 well plates and luciferase signal was read after 48hrs (Table 2, FIGS. 4 and 5).
  • AB iuc3 and AB luc4 were produced in 293 cells after transfection with the l6sheLL plasmid as pseudovirions (PSV) already encapsulating the payload plasmid (pi iucL) (Buck, C, B., et al. (2005) Methods Mol Med., 119: 445-462). Results showed superior transfer of plasmid when the reassembly loading method was used (AB 1 -2) compared with VLPs that were loaded through packaging of plasmid in the host cells (AB luc 3 and AB luc 4).
  • PSV pseudovirions
  • Alopecia means loss of hair from the head or body and includes baldness, a term generally reserved for pattern alopecia or androgenic alopecia (AG A).
  • AGA is the most common cause of hair loss in humans, both male and female and its incidence is increasing 10% per decade.
  • DHT dihydrotestosterone
  • Men with androgenic alopecia typically have higher levels of 5-aipha-reductase, lower levels of total testosterone, higher levels of unbound/free testosterone, and higher levels of total free androgens including DHT (Denmark- Wahnefried 1997).
  • 5-aipha-reductase is responsible for converting free testosterone into DHT.
  • 5-alpha-reductase The genes for 5-alpha-reductase are known (Ellis 2005), The enzymes are present predominantly in the scalp and prostate. Levels of 5-alpha- reductase are one factor in determining levels of DHT in the scalp and drugs which interfere with 5-alpha-reductase (such as Finasteride, which inhibits the predominant type 2 isoform) have been approved by the FDA as treatments for hair loss.
  • the adverse side effects of the Minoxidil dosing include scalp irrita tion and allergic contact dermatitis.
  • the side effects associated with the DHT inhibitor, Finasteride, which when applied topically has very little penetration are more serious.
  • the compounds and formulas of U.S. Patent No. 6,268,377 which is hereby incorporated by reference, for the treatment of hyperandrogenic condition include the 5-alpha-reductase inhibitors and calcium channel blocking agents which may be combined in a single oral, topical, systemic or parenteral dosage formulation.
  • the composition has a number of side effects that include: erectile dysfunction (ED) or impotence in up to 18.5 percent of people, decreased libido up to 10 percent, abnormal ejaculation, including decreased ejaculate amount, up to 7.2 percent, and breast enlargement up to 2.2 percent. .
  • ED erectile dysfunction
  • impotence in up to 18.5 percent of people
  • libido up to 10 percent
  • abnormal ejaculation including decreased ejaculate amount, up to 7.2 percent
  • breast enlargement up to 2.2 percent.
  • Propecia blocks the fomiation of DHT which is intended to inhibit the development of male pattern baldness.
  • the ineffectiveness of Propecia in treating male patterned baldness and the adverse effects associated with the currently prescribed method of dosing and treating male pattern baldness indicate that a more effective treatment is needed.
  • a preferred method for treating alopecia preferably includes using a combination of betapapillomavirus viral shells (L1/L2) to deliver a DHT inhibitor as a therapeutic agent.
  • the method preferably includes the steps of constructing a recombinant DNA molecule which contains a sequence encoding a papillomavirus LI protein or a papillomavims L2 protein or a combination of L I and 1.2 proteins and transfecting a host cell with the recombinant DNA molecule.
  • the virus like particles may express papillomavirus L I protein or L2 protein or a combination of LI and L2 proteins in the host cell.
  • the papillomavirus virus-like particles obtained from the transfected host cell may be purified which will cause the disassembling the LI and L2 capsid proteins of the virus-like particles into smaller units.
  • it is these smaller disassembled LI and L2 capsid proteins which may be loaded with a DHT inhibitor.
  • the loaded proteins may be reassembled to form a loaded virus-like particles comprising HPV protein with the DHT inhibitor and administered to the skin of an animal or a human subject.
  • the method can be applied to enhance topical deliver ⁇ ' of Minoxidil , Finasteride or other drugs to treat AGA topically through the skin and through the hair follicle.
  • the present invention provides a method for treating alopecia 1200, which includes a first step in which a recombinant DNA molecule is contracted which contains a sequence for encoding a papillomavims LI protein or a papillomavirus 1.2 protein, or a combination of papillomavirus L I and L2 proteins 1220. Thereafter, a host cell will be transfected with the recombinant DNA molecule 1230.
  • the transfected host cell will be treated to purify the papillomavirus vims-like particles causing the LI and L2 capsid proteins to disassemble into smaller units 1240, At which time, an appropriate therapeutic agent or drag for treating AGA will be introduced into the proximity of the virus-like particle where the agent or drag for treatment will be loaded into the virus-like particles 1250. Thereafter, the loaded virus-like particles comprising HPV protein with an effective DHT inhibitor for treating alopecia may be reassembled 1260. Finally, the treatment may preferably be topically applied through the skin and the hair follicles 1270 for the treatment of alopecia ,
  • the above method further includes a step to pre-treat the area of the skin.
  • the area of the scalp to be treated for alopecia may preferably be prepared by pre-administering a delipidating substance to the region of the scalp before applying the treatment of HPV pseudo-virus particles loaded with a DHT inhibitor to the scalp of a human subject,
  • the above method also further includes another pre -treatment of the scalp.
  • the area of the scalp to be treated for alopecia may be pre -treated by micro-abrasion before applying the treatment of HPV pseudo-virus particles loaded with a DHT inhibitor to the scalp of a human subject
  • RNA short interfering nucleic acid
  • a siRNA (short interfering nucleic acid) molecule may be formulated as a composition as described in U.S. Patent Application Publication No. 200770179104 A l (which is hereby incorporated by reference herein).
  • the formulated composition which may be loaded into the betaHPV pseudovirions and transdermally administered may include such siRNA formulations which are generally referred to as "lipid nucleic acid particles" (LNP) which include membrane disruptive agents in which the siRNA molecule is complexed with a cationic lipid and helper lipid molecule; or formulated with polyethylenimine derivatives, including for example grated PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEL and polyethylene glycol PEI (PEG-PEI) and/or derivatives thereof,
  • LNP lipid nucleic acid particles
  • PEG-PEI polyethylene glycol PEI
  • a method for topically treating non-melanoma skin cancer using a combination of betapapillomavirus viral shells (L.1/L2) to deliver a siRNA against the transcription factor protein for GH2 is provided.
  • other HPV viral shells may also be used.
  • HPV5 viral shells may be used.
  • a herpes viral shell may also be use.
  • a first step in this preferred emboidement includes constructing a recombinant DNA molecule that contains a sequence encoding a papillomavirus LI protein or a papillomavirus L2 protein or a combination of LI and L2 proteins and then transfecting a host cell with the recombinant DNA molecule.
  • the virus like particles may express papillomavirus LI protein or L2 protein or a combination of L I and L2 proteins in the host cell.
  • the betapapillomavirus virus-like particles obtained from the transfected host cell may be purified which will cause the disassembling of the LI and L2 capsid proteins of the vims-like particles into smaller units.
  • L I and L2 capsid proteins which may be combmed with a siRNA against the transcription factor protein for GH2.
  • the combination of siRNA with proteins may be reassembled to form loaded vims-like particles comprising HPV protein with the siRNA against the transcription factor protein for GU2 and administered to the skin of an animal or a human subject.
  • the present invention provides a method for treating non-melanoma skin cancer 800, which includes a first step in which a recombinant DNA molecule is contracted which contains a sequence for encoding a papillomavirus LI protein or a papillomavirus L2 protein, or a combination of papillomavirus LI and L2 proteins 820. Thereafter, a host cell will be transfected with the recombinant DNA molecule 830. After which, the transfected host ceil will be treated to purify the LI and L2 capsid proteins into smaller units 840.
  • an appropriate therapeutic agent or drug for treating non-melanoma skin cancer will be introduced into the proximity of the proteins where the agent or drug for treatment will be combined with the Nanosphere particle 850. Thereafter, the particles comprisingsiRNA against the transcription factor protein for GH2 may be reassembled 860, Finally, the treatment may preferably be topically applied through the skin 870 for the treatment of non-melanoma skin cancer.
  • these smaller units will include intermediate structure and capsomers.
  • these smaller units of capsid proteins, intermediate structures and capsomers are purified and then combined with a siRNA targeting transciption factor Gli2. Thereafter, reassembly of the capsid proteins is initiated to form loaded virus-like particles which preferably include PV proteins with attached siRNA targeting transcription factor GH2.
  • a method for topically treating Pachyonychia Congenita using a combination of betapapillomavirus viral shells (L1/L2) to deliver a siRNA targeting the N17K1 mutation in the keratin 6a gene is provided,
  • the siRNA of present invention may alternatively target known mutations in the genes encoding K6b, K16 and/or K17A.
  • the present invention provides a method for treating Pachyonychia Congenita 900, which includes a first step in which a recombinant DNA molecule is contracted which contains a sequence for encoding a papillomavirus LI protein or a papillomavirus L2 protein, or a combination of papillomavirus LI and L2 proteins 920. Thereafter, a host cell will be transiected with the recombinant DNA molecule 930.
  • the transiected host cell will be treated to purify the papillomavirus virus-like particles causing the LI and L2 capsid proteins to disassemble into smaller units 940.
  • an appropriate therapeutic agent or drug for treating Pachyonychia Congenita will be introduced into the proximity of the virus-like particle where the agent or drug for treatment will be loaded into the viras-like particles 950.
  • the loaded vims-like particles enclosing siRNA targeting the N17K1 mutation in keratin 6a may be reassembled 960.
  • the treatment may preferably be topically applied through the skin 970 for the treatment of Pachyonychia Congenita.
  • Table 1 shows a list of additional genetic skin related disorders which may be considered treatable in accordance with the present invention:
  • ADULT syndrome Acro-dermato-ungual-lacrimal-tooth syndrome
  • Ankyloblepharon-ectodermal defects-cleft lip and palate syndrome (AEC syndrome) Arthrogryposis and ectodermal dysplasia
  • CHS Curly hair-ankyloblepharon-nail dysplasia
  • Oculodentodigital displasia ODDD
  • a method for topically treating Psoriasis using a combination of betapapillomavirus viral shells (L1/L2) to deliver a siRNA against cytokines is provided.
  • a first step in this preferred embodiment includes constructing a recombinant DNA molecule that contains a sequence encoding a papillomavirus LI protein or a papillomavirus L2 protein or a combination of LI and L2 proteins and then transfecting a host cell with the recombinant DNA molecule.
  • the virus like particles may express papillomavirus LI protein or L2 protein or a combination of LI and L2 proteins in the host ceil.
  • the betapapillomavirus virus-like particles obtained from the transfected host cell may be purified which will cause the disassembling of the LI and L2 capsid proteins of the viras-like particles into smaller units.
  • these smaller disassembled LI and 1.2 capsid proteins which may be loaded with a si R NA against cytokines.
  • the loaded protems may be reassembled to form loaded virus-like particles comprising HPV protein with the siRNA against cytokines and administered to the skin of an animal or a human subject.
  • the present invention provides a method for treating Psoriasis 1 100, which includes a first step in which a recombinant DNA molecule is contracted which contains a sequence for encoding a papillomavirus LI protein or a papillomavirus L2 protein, or a combination of papillomavirus LI and L2 proteins 1 120. Thereafter, a host cell will be transfected with the recombinant DN A molecule 1 130.
  • the transfected host cell will be treated to purify the papillomavirus virus-like particles causing the LI and L2 capsid proteins to disassemble into smaller units 1 140.
  • an appropriate therapeutic agent or drag for treating Psoriasis will be introduced into the proximity of the virus-like particle where die agent or drug for treatment will be loaded into the virus-like particles 1 150.
  • the loaded virus-like particles enclosing siRNA against cytokines may be reassembled 1160.
  • the treatment may preferably be topically applied through the skin 1170 for the treatment of psoriasis.
  • the RNA which inhibits the expression of cytokines is an siRNA which inhibits the expression of TNF-a for the treatment of psoriasis.
  • alternati ve viral vectors may be used in place of the betapapillomavirus.
  • alternative viral vector may include herpes virus vectors.

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Abstract

La présente invention a pour objet un système d'administration transdermique pour le traitement de maladies cutanées utilisant des nanoparticules de protéine dérivée du papillome pour administrer des médicaments aux kératinocytes et aux cellules de la membrane basale pour le traitement de maladies cutanées. La présente invention concerne une méthode efficace pour administrer de petites molécules d'acides nucléiques aux cellules épidermiques.
PCT/US2012/045989 2011-07-10 2012-07-09 Nanoparticules de protéine virionique pour l'administration d'agents diagnostiques ou thérapeutiques pour le traitement de maladies cutanées WO2013009717A1 (fr)

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