WO2005113781A2 - Vectors for use in gene and cancer therapy - Google Patents

Vectors for use in gene and cancer therapy Download PDF

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Publication number
WO2005113781A2
WO2005113781A2 PCT/GB2005/002016 GB2005002016W WO2005113781A2 WO 2005113781 A2 WO2005113781 A2 WO 2005113781A2 GB 2005002016 W GB2005002016 W GB 2005002016W WO 2005113781 A2 WO2005113781 A2 WO 2005113781A2
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Prior art keywords
peptide
vector
adgfp
knob
cells
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PCT/GB2005/002016
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French (fr)
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WO2005113781A3 (en
Inventor
Pierre Boulanger
Leif Lindholm
Maria Magnusson
Saw See Hong
Manuel Rosa-Calatrava
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Got-A-Gene Ab
Habib, Nagy
Centre National De La Recherche Scientifique
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Publication of WO2005113781A2 publication Critical patent/WO2005113781A2/en
Publication of WO2005113781A3 publication Critical patent/WO2005113781A3/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein

Definitions

  • Vectors The present invention relates to vectors, in particular to modified or engineered vectors with good gene transfer efficiency, and to the use of such vectors in gene therapy.
  • Viruses are now widely used therapeutic agents in the fight against disease, both in non-human animals and humans. Particularly, viruses are utilised as vectors for gene therapy. Alternatively, cytolytic viruses have been used to target and kill cancer or unwanted proliferating cells, such therapy is also known as "virotherapy" .
  • cytolytic viruses have been used to target and kill cancer or unwanted proliferating cells, such therapy is also known as "virotherapy" .
  • the direct use of viruses in medical treatments is a widely growing area, and new techniques and uses involving viruses in treatment and therapy are being developed.
  • Viruses are highly evolved biological entities that efficiently gain access to their host cells and exploit the cellular machinery of the cell to facilitate their replication.
  • genes are heralded as ideal gene therapy vectors since foreign/heterologous genes or coding sequences may be inserted into the viral genome and infection thus allows the foreign gene to be delivered to the nucleus of the host cell .
  • Gene therapy was first conceived in order to treat genetic diseases where the defect lay in a hereditary single-gene defect, for example severe combined immunodeficiency disease (SCID) .
  • SCID severe combined immunodeficiency disease
  • viruses may be utilised to deliver genes for acquired diseases such as cancer, cardiovascular disease, neurodegenerative disorders, inflammation and even infectious disease.
  • viral vectors There are currently five main classes of clinically applicable viral vectors that are derived from oncoretroviruses, lentivirus, adenovirus, adeno- associated - virus (AAVs) and herpes-simplex-1 viruses (HSV-ls) .
  • Each class of vector is characterised by a set of different properties that make it suitable for use in certain applications, and unsuitable for others.
  • the five main classes of viral vector can be categorized in two groups according to whether their genomes integrate into host DNA (oncoretroviruses, lentiviruses and AAVs) or persist in the cell nucleus predominantly as extrachromosomal episomes (adenoviruses and herpes viruses) .
  • Non-integrating vectors can, under certain circumstances, mediate persistent transgene expression in non-proliferating cells, but integrating vectors are, at present, the tools of choice if stable genetic alteration needs to be maintained in dividing cells .
  • Human adenovirus (Ad) which is now commonly used as a gene vector, is a non-enveloped virus, whose capsid is constituted of 240 hexons and 12 pentons located at the
  • the penton capsomer is formed of two components, the fiber and the penton base.
  • the fiber has three structural domains, the tail, which is anchored to the penton base, the shaft, constituted of several repeats of a 15-amino acid motif, and a terminal globular structure, called the 'knob 1 .
  • Attachment of Ad of species C e.g. Ad2, Ad5
  • Ad of other species use different attachment receptors, recently identified as sialic acid residues for species D Ad37 (Arnberg, N.
  • the apparent simplest strategy would be to delete the knob domain, and replace it by the desired cell ligand, to ablate the natural tropism of Ad and confer upon the redesigned Ad a new cell tropism.
  • deletions or insertions in the knob domains except those involving some flexible loops, frequently result in nonviable viruses.
  • this strategy requires the generation of receptor-expressing, trans-complementing cell lines, i.e. cells expressing the surface receptor for the new ligand, or expressing wildtype (WT) fiber protein, in order to produce recombinant Ad in high yields .
  • Ad5 is a particularly useful and well studied vector for gene transfer as it has a high growth rate, high productivity and a high infectivity index. As discussed by Miyazawa et al. in J. Virol. (2001) Vol 75, No. 3, pp 1387-1400 (see for example Figure 10 therein) , Ad5 utilizes the intracellular microtubules to achieve nuclear localization following receptor-mediated enclocytosis and escape to the cytosol.
  • Ad7 is thought to achieve nuclear localization by remaining in late endosomes or lysosomes before finally escaping to the cytosol and translocating to the nucleus, this pathway is referred to herein as the lysosomal pathway.
  • the present inventors have surprisingly discovered that it is possible to re-direct vectors which do not normally utilize the lysosomal pathway to this pathway.
  • a class of peptides suitable for this re-directing have been identified and it has been shown that vectors incorporating such a peptide ligand have a greater efficiency of gene transfer than similar non-liganded vectors.
  • the present invention provides a vector comprising a nucleic acid molecule and a peptide or peptide derivative, said peptide or peptide derivative comprising a peptide of general Formula (I) : R- L -R ⁇ R J -R ⁇ R S -R S (I)
  • R x is arginine or lysine
  • R 2 is glutamine or asparagine
  • R 3 is methionine
  • R 4 is serine or threonine
  • R 5 is histidine, tryptophan, arginine or lysine
  • R s is valine, leucine or isoleucine or wherein one or more of R ⁇ -R s is replaced by a non-genetically coded amino acid residue which is equivalent in terms of its physical properties, e.g. in terms of its general size, charge and hydrophobicity, to one of the genetically coded residues listed above for that same position.
  • the peptide may incorporate one or more post-translational modifications, e.g.
  • peptide hexamer and other peptides described below may be replaced as a whole or in part by a peptidomimetic, non-peptide organic molecules which nevertheless incorporate the groups which contribute the same properties as the amino acid side chains by way of spatial configuration, charge and hydrophobicity etc.
  • Suitable replacement amino acids for the genetically coded amino acids and peptidomimetic molecules and groups are known in the art .
  • Preferred examples of the peptide of general Formula (I) are as follows, the standard single letter code for amino acids being used for convenience:
  • the peptide or peptide derivative may comprise flanking amino acids at the N- and/or C-terminus of the hexapeptides described above. More specifically, the peptide or peptide derivative of the vector will preferably comprise or consist of a peptide of general Formula (II) : R 7 -R S -R 9 -(I)-R 10 (II) wherein :
  • (I) is a peptide of general Formula (I) as defined above;
  • R 7 is glycine or alanine;
  • R 8 is histidine, tryptophan, arginine or lysine;
  • R 3 is proline;
  • R 10 is tyrosine or phenylalanine; or, as described above, one or more of R 7 -R 10 is replaced by an equivalent non-genetically-coded amino acids or the whole or part of the peptide is replaced by a peptidomimetic.
  • the peptide or peptide derivative will preferably comprise a decamer of Formula (II) .
  • Further preferred peptides will be made up of fragments of the peptide of Formula II.
  • R 10 may be absent.
  • R 7 may also be absent and if it is present, one or more of R 8 and R 9 , preferably both R B and R 9 are present.
  • R 8 may be absent and in that case R 7 will typically be absent, if R 8 is present then R 9 will preferably also be present .
  • R 9 may be absent in which case R 7 and R 8 will typically also be absent .
  • a particularly preferred decamer for incorporation into the vectors of the invention is G-H-P-(I) -Y, more particularly G-H-P-R-Q-M-S-H-V-Y, and variants of it including:
  • the peptide or peptide derivative of the vector may contain any functionally active fragment of a peptide of general formula (II) .
  • Such fragments will typically comprise at least 4, preferably at least 5 or 6, more preferably at least 7 or 8 and most preferably 9 amino acids.
  • the fragments are 'functionally active 1 if they can (as part of a vector) increase gene transduction (transfer) efficiency as compared to a vector which lacks the targeting peptide fragment.
  • a suitable Ad-mediated gene transduction assay is described in the Examples herein.
  • the vectors according to the present invention will result in at least 5%, preferably at least 10%, more preferably at least 20 or 30%, most preferably at least 50% greater gene transduction efficiency as compared to a vector which lacks the peptide fragment.
  • functionally active fragments are those which are able to redirect the vector of which they form a part to the lysosomal pathway.
  • the Examples section herein teaches how localisation may be measured (e.g. by confocal immunofluorescence microscopy or electron- microscopy) and 'active' fragments are those which result in a measurably greater amount of vector being re-directed to the lysosomal pathway as compared to a vector lacking that fragment but the same in all other respects.
  • the proportion of vector using the lysosomal pathway will be increased by at least 20%, preferably at least 30%, e.g. at least 50%, by an active fragment of a peptide of general formula (II) .
  • the present invention provides a vector comprising a nucleic acid molecule and a peptide or peptide derivative, said peptide or peptide derivative comprising a peptide of general Formula (III) : R 1 -R 2 -R 3 -R 4 -R s -R s -R 7 -R g -R 9 -R 1 ( I I I )
  • the peptide may incorporate one or more post-translational modifications, e.g.
  • glycosylation, phosphorylation, myristylation, pal itylation, isoprenylation or biotinylation may be replaced as a whole or in part by a peptidomimetic, non-peptide organic molecules which nevertheless incorporate the groups which contribute the same properties as the amino acid side chains by way of spatial configuration, charge and hydrophobicity etc.
  • Suitable replacement amino acids for the genetically coded amino acids and peptidomimetic molecules and groups are known in the art.
  • R 7 and R 8 of the peptide of general Formula (I) are as follows, R 7 is methionine, leucine, isoleucine, valine, threonine, tryptophan or phenylalanine; and R 8 is lysine or arginine
  • R 7 is methionine; and R 8 is lysine
  • the peptide or peptide derivative may comprise flanking amino acids at the N- and/or C-terminus of the decapeptides described above. More specifically, the peptide or peptide derivative of the vector will preferably comprise a peptide of general Formula (IV) :
  • (III) is a peptide of general Formula (III) as defined above;
  • R u is lysine or arginine
  • R 12 is phenylalanine or tyrosine or, as described above, one or more of R X1 -R 12 is replaced by an equivalent non-genetically-coded amino acids or the whole or part of the peptide is replaced by a peptidomimetic.
  • the peptide or peptide derivative will preferably comprise a dodecamer of Formula (IV) .
  • Further preferred peptides will be made up of fragments of the peptide of Formula IV.
  • R 12 may be absent.
  • R 1X may also be absent however R u may be absent and R e present.
  • a particularly preferred dodecamer for incorporation into the vectors of the invention is (IV)-K-F, more particularly T-A-Y-S-S-X-X-G-G-K-F (wherein X means any amino acid) , and most particularity T-A-Y-S-S-M-K-G-G-K-F.
  • the peptide or peptide derivative of the vector may contain any functionally active fragment of a peptide of general formula (IV) .
  • Such fragments will typically comprise at ' least 4, preferably at least 5 or 6, more preferably at least 7 or 8 and most preferably 9 amino acids.
  • the fragments are 'functionally active' if they can (as part of a vector) increase gene transduction (transfer) efficiency as compared to a vector which lacks the targeting peptide fragment .
  • a suitable Ad-mediated gene transduction assay is described in the Examples herein.
  • the vectors according to the present invention will result in at least 5%, preferably at least 10%, more preferably at least 20 or 30%, most preferably at least 50% greater gene transduction efficiency as compared to a vector which lacks the peptide fragment.
  • the vectors of the invention are typically synthetic vectors in that they are not naturally occurring but have been engineered to incorporate a peptide or peptide derivative as defined herein.
  • the vector may preferably be a modification of a naturally occurring vector, e.g. a modified viral vector or may have been synthesised essentially de novo, e.g. made up of a simple peptide/nucleic acid complex.
  • Viral vectors are preferred, in particular adenoviral vectors, especially those of subgroup C, most especially Ad5.
  • the present invention provides a modified adenovirus (Ad) incorporating in one or more of its capsid proteins a peptide of Formula (I) , preferably a peptide of Formula (II) or an active fragment thereof as discussed and defined above.
  • the present invention provides a modified adenovirus (Ad) incorporating in one or more of its capsid proteins a peptide of Formula (III) , preferably a peptide of Formula (IV) or an active fragment thereof as discussed and defined above.
  • the peptides of formula (I), (II), (III) and (IV) may be incorporated into any of the capsid proteins of the adenovirus, e.g.
  • penton capsomer is made up of penton base plus fiber
  • pIX hexon or protein IX
  • the penton has often been preferred because of the natural role in receptor recognition played by the fiber knob, and in integrin binding and endocytosis played by the penton base. If the penton is used the fiber domain will be preferred for insertion due to its exposure and high accessibility.
  • the peptide would not normally be incorporated into the tail, since this could be detrimental to the interaction of the fiber with the virion, but it can conveniently be inserted into the shaft or the globular 'knob'. Insertion into the shaft will be preferred since genetic or proteolytic de-knobbing will be possible and will result in the detargeting of the virus. Detargeting the virus will prevent it from binding to its natural receptors and infecting non-desired cells.
  • the knob is not required for effective gene transfer and that the Ad may incorporate further modifications, e.g. deletion of some of the shaft as well as removal of the knob .
  • insertion of the peptides of the invention into the C-terminal portion of protein IX is preferred. Preferably between amino acid position 100 and the C-terminal residue, more preferably between amino acid 125 and the C-terminal residue and most preferably between residues 131 and 132.
  • the peptides of the invention can be inserted into a protein IX containing other mutations/modifications in its C-terminal domain which would permit greater accessibility and/or functionality of the ligands.
  • protein IX could contain an extra amino acid sequence, a "linker' at its C-terminus, which would allow the peptides of the invention to protrude further at the surface of viral capsid.
  • This linker could have different lengths in terms of amino acid number.
  • Peptides (III) or (IV) or an active fragment thereof as discussed and defined above are particularily efficient in enhancing transduction of melanoma cells and other tumour cells, including those which are not usually permissive to adenovirus infection. The skilled man will be aware of and be able to determine cells which are not permissive to adenovirus infection.
  • insertion into the pIX capsid protein is most prefered.
  • non-Ad viral vectors such as oncovetroviruses or lentiviruses which are enveloped
  • the peptide may conveniently be inserted into the accessible domains of the envelope glycoproteins.
  • accessible domains of the major capsid protein can be used.
  • a variety of non-viral vectors also called synthetic gene delivery systems) are known and are regularly reported and reviewed, for example in the
  • non-viral approach for gene delivery may consist merely of introducing naked DNA into cells, e.g. using physical or chemical techniques such as electroporation or ultrasound. Plasmids are particularly preferred for use as or in non-viral vectors .
  • the non-viral vectors of the present invention will thus typically comprise functional conjugates of nucleic acid, particularly DNA, and a peptide or peptide derivative incorporating a peptide of Formula (I), (II) (III) or (IV) described above.
  • Suitable conjugates comprise plasmid DNA and a bifunctional peptide which comprises a lysine rich part (e.g.
  • oligolysine which binds to the plasmid DNA and may pack the complex and a peptide or peptide derivative as described above which is able to direct the vector to the desired lysosomal pathway after the vector has been taken up by the cell .
  • liposomes may be used which encapsulate the vector nucleic acid and have a peptide or peptide derivative inserted into the phospholipid of the liposome.
  • a peptide derivative comprising a peptide portion for intracellular redirection of the vector and a hydrophobic tail can be made (as described in Molinier-Frenkel et al. (2002) J. Virol. Vol 76, No. 1, pp 127-135) .
  • the tail which may conveniently comprise a fatty acid chain such as palmitate, can be anchored in the liposome leaving the peptide part of the molecule free for intracellular interaction.
  • the nucleic acid molecule of the vector is typically DNA but may, for example where the vector is an RNA virus, be RNA.
  • Non-viral vectors may contain cDNA and the nucleic acid may be linear or circular, e.g. as with plasmid DNA. DNA may be single or double stranded.
  • the vectors of the invention are typically for use in gene transfer, i.e. the introduction of genetic material into a target cell population, in particular in vivo into the cells of a subject or into ex vivo cell cultures.
  • the nucleic acid molecule of the vector will typically encode a therapeutic gene, i.e. a gene which encodes a protein which is directly or indirectly involved in treating the subject or in affecting the cellular processes or properties in an ex vivo cell culture.
  • Genes which may be administered as part of a regimen of gene or cancer therapy are well known in the art and the list is increasing all the time.
  • the nucleic acid sequence which it is desired to introduce into the cells is a regulatory region rather than a region which encodes a protein product. Such a region may then be able to modulate expression of one of the target cell's own genes through integration into the host genome and thus a cis-acting effect.
  • the vector may carry a gene whose protein product is able to have a trans-acting effect by up- or down-regulating the expression of a target gene within the host cell.
  • the vector nucleic acid encodes a protein which it is desired to express in the infected cells
  • the nucleic acid molecule will typically also comprise an operably linked promotor and other regulatory sequences .
  • the nucleic acid of the vector will also encode structural and other proteins involved in the generation of further vectors which can go on to infect other cells, e.g. the gag, pol and env genes of a retrovirus.
  • the nucleic acid molecules are recombinant in that they are not naturally occurring but the product of genetic engineering techniques, e.g. to modify a viral genome or to modify or generate a plasmid.
  • the vectors of the invention incorporate a peptide or peptide derivative which is able to increase gene transfer efficiency as compared to vectors which lack that ligand. Without wishing to be bound by a particular theory, this is thought to be achieved by the ability of the ligand (at least in the case of ligands of formulae I and II) to redirect the vector to the lysosomal pathway within infected cells .
  • the observed enhancement of gene transfer was not cell-specific and the results indicate that the peptides (e.g.
  • QM10 do not confer any novel cell-binding capacity on the vector but intervene at the step of endocytosis by redirecting the vector to an endocytic compartment different from the class of early endosomes used by adenovirus type C members. In both human and simian cells lines this gives significant advantages in terms of gene transfer. As postulated in Miyazawa (supra) such redirection also being of utility with non-viral vectors.
  • the peptide or peptide derivative discussed above can be considered a redirecting or internal targeting moiety which incorporates a ligand, e.g. a peptide of formula (I) or (II) or a fragment thereof which provides the redirecting or internal targeting function.
  • the targeting peptide or peptide derivative may form all or part of a viral protein, e.g. it may replace an Ad penton fibre knob or be inserted in the fiber shaft.
  • Standard recombinant techniques may be used to generate chimeric molecules which include the ligands for intra-cellular re-targeting.
  • the peptides may also be chimeric molecules, with a targeting portion and a portion which links the chimera with the rest of the vector, e.g. a lysine rich part which binds to the vector nucleic acid or a hydrophobic part (which may not be made up of amino acids) to anchor the chimera to a liposome.
  • a peptide/fatty acid conjugate which may be used in a liposome based vector can be considered a peptide derivative.
  • Further derivatives may include molecules which include regions which act as peptidomimetics, i.e. not made up solely of amino acids but having chemical groups which can mimic the physical properties of amino acids, e.g. in terms of charge and hydrophobicity.
  • One particularly preferred non-viral conjugate comprises a biotinylated peptide (e.g. QM10) , streptavidin and a biotinylated (DNA) plasmid carrying a therapeutic gene. The streptavidin bridges the nucleic acid molecule to the targeting peptide.
  • a preferred class of vectors are those which comprise a nucleic acid molecule (e.g. a plasmid) joined by a linker moiety (e.g. a streptavidin-biotin linker but other suitable linkers are known) to a peptide or peptide derivative as defined and discussed above.
  • a linker moiety e.g. a streptavidin-biotin linker but other suitable linkers are known
  • the non-native portion which incorporates a peptide of formula (I) -(IV) or a fragment thereof is typically no more than 100 amino acids or equivalent in length, preferably no more than 80 or 60 amino acids in length.
  • peptide ligand e.g. of formula (I) -(IV)
  • the vector e.g. into the fiber shaft or other Ad capsid protein.
  • 2-6 copies, preferably 2-4 copies, may be incorporated, preferably each separated by a linker sequence of 2-5 neutral amino acid residues (such as G-S or G-S-G-S-G) to increase the flexibility and accessibility of the targeting ligand.
  • Preferred embodiments of the invention will not include the incorporation of large proteins such as ⁇ 2MG, which as described in the Examples, is normally found in the late endosomes of the lysosomal pathway.
  • the targeting peptide or peptide derivative will be arranged such that the peptide of Formula (I) or (III) which it contains is available for interaction with intra-cellular components after internalization of the vector.
  • Intra-cellular components may include intra- endosomal components.
  • the targeting peptide or peptide derivative or at least that part of it containing the peptide of Formula (I) or (III) will therefore be accessible on the surface of the vector, e.g. as part of an adenoviral capsid protein or protruding on the outside of a liposome. It may be immediately accessible or after an in vivo processing step, e.g.
  • Directing refers to the different intra-cellular pathways from endocytosis to nucleus and not to the targeting of specific cell types. Methods of re-targeting adenoviruses to particular cell types are known in the art, see for example WO 02/08263, and thus vectors of the invention may also incorporate moieties for cell-specific targeting.
  • Peptides and peptide derivatives incorporating a region having the sequence of Formula (I) , preferably of Formula (II) , particularly the sequence GHPRQMSHVY, constitute a further aspect of the present invention.
  • Peptides and peptide derivatives incorporating a region having the sequence of Formula (III) , preferably of Formula (IV) , particularly the sequence T-A-Y-S-S-M-K-G- G-K-F constitute a still further aspect of the present invention.
  • Such peptides will preferably be modified adenoviral capsid peptides and thus include flanking regions which are native sequences of said capsid proteins. Nucleic acid molecules encoding said peptides constitute further aspects of the invention.
  • the present invention provides a method of gene delivery which comprises contacting a cell population ex vivo with a vector of the invention as defined above or administering a vector of the invention as defined above to a subject.
  • the subject will typically be mammalian and in-vitro cell lines will typically be of mammalian origin, humans and human cell lines being especially preferred.
  • the invention provides a vector of the invention as defined above for use in therapy, in particular for use in gene therapy or cancer treatment, particularly for use in the treatment of cystic fibrosis or melanoma.
  • a further aspect of the invention are formulations comprising a vector of the invention as defined above together with a pharmaceutically acceptable diluent or carrier.
  • a method of gene therapy or cancer treatment which comprises administration to a patient of a vector of the invention as described above constitutes a further aspect of the invention.
  • FIG. 1 is a schematic representation of genetic constructs of Ad5-based vectors (AdGFP) carrying wild- type (WT) fiber (a) , or recombinant fibers (b-e) .
  • AdGFP Ad5-based vectors
  • WT wild- type
  • b-e recombinant fibers
  • the tail domain bound to penton base capsomer (shown on the left)
  • the shaft domain is represented by a stippled box
  • the shaft domain by a zig-zagged box
  • the last shaft repeat by a black box and the knob symbolized by an open circle
  • R7 In short-shafted recombinant fibers with seven repeat shaft (R7) , the different fiber domains are represented as in (a) .
  • the extrinsic trimerisation motif, inserted downstream of amino acid residue 158 of the shaft, the Factor-Xa cleavage site and the cell ligand were represented by different symbols, as indicated in the figure.
  • Figure 2 provides a graphical comparison of gene transfer efficiency (GTE) between the QMlO-liganded vector AdGFP- QMlO-knob (black bars) and the non-liganded control vectors AdGFP-WTFi (long-shafted fibers; striped bars) and AdGFP-R7-knob (short-shafted fibers; grey bars) in various cell lines at a MOI of 20 PFU/cell.
  • GFP expression was analyzed by FACS, and the results were expressed as the percentage of GFP-positive cells (a) , and GTE index (b) , as described in Materials and Methods (AU, arbitrary units) .
  • Figure 3 shows the effect of vector de-knobbing on gene transduction of various cell lines mediated by knobless or knob-carrying versions of AdGFP vectors.
  • HeLa and A549 cells CAR+, CFTR+
  • glandular cells MM39 CAR-, CFTR+
  • CF-KM4 CAR-, CFTR-
  • AdGFP-QM10-knob e, f, i
  • AdGFP-QM10 g, h, i
  • AdGFP- SY12-knob AdGFP-QMSY12 (i)
  • GFP expression was estimated at 36 h pi by IF microscopy (b, d, f , h) , and quantitated by FACS analysis (i).
  • a, c, e, g) DAPI staining of cell samples shown in (b, d, f , h) .
  • GTE gene transfer efficiency
  • AdGFP-R7 versus AdGFP-R7-knob
  • AdGFP-QMlO versus AdGFP-QMlO-knob
  • AdGFP-SY12 versus AdGFP-SY12-knob
  • Figure 4 comprises graphs showing the results of cell- binding competition assays between Ad5 capsid components and knobbed or deknobbed versions of non-liganded or liganded AdGFP vectors.
  • CF-KM4 (a) and A549 (b) cell monolayers were incubated with a mixture of AdGFP vector and a large excess of capsid protein, under conditions allowing for cell attachment but not cell entry. After removal of unadsorbed virus, cells were transferred to 37°C for 24 h and the amount of cell-bound virus was indirectly assayed by the level of GFP expression, determined by FACS analysis. Data, from two separate experiments, were expressed as GTE indices, given in arbitrary units (AU) , + SD.
  • Figure 5 comprises graphs showing endocytosis assays of knobbed versus deknobbed versions of non-liganded vector AdGFP-R7 ⁇ knob, and liganded vectors AdGFP-QMlO ⁇ knob and AdGFP-SY12 ⁇ knob in A549 (a), MM39 (b) and CF-KM4 (c) cells.
  • Cy3-labeled vector particles were incubated with cell monolayers at 37°C for 1 h, cell surface-bound virus detached by EDTA-trypsin treatment, and cells assayed for intracellular fluorescent signal by FACS analysis. Data, from three separate experiments, were expressed as endocytic indices, given in arbitrary units (AU) , as described in the Examples .
  • Figure 6 comprises graphs showing the results of endosomolysis assays.
  • Cells were co-incubated with toxin (Ricin A, RcA) and AdGFP vectors for 1 h at 37°C, at different toxin-to-cell ratios and a constant vector input (10 4 PP/cell) .
  • Vesicular escape and cell internalization of co-endocytosed RcA and AdGFP vector were assayed in A549 (a) , MM39 (b) and CF-KM4 (c) cells, using toxin-induced inhibition of host-cell protein synthesis.
  • Data indicated on the x-axis represented the values of RcA concentration (in pg/cell) which provoked a 50% inhibition of cell protein synthesis (IC50) .
  • Data presented are the means of four separate experiments, ⁇ SD.
  • Figure 7 shows in photos the intracellular localization of liganded vectors AdGFP-QM10-knob and AdGFP-SY12-knob, in comparison with that of control, non-liganded vector AdGFP-R7-knob.
  • Vectors were co-incubated pairwise with CF-KM4 cells and analyzed by confocal IF microscopy at 45 min after input, (a-c) , Cy2-labeled AdGFP-QM10-knob and Cy3-labeled AdGFP-SY12-knob; (d-f) , Cy2-labeled AdGFP-R7- knob and Cy3-labeled AdGFP-SY12-knob; (g-i) , Cy2-labeled AdGFP-QM10-knob and Cy3-labeled AdGFP-R7-knob; (j-1), Cy2-labeled, knobless AdGFP-QMlO and Cy3-labeled AdGFP- R7-knob. Overlay pictures are shown in (c) , (f) , (i) and 5 (1) .
  • Figure 8 shows in photos the cell compartmentalization of non-liganded vector AdGFP-R7-knob, and liganded vector AdGFP-QMlO-knob in CF-KM4 cells, analyzed by confocal IF
  • Figure 9 shows in photos the colocalization of human 25 alpha-2 acroglobulin (a2M) and QMlO-liganded AdGFP vector in its knob-carrying version AdGFP-QMlO-knob (a-c) and its knobless version AdGFP-QMlO (d-f) in A549 cells at 45 min after input, analyzed by confocal IF microscopy.
  • the left panels (a, d) show the signal of 30 FITC-labeled a2M, the middle panels (b, e) the signal of Cy3-labeled vector particles, and the right panels (c, f) the signal overlay.
  • Figure 10 provides electron microscope analysis of early 35 events in the interaction of AdGFP-QMlO-knob vector with A549 cells.
  • Cells were incubated with a viral input of 10 4 virions per cell for 1 h at 37°C.
  • (a) General view of the cellular apical surface;
  • (b, c) enlargements of plasma membrane-bound virions.
  • (d) Endocytosed virion.
  • Arrows in (b) and (c) show filaments linking the virus to the cell surface, whose length was compatible with that of a short-shafted fiber of seven repeats. Bar represents 200 nm in (a) , (c) and (d) and 100 nm in (b) .
  • Figure 11 provides electron microscope analysis of endocytosed virions of AdGFP-QMlO-knob in A549 cells after 1 h infection at 37°C, at an input multiplicity of 10 4 virions per cell
  • Curved arrows show two small intracytoplasmic vesicles opening into a larger one, containing multiple virions and heterogeneous, electron- dense material
  • (b) Vesicle containing one virion and heterogeneous and amorphous material. Arrows indicate microtubules apparently connecting endosomal vesicle to the nuclear envelope ; NPC, nuclear pore complex, (c-e) , Arrows point to junctions of intravesicular virions to the endosomal leaflet.
  • Panels (d) and (e) are enlargements of areas with intravesicular virions shown in (c) .
  • Bar represents 100 nm in (a) , (b) , (d) and (e) , and 200 nm in (c) .
  • Figure 12 shows electron microscope analysis of intracellular virions of knobless AdGFP-QMlO-knob in A549 cells.
  • Cells were incubated with vector for 1 h at 37°C, and at an input multiplicity of 104 virus particles per cell.
  • Virions (arrows) were seen associated with multivesicular bodies (a, b) , or with multilamellar inclusion bodies (c) characteristic of A549 cells. Bar represents 200 nm.
  • Figure 13 shows electron microscope analysis of early virus-cell interaction between CF-KM4 cells and QM10- liganded or control vectors.
  • Cells were incubated with vector particles for 1 h at 37°C, and at an input multiplicity of 104 virions per cell, (a-c), AdGFP-R7- knob ; (d, e) , AdGFP-WTFi ; (f) , AdGFP-QMlO-knob ; (g) , AdGFP-QMlO.
  • Arrows in (a) point to an electron-dense layer of proteinic material, most likely clathrin, at the virus-attachment site on the plasma membrane. Bar represents 100 nm in (a-e) , (f) , (g) , and 200 nm in (d) , (e) .
  • Figure 14 shows the infectivity and transducing capacity of Ad ⁇ Gal-pIX-QMlO for melanoma cells Ml, M2, M3 and M4.
  • Adenoviral vectors in the range of concentrations of 100, 500 and 2,000 virus particles/cell were added to target cell monolayers for 1 h at 4°C. The inoculum was removed and the cells were washed twice with cold medium, then further incubated for 48 h at 37°C. Cells were then fixed and stained to determine beta-galactosidase activity, using a beta-galactosidase staining kit (Chemicon) . Infected cells are visible as blue cells (adenovirus- positive cells)
  • Figure 15 shows the infectivity and transducing capacity of Ad ⁇ Gal-pIX-QMlO and Ad ⁇ Gal-pIX-SY12 for melanoma cells M8.
  • Adenoviral vectors in the range of concentrations of 100, 500 and 2,000 virus particles/cell were added to target cell monolayers for 1 h at 4°C. The inoculum was removed and the cells were washed twice with cold medium, then further incubated for 48 h at 37°C. Cells were then fixed and stained to determine beta-galactosidase activity, using a beta-galactosidase staining kit (Chemicon) . Infected cells are visible as blue.
  • Ad ⁇ Gal-pIX-QMlO and Ad ⁇ Gal-pIX-SY12 for the melanoma cell line MeWo cells M8 Ad ⁇ Gal-pIX-QMlO and Ad ⁇ Gal-pIX-SY12 for the melanoma cell line MeWo cells M8.
  • Adenoviral vectors in the range of concentrations of 200 and 2,000 virus particles/cell were added to target cell monolayers for 1 h at 4°C. The inoculum was removed and the cells were washed twice with cold medium, then further incubated for 48 h at 37°C. Cells were then fixed and stained to determine beta- galactosidase activity, using a beta-galactosidase staining kit (Chemicon) . Infected cells are visible as blue .
  • the SV40-immortalized human tracheal gland cell lines MM39 (normal) and CFKM4 (CFTR-deficient) were maintained as monolayers on collagen-I-coated flasks (Biocoat; Becton-Dickinson, Bedford, MA) in Dulbecco's modified Eagles ' s medium-Ham's F12 (DMEM-F12) supplemented with 1% Ultroser G (Gibco-Invitrogen, Rockville, MD) , penicillin (200 U/ml) , streptomycin (200 ⁇ g/ml) and epinephrin (3 ⁇ M; Sigma, St Louis, MO) .
  • DMEM-F12 Dulbecco's modified Eagles ' s medium-Ham's F12
  • the E1A+E1B-trans-complementing HEK-293 cell line (abbreviated 293) was obtained from ATCC (Manassas, VA, USA; CRL 1573) , and ElA+ElB+fiber-trans-complementing 293 cells (293-Fibre ; (Legrand, V. et al (1999) J. Virol.
  • Transgene SA Strasbourg, France
  • Cells were cultured as monolayers in DMEM medium (Gibco-Invitrogen) , supplemented with 10% fetal calf serum (FCS; Sigma) , penicillin (200 U/ml) and streptomycin (200 ⁇ g/ml; Gibco-Invitrogen) at 37°C and 5% C0 2 , and, for 293-Fibre cells, hygromycin was added at 350 ⁇ g/mL.
  • FCS fetal calf serum
  • HeLa cervical epitheliod carcinoma
  • A549 lung alveolar carcinoma ; CCL 185)
  • HEp2 larynx epidermoid carcinoma ; CCL 23
  • MRC-5 foetal lung
  • HRT-18 ileocecal adenocarcinoma ; CCL 244
  • PLC/PRF/5 hepatoma ,- CRL 8024
  • RD rhabdomyosarcoma
  • Vero African green monkey kidney ; CCL 81
  • LLC-MIC LLC-MIC
  • BGM Buffalo green monkey kidney epithelial cells
  • Daudi cells (EBV-transformed B lymphoma cells) were cultured in RPMI medium supplemented with 10% FCS (Hong, S.S. et al (1997) EMBO J. 16:2294-2306).
  • FCS fetal bovine serum
  • Spodoptera frugiperda cells Sf9 subclone were propagated in TNM medium (Gibco- Invitrogen) and cultured as monolayers with 10% FCS, penicillin and streptomycin as mentioned above, and maintained at 28°C.
  • the cells were incubated with 0.25 ml elution buffer (0.1 M glycine-HCl buffer, pH 3.0, 2 M urea) for 10 mn at RT .
  • 0.25 ml elution buffer 0.1 M glycine-HCl buffer, pH 3.0, 2 M urea
  • the cells were then harvested by scraping with a rubber policeman, pelleted at 2,000 x g for 3 mn, and lysed by swelling and vortexing in 0.1 ml hypotonic buffer (5 mM Tris-HCl buffer pH 7.5, 1 mM Na 2 EDTA) .
  • Ad vectors Adenoviruses (Ad) and Ad vectors
  • FIG. 1 Wild-type (WT) Ad serotype 5 (Ad5) , and replication-competent vector Ad5Luc3 (Mittal, S. K. et al (1993) Virus Res. 28:67-90), harboring the firefly luciferase gene (luc) under the control of the SV40 early promoter inserted in the E3 region of the Ad5 genome, were propagated in HeLa cells.
  • El-deleted recombinant Ad5 vectors with WT fibers were propagated in 293 cells, whereas El-deleted, fiber-modified Ad5 vectors were grown in 293-Fibre cells, until the last amplification step of the vector stock, which was performed in 293 cells.
  • AdGFP-R7- knob was an Ad5-based vector with its deleted El region replaced by a GFP expression cassette under the CMV promoter, and a genetically-modified fiber which contained the following domains, from the N- to C- terminus : (i) the tail, (ii) the N-terminal seven shaft repeats (R7) , (iii) a trimerisation signal (PDVASLRQQVAELQGQVQHLQAAFSQYKKVELFPNG) called the neck region peptide (NRP) from the human lung surfactant protein D, (iv) a tridecapeptide linker (AKKLNDAQAPKSD) , (v) the desired cell ligand, (Xa) -knob (abbreviated AdGFP-R7- knob) , was an Ad5-based vector with its deleted El region replaced by a GFP expression cassette under the CMV promoter, and a genetically-modified fiber which contained the following domains, from the N- to C- terminus
  • the cleavage site for Factor Xa consisted of the tetrapeptide IEGR, and the cleavage occurred after the arginine residue. Recombinant fibers were rescued into the Ad5-GFP genome as previously described (Magnusson, M. K. et al (2001) J. Virol. 75:7280-7289).
  • the backbone vector AdGFP-R7-knob which carried non-liganded, shortshafted fibers with NRP, Factor Xa site, the terminal knob but no specific cell ligand (Fig. l b, c) , was used as the control vector in our experiments, with or without cleavage by Factor Xa.
  • AdGFP-R7-knob vector For construction of liganded AdGFP-R7-knob vector with new cell tropism, three peptide ligands potentially involved in phage endocytosis, and abbreviated QM10, SY12 and LAP25, respectively, were individually inserted into the fiber shaft domain, upstream to the Factor Xa cleavage site.
  • QM10, SY12 and LAP25 were individually inserted into the fiber shaft domain, upstream to the Factor Xa cleavage site.
  • virus samples were treated with 1 U Factor Xa per 10 9 physical virus particles in cleavage buffer (50 mM Tris pH 8.0, 100 mM NaCI, 5 mM CaCl 2 ) for 16 h at 22 °C.
  • the concentration in infectious viral particles of CsCl-purified Ad stocks could be determined and compared using plaque assays in 293 cell monolayers, according to conventional methods (Magnusson, M. K. et al (2001) J. Virol. 75:7280-7289), and the infectivity titers expressed as plaque-forming units per mL (PFU/mL) .
  • the concentration in physical particles (PP) was deduced from the protein concentration determined in the same CsCl-purified Ad stocks, using the Bradford protein assay (BioRad) with bovine serum albumin (2x crystallized BSA, BioRad, Hercules, CA) as the standard.
  • the number of PP was calculated from the total protein content of the sample, taking the mass of 2.91 x 10 "16 g for one single virion, i.e. 3.4 x 10 12 virions per mg protein.
  • the infectivity index represented the ratio of infectious to physical particles (PFU/PP) , and usually ranged between 1:25 to 1:50 for WT Ad5.
  • PFU/PP physical particles
  • WT Ad5 WT Ad5.
  • MOI multiplicity of infection
  • the MOI used varied from 5 x 10 2 to 5 x 10 4 PP/cell.
  • Ad penton base and fiber proteins were isolated in native and soluble form from lysates of Ad5- infected HeLa cells or baculovirus-infected Sf9 cells, and purified according to a previously published method (Boulanger, P. et al (1973) Eur. J. Biochem. 39:37-42), adapted to FPLC (Molinier-Frenkel, V. et al (2002) J. Virol. 76:127-135.).
  • the genetic constructions of recombinant Autographa californica Multiple Nuclear Polyhedrosis Viruses (AcMNPV) expressing Ad5 full-length fiber or fiber knob domain have been described in detail elsewhere (Novelli, A.
  • Ad protein concentration was estimated by the intensity of Coomassie blue staining of protein band in SDS-gel, measured by scanning at 610 nm in an automatic densitometer (REP-EDC, Helena Laboratories, Beaumont, TX) , using a range of known BSA concentrations for calibration.
  • Cell-binding competition assays were performed using purified hexon, penton base, full-length fiber and knob proteins, and penton base mutant R340E. Each capsid protein was individually mixed with each vector, in large excess over its copy number in the Ad virion, and incubated at 4°C with cells, to allow attachment but not entry.
  • hexon protein was added in only 100-fold excess over the hexon content of the virions, whereas penton base, fiber and knob proteins were added in 10 3 -fold excess. Unadsorbed virus was rinsed off, and cells assayed for GFP expression at 28 h pi.
  • Antibodies . 1D6.14 an anti-Ad5 fiber knob monoclonal antibody (mAb) with CAR-blocking activity, was supplied by D.T. Curiel (University of Alabama at Birmingham, UAB, AL; (Magnusson, M. K. et al (2001) J. Virol. 75:7280-7289)).
  • MAb 4D2.5 directed towards the conserved fiber tail motif (Hong, J.S. et al (1991) Virology, 185:758-767), was obtained from J.A. Engler (UAB, AL) .
  • Other antibodies against Ad proteins have been described in a previous study (Gaden, F. et al (2002) Am. J. Respir. Cell Mol. Biol.
  • the main modification of the protocol consisted of replacing the step of dialysis, which often provokes the precipitation of virions and macromolecules , or the gel-exclusion chromatography, which dilutes the virus inoculum, by blocking the excess of coupling reagent with 20 mM lysine, followed by dilution of the fluorescent dye- conjugated ligands in culture medium.
  • Cy2- or Cy3-labeled Ad were used within 2 h after coupling.
  • Human alpha-2 macroglobulin ( ⁇ 2M ; Sigma) was conjugated with fluorescent isothiocyanate (FITC) using the EZ-labelTM
  • FITC protein labeling kit Pieris, Rockford, IL
  • protocol recommended by the manufacturer IF microscopy of cellbound virus was performed using an Axiovert 135 microscope (Zeiss) , equipped with an AxioCam videocamera and a quantitative image analysis program.
  • Ad-mediated endosomolysis , vesicular escape and virus internalization was determined using a toxin assay, as previously described (Gaden, F. et al (2002) Am. J. Respir. Cell Mol. Biol. 27:628-640). In brief, cell monolayers were preincubated with ricin agglutinin (RcA of 120,000 mol wt . ; Sigma) at concentrations ranging from 0 to 10 ⁇ g of RcA per aliquot of 5x10 s cells for 1 h at 37°C, in methionine- and cysteine-free culture medium, and in the presence (or absence) of Ad vector, at a constant input of 10 4 PP/cell.
  • RcA ricin agglutinin
  • Ad+RcA- mediated inhibition of cell protein synthesis was evaluated from TCA-precipitable radioactivity, determined by scintillation counting in a liquid scintillation spectrometer (Beckman LS6500, Beckman Coulter Inc., Fullerton, CA) .
  • Ad5 vector carrying the reporter gene luc or gfp
  • Luciferase activity was assayed in cell lysates using luciferase substrate solution (Promega) in a Lumat LB-9501 luminometer (Berthold Bioanalytical, Germany) . The results were expressed in relative light units (RLU) per mg of whole protein present in cell lysates (Hong, S.S. et al (1999) Virology, 262:163-177).
  • RLU relative light units
  • GFP activity was determined using FACS analysis or IF microscopy with quantitative image analysis program, as described above.
  • Electron microscopy (EM) Mock- or AdGFP vector-transduced cells were harvested at early times after infection (0.5 to 1 h pi), pelleted, fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and post-fixed with osmium tetroxide (1% in 0.1 M cacodylate buffer, pH 7.4).
  • Cell specimens were dehydrated and embedded in Epon (Epon-812; Fulha , Latham, NY) . Sections were stained with 7% uranyl acetate in methanol, and post-stained with 2.6% alkaline lead citrate in H 2 0.
  • Specimens were examined under a Jeol 1200-EX electron microscope, equipped with a MegaView II high resolution TEM camera and a Soft Imaging System of analysis (Elo ⁇ se, Roissy, France) .
  • CF-KM4 cells do not express CAR, the high affinity receptor for Ad5, we looked for other surface molecules of CF-KM4 cells which could act as substitute receptors for Ad5, and be targeted by recombinant Ad5 vectors .
  • Ad5 vectors As there was no direct screening method available for identifying ligands of CF-KM4 cells, we applied the indirect approach of biopanning monolayers of living CF-KM4 cells (Fontana, L. et al (2003) J. Virol. 77:11094-11104; Smothers, J.F. et al (2002) Science, 298:621-622) using a phage-displayed hexapeptide library. Two classes of phage populations were isolated.
  • the cell binders carried peptide aptamers corresponding to ligands of surface exposed domains of plasma membrane molecules which would not necessarily lead to phage endocytosis and entry.
  • the other population of phages recovered after cell lysis corresponded to intracellular phages. It has been shown that recombinant bacteriophages carrying a portion of the Ad penton base sequence overlapping the RGD peptide motif were capable of binding to ⁇ v integrins of mammalian cell plasma membrane, and were found to be endocytosed into endosomal vesicles.
  • the intracellular phages were thus isolated, amplified and sequenced, and the most represented phagotopes were grouped in families according to their sequence homology. Alignment of overlapping peptides sharing common motifs allowed us to design consensus oligopeptides of longer sequences than the original hexapeptide phagotopes, e.g. 10- or 12-mer. Three major different ligands emerged from this analysis.
  • the first family of intracellular phages contained recurrent peptide motifs like LLTV, RMQ and QPPG.
  • LAP lysosomial acid phosphatase
  • the two other phagotope families were represented by the decapeptide GHPRQMSHVY (abbreviated QM10) and the dodecapeptide TAYSSYMKGGKF (abbreviated SY12) , respectively. Sequence comparison with proteins in data banks did not elicit any plasma membrane protein or protein domain with any known receptor activity or potentiality. Construction and nomenclature of AdGFP vectors carrying liganded fibers with scissile knobs (Fig. 1) .
  • Each of the three ligands LAP25, QM10 and SY12 was inserted into the fiber of the backbone vector AdGFP-R7- knob, upstream of the Factor Xa cleavage site, to generate recombinant vectors AdGFP-LAP25-knob, AdGFP- QMlO-knob and AdGFP-SY12-knob, respectively.
  • Trans- complementing HEK-293-Fiber cells were transfected with each of the Ad DNA genomes recombined with liganded fiber genes, and plaques isolated and amplified.
  • Ad5 vector Virion titer (b) Infectious Ratio PP/mL titer (c) PP : PFU PFU/mL
  • AdGFP- 7-knob ( ) 1 . 00 X 10 E 12 3 . 75 x 10 E 9 266
  • AdGFP -SY12- knob 1 10 X 10 E 12 1. 10 x 10 12 38
  • the two liganded vectors were designated as AdGFP-QMlO and AdGFP-SY12, and the non-liganded, control vector as AdGFP-R7 (Fig. 1 c, e) .
  • AdGFP-QMlO and AdGFP-SY12 the non-liganded, control vector as AdGFP-R7
  • the three recombinant vectors showed fiber protein and fiber cleavage products migrating with the apparent molecular mass expected before and after cleavage by Factor Xa.
  • AdGFP-QMlO-knob compared to its non-liganded version AdGFP-R7-knob, showed that the QM10 ligand consistently and significantly augmented the GTE in all the cell lines tested but to various degrees (Fig. 2 a, b) .
  • This enhancement ranged from 2- to 40-fold, depending on the
  • AdGFP-SY12-knob gave similar levels of gene transfer as AdGFP-R7-knob (data not shown) , indicating that the effect observed with the decapeptide ligand GHPRQMSHVY was not simply a property of any foreign peptide inserted at that site in Ad5 vectors, but was sequence-related. However, our results also implied that QM10, although being isolated from CF-KM4 cell- internalized phages, was not tissue-specific or even species-specific.
  • QM10 was more efficient in promoting gene transduction of A549 cells (terminal bronchial cells from human lung carcinoma) , MRC5 (primary fibroblastic cells from human foetal lung) , HRT-18 (human ileocecal adenocarcinoma cells) and RD cells (from human rhabdomyosarcoma) , as well as LLC-MK2 cells (simian kidney cells) , than the human tracheal glandular cells CF-KM4 from which it was issued.
  • A549 cells terminal bronchial cells from human lung carcinoma
  • MRC5 primary fibroblastic cells from human foetal lung
  • HRT-18 human ileocecal adenocarcinoma cells
  • RD cells from human rhabdomyosarcoma
  • AdGFP-QMlO-knob A nearly linear dose-response was observed with AdGFP-QMlO-knob between 20 and 200 PFU/cell, and almost 100 % cells were found to be GFP- positive at the highest MOI, whereas GFP expression reached a plateau with AdGFP-WTFi (50 %) and AdGFP-R7- knob (35 %) for MOI higher than 100 PFU/cell (Fig. 2 c) .
  • knob domain was dispensable for achieving the early steps of the virus cycle which led to gene delivery by AdGFP-QMlO vector into CAR+ and CAR- cells .
  • liganded vectors such as AdGFP-SY12 and AdGFP-QMlO, considering that the proteolytic removal of the knob had exposed the peptide ligands of the fiber shafts and made them directly accessible to their specific receptor molecules at the cell surface.
  • knob domain was dispensable for the step of cell attachment of the vectors, as already observed for the overall gene transduction process (refer to Fig. 3) .
  • Antibody-mediated competition for cellular attachment of vectors via the knob domain In our previous study, experiments using anti-knob 1D6.14 mAb have suggested that the fiber knob domain was involved in the binding of Ad5 to surface SA-GP molecules on CF-KM4, and to HS-GAG on MM39 cells.
  • knob-blocking antibody 1D6.14 had a much lower inhibitory effect on the cell attachment of short-shafted fiber vectors AdGFP-R7- knob and AdGFP-SY12-knob (about 20 % and 50 % inhibition, respectively) , and an even lesser effect ( " 10 % inhibition only) on AdGFP-QMlO-knob (Table 2) .
  • AdGFP-R7- knob and AdGFP-SY12-knob about 20 % and 50 % inhibition, respectively
  • AdGFP-QMlO-knob Table 2
  • virus-cell binding could be mediated by viral capsid proteins or domains different from the fiber knob, e.g penton base, as for the non-liganded vector, or the new cellular ligand inserted into the fiber, as for AdGFP-QMlO-knob, or several binding sites simultaneously. In any case, this confirmed the major role played by the fiber shaft in controlling the interaction of Ad virions with cell surface receptors.
  • Cell-binding competition assays were performed in the presence of purified Ad proteins, hexon, full-length fiber, knob, or penton base (WT or RGD-mutant R340E) , individually mixed with each vector, and added in large excess over their copy numbers in Ad virion.
  • Vector and competitor were co-incubated at 4°C with CF-KM4 or A549 cells, to allow attachment but not entry. Unadsorbed virus was rinsed off, and cells assayed for GFP expression at 28 h pi.
  • Cell attachment of AdGFP-QMlO-knob to CF-KM4 cells was not influenced by an excess of hexon or penton base proteins (Fig. 4a) .
  • the fiber shaft domain of our AdGFP vectors still carried the conserved basic peptide KKTK at position 91-94, a motif which has been proposed to be responsible for the binding of Ad fiber to heparan sulfate proteoglycans and acidic carbohydrates.
  • some cell-binding activity for the nonviral tri erization peptide from the neck region of the human lung surfactant protein D inserted at the C- terminal end of the fiber shaft of our vectors could not be excluded. This would explain the absence of significant change in the nonliganded AdGFP-R7 vector infectivity after de-knobbing, as shown in Fig. 3.
  • Ad-mediated endosomolysis and vesicular escape of Ad virions was determined from the degree of Ad-mediated augmentation of cell protein synthesis inhibition by the toxin Ricin A (RcA) , as a result of their co-endocytosis and subsequent vesicular release.
  • RcA agglutinin blocks the cell protein synthesis and its effect is enhanced by the endosomal escape of co- endocytosed Ad virions into the cytosol.
  • AdGFP-R7- nob, AdGFP-SY12-knob and AdGFP-QMlO-knob were investigated in CF-KM4 and A549 cells.
  • Cells were co- incubated with aliquots of Cy3- and Cy2-labeled particles of each vector pairwise, and analyzed by confocal IF microscopy. Only partial co-localization was observed for AdGFP-QMlO-knob and AdGFP-SY12-knob (Fig. 7 a-c) , whereas the majority of AdGFP-R7-knob and AdGFP-SY12-knob virions were found to co-localize in the same vesicular compartment (Fig.
  • AdGFP-R7-knob and AdGFP-SY12-knob co- localized with the IF signals of TF R (Fig. 8 a-c) , and Rab5, but not with LAMP-1 (Fig. 8 g-i) , confirming the role of clathrin-coated vesicles and early endosomes in early steps of endocytosis and entry of Ad5, a prototype member of species C Ad.
  • no significant occurrence of AdGFP-QMlO-knob within the TF R /Rab5 endosomal compartment could be detected at 45 min (Fig. 8d-f) .
  • AdGFP-QMlO-knob and LAMP-1 signals were observed with a high a frequency (Fig. 8 j-1) .
  • Fig. 8 j-1 This indicated that a significant proportion of AdGFP-QMlO-knob vector particles followed an endocytic pathway different from that of AdGFP-R7-knob and AdGFP- SY12-knob, and were preferentially endocytosed and addressed to the late, acidic endosomal compartment.
  • Tf ligands have been shown to be directed to early and recycling endosomes that are relatively alkaline (pH " 6.0-6.8), whereas ⁇ 2M ligands do not recycle to the cell surface but progress rapidly through the endosomal pathway to late, relatively acidic (pH ⁇ 5) endosomes (Sonowane et al. 2003 J. Cell Biol. 160: 1129-1138).
  • AdGFP- QMlO- knob vector particles were found to reside in the same vesicular compartment as ⁇ 2M after 45-60 min incubation at 37°C (Fig. 9 a-c) .
  • the events of co-localization of AdGFP-QMlO with oc2M also occurred with a high frequency (Fig. 9 d-f) .
  • AdGFP-QMlO-knob virions were found in greater numbers both at the cell surface (Fig. 10, a-c) , and in intracellular compartments (Fig. 11) .
  • knob diameter of 49 A and the shaft repeat increment of 13.4 to 13.5 A per repeat gave a theoretical value of 94 A for the R7-shaft, and of 143 A for the R7-knob fiber.
  • AdGFP-QMlO-knob virions were seen within A459 cells in two unequally represented types of intravesicular compartments: on rare occasions, single virions occurred in vesicles with discontinuities in the vesicular membrane leaflet (Fig. 10, d) , resembling the vesicles in which the control vector AdGFP-R7-knob was endocytosed.
  • the most frequent events consisted of several AdGFP-QMlO-knob virions present in the same vesicles containing electron- dense heterogeneous material (Fig. 11, a) , amorphous inclusions or both (Fig.
  • AdGFP-QMlO-knob and AdGFP-QMlO could follow the same endocytic pathway as Ad5WT through early endosomes leading to their endosomal escape, but that late endosomes and lysosomes represented the preferred endocytic compartment whereto QMlO-liganded vectors were rapidly redirected and accumulated.
  • the cytoplasm of CF-KM4 cells has been shown to contain a very rich vesicular network, and this made difficult to distinguish between early and late endosomal compartments in these particular cell line by using conventional EM.
  • AdGFP- QMlO-knob and AdGFP-QMlO presented a blurred aspect and/or irregular morphology
  • AdGFP-WTFi and AdGFP-R7-knob virions had retained their sharp contour and regular, icosahedral shape (Fig . 13, compare panels a-e to f-g) .
  • Ml melanoma cells derived from human patient explants referred to as Ml, M2, M3, M4 (a gift from the Nursings Civils de Lyon) , and M8 (a gift from Dr. A. Blondel, Institut Cochin, Paris) and human melanoma cell line MeWo (from ATCC) were grown at 37°C in RPMI supplemented with 10% Fetal Calf Serum.
  • Ligand insertion Insertion of SY12 or QM10 peptide ligands into the gene encoding the human adenovirus serotype 5 pIX protein was performed as follows.
  • the pIX gene was mutated using the QuickChange site-directed mutagenesis system (Stratagene) , to introduce mutations and additional sequence (s) into the pIX coding sequence, into a novel Bam H I site inserted between the base codons encoding Leu-131 and Lys-132 residues within the C-terminal moiety of pIX.
  • the following sense and antisense oligonucleotides were used : 5'-cgc cag cag gtt tct gcc ctg gga tec aag get tec tec cct ccc aat gcg g3 ' and 5'-c cgc att ggg agg gga gga age ctt gga tec cag ggc aga aac ctg ctg gcg-3'.
  • the oligonucleotides 5 '-cgc gga tec gga cac ccc cga cag atg tea cac gtc tac tag gga tec gcg-3' and 5 ' -gcg gga tec eta gta cac gtg tga cat ctg teg ggg gtg tec gga tec gcg-3 ' were hybridised to each other and cloned into the Bam HI restriction site to directly introduce the nucleotide coding sequence of QM10 peptide within the C-terminal portion of pIX.
  • the resulting amino acid sequence of the C-terminal domain of the modified pIX protein is as follows, starting from the Leu residue at position 100: 100-LTALLAQLDSLTRELNWSQQLLDLRQQVSALGHPRQMSHVY-C0 2 H for the pIX-QMlO peptide, and 100-LTALLAQLDSLTRELNWSQQLLDLRQQVSALTAYSSYMKGGKF-C0 2 H for the pIX-SY12 peptide.
  • an adenoviral vector carrying pIX proteins liganded with the model peptide ligand 7K was used.
  • oligo-lysine peptide ligand (Lys- Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys, abbreviated 7K) in the pIX protein between Leu-131 and Lys-132 residues within the C-terminal domain of pIX was performed using the same protocol as for pIX-QMlO and pIX-SY12.
  • All adenoviral vectors contained, in addition to alterations of the pIX-encoding gene, a deletion in El and in E3, and carry the LacZ gene (encoding the bacterial beta-galactosidase) driven by the CMV promoter, in lieu of the deleted El gene-encoding region.
  • the vectors were referred to as Ad ⁇ Gal-pIX-QMlO, Ad ⁇ Gal-pIX-
  • the gene transduction efficiency of the QM10- or SY12-liganded pIX constructs, Ad ⁇ Gal-pIX-QMlO and Ad ⁇ Gal- pIX-SY12 was evaluated in a number of melanoma cell explants issued from various patients, or in a human melanoma cell line. Efficiency of gene transduction was compared with (i) Ad ⁇ Gal-pIX-WT, an adenoviral vector carrying unmodified capsid proteins pIX, and (ii) Ad ⁇ Gal- pIX-7K, an adenoviral vector carrying pIX proteins liganded with the model peptide ligand 7K.
  • the oligo- lysine ligand is supposed to bind nonspecifically to sialic acid residues of most sialylated cell surface glycoproteins, or/and to acidic glycosamino-glycans such as heparan sulfate, also present on plasma membrane of most cell types.
  • Adenoviral vectors in the range of concentrations of 100, 200, 500 and 2,000 virus particles/cell were added to target cell monolayers for 1 h at 4°C. The inoculum was removed and the cells were washed twice with cold medium, then further incubated for 48 h at 37°C. Cells were then fixed and stained to determine beta- galactosidase activity, using a beta-galactosidase staining kit (Chemicon) . Infected cells, visible as blue cells (adenovirus-positive cells) were then counted under the light microscope.
  • beta- galactosidase activity was determined on the whole cell lysate, and monitored using a chemiluminescent substrate (luminescent beta-galactosidase detection kit, Clontech, Palo Alto) .
  • Ad ⁇ Gal-pIX-QMlO For melanoma cells Ml, M2, M3 and M4, the infectivity and transducing capacity of Ad ⁇ Gal-pIX-QMlO was significantly higher than those of other vectors : a factor of 10- to 100-fold was obtained compared to the model vector Ad ⁇ Gal-pIX-7K, and a factor of 1, 000-fold, compared to Ad ⁇ Gal-pIX-WT carrying unmodified capsid protein pIX ( Figure 14) .
  • Ad ⁇ Gal-pIX-QMlO and Ad ⁇ Gal-pIX- SY12 were equivalent, but significantly higher (by a factor of 10- to 100-fold) than those of Ad ⁇ Gal-pIX-WT ( Figure 15) .
  • Ad ⁇ Gal-pIX-SY12 infects and transduces melanoma cell line MeWo with a 100-fold higher efficiency than of Ad ⁇ Gal-pIX-QMlO, and 1, 000-fold higher than Ad ⁇ Gal-pIX-WT ( Figure 16) .
  • Ad ⁇ Gal-pIX-QMlO and Ad ⁇ Gal-pIX-SY12 show a similar level of infectivity and gene transduction capacity in a variety of human melanoma cells, a cell type which is known to be non permissive or poorly permissive to adenovirus, compared to a non-liganded vector (Ad ⁇ Gal-pIX-WT) or a vector liganded by the nonspecific oligo-lysine peptide (Ad ⁇ Gal-pIX-7K) .
  • pIX could contain an extra amino acid sequence, a ⁇ linker', at its C-terminus, which would allow the QMIO or SY12 to protrude further at the surface of viral capsid.
  • This linker could have different lengths in terms of amino acid number.

Abstract

The present invention relates to a vector comprising a nucleic acid molecule and a peptide or peptide derivative, said peptide or peptide derivative comprising a peptide of general Formula (I) R1-R2-R3-R4-R5-R6 wherein: R1 is arginine or lysine; R2 is glutamine or asparagine; R3 is methionine; R4 is serine Or threonine; R5 is histidine, tryptophan, arginine or lysine; and R6 is valine, leucine or isoleucine; or a functionally active fragment thereof and a vector comprising a nucleic acid molecule and a peptide or peptide derivative, said peptide or peptide derivative comprising a peptide of general Formula (III) R1-R2-R3-R4-R5-R6-R7-R8-R9-R10 wherein: R1 is threonine; R2 is alanine; R3 is tyrosine; R4 is Serine; R5 is serine R6 is tyrosine R7 is any amino acid, R8 is any amino acid; R9 is glycine; and R10 is glycine or a functionally active fragment thereof and uses thereof in gene cancer

Description

Vectors The present invention relates to vectors, in particular to modified or engineered vectors with good gene transfer efficiency, and to the use of such vectors in gene therapy. Viruses are now widely used therapeutic agents in the fight against disease, both in non-human animals and humans. Particularly, viruses are utilised as vectors for gene therapy. Alternatively, cytolytic viruses have been used to target and kill cancer or unwanted proliferating cells, such therapy is also known as "virotherapy" . Thus, the direct use of viruses in medical treatments is a widely growing area, and new techniques and uses involving viruses in treatment and therapy are being developed. Viruses are highly evolved biological entities that efficiently gain access to their host cells and exploit the cellular machinery of the cell to facilitate their replication. As such, they are heralded as ideal gene therapy vectors since foreign/heterologous genes or coding sequences may be inserted into the viral genome and infection thus allows the foreign gene to be delivered to the nucleus of the host cell . Gene therapy was first conceived in order to treat genetic diseases where the defect lay in a hereditary single-gene defect, for example severe combined immunodeficiency disease (SCID) . However, the scope of gene therapy is now much broader, and it is envisaged that viruses may be utilised to deliver genes for acquired diseases such as cancer, cardiovascular disease, neurodegenerative disorders, inflammation and even infectious disease. There are currently five main classes of clinically applicable viral vectors that are derived from oncoretroviruses, lentivirus, adenovirus, adeno- associated - virus (AAVs) and herpes-simplex-1 viruses (HSV-ls) . Each class of vector is characterised by a set of different properties that make it suitable for use in certain applications, and unsuitable for others. The five main classes of viral vector can be categorized in two groups according to whether their genomes integrate into host DNA (oncoretroviruses, lentiviruses and AAVs) or persist in the cell nucleus predominantly as extrachromosomal episomes (adenoviruses and herpes viruses) . This distinction is an important determinant of the suitability of each vector for particular applications; non-integrating vectors can, under certain circumstances, mediate persistent transgene expression in non-proliferating cells, but integrating vectors are, at present, the tools of choice if stable genetic alteration needs to be maintained in dividing cells . Human adenovirus (Ad) , which is now commonly used as a gene vector, is a non-enveloped virus, whose capsid is constituted of 240 hexons and 12 pentons located at the
12 apices of the icosahedral capsid. The penton capsomer is formed of two components, the fiber and the penton base. The fiber has three structural domains, the tail, which is anchored to the penton base, the shaft, constituted of several repeats of a 15-amino acid motif, and a terminal globular structure, called the 'knob1. Attachment of Ad of species C (e.g. Ad2, Ad5) to the cell surface is mediated by a high affinity interaction between the knob domain of the fiber and the CAR protein (Coxsackie-Adenovirus Receptor) . However, Ad of other species use different attachment receptors, recently identified as sialic acid residues for species D Ad37 (Arnberg, N. et al . (2000) J. Virol. 74:42-8), and CD46 for species B members Adll and Ad35 (Gaggar, A. et al. (2003) Nature Med. 8, 746-755 and Segerman et al . J. Virol. 77: 9183-9191). Following attachment, the interaction between the RGD motifs of penton base with αvβ3/5 integrin molecules promotes endocytosis and internalization of the virus, and initiates cell signalling. The absence or low level of expression of CAR, the high affinity receptor for most Ad serotypes, or its cellular location at the basolateral tight junctions of the airway epithelium cells, are two parameters which are detrimental to the usage of unmodified Ad vectors in gene therapy protocols aiming at the airways or lung tissues. Thus there is a particular need for vectors with an enhanced ability to transfer genes to cells of the airways or lungs, e.g. to CFTR (Cystic Fibrosis Transmembrane-Conductance Regulator) - deficient airway cells. As the fiber knob domain is essentially responsible for the cell tropism of Ad, the apparent simplest strategy would be to delete the knob domain, and replace it by the desired cell ligand, to ablate the natural tropism of Ad and confer upon the redesigned Ad a new cell tropism. Unfortunately, deletions or insertions in the knob domains, except those involving some flexible loops, frequently result in nonviable viruses. When viable Ad are obtained, this strategy requires the generation of receptor-expressing, trans-complementing cell lines, i.e. cells expressing the surface receptor for the new ligand, or expressing wildtype (WT) fiber protein, in order to produce recombinant Ad in high yields . More generally, improving gene transfer efficiency is a goal in all methods which use vectors to deliver target nucleic acid to the cells of a subject or to cells in an in vitro cell culture. Ad5 is a particularly useful and well studied vector for gene transfer as it has a high growth rate, high productivity and a high infectivity index. As discussed by Miyazawa et al. in J. Virol. (2001) Vol 75, No. 3, pp 1387-1400 (see for example Figure 10 therein) , Ad5 utilizes the intracellular microtubules to achieve nuclear localization following receptor-mediated enclocytosis and escape to the cytosol. In contrast, Ad7 is thought to achieve nuclear localization by remaining in late endosomes or lysosomes before finally escaping to the cytosol and translocating to the nucleus, this pathway is referred to herein as the lysosomal pathway. The present inventors have surprisingly discovered that it is possible to re-direct vectors which do not normally utilize the lysosomal pathway to this pathway. Specifically, a class of peptides suitable for this re-directing have been identified and it has been shown that vectors incorporating such a peptide ligand have a greater efficiency of gene transfer than similar non-liganded vectors. Thus, according to one aspect, the present invention provides a vector comprising a nucleic acid molecule and a peptide or peptide derivative, said peptide or peptide derivative comprising a peptide of general Formula (I) : R-L-R^RJ-R^RS-RS (I)
wherein:
Rx is arginine or lysine; R2 is glutamine or asparagine; R3 is methionine; R4 is serine or threonine; R5 is histidine, tryptophan, arginine or lysine; and Rs is valine, leucine or isoleucine or wherein one or more of Rι-Rs is replaced by a non-genetically coded amino acid residue which is equivalent in terms of its physical properties, e.g. in terms of its general size, charge and hydrophobicity, to one of the genetically coded residues listed above for that same position. The peptide may incorporate one or more post-translational modifications, e.g. glycosylation, phosphorylation, myristylation, palmitylation, isoprenylation or biotinylation. Alternatively, the above peptide hexamer and other peptides described below may be replaced as a whole or in part by a peptidomimetic, non-peptide organic molecules which nevertheless incorporate the groups which contribute the same properties as the amino acid side chains by way of spatial configuration, charge and hydrophobicity etc. Suitable replacement amino acids for the genetically coded amino acids and peptidomimetic molecules and groups are known in the art . Preferred examples of the peptide of general Formula (I) are as follows, the standard single letter code for amino acids being used for convenience:
R-Q-M- S-H-V R-Q-M- T-W-I R-Q-M- S-H-L R-Q-M- T-R-I R-Q-M- S-H-I R-Q-M- T-K-V R-Q-M- S-W-V R-Q-M- T-K-L R-Q-M- S-R-L R-Q-M- T-K-I R-Q-M- S-R-V R-N-M- S-H-V RrQ-M- S-W-L R-N-M- S-H-L R-Q-M- S-W-I R-N-M- S-H-I R-Q-M- S-R-I R-N-M- S-W-V R-Q-M- S-K-V R-N-M- S-R-L R-Q-M- S-K-L R-N-M- S-R-V R-Q-M- S-K-I R-N-M- S-W-L R-Q-M- T-H-V R-N-M- S-W-I R-Q-M- T-H-L R-N-M- S-R-I R-Q-M- •T-H-I R-N-M- S-K-V R-Q-M- ■T-W-V R-N-M-•S-K-L R-Q-M- ■T-R-L R-N-M-■S-K-I R-Q-M- ■T-R-V R-N-M-■T-H-V R-Q-M- •T-W-L R-N-M-•T-H-L R-N-M-T-H-I K-Q-M-T-R-I R-N-M-T-W-V K-Q-M-T-K-V R-N-M-T-R-L K-Q-M-T-K-L R-N-M-T-R-V K-Q-M-T-K-I R-N-M-T-W-L K-N-M-S-H-V R-N-M-T-W-I K-N-M-S-H-L R-N-M-T-R-I K-N-M-S-H-I R-N-M-T-K-V K-N-M-S-W-V R-N-M-T-K-L K-N-M-S-R-L R-N-M-T-K-I K-N-M-S-R-V K-Q-M-S-H-V K-N-M-S-W-L K-Q-M-S-H-L K-N-M-S-W-I K-Q-M-S-H-I K-N-M-S-R-I K-Q-M-S-W-V K-N-M-S-K-V K-Q-M-S-R-L K-N-M-S-K-L K-Q-M-S-R-V K-N-M-S-K-I K-Q-M-S-W-L K-N-M-T-H-V K-Q-M-S-W-I K-N-M-T-H-L K-Q-M-S-R-I K-N-M-T-H-I K-Q-M-S-K-V K-N-M-T-W-V K-Q-M-S-K-L K-N-M-T-R-L K-Q-M-S-K-I K-N-M-T-R-V K-Q-M-T-H-V K-N-M-T-W-L K-Q-M-T-H-L K-N-M-T-W-I K-Q-M-T-H-I K-N-M-T-R-I K-Q-M-T-W-V K-N-M-T-K-V K-Q-M-T-R-L K-N-M-T-K-L K-Q-M-T-R-V K-N-M-T-K-I K-Q-M-T-W-L K-Q-M-T-W-I Peptide RQMSHV being particularly preferred. The peptide or peptide derivative may comprise flanking amino acids at the N- and/or C-terminus of the hexapeptides described above. More specifically, the peptide or peptide derivative of the vector will preferably comprise or consist of a peptide of general Formula (II) : R7-RS-R9-(I)-R10 (II) wherein :
(I) is a peptide of general Formula (I) as defined above; R7 is glycine or alanine; R8 is histidine, tryptophan, arginine or lysine; R3 is proline; R10 is tyrosine or phenylalanine; or, as described above, one or more of R7-R10 is replaced by an equivalent non-genetically-coded amino acids or the whole or part of the peptide is replaced by a peptidomimetic. Thus the peptide or peptide derivative will preferably comprise a decamer of Formula (II) . Further preferred peptides will be made up of fragments of the peptide of Formula II. Thus R10 may be absent. R7 may also be absent and if it is present, one or more of R8 and R9, preferably both RB and R9 are present. R8 may be absent and in that case R7 will typically be absent, if R8 is present then R9 will preferably also be present . R9 may be absent in which case R7 and R8 will typically also be absent . A particularly preferred decamer for incorporation into the vectors of the invention is G-H-P-(I) -Y, more particularly G-H-P-R-Q-M-S-H-V-Y, and variants of it including:
A-H-P-(I)-Y G-H-P-(I)-F G-W-P-(I)-Y A-H-P-(I) -F G-R-P-(I)-Y G-W-P-(I)-F A-R-P-(I)-Y G-R-P-(I) -F A-W-P-(I)-Y A-R-P-(I)-F G-K-P-(I)-Y A-W-P-(I)-F A-K-P-(I)-Y G-K-P-(I)-F A-K-P-(I) -F
The peptide or peptide derivative of the vector may contain any functionally active fragment of a peptide of general formula (II) . Such fragments will typically comprise at least 4, preferably at least 5 or 6, more preferably at least 7 or 8 and most preferably 9 amino acids. The fragments are 'functionally active1 if they can (as part of a vector) increase gene transduction (transfer) efficiency as compared to a vector which lacks the targeting peptide fragment. A suitable Ad-mediated gene transduction assay is described in the Examples herein. Typically the vectors according to the present invention will result in at least 5%, preferably at least 10%, more preferably at least 20 or 30%, most preferably at least 50% greater gene transduction efficiency as compared to a vector which lacks the peptide fragment. Preferably functionally active fragments are those which are able to redirect the vector of which they form a part to the lysosomal pathway. The Examples section herein teaches how localisation may be measured (e.g. by confocal immunofluorescence microscopy or electron- microscopy) and 'active' fragments are those which result in a measurably greater amount of vector being re-directed to the lysosomal pathway as compared to a vector lacking that fragment but the same in all other respects. Preferably the proportion of vector using the lysosomal pathway will be increased by at least 20%, preferably at least 30%, e.g. at least 50%, by an active fragment of a peptide of general formula (II) . According to a further aspect, the present invention provides a vector comprising a nucleic acid molecule and a peptide or peptide derivative, said peptide or peptide derivative comprising a peptide of general Formula (III) : R1-R2 -R3 -R4-Rs -Rs -R7 -Rg-R9 -R1 ( I I I )
wherein :
Rx is threonine; R2 is alanine; R3 is tyrosine; R4 is serine; Rs is serine Re is tyrosine R7 is any amino acid, R8 is any amino acid; R9 is glycine; and R10 is glycine or wherein one or more of RX-RK, is replaced by a non-genetically coded amino acid residue which is equivalent in terms of its physical properties, e.g. in terms of its general size, charge and hydrophobicity, to one of the genetically coded residues listed above for that same position. The peptide may incorporate one or more post-translational modifications, e.g. glycosylation, phosphorylation, myristylation, pal itylation, isoprenylation or biotinylation. Alternatively, the above peptide decamer and other peptides described below may be replaced as a whole or in part by a peptidomimetic, non-peptide organic molecules which nevertheless incorporate the groups which contribute the same properties as the amino acid side chains by way of spatial configuration, charge and hydrophobicity etc. Suitable replacement amino acids for the genetically coded amino acids and peptidomimetic molecules and groups are known in the art. Preferably R7 and R8 of the peptide of general Formula (I) are as follows, R7 is methionine, leucine, isoleucine, valine, threonine, tryptophan or phenylalanine; and R8 is lysine or arginine
Most preferably
R7 is methionine; and R8 is lysine
Peptide T-A-Y-S-S-Y-M-K-G-G being particularly preferred. The peptide or peptide derivative may comprise flanking amino acids at the N- and/or C-terminus of the decapeptides described above. More specifically, the peptide or peptide derivative of the vector will preferably comprise a peptide of general Formula (IV) :
(IID-Ru-Ru (IV) wherein:
(III) is a peptide of general Formula (III) as defined above; Ru is lysine or arginine R12 is phenylalanine or tyrosine or, as described above, one or more of RX1-R12 is replaced by an equivalent non-genetically-coded amino acids or the whole or part of the peptide is replaced by a peptidomimetic. Thus the peptide or peptide derivative will preferably comprise a dodecamer of Formula (IV) . Further preferred peptides will be made up of fragments of the peptide of Formula IV. Thus R12 may be absent. R1X may also be absent however Ru may be absent and Represent. A particularly preferred dodecamer for incorporation into the vectors of the invention is (IV)-K-F, more particularly T-A-Y-S-S-X-X-G-G-K-F (wherein X means any amino acid) , and most particularity T-A-Y-S-S-M-K-G-G-K-F.
The peptide or peptide derivative of the vector may contain any functionally active fragment of a peptide of general formula (IV) . Such fragments will typically comprise at' least 4, preferably at least 5 or 6, more preferably at least 7 or 8 and most preferably 9 amino acids. The fragments are 'functionally active' if they can (as part of a vector) increase gene transduction (transfer) efficiency as compared to a vector which lacks the targeting peptide fragment . A suitable Ad-mediated gene transduction assay is described in the Examples herein. Typically the vectors according to the present invention will result in at least 5%, preferably at least 10%, more preferably at least 20 or 30%, most preferably at least 50% greater gene transduction efficiency as compared to a vector which lacks the peptide fragment. The vectors of the invention are typically synthetic vectors in that they are not naturally occurring but have been engineered to incorporate a peptide or peptide derivative as defined herein. The vector may preferably be a modification of a naturally occurring vector, e.g. a modified viral vector or may have been synthesised essentially de novo, e.g. made up of a simple peptide/nucleic acid complex. Viral vectors are preferred, in particular adenoviral vectors, especially those of subgroup C, most especially Ad5. Thus in a preferred embodiment, the present invention provides a modified adenovirus (Ad) incorporating in one or more of its capsid proteins a peptide of Formula (I) , preferably a peptide of Formula (II) or an active fragment thereof as discussed and defined above. In another preferred embodiment the present invention provides a modified adenovirus (Ad) incorporating in one or more of its capsid proteins a peptide of Formula (III) , preferably a peptide of Formula (IV) or an active fragment thereof as discussed and defined above. The peptides of formula (I), (II), (III) and (IV) may be incorporated into any of the capsid proteins of the adenovirus, e.g. into the fiber domain of the penton capsomer (penton is made up of penton base plus fiber) as shown in the accompanying Examples, or the hexon or protein IX (pIX) , or into a combination of the capsid proteins simultaneously. Insertion into flexible and accessible loops of the hexon, or at the C-terminus of protein IX (protruding outwards from the capsid) are preferred locations since the virion is composed of 240 x 3 = 720 copies of hexon subunits (hexon is a trimer, and there are 240 hexons per capsid) and 240 copies of protein IX, versus only 3 x 12 = 36 copies of fiber subunits (the fiber is also a trimer) . The penton has often been preferred because of the natural role in receptor recognition played by the fiber knob, and in integrin binding and endocytosis played by the penton base. If the penton is used the fiber domain will be preferred for insertion due to its exposure and high accessibility. The peptide would not normally be incorporated into the tail, since this could be detrimental to the interaction of the fiber with the virion, but it can conveniently be inserted into the shaft or the globular 'knob'. Insertion into the shaft will be preferred since genetic or proteolytic de-knobbing will be possible and will result in the detargeting of the virus. Detargeting the virus will prevent it from binding to its natural receptors and infecting non-desired cells. The Examples herein demonstrate that the knob is not required for effective gene transfer and that the Ad may incorporate further modifications, e.g. deletion of some of the shaft as well as removal of the knob . In the case of protein IX, insertion of the peptides of the invention into the C-terminal portion of protein IX is preferred. Preferably between amino acid position 100 and the C-terminal residue, more preferably between amino acid 125 and the C-terminal residue and most preferably between residues 131 and 132. Alternatively the peptides of the invention can be inserted into a protein IX containing other mutations/modifications in its C-terminal domain which would permit greater accessibility and/or functionality of the ligands. By way of example, protein IX could contain an extra amino acid sequence, a "linker' at its C-terminus, which would allow the peptides of the invention to protrude further at the surface of viral capsid. This linker could have different lengths in terms of amino acid number. Peptides (III) or (IV) or an active fragment thereof as discussed and defined above are particularily efficient in enhancing transduction of melanoma cells and other tumour cells, including those which are not usually permissive to adenovirus infection. The skilled man will be aware of and be able to determine cells which are not permissive to adenovirus infection. In the case of peptides (III) or (IV) or an active fragment thereof as discussed and defined above, insertion into the pIX capsid protein is most prefered. In non-Ad viral vectors such as oncovetroviruses or lentiviruses which are enveloped, the peptide may conveniently be inserted into the accessible domains of the envelope glycoproteins. For other nonenveloped virus vectors such as AAV, accessible domains of the major capsid protein can be used. A variety of non-viral vectors (also called synthetic gene delivery systems) are known and are regularly reported and reviewed, for example in the
Nature publication "Gene Therapy". In its simplest, a non-viral approach for gene delivery may consist merely of introducing naked DNA into cells, e.g. using physical or chemical techniques such as electroporation or ultrasound. Plasmids are particularly preferred for use as or in non-viral vectors . The non-viral vectors of the present invention will thus typically comprise functional conjugates of nucleic acid, particularly DNA, and a peptide or peptide derivative incorporating a peptide of Formula (I), (II) (III) or (IV) described above. Suitable conjugates comprise plasmid DNA and a bifunctional peptide which comprises a lysine rich part (e.g. oligolysine) which binds to the plasmid DNA and may pack the complex and a peptide or peptide derivative as described above which is able to direct the vector to the desired lysosomal pathway after the vector has been taken up by the cell . Alternatively, liposomes may be used which encapsulate the vector nucleic acid and have a peptide or peptide derivative inserted into the phospholipid of the liposome. For example, a peptide derivative comprising a peptide portion for intracellular redirection of the vector and a hydrophobic tail can be made (as described in Molinier-Frenkel et al. (2002) J. Virol. Vol 76, No. 1, pp 127-135) . The tail, which may conveniently comprise a fatty acid chain such as palmitate, can be anchored in the liposome leaving the peptide part of the molecule free for intracellular interaction. The nucleic acid molecule of the vector is typically DNA but may, for example where the vector is an RNA virus, be RNA. Non-viral vectors may contain cDNA and the nucleic acid may be linear or circular, e.g. as with plasmid DNA. DNA may be single or double stranded. The vectors of the invention are typically for use in gene transfer, i.e. the introduction of genetic material into a target cell population, in particular in vivo into the cells of a subject or into ex vivo cell cultures. Thus the nucleic acid molecule of the vector will typically encode a therapeutic gene, i.e. a gene which encodes a protein which is directly or indirectly involved in treating the subject or in affecting the cellular processes or properties in an ex vivo cell culture. Genes which may be administered as part of a regimen of gene or cancer therapy are well known in the art and the list is increasing all the time. In certain circumstances the nucleic acid sequence which it is desired to introduce into the cells is a regulatory region rather than a region which encodes a protein product. Such a region may then be able to modulate expression of one of the target cell's own genes through integration into the host genome and thus a cis-acting effect. Alternatively, according to a preferred embodiment, the vector may carry a gene whose protein product is able to have a trans-acting effect by up- or down-regulating the expression of a target gene within the host cell. Where the vector nucleic acid encodes a protein which it is desired to express in the infected cells, the nucleic acid molecule will typically also comprise an operably linked promotor and other regulatory sequences . For certain vectors, in particular viral vectors, the nucleic acid of the vector will also encode structural and other proteins involved in the generation of further vectors which can go on to infect other cells, e.g. the gag, pol and env genes of a retrovirus. The nucleic acid molecules are recombinant in that they are not naturally occurring but the product of genetic engineering techniques, e.g. to modify a viral genome or to modify or generate a plasmid. The vectors of the invention incorporate a peptide or peptide derivative which is able to increase gene transfer efficiency as compared to vectors which lack that ligand. Without wishing to be bound by a particular theory, this is thought to be achieved by the ability of the ligand (at least in the case of ligands of formulae I and II) to redirect the vector to the lysosomal pathway within infected cells . The observed enhancement of gene transfer was not cell-specific and the results indicate that the peptides (e.g. QM10) do not confer any novel cell-binding capacity on the vector but intervene at the step of endocytosis by redirecting the vector to an endocytic compartment different from the class of early endosomes used by adenovirus type C members. In both human and simian cells lines this gives significant advantages in terms of gene transfer. As postulated in Miyazawa (supra) such redirection also being of utility with non-viral vectors. The peptide or peptide derivative discussed above can be considered a redirecting or internal targeting moiety which incorporates a ligand, e.g. a peptide of formula (I) or (II) or a fragment thereof which provides the redirecting or internal targeting function. The targeting peptide or peptide derivative may form all or part of a viral protein, e.g. it may replace an Ad penton fibre knob or be inserted in the fiber shaft. Standard recombinant techniques may be used to generate chimeric molecules which include the ligands for intra-cellular re-targeting. In non-viral vectors the peptides may also be chimeric molecules, with a targeting portion and a portion which links the chimera with the rest of the vector, e.g. a lysine rich part which binds to the vector nucleic acid or a hydrophobic part (which may not be made up of amino acids) to anchor the chimera to a liposome. A peptide/fatty acid conjugate which may be used in a liposome based vector can be considered a peptide derivative. Further derivatives may include molecules which include regions which act as peptidomimetics, i.e. not made up solely of amino acids but having chemical groups which can mimic the physical properties of amino acids, e.g. in terms of charge and hydrophobicity. One particularly preferred non-viral conjugate comprises a biotinylated peptide (e.g. QM10) , streptavidin and a biotinylated (DNA) plasmid carrying a therapeutic gene. The streptavidin bridges the nucleic acid molecule to the targeting peptide. Thus a preferred class of vectors are those which comprise a nucleic acid molecule (e.g. a plasmid) joined by a linker moiety (e.g. a streptavidin-biotin linker but other suitable linkers are known) to a peptide or peptide derivative as defined and discussed above. Where the peptide or peptide derivative is a modified version of a naturally occurring vector protein, e.g. an Ad capsid protein, the non-native portion which incorporates a peptide of formula (I) -(IV) or a fragment thereof, is typically no more than 100 amino acids or equivalent in length, preferably no more than 80 or 60 amino acids in length. Nevertheless, it may be beneficial to include multiple copies of the peptide ligand, e.g. of formula (I) -(IV), into the vector, e.g. into the fiber shaft or other Ad capsid protein. Thus 2-6 copies, preferably 2-4 copies, may be incorporated, preferably each separated by a linker sequence of 2-5 neutral amino acid residues (such as G-S or G-S-G-S-G) to increase the flexibility and accessibility of the targeting ligand. Preferred embodiments of the invention will not include the incorporation of large proteins such as α2MG, which as described in the Examples, is normally found in the late endosomes of the lysosomal pathway. The targeting peptide or peptide derivative will be arranged such that the peptide of Formula (I) or (III) which it contains is available for interaction with intra-cellular components after internalization of the vector. Intra-cellular components may include intra- endosomal components. The targeting peptide or peptide derivative or at least that part of it containing the peptide of Formula (I) or (III) will therefore be accessible on the surface of the vector, e.g. as part of an adenoviral capsid protein or protruding on the outside of a liposome. It may be immediately accessible or after an in vivo processing step, e.g. after unmasking by proteolytic cleavage of the shaft and removal of the knob domain (when inserted in the fiber shaft, upstream of a protease cleavage site) . "Directing" or "targeting" as generally used herein refers to the different intra-cellular pathways from endocytosis to nucleus and not to the targeting of specific cell types. Methods of re-targeting adenoviruses to particular cell types are known in the art, see for example WO 02/08263, and thus vectors of the invention may also incorporate moieties for cell-specific targeting. Peptides and peptide derivatives incorporating a region having the sequence of Formula (I) , preferably of Formula (II) , particularly the sequence GHPRQMSHVY, constitute a further aspect of the present invention. Peptides and peptide derivatives incorporating a region having the sequence of Formula (III) , preferably of Formula (IV) , particularly the sequence T-A-Y-S-S-M-K-G- G-K-F, constitute a still further aspect of the present invention. Such peptides will preferably be modified adenoviral capsid peptides and thus include flanking regions which are native sequences of said capsid proteins. Nucleic acid molecules encoding said peptides constitute further aspects of the invention. In a still further aspect the present invention provides a method of gene delivery which comprises contacting a cell population ex vivo with a vector of the invention as defined above or administering a vector of the invention as defined above to a subject. The subject will typically be mammalian and in-vitro cell lines will typically be of mammalian origin, humans and human cell lines being especially preferred. In further aspects the invention provides a vector of the invention as defined above for use in therapy, in particular for use in gene therapy or cancer treatment, particularly for use in the treatment of cystic fibrosis or melanoma. A further aspect of the invention are formulations comprising a vector of the invention as defined above together with a pharmaceutically acceptable diluent or carrier. A method of gene therapy or cancer treatment which comprises administration to a patient of a vector of the invention as described above constitutes a further aspect of the invention. The invention will now be further described in the following Examples and by reference to the Figures in which;
Figure 1 is a schematic representation of genetic constructs of Ad5-based vectors (AdGFP) carrying wild- type (WT) fiber (a) , or recombinant fibers (b-e) . (a) , In WT fiber, the tail domain, bound to penton base capsomer (shown on the left) , is represented by a stippled box, the shaft domain by a zig-zagged box, the last shaft repeat by a black box, and the knob symbolized by an open circle, (b-e) , In short-shafted recombinant fibers with seven repeat shaft (R7) , the different fiber domains are represented as in (a) . The extrinsic trimerisation motif, inserted downstream of amino acid residue 158 of the shaft, the Factor-Xa cleavage site and the cell ligand were represented by different symbols, as indicated in the figure.
Figure 2 provides a graphical comparison of gene transfer efficiency (GTE) between the QMlO-liganded vector AdGFP- QMlO-knob (black bars) and the non-liganded control vectors AdGFP-WTFi (long-shafted fibers; striped bars) and AdGFP-R7-knob (short-shafted fibers; grey bars) in various cell lines at a MOI of 20 PFU/cell. GFP expression was analyzed by FACS, and the results were expressed as the percentage of GFP-positive cells (a) , and GTE index (b) , as described in Materials and Methods (AU, arbitrary units) . (c, d) , Dose-dependence of gene transduction mediated by AdGFP-WTFi (open square symbols) , AdGFP-R7-knob (solid triangles) and AdGFP-QMlO- knob (solid circles) in A549 (c) and CF-KM4 cells (d) .
Cells were transduced at various MOI, as indicated in the x-axis, and GFP expression determined by counting GFP- positive cells in IF microscopy.
Figure 3 shows the effect of vector de-knobbing on gene transduction of various cell lines mediated by knobless or knob-carrying versions of AdGFP vectors. HeLa and A549 cells (CAR+, CFTR+) , glandular cells MM39 (CAR-, CFTR+) and CF-KM4 (CAR-, CFTR-) were infected by AdGFP-R7-knob (a, b, i) or its knobless version AdGFP-R7 (c, d, i) ,
AdGFP-QM10-knob (e, f, i) or AdGFP-QM10 (g, h, i) , AdGFP- SY12-knob or AdGFP-QMSY12 (i) , at constant MOI (30 PFU/cell) . GFP expression was estimated at 36 h pi by IF microscopy (b, d, f , h) , and quantitated by FACS analysis (i). (a, c, e, g), DAPI staining of cell samples shown in (b, d, f , h) . In (i) , the gene transfer efficiency (GTE) was expressed as the ratios of GTE indices, i.e. AdGFP-R7 versus AdGFP-R7-knob, AdGFP-QMlO versus AdGFP-QMlO-knob, and AdGFP-SY12 versus AdGFP-SY12-knob, respectively. Data shown were from three separate experiments, + standard deviation (SD) .
Figure 4 comprises graphs showing the results of cell- binding competition assays between Ad5 capsid components and knobbed or deknobbed versions of non-liganded or liganded AdGFP vectors. CF-KM4 (a) and A549 (b) cell monolayers were incubated with a mixture of AdGFP vector and a large excess of capsid protein, under conditions allowing for cell attachment but not cell entry. After removal of unadsorbed virus, cells were transferred to 37°C for 24 h and the amount of cell-bound virus was indirectly assayed by the level of GFP expression, determined by FACS analysis. Data, from two separate experiments, were expressed as GTE indices, given in arbitrary units (AU) , + SD.
Figure 5 comprises graphs showing endocytosis assays of knobbed versus deknobbed versions of non-liganded vector AdGFP-R7±knob, and liganded vectors AdGFP-QMlO±knob and AdGFP-SY12±knob in A549 (a), MM39 (b) and CF-KM4 (c) cells. Cy3-labeled vector particles were incubated with cell monolayers at 37°C for 1 h, cell surface-bound virus detached by EDTA-trypsin treatment, and cells assayed for intracellular fluorescent signal by FACS analysis. Data, from three separate experiments, were expressed as endocytic indices, given in arbitrary units (AU) , as described in the Examples .
Figure 6 comprises graphs showing the results of endosomolysis assays. Cells were co-incubated with toxin (Ricin A, RcA) and AdGFP vectors for 1 h at 37°C, at different toxin-to-cell ratios and a constant vector input (104 PP/cell) . Vesicular escape and cell internalization of co-endocytosed RcA and AdGFP vector were assayed in A549 (a) , MM39 (b) and CF-KM4 (c) cells, using toxin-induced inhibition of host-cell protein synthesis. Data indicated on the x-axis represented the values of RcA concentration (in pg/cell) which provoked a 50% inhibition of cell protein synthesis (IC50) . Data presented are the means of four separate experiments, ± SD.
Figure 7 shows in photos the intracellular localization of liganded vectors AdGFP-QM10-knob and AdGFP-SY12-knob, in comparison with that of control, non-liganded vector AdGFP-R7-knob. Vectors were co-incubated pairwise with CF-KM4 cells and analyzed by confocal IF microscopy at 45 min after input, (a-c) , Cy2-labeled AdGFP-QM10-knob and Cy3-labeled AdGFP-SY12-knob; (d-f) , Cy2-labeled AdGFP-R7- knob and Cy3-labeled AdGFP-SY12-knob; (g-i) , Cy2-labeled AdGFP-QM10-knob and Cy3-labeled AdGFP-R7-knob; (j-1), Cy2-labeled, knobless AdGFP-QMlO and Cy3-labeled AdGFP- R7-knob. Overlay pictures are shown in (c) , (f) , (i) and 5 (1) .
Figure 8 shows in photos the cell compartmentalization of non-liganded vector AdGFP-R7-knob, and liganded vector AdGFP-QMlO-knob in CF-KM4 cells, analyzed by confocal IF
1.0 microscopy at 45 min after input. After fixation and permeabilization, cells were reacted with FITC-labeled, anti-TfR (A) or anti-LAMP-1 antibody (B) . The left column (panels a, d, g, j) shows the signal of FITC-labeled antibodies, the middle column (panels b, e, h, k) the
15 signal of Cy3-labeled vector particles, and the right column (panels c, f, i, 1) the merging of the two signals, (a-c) , FITC-labeled anti-TfR antibody and Cy3- labeled AdGFP-R7-knob ; (d-f) , FITC-labeled anti-TfR antibody and Cy3-labeled AdGFP-QMlO-knob ; (g-i) , FITC-
20 labeled anti-LAMP-1 antibody and Cy3-labeled AdGFP-R7- knob ; (j-1), FITC-labeled anti-LAMP-1 antibody and Cy3- labeled AdGFP-QMlO-knob.
Figure 9 shows in photos the colocalization of human 25 alpha-2 acroglobulin (a2M) and QMlO-liganded AdGFP vector in its knob-carrying version AdGFP-QMlO-knob (a-c) and its knobless version AdGFP-QMlO (d-f) in A549 cells at 45 min after input, analyzed by confocal IF microscopy. The left panels (a, d) show the signal of 30 FITC-labeled a2M, the middle panels (b, e) the signal of Cy3-labeled vector particles, and the right panels (c, f) the signal overlay.
Figure 10 provides electron microscope analysis of early 35 events in the interaction of AdGFP-QMlO-knob vector with A549 cells. Cells were incubated with a viral input of 104 virions per cell for 1 h at 37°C. (a) , General view of the cellular apical surface; (b, c) , enlargements of plasma membrane-bound virions. (d) , Endocytosed virion. Arrows in (b) and (c) show filaments linking the virus to the cell surface, whose length was compatible with that of a short-shafted fiber of seven repeats. Bar represents 200 nm in (a) , (c) and (d) and 100 nm in (b) .
Figure 11 provides electron microscope analysis of endocytosed virions of AdGFP-QMlO-knob in A549 cells after 1 h infection at 37°C, at an input multiplicity of 104 virions per cell, (a), Curved arrows show two small intracytoplasmic vesicles opening into a larger one, containing multiple virions and heterogeneous, electron- dense material, (b) , Vesicle containing one virion and heterogeneous and amorphous material. Arrows indicate microtubules apparently connecting endosomal vesicle to the nuclear envelope ; NPC, nuclear pore complex, (c-e) , Arrows point to junctions of intravesicular virions to the endosomal leaflet. Panels (d) and (e) are enlargements of areas with intravesicular virions shown in (c) . Bar represents 100 nm in (a) , (b) , (d) and (e) , and 200 nm in (c) .
Figure 12 shows electron microscope analysis of intracellular virions of knobless AdGFP-QMlO-knob in A549 cells. Cells were incubated with vector for 1 h at 37°C, and at an input multiplicity of 104 virus particles per cell. Virions (arrows) were seen associated with multivesicular bodies (a, b) , or with multilamellar inclusion bodies (c) characteristic of A549 cells. Bar represents 200 nm.
Figure 13 shows electron microscope analysis of early virus-cell interaction between CF-KM4 cells and QM10- liganded or control vectors. Cells were incubated with vector particles for 1 h at 37°C, and at an input multiplicity of 104 virions per cell, (a-c), AdGFP-R7- knob ; (d, e) , AdGFP-WTFi ; (f) , AdGFP-QMlO-knob ; (g) , AdGFP-QMlO. Arrows in (a) point to an electron-dense layer of proteinic material, most likely clathrin, at the virus-attachment site on the plasma membrane. Bar represents 100 nm in (a-e) , (f) , (g) , and 200 nm in (d) , (e) .
Figure 14 shows the infectivity and transducing capacity of AdβGal-pIX-QMlO for melanoma cells Ml, M2, M3 and M4. Adenoviral vectors in the range of concentrations of 100, 500 and 2,000 virus particles/cell were added to target cell monolayers for 1 h at 4°C. The inoculum was removed and the cells were washed twice with cold medium, then further incubated for 48 h at 37°C. Cells were then fixed and stained to determine beta-galactosidase activity, using a beta-galactosidase staining kit (Chemicon) . Infected cells are visible as blue cells (adenovirus- positive cells)
Figure 15 shows the infectivity and transducing capacity of AdβGal-pIX-QMlO and AdβGal-pIX-SY12 for melanoma cells M8. Adenoviral vectors in the range of concentrations of 100, 500 and 2,000 virus particles/cell were added to target cell monolayers for 1 h at 4°C. The inoculum was removed and the cells were washed twice with cold medium, then further incubated for 48 h at 37°C. Cells were then fixed and stained to determine beta-galactosidase activity, using a beta-galactosidase staining kit (Chemicon) . Infected cells are visible as blue.
Figure 16 the infectivity and transducing capacity of
AdβGal-pIX-QMlO and AdβGal-pIX-SY12 for the melanoma cell line MeWo cells M8. Adenoviral vectors in the range of concentrations of 200 and 2,000 virus particles/cell were added to target cell monolayers for 1 h at 4°C. The inoculum was removed and the cells were washed twice with cold medium, then further incubated for 48 h at 37°C. Cells were then fixed and stained to determine beta- galactosidase activity, using a beta-galactosidase staining kit (Chemicon) . Infected cells are visible as blue .
EXAMPLE 1
Ligand insertion into the adenoviral fiber protein
Materials and Methods
Cells
The SV40-immortalized human tracheal gland cell lines MM39 (normal) and CFKM4 (CFTR-deficient) were maintained as monolayers on collagen-I-coated flasks (Biocoat; Becton-Dickinson, Bedford, MA) in Dulbecco's modified Eagles ' s medium-Ham's F12 (DMEM-F12) supplemented with 1% Ultroser G (Gibco-Invitrogen, Rockville, MD) , penicillin (200 U/ml) , streptomycin (200 μg/ml) and epinephrin (3 μM; Sigma, St Louis, MO) . The E1A+E1B-trans-complementing HEK-293 cell line (abbreviated 293) was obtained from ATCC (Manassas, VA, USA; CRL 1573) , and ElA+ElB+fiber-trans-complementing 293 cells (293-Fibre ; (Legrand, V. et al (1999) J. Virol.
73:907-919)) were obtained from Transgene SA (Strasbourg, France) . Cells were cultured as monolayers in DMEM medium (Gibco-Invitrogen) , supplemented with 10% fetal calf serum (FCS; Sigma) , penicillin (200 U/ml) and streptomycin (200 μg/ml; Gibco-Invitrogen) at 37°C and 5% C02, and, for 293-Fibre cells, hygromycin was added at 350 μg/mL. Human cell lines HeLa (cervix epitheliod carcinoma; CCL 2) , A549 (lung alveolar carcinoma ; CCL 185) , HEp2 (larynx epidermoid carcinoma ; CCL 23) , MRC-5 (foetal lung) , HRT-18 (ileocecal adenocarcinoma ; CCL 244) , PLC/PRF/5 (hepatoma ,- CRL 8024) and RD (rhabdomyosarcoma; CCL 136) , simian cell lines Vero (African green monkey kidney ; CCL 81) , LLC-MIC, (Rhesus monkey kidney ; CCL 7) and BGM (Buffalo green monkey kidney epithelial cells; (Kok, T. W. et al (1998) J. Clin. Virol. 11:61-65)), canine MDCK (dog kidney ; CCL 34) , hamster CH0K1 (Chinese hamster ovary ; CCL 61) and mouse L20B cells (recombinant murine cell line expressing human poliovirus receptor CD155 on the cell surface; (Wood, D.J. et al (1999) J. Med. Virol. 58:188-192)) were provided by the Clinical Virology Laboratory (Hospices Civils de Lyon, Domaine Rockefeller, Lyon, France) . Except MRC-5, BGM and L20B cells, they were originally from ATCC. Cells were cultured as monolayers in DMEM medium as above. Daudi cells (EBV-transformed B lymphoma cells) were cultured in RPMI medium supplemented with 10% FCS (Hong, S.S. et al (1997) EMBO J. 16:2294-2306). For production of recombinant Ad proteins Spodoptera frugiperda cells (Sf9 subclone) were propagated in TNM medium (Gibco- Invitrogen) and cultured as monolayers with 10% FCS, penicillin and streptomycin as mentioned above, and maintained at 28°C.
Identification of CF-KM4 cell ligands Confluent monolayers of CF-KM4 cells (4 x 105 cells) were incubated with an aliquot (3 x 1010 phages in 0.25 ml TBS) of a phage-displayed hexapeptide library (Smith, G.P. et al (1993) Method Enzymol . 217:228-257), for 20 mn at 37°C, then rinsed twice in TBS at room temperature (RT) . In order to detach cell surface-adsorbed phages, the cells were incubated with 0.25 ml elution buffer (0.1 M glycine-HCl buffer, pH 3.0, 2 M urea) for 10 mn at RT . The cells were then harvested by scraping with a rubber policeman, pelleted at 2,000 x g for 3 mn, and lysed by swelling and vortexing in 0.1 ml hypotonic buffer (5 mM Tris-HCl buffer pH 7.5, 1 mM Na2EDTA) . Cell debris were eliminated by low-speed centrifugation (500 x g for 3 ins) , and the supernatant containing cell-internalized phages was recuperated for phage isolation, amplification and phagotope sequencing, according to previously published methods (Hong, S.S. et al (1995) EMBO J. 14:4714-4727; Hong, S.S. et al (1997) EMBO J. 16:2294- 2306) .
Adenoviruses (Ad) and Ad vectors
(Fig. 1) . Wild-type (WT) Ad serotype 5 (Ad5) , and replication-competent vector Ad5Luc3 (Mittal, S. K. et al (1993) Virus Res. 28:67-90), harboring the firefly luciferase gene (luc) under the control of the SV40 early promoter inserted in the E3 region of the Ad5 genome, were propagated in HeLa cells. El-deleted recombinant Ad5 vectors with WT fibers were propagated in 293 cells, whereas El-deleted, fiber-modified Ad5 vectors were grown in 293-Fibre cells, until the last amplification step of the vector stock, which was performed in 293 cells. Virus stocks were purified by CsCl gradient ultracentrifugation, according to conventional methods (Defer, C. et al (1990) J. Virol. 64:3661-3673). The backbone vector AdGFP-R7- (Xa) -knob (abbreviated AdGFP-R7- knob) , was an Ad5-based vector with its deleted El region replaced by a GFP expression cassette under the CMV promoter, and a genetically-modified fiber which contained the following domains, from the N- to C- terminus : (i) the tail, (ii) the N-terminal seven shaft repeats (R7) , (iii) a trimerisation signal (PDVASLRQQVAELQGQVQHLQAAFSQYKKVELFPNG) called the neck region peptide (NRP) from the human lung surfactant protein D, (iv) a tridecapeptide linker (AKKLNDAQAPKSD) , (v) the desired cell ligand, (vi) the cleavage site for Factor Xa protease, and (vii) the last shaft repeat and the terminal knob domain (Fig. 1) . The cleavage site for Factor Xa consisted of the tetrapeptide IEGR, and the cleavage occurred after the arginine residue. Recombinant fibers were rescued into the Ad5-GFP genome as previously described (Magnusson, M. K. et al (2001) J. Virol. 75:7280-7289). The backbone vector AdGFP-R7-knob, which carried non-liganded, shortshafted fibers with NRP, Factor Xa site, the terminal knob but no specific cell ligand (Fig. l b, c) , was used as the control vector in our experiments, with or without cleavage by Factor Xa. For construction of liganded AdGFP-R7-knob vector with new cell tropism, three peptide ligands potentially involved in phage endocytosis, and abbreviated QM10, SY12 and LAP25, respectively, were individually inserted into the fiber shaft domain, upstream to the Factor Xa cleavage site. For proteolytic de-knobbing of Ad5 vectors, virus samples were treated with 1 U Factor Xa per 109 physical virus particles in cleavage buffer (50 mM Tris pH 8.0, 100 mM NaCI, 5 mM CaCl2) for 16 h at 22 °C.
Ad titration
Since all our uncleaved Ad5 clones carried the fiber knob domain and bound to CAR, the concentration in infectious viral particles of CsCl-purified Ad stocks could be determined and compared using plaque assays in 293 cell monolayers, according to conventional methods (Magnusson, M. K. et al (2001) J. Virol. 75:7280-7289), and the infectivity titers expressed as plaque-forming units per mL (PFU/mL) . The concentration in physical particles (PP) was deduced from the protein concentration determined in the same CsCl-purified Ad stocks, using the Bradford protein assay (BioRad) with bovine serum albumin (2x crystallized BSA, BioRad, Hercules, CA) as the standard. The number of PP was calculated from the total protein content of the sample, taking the mass of 2.91 x 10"16 g for one single virion, i.e. 3.4 x 1012 virions per mg protein. The infectivity index represented the ratio of infectious to physical particles (PFU/PP) , and usually ranged between 1:25 to 1:50 for WT Ad5. For propagation of Ad virus clones, we used a multiplicity of infection (MOI) ranging from 2 to 10 PFU/cell, corresponding to 50 to 500 PP/cell. For probing cellular functions (e.g., attachment, endocytosis, vesicular escape) , the MOI used varied from 5 x 102 to 5 x 104 PP/cell.
Recombinant Ad proteins and competition assays
Recombinant Ad penton base and fiber proteins were isolated in native and soluble form from lysates of Ad5- infected HeLa cells or baculovirus-infected Sf9 cells, and purified according to a previously published method (Boulanger, P. et al (1973) Eur. J. Biochem. 39:37-42), adapted to FPLC (Molinier-Frenkel, V. et al (2002) J. Virol. 76:127-135.). The genetic constructions of recombinant Autographa californica Multiple Nuclear Polyhedrosis Viruses (AcMNPV) expressing Ad5 full-length fiber or fiber knob domain have been described in detail elsewhere (Novelli, A. et al (1991) Virology, 185:365- 376) . Since penton base proteins from serotypes 2 and 5 Ad have 98% amino acid sequence identity, our recombinant Ad2 penton base proteins, WT and RGD-mutant R340E, isolated from AcMNPV-infected Sf9 cells were used in competition experiments with Ad5 virions. Protein samples were analyzed by conventional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) , and imunoblotting using the required antibodies, as already described (Novelli, A. et al (1991) Virology, 185:365-376). Ad protein concentration was estimated by the intensity of Coomassie blue staining of protein band in SDS-gel, measured by scanning at 610 nm in an automatic densitometer (REP-EDC, Helena Laboratories, Beaumont, TX) , using a range of known BSA concentrations for calibration. Cell-binding competition assays were performed using purified hexon, penton base, full-length fiber and knob proteins, and penton base mutant R340E. Each capsid protein was individually mixed with each vector, in large excess over its copy number in the Ad virion, and incubated at 4°C with cells, to allow attachment but not entry. For solubility reasons, hexon protein was added in only 100-fold excess over the hexon content of the virions, whereas penton base, fiber and knob proteins were added in 103-fold excess. Unadsorbed virus was rinsed off, and cells assayed for GFP expression at 28 h pi.
Immunological reagents and flow cytometry
(i) Antibodies . 1D6.14, an anti-Ad5 fiber knob monoclonal antibody (mAb) with CAR-blocking activity, was supplied by D.T. Curiel (University of Alabama at Birmingham, UAB, AL; (Magnusson, M. K. et al (2001) J. Virol. 75:7280-7289)). MAb 4D2.5 , directed towards the conserved fiber tail motif (Hong, J.S. et al (1991) Virology, 185:758-767), was obtained from J.A. Engler (UAB, AL) . Other antibodies against Ad proteins have been described in a previous study (Gaden, F. et al (2002) Am. J. Respir. Cell Mol. Biol. 27:628-640). (ii ) Flow cytometry. Confluent or subconfluent cells were rinsed with phosphate buffered saline (PBS) , detached with PBS containing 1 mM Na2 EDTA and trypsin (0.1 mg/mL), fixed with paraformaldehyde (3% PFA in PBS) for 10 mn at RT, and resuspended in PBS containing BSA at 0.1 mg/mL. Cell samples (10s cells/0.1 mL) were analyzed by flow cytometry, using a FACScalibur flow cytometer (Becton Dickinson) .
Fluorescent probes and immunofluorescence (IF) microscopy
(i) Labeling of cell ligands . To generate fluorescent Cy2-Ad or Cy3-Ad vectors, Ad virions were covalently conjugated with two different fluorophores, carbocyanine bisfunctional dyes Cy™2 or Cy™3 (Amersham Biosciences, Little Chalfont, UK) , according to a published protocol (Leopold, P.L. et al (1998) Hum. Gene Ther. 9:367-378), with some modifications. The main modification of the protocol consisted of replacing the step of dialysis, which often provokes the precipitation of virions and macromolecules , or the gel-exclusion chromatography, which dilutes the virus inoculum, by blocking the excess of coupling reagent with 20 mM lysine, followed by dilution of the fluorescent dye- conjugated ligands in culture medium. Cy2- or Cy3-labeled Ad were used within 2 h after coupling. Human alpha-2 macroglobulin (α2M ; Sigma) was conjugated with fluorescent isothiocyanate (FITC) using the EZ-label™
FITC protein labeling kit (Pierce, Rockford, IL) , and the protocol recommended by the manufacturer. IF microscopy of cellbound virus was performed using an Axiovert 135 microscope (Zeiss) , equipped with an AxioCam videocamera and a quantitative image analysis program.
(ii) Endosomal compartment markers . Mouse mAb to transferrin receptor (anti-CD71) , rabbit antibodies against Rab proteins Rab4 and Rabll were purchased from Santa Cruz Biotechnoloy (Santa Cruz, CA) , Inc. Mouse mAb to LAMP-1 (anti-CD107a) was from BD PharMingen (San Diego, CA) , and rabbit antibody against Rab5 protein was from Stressgen Biotechnologies (Victoria, BC, Canada) . FITC-conjugated anti-mouse IgG was from Pierce, and FITCconjugated anti-rabbit IgG was from Santa Cruz
Biotechnoloy. Internalized ligands and cell compartments were analyzed in IF microscopy, using a Zeiss LSM 510 META confocal microscope.
Assays for cell attachment, endocytosis and vesicular escape of Ad5 vectors
(i) Cell attachment . Confluent cell monolayers were incubated with aliquots of Cy3-Ad vectors ranging from 5 x 103 to 5 x 104 particles per cell at 0°C for 1 h. After rinsing, cell adsorbed fluorescent signal was quantitated by IF microscopy. The cell-binding index, expressed as arbitrary units (AU) , was calculated using the formula : number of positive, fluorescent cells per field x (mean fluorescent intensity per cell - mean background fluorescent signal of control cells) . The number of individual cells analyzed ranged within 100-200 in each separate experiment.
(ii) Endocytosis . Confluent cell monolayers were incubated with aliquots of Cy2- or Cy3-Ad vectors ranging from 5 x 103 to 5 x 104 particles per cell at 37°C for 1 h, then treated with 0.05 % trypsin and 0.53 mM Na4EDTA in Hanks ' s balanced salt solution (Hank's BSS-trypsin-EDTA IX ; Gibco Invitrogen Corp.) at 37°C for 15 min to detach possible surface-sequestered viruses. After fixation in 3% paraformaldehyde in PBS, the intensity of internalized fluorescent signal was determined by FACS analysis, as above. The endocytic index, also expressed as AU, was calculated as above, using the equation : percentage of fluorescent cells x (mean fluorescent intensity per cell - mean background fluorescent signal of control cells) .
(iii) Ad-mediated endosomolysis , vesicular escape and virus internalization . This was determined using a toxin assay, as previously described (Gaden, F. et al (2002) Am. J. Respir. Cell Mol. Biol. 27:628-640). In brief, cell monolayers were preincubated with ricin agglutinin (RcA of 120,000 mol wt . ; Sigma) at concentrations ranging from 0 to 10 μg of RcA per aliquot of 5x10s cells for 1 h at 37°C, in methionine- and cysteine-free culture medium, and in the presence (or absence) of Ad vector, at a constant input of 104 PP/cell. (35S) -methionine and (35S) -cysteine (> 1,000 Ci/mM; PRO- MIX; Amersham) was added at 15 μCi per 5xl05 cell sample, and incubation further proceeded for 2 h at 37°C. Cells were then rinsed with culture medium, detached from the support and dissolved in 0.2 N NaOH, 1% SDS. Cellular proteins were precipitated by addition of 10 vol trichloroacetic acid (TCA) at 10%, and precipitates retained by filtration on GF/C glass filters. Ad+RcA- mediated inhibition of cell protein synthesis was evaluated from TCA-precipitable radioactivity, determined by scintillation counting in a liquid scintillation spectrometer (Beckman LS6500, Beckman Coulter Inc., Fullerton, CA) .
Ad-mediated gene transduction assays
Ad5 vector, carrying the reporter gene luc or gfp, was added to treated or mock-treated cells, in a final vol of 200 μl in culture medium and at MOI ranging from 0 to 30 PFU/cell. After incubation for 1 h at 0°C with intermittent rocking, the cell monolayers were washed with cold medium to remove unadsorbed virus . Prewarmed medium was then added and the cells transferred to 37°C for 24-36 h. The cells were then harvested, and the efficiency of Ad-mediated gene transfer was estimated by the level of reporter gene product expression, luciferase or GFP. Luciferase activity was assayed in cell lysates using luciferase substrate solution (Promega) in a Lumat LB-9501 luminometer (Berthold Bioanalytical, Germany) . The results were expressed in relative light units (RLU) per mg of whole protein present in cell lysates (Hong, S.S. et al (1999) Virology, 262:163-177). GFP activity was determined using FACS analysis or IF microscopy with quantitative image analysis program, as described above. The index of gene transfer efficiency (GTE) , expressed as AU, was measured by the following equation : GTE index = percentage of GFP-positive cells x MFI (mean fluorescent intensity per cell - mean background fluorescent signal of control cells) .
Electron microscopy (EM) Mock- or AdGFP vector-transduced cells were harvested at early times after infection (0.5 to 1 h pi), pelleted, fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and post-fixed with osmium tetroxide (1% in 0.1 M cacodylate buffer, pH 7.4). Cell specimens were dehydrated and embedded in Epon (Epon-812; Fulha , Latham, NY) . Sections were stained with 7% uranyl acetate in methanol, and post-stained with 2.6% alkaline lead citrate in H20. Specimens were examined under a Jeol 1200-EX electron microscope, equipped with a MegaView II high resolution TEM camera and a Soft Imaging System of analysis (Eloϊse, Roissy, France) .
RESULTS
Screening for peptide ligands of cell-internalization receptors in CF- M4 cells
Since CF-KM4 cells do not express CAR, the high affinity receptor for Ad5, we looked for other surface molecules of CF-KM4 cells which could act as substitute receptors for Ad5, and be targeted by recombinant Ad5 vectors . As there was no direct screening method available for identifying ligands of CF-KM4 cells, we applied the indirect approach of biopanning monolayers of living CF-KM4 cells (Fontana, L. et al (2003) J. Virol. 77:11094-11104; Smothers, J.F. et al (2002) Science, 298:621-622) using a phage-displayed hexapeptide library. Two classes of phage populations were isolated. One consisted of extracellular phages, eluted from the surface of intact cells and referred to as 'cell binders'. The cell binders carried peptide aptamers corresponding to ligands of surface exposed domains of plasma membrane molecules which would not necessarily lead to phage endocytosis and entry. The other population of phages recovered after cell lysis corresponded to intracellular phages. It has been shown that recombinant bacteriophages carrying a portion of the Ad penton base sequence overlapping the RGD peptide motif were capable of binding to αv integrins of mammalian cell plasma membrane, and were found to be endocytosed into endosomal vesicles. We then postulated that intracellular phages recovered from CF-KM4 cells were competent for cell entry, and that this novel competency, conferred upon the bacteriophages by the extrinsic peptides displayed on their piII attachment proteins, could also be conferred upon recombinant Ad vectors by insertion of the same peptides into their fibers.
The intracellular phages were thus isolated, amplified and sequenced, and the most represented phagotopes were grouped in families according to their sequence homology. Alignment of overlapping peptides sharing common motifs allowed us to design consensus oligopeptides of longer sequences than the original hexapeptide phagotopes, e.g. 10- or 12-mer. Three major different ligands emerged from this analysis. The first family of intracellular phages contained recurrent peptide motifs like LLTV, RMQ and QPPG. The search in data banks revealed the same motifs in a lysosomial acid phosphatase (LAP) , which has the property of recirculating between the plasma membrane and the lysosomial membrane (46) (Obermuller, S. et al (2002) J. Cell Sci. 115:185-194). Since the phagotopes overlapped the LAP sequence between residues 399-423 (LLTVLFRMQAQPPGYRHVADGQDHA) , it was referred to as LAP25 in the present study. The two other phagotope families were represented by the decapeptide GHPRQMSHVY (abbreviated QM10) and the dodecapeptide TAYSSYMKGGKF (abbreviated SY12) , respectively. Sequence comparison with proteins in data banks did not elicit any plasma membrane protein or protein domain with any known receptor activity or potentiality. Construction and nomenclature of AdGFP vectors carrying liganded fibers with scissile knobs (Fig. 1) .
Each of the three ligands LAP25, QM10 and SY12, was inserted into the fiber of the backbone vector AdGFP-R7- knob, upstream of the Factor Xa cleavage site, to generate recombinant vectors AdGFP-LAP25-knob, AdGFP- QMlO-knob and AdGFP-SY12-knob, respectively. Trans- complementing HEK-293-Fiber cells were transfected with each of the Ad DNA genomes recombined with liganded fiber genes, and plaques isolated and amplified. However, we were not able to amplify the plaques obtained after transfection with AdGFP-LAP25-knob DNA, likely due to the cytotoxic effect provoked by the LAP25-liganded recombinant fiber. Normal virus growth and infectious virus stocks with high titers in trans-complementing 293 cells were obtained with AdGFPQMlO-knob and AdGFP-SY12- knob vectors, see Table 1 below.
Table 1. Biological characteristics of Ad5 vectors carrying unliganded or liganded fibers(a) .
Ad5 vector Virion titer(b) Infectious Ratio PP/mL titer(c) PP : PFU PFU/mL
AdGFP -WTFi (d) 1 . 00 x 10E12 1 . 50 x 10E11 7
AdGFP- 7-knob (=) 1 . 00 X 10E12 3 . 75 x 10E9 266
AdGFP-QMlO-knob 3 .20 x 10B11 4 . 00 x 10E9 80
AdGFP -SY12- knob 1 .10 X 10E12 1. 10 x 10 12 38
AdGFP - LAP25 - nob NA NA NA
<a) Samples of Ad5 vector, purified by ultracentrifugation in CsCl gradient, were assayed for infectivity and virion content . (b) The number of virions present in each sample, comprising of infectious and uninfectious virus particles, was calculated from the penton base protein content of the samples, determined by western blot analysis (36) , and the titer expressed as physical particles (PP) per mL.
(α' The titer in infectious particles was determined by plaque titration in 293 cells, and expressed as plaque forming units (PFU) per mL. (d) Control vector with wild-type, non-liganded, long-shafted fibres.
(e> Control vector with non-liganded, short-shafted fibers . The infectivity index (estimated from the ratio of physical to infectious particles per mL) of AdGFP-SY12- knob and AdGFP-QMlO-knob was 5- to 10-fold lower than that of the normal, long-shafted fiber-carrying vector AdGFP-WTFi, but 6- to 3-fold higher than the corresponding control vector AdGFP-R7-knob which carried non-liganded fibers of the same length (Table 1) .
After ablation of the knob by Factor-Xa digestion, the two liganded vectors were designated as AdGFP-QMlO and AdGFP-SY12, and the non-liganded, control vector as AdGFP-R7 (Fig. 1 c, e) . When analyzed by SDS-PAGE and immunoblotting, the three recombinant vectors showed fiber protein and fiber cleavage products migrating with the apparent molecular mass expected before and after cleavage by Factor Xa.
Efficiency and cell-specificity of gene transduction mediated by liganded AdGFP vectors The functionality of the fiber-inserted ligands was evaluated for AdGFP-QMlO-knob and AdGFP-SY12-knob in transduction assays of CF-KM4 cells, control MM39 cells and a variety of other cells from different species as targets, in comparison with their non-liganded counterpart AdGFP-R7-knob and with AdGFP-WTFi. Gene transfer efficiency (GTE) was expressed as the percentage of GFP-positive cells (Fig. 2 a) , but also as GTE index, as shown in Fig. 2 b. Since some cell populations were found to be heterogeneous in their levels of GFP expression, which was particularly high in a limited number of cells, the parameter of mean fluorescent intensity (MFI) was taken into account and included in the calculations of the GTE index. In most cells, gene transduction was 4- to 60-fold lower with the non-liganded, short-shafted vector AdGFP- R7-knob than with AdGFP-WTFi, as expressed by the percentage of GFP-positive cells (Fig. 2 a) , and the difference was even more pronounced as measured by the GTE index, ranging from one to three orders of magnitude (Fig. 2 b) . These results indicated that the length of the fiber shaft greatly influenced the mechanism of Ad- cell recognition. This was consistent with previous observations and with a recent modelization of the interaction between cell receptor and Ad capsid in the case of long and flexible, versus short and rigid fibers, showing that the length and flexibility of the fiber shaft are critical parameters for the usage of adenoviral receptors such as integrins or CAR, as well as for virus infectivity. However, the GTE was higher with AdGFP-R7- knob than with AdGFP-WTFi in MRC5 and CHO cells, and equivalent with both vectors in L20B cells (Fig. 2 a, b) . The level of GFP expression in cells transduced by
AdGFP-QMlO-knob, compared to its non-liganded version AdGFP-R7-knob, showed that the QM10 ligand consistently and significantly augmented the GTE in all the cell lines tested but to various degrees (Fig. 2 a, b) . This enhancement ranged from 2- to 40-fold, depending on the
MOI and the cell type. In LLC-MK2 and RD cells, GFP level was equivalent to that mediated by AdGFP-WTFi, whereas in other cell lines (A549, MRC5, HRT-18, L20B and CHO) it was 3- to 10-fold higher. In tracheal glandular cells, the difference between AdGFP-QMlO-knob and AdGFP-R7-knob was not significant for MM39 cells, and less than 3 -fold for CF-KM4 cells. AdGFP-SY12-knob gave similar levels of gene transfer as AdGFP-R7-knob (data not shown) , indicating that the effect observed with the decapeptide ligand GHPRQMSHVY was not simply a property of any foreign peptide inserted at that site in Ad5 vectors, but was sequence-related. However, our results also implied that QM10, although being isolated from CF-KM4 cell- internalized phages, was not tissue-specific or even species-specific. In fact, QM10 was more efficient in promoting gene transduction of A549 cells (terminal bronchial cells from human lung carcinoma) , MRC5 (primary fibroblastic cells from human foetal lung) , HRT-18 (human ileocecal adenocarcinoma cells) and RD cells (from human rhabdomyosarcoma) , as well as LLC-MK2 cells (simian kidney cells) , than the human tracheal glandular cells CF-KM4 from which it was issued.
Dose-dependence of gene transfer by the different vectors in airway cells . Since the possible low density of some cell surface receptors could mask the usefulness of the modifications of fibers at low vector doses, gene transduction was assayed at various MOI of AdGFP-WTFi, AdGFP-R7-knob and AdGFP-QMlO-knob in cell lines with different QMlO-ligand responsiveness, A549 (high) and CF-KM4 cells (low) , respectively (Fig. 2 a, b) . No difference in the levels of gene transfer was detectable at low infectious doses of the different vectors. At higher doses however, gene transduction of A549 cells was found to increase in a dose-dependent manner. A nearly linear dose-response was observed with AdGFP-QMlO-knob between 20 and 200 PFU/cell, and almost 100 % cells were found to be GFP- positive at the highest MOI, whereas GFP expression reached a plateau with AdGFP-WTFi (50 %) and AdGFP-R7- knob (35 %) for MOI higher than 100 PFU/cell (Fig. 2 c) . In CF-KM4 cells, a plateau was also reached at MOI-100 with AdGFP-R7-knob (25 %) and AdGFP-QMlO-knob (50 %) , whereas AdGFP-WTFi-mediated gene transduction increased linearly between 20 and 200 PFU/cell, with 90 % transduced cells at MOI-200 (Fig. 2 d) . Thus, the difference in the efficiency of gene transduction between WT and fiber-modified AdGFP vectors was better detected at relatively high vector doses. These results also suggested that limiting factors other than the number of cell receptors could exist in the CAR/integrins entry pathway, as for AdGFP-WTFi in A549 cells, as well as with vectors using alternative pathways, as for AdGFP- QMlO-knob in CF-KM4 cells
Absence of knob requirement for gene transfer mediated by AdGFP vectors carrying short-shafted, liganded fibers
We then determined whether the terminal knob domain of the fiber was required for gene transduction by our liganded vectors, and how efficient knobless vectors would be in this process. A549, HeLa, CF-KM4 and MM39 cells were incubated at 37°C for 1 h with each vector, and the gene transduction efficiency (GTE) index was compared between AdGFP-R7-knob and AdGFP-R7, AdGFP-QMlO- knob and AdGFP-QMlO, and AdGFP-SY12-knob and AdGFP-SY12, respectively. The ratios of GTE indices of the knobbed versus de-knobbed version of each vector gave an insight on the role of the knob domain in the interaction of liganded versus non-liganded vectors with the different cell targets. As a control for nonspecific proteolytic effect, AdGFP-WTFi vector carrying non-scissible WT fibers was subjected to Factor Xa digestion under the same conditions. Except for MM39 cells and control vector AdGFP-R7, for which de-knobbing drastically reduced the gene transduction efficiency, the other pairs of cell- vector involving AdGFP-R7 (±knob) or AdGFP-SY12 (±knob) seemed to be relatively unaffected by the presence or absence of the knob domain (GTE ratio of about 1 ; Fig. 3 b, d, i) . In contrast, ablation of the knob domain in AdGFP-QMlO resulted in a significant enhancement of gene transduction of the four cell lines, and in particular A549, HeLa and MM39 cells (5- to 8-fold; Fig. 3 f , h, i) . This implied that the knob domain was dispensable for achieving the early steps of the virus cycle which led to gene delivery by AdGFP-QMlO vector into CAR+ and CAR- cells . These results were conceivable for liganded vectors such as AdGFP-SY12 and AdGFP-QMlO, considering that the proteolytic removal of the knob had exposed the peptide ligands of the fiber shafts and made them directly accessible to their specific receptor molecules at the cell surface. However, the finding that the control, non- liganded vector AdGFP-R7 still transduced cells in its de-knobbed version indicated that our short-shafted AdGFP vectors, regardless of the occurrence of the knob or a cell ligand at the extremity of their short-shafted fibers, bound to the cell surface via domains of capsid components other than the fiber knob. The next set of experiments were designed to analyze the mechanism of the absence of knob requirement for gene transduction by our non-liganded control vector, and also to determine at which step(s) of the viral infection the QM10 ligand enhanced the efficiency of Ad-mediated gene transfer, attachment, endocytosis, endosomal release, nuclear addressing, or several of these four steps successively.
Role of cell ligands and knob domains in AdGFP-cell interaction
(i) Cell binding of liganded versus non-liganded vectors . Virus attachment to the cell surface was assayed at 4°C on CF-KM4, MM-39 and A549 cells, using Cy3-labeled virions of AdGFP-R7 (±knob) , AdGFP-QMlO (±knob) and AdGFP-SY12 (±knob) . Cell-bound fluorescence was determined by quantitative imaging in IF microscopy, and compared between knobbed and de-knobbed versions of each vector. The intensity of the signal of cell-bound viruses was very similar for the three knob-bearing vectors in each cell line, and the variations were within the range of experimental error (data not shown) . In no case the level of cell-binding of AdGFP-QMlO-knob could account for the 40-fold higer level of gene transfer observed with this vector in A549 cells, compared to that of the non-liganded vector AdGFP-R7-knob, or for its 3-fold higher level in CF-KM4 cells (refer to Fig. 2) . This suggested that the QM10 and SY12 peptides did not function as ligands of virus-attachment receptors in A549, CF-KM4 and MM39 cells, but acted at later steps, at least in the presence of the knob domain. This was consistent with the fact that QM10 and SY12 were two peptides derived from cell-internalized bacteriophages, and not from extracellular, plasma membrane-bound phages.
(ii) Cell binding of knobbed versus de -knobbed vectors . After ablation of the knob, a slight augmentation of the cell-binding level (ca. 2-fold) was observed for AdGFP-QMlO vector in the three cell lines, compared to AdGFP-QMlO-knob, but no significant variations were observed with non-liganded or SY12- liganded vectors, with or without their knobs (data not shown) . These results suggested that the mechanism of increase in gene transfer efficiency shown by AdGFP-QMlO- knob did not take place at the cell-attachment step, at least in a quantitative manner. They also suggested that the knob domain was dispensable for the step of cell attachment of the vectors, as already observed for the overall gene transduction process (refer to Fig. 3) . (iii) Antibody-mediated competition for cellular attachment of vectors via the knob domain . In our previous study, experiments using anti-knob 1D6.14 mAb have suggested that the fiber knob domain was involved in the binding of Ad5 to surface SA-GP molecules on CF-KM4, and to HS-GAG on MM39 cells. This was confirmed in the present study using AdGFP-WTFi vector carrying its natural, long-shafted fibers : a 98 % inhibition of attachment to CF-KM4 cells was observed in the presence of a large excess of 1D6.14 mAb, see Table 2 below. Table 2. Effect of blocking anti-knob monoclonal antibody (mAb) 1D6.14 on the attachment of non-liganded and liganded AdGFP vectors to CF-KM4 cells (a) .
GFP expression
Ad5 vector without with 1D6.14 Inhib tion of 1D6.14 cell- -binding (%)
AdGFP-WTFi 100 2 ± 1 98
AdGFP-R7- nob 100 81 + 12 19
AdGFP-QM10- -knob 100 89 ± 11 11
AdGFP-SY12- -knob 100 45 ± 8 55
(a) Cell attachment of Ad vectors was assayed by gene transduction following virus-cell binding experiments conducted at low temperature to prevent virus entry. Cell attachment was performed at a MOI of 100 for 2 h at 4°C, in the absence (control samples) or presence of aliquots of 1D6.14 ascite fluid corresponding to 15 μg of monoclonal IgG (ca. a 103-fold excess over the fiber content of the vectors) . The inoculum was then removed and cells post-incubated for 24 h at 37°C. GFP expression was quantitatively determined by FACS analysis, and its level expressed as the percentage of the values in corresponding control samples . Data presented were the mean of three separate experiments, ± SD. However, under the same conditions, knob-blocking antibody 1D6.14 had a much lower inhibitory effect on the cell attachment of short-shafted fiber vectors AdGFP-R7- knob and AdGFP-SY12-knob (about 20 % and 50 % inhibition, respectively) , and an even lesser effect ("10 % inhibition only) on AdGFP-QMlO-knob (Table 2) . This suggested that recombinant Ad5 carrying short-shafted fibers bound to cell surface molecules via sites of the knob domain different from the 1D6.14 epitope. Alternatively but not exclusively, the virus-cell binding could be mediated by viral capsid proteins or domains different from the fiber knob, e.g penton base, as for the non-liganded vector, or the new cellular ligand inserted into the fiber, as for AdGFP-QMlO-knob, or several binding sites simultaneously. In any case, this confirmed the major role played by the fiber shaft in controlling the interaction of Ad virions with cell surface receptors. The following experiments were aimed at determining which viral capsid protein (s) , or specific domain (s) thereof, could compensate for the absence of the knob or its blockade by mAb, and acted as alternative or/and auxiliary cell-attachment component (s) in the knobbed or de-knobbed versions of our AdGFP vectors .
Receptor-binding domains in the capsid of knobbed and deknobbed AdGFP vectors
Cell-binding competition assays were performed in the presence of purified Ad proteins, hexon, full-length fiber, knob, or penton base (WT or RGD-mutant R340E) , individually mixed with each vector, and added in large excess over their copy numbers in Ad virion. Vector and competitor were co-incubated at 4°C with CF-KM4 or A549 cells, to allow attachment but not entry. Unadsorbed virus was rinsed off, and cells assayed for GFP expression at 28 h pi. Cell attachment of AdGFP-QMlO-knob to CF-KM4 cells was not influenced by an excess of hexon or penton base proteins (Fig. 4a) . However, it was competed by knob protein to significant levels (~50%) , and to a lesser degree (25-30%) by full-length fiber, a difference which might reflect variations in accessibility of different sites on the viral capsid proteins. In its knobless version, AdGFP-QMlO was not competed by knob or fiber, as expected, but was competed to ~50% by WT penton base and R340E mutant. Similar patterns of competition for binding to CF-KM4 cells were observed with control vectors AdGFP-R7-knob and AdGFP-R7 (Fig. 4 a) . Only slight difference in cell-binding competition assays was observed between CF-KM4 and A549 cells for the different vectors (Fig. 4 b) : full-length fiber was apparently more efficient in competing with the knobbed vectors AdGFP-R7-knob and AdGFP-QMlO-knob for attachment to A549 cells, a result which might be due to the high accessibility of CAR to its fiber ligand. Taken together, our results suggested that the lack of knob in our short-shafted AdGFP vectors could be compensated for by other components of the viral capsid, mainly the penton base. The data obtained with penton base R340E mutant implied that binding site(s) on the penton base capsomer did not involve the RGD motif . This was reminiscent of our previous observation that in CF-KM4 cells, cell endocytosis and entry of Ad5Luc3 vector were apparently independent of penton base and RGD ligand. In A549 cell-binding assays however, penton base competed as poorly as fiber protein with the control knobless vector AdGFP-R7 (Fig. 4 b) . This pattern, and the fact that a maximum level of only 50 % inhibition was reached in other cell-binding competition assays using a 3 -log excess knob protein over knob-carrying vectors, as well as using a 3 -log excess excess penton base protein over knobless vectors, strongly suggested that several different domains on the viral capsid of short-shafted fiber vectors could serve alternatively or even simultaneously as cell-binding sites. Of note, cellular binding through hexon capsomer has been reported, although no significant competition effect was observed with hexon protein in our experiments (Fig. 4 a) . Likewise, the fiber shaft could participate in the cell- binding process to some extent. In particular, the fiber shaft domain of our AdGFP vectors still carried the conserved basic peptide KKTK at position 91-94, a motif which has been proposed to be responsible for the binding of Ad fiber to heparan sulfate proteoglycans and acidic carbohydrates. Moreover, some cell-binding activity for the nonviral tri erization peptide from the neck region of the human lung surfactant protein D inserted at the C- terminal end of the fiber shaft of our vectors could not be excluded. This would explain the absence of significant change in the nonliganded AdGFP-R7 vector infectivity after de-knobbing, as shown in Fig. 3.
Cellular uptake of knobbed versus deknobbed AdGFP vectors The mechanism of gene transfer enhancement mediated by the QM10 ligand in different cell lines was then investigated at the steps of endocytosis and entry of Ad virions . (i ) Endocytosis . Endocytosis of AdGFP-QMlO-knob, AdGFP-SY12-knob and AdGFP-R7-knob in their knobbed and deknobbed versions was then assayed in A549, MM39 and CFKM4 cells, using flow cytometry analysis of cells rendered fluorescent by intracellular Cy3-labeled virions. In the three cell lines, the number of internalized vector particles was not significantly different for AdGFP-R7-knob and AdGFP-SY12-knob, and deknobbing of these two vectors resulted in a slight increase in endocytosis in MM39 and CF-KM4 cells, and a decrease in A549 cells (Fig. 5a) . However, the difference in the number of endocytosed virions between AdGFP-QMlO- knob and AdGFP-QMlO was significant in A549 cells (2- fold; Fig. 5 a) , and CF-KM4 cells (5-fold ; Fig. 5 c) . This suggested that deknobbing of QM10 fibers resulted in a better exposure of the QM10 motifs, which in turn increased the efficacy of endocytosis and/or possibly further steps.
(ii ) Endosomolysis . Ad-mediated endosomolysis and vesicular escape of Ad virions was determined from the degree of Ad-mediated augmentation of cell protein synthesis inhibition by the toxin Ricin A (RcA) , as a result of their co-endocytosis and subsequent vesicular release. RcA agglutinin blocks the cell protein synthesis and its effect is enhanced by the endosomal escape of co- endocytosed Ad virions into the cytosol. The kinetics of protein synthesis inhibition was analyzed after incubation of cells with RcA and each of the three Ad vectors for 2 h at 37°C, at different RcA concentrations and constant input of Ad vector particles, and the 50%- inhibition value (IC50) was determined. In their knobbed versions, all three vectors showed similar curves of RcA dose-dependent inhibitory effect, with 10-20% residual protein synthesis at 20 pg RcA and 104 virions per cell, and IC50 values ranging within 0.8 to 4.0 pg RcA and 104 virions per cell, depending on the cell type (Fig. 6) .
Interestingly, the inhibitory effect of RcA on host- cell protein synthesis was consistently lower in the presence of knobless vectors AdGFP-R7, AdGFP-QMlO or AdGFP-SY12, compared to their knobbed versions, irrespective of the cell ligand. However, the difference was particularly striking with AdGFP-QMlO vector in CF- KM4 cells, in which a 15- to 20-fold augmentation of the IC50 value was observed, and in A549 cells, showing a 4- to 5-fold increase (Fig. 6) . Since de-knobbing did not negatively affect the cell attachment of AdGFP-QMlO vector and positively influenced its endocytosis, these results suggested that the fiber knob domain played a role in the endosomal release of coendocytosed Ad virions and toxin. The association of RcA + AdGFP-QMlO-knob was the pair of co-endocytosed macromolecules which was apparently the most sensitive to the de-knobbing process in CF-KM4 and A549 cells, suggesting that the QM10 ligand acted mainly at the step of Ad endocytosis and cell entry. This could theoretically be explained by at least two mechanisms: (i) QM10 ligand decreased the level of endocytosis of Ad vector, but this hypothesis could be excluded since it was in contradiction with the FACS analysis of intracellular fluorescent-labeled virions (refer to Fig 5) , and by the positive effect of QM10 on the efficiency of Ad-mediated gene transfer, (refer to Fig. 2) . (ii) A significant proportion of AdGFP-QM10±knob was endocytosed in an endocytic compartment different from the one in which most RcA and AdGFP-R7-knob, or RcA and AdGFP-SY12-knob, were co-endocytosed.
Endocytic compartments of non-liganded and liganded AdGFP vectors
The cellular compartments in which AdGFP-R7- nob, AdGFP-SY12-knob and AdGFP-QMlO-knob were endocytosed were investigated in CF-KM4 and A549 cells. Cells were co- incubated with aliquots of Cy3- and Cy2-labeled particles of each vector pairwise, and analyzed by confocal IF microscopy. Only partial co-localization was observed for AdGFP-QMlO-knob and AdGFP-SY12-knob (Fig. 7 a-c) , whereas the majority of AdGFP-R7-knob and AdGFP-SY12-knob virions were found to co-localize in the same vesicular compartment (Fig. 7 d-f) . Co-localization events were very rare with AdGFP-QMlO-knob and control vector AdGFP- R7-knob (Fig. 7 g-i) , and even more rarely observed in cells co-infected with knobless AdGFP-QMlO and AdGFP-R7- knob, where most of the Cy2 and Cy3 signals emanated from separate compartments (Fig. 7 j-1). These patterns suggested that our vectors used different endocytic pathways, one common to AdGFP-R7-knob and AdGFP-SY12- knob, another independent one used by AdGFP-QMlO-knob and its knobless version. The nature of the compartments in which vectors resided was then analyzed using fluorescent probes of endosomal vesicles. Cells incubated at 37°C for 45 min with Cy3 -labeled vectors were harvested, fixed and permeabilized, then reacted with FITC-labeled antibodies specific for endosomal compartment markers, and examined in confocal IF microscopy. Transferrin receptor (TFR) and Rab protein Rab5 were both used as markers of clathrin- coated vesicles and early endosomes, Rab4 for early and recycling endosomes, Rabll for recycling endosomes of epithelial cells, and LAMP-1 for late endosomes and lysosomes. Both AdGFP-R7-knob and AdGFP-SY12-knob co- localized with the IF signals of TFR (Fig. 8 a-c) , and Rab5, but not with LAMP-1 (Fig. 8 g-i) , confirming the role of clathrin-coated vesicles and early endosomes in early steps of endocytosis and entry of Ad5, a prototype member of species C Ad. In contrast, no significant occurrence of AdGFP-QMlO-knob within the TFR/Rab5 endosomal compartment could be detected at 45 min (Fig. 8d-f) . Instead, co-localization of AdGFP-QMlO-knob and LAMP-1 signals was observed with a high a frequency (Fig. 8 j-1) . This indicated that a significant proportion of AdGFP-QMlO-knob vector particles followed an endocytic pathway different from that of AdGFP-R7-knob and AdGFP- SY12-knob, and were preferentially endocytosed and addressed to the late, acidic endosomal compartment.
This was confirmed by the results of co-incubation of our vectors with human α2-macroglobulin (α2M) , a ligand of cell receptors targeted to one of the two well characterized endosomal pathways. Tf ligands have been shown to be directed to early and recycling endosomes that are relatively alkaline (pH "6.0-6.8), whereas α2M ligands do not recycle to the cell surface but progress rapidly through the endosomal pathway to late, relatively acidic (pH ~5) endosomes (Sonowane et al. 2003 J. Cell Biol. 160: 1129-1138). FITC-labeled α.2M and each of our three Cy3-labeled AdGFP vectors were therefore incubated with CF-KM4 and A549 cells, and their possible co- localization analyzed by confocal IF microscopy. AdGFP- R7-knob and AdGFP-SY12-knob were very rarely found to co- localize with α2M. By contrast, a majority of AdGFP-QMlO- knob vector particles were found to reside in the same vesicular compartment as α2M after 45-60 min incubation at 37°C (Fig. 9 a-c) . In its knobless version, the events of co-localization of AdGFP-QMlO with oc2M also occurred with a high frequency (Fig. 9 d-f) .
EM analysis of intracellular vector particles
Samples of A549 and CF-KM4 cells were incubated at 37°C for 1 h with AdGFP-R7-knob and AdGFP-QMlO-knob at 104 virions per cell, and processed for EM. The average number of cell-associated virions, determined from at least 30 sections of individual cells, was about 10-fold higher for AdGFP-QMlO-knob than for non-liganded AdGFP- R7-knob, a result consistent with our FACS analysis. As expected, most of the cell-associated virions of AdGFP- R7-knob were found in typical endosomal vesicles (not shown ; refer to Fig. 10, d) , and rare particles were visible at the cell surface. In contrast, AdGFP-QMlO-knob virions were found in greater numbers both at the cell surface (Fig. 10, a-c) , and in intracellular compartments (Fig. 11) . Enlargement of plasma membrane-bound AdGFP- QMlO-knob virions often revealed thin filaments linking one or more vertices of the vector capsid to the cell surface, as depicted in Fig. 10, b, c. The average length of these links, 145 ± 6 A (n = 12) , was compatible with the dimensions of the short-shafted fibers of our vectors (seven repeats; R7) , calculated from experimental determinations. Without taking into account the 36 residues of our trimerisation motif and the 13 residues of the linker, the knob diameter of 49 A and the shaft repeat increment of 13.4 to 13.5 A per repeat gave a theoretical value of 94 A for the R7-shaft, and of 143 A for the R7-knob fiber.
The intracellular virions of AdGFP-QMlO-knob virions were seen within A459 cells in two unequally represented types of intravesicular compartments: on rare occasions, single virions occurred in vesicles with discontinuities in the vesicular membrane leaflet (Fig. 10, d) , resembling the vesicles in which the control vector AdGFP-R7-knob was endocytosed. However, the most frequent events consisted of several AdGFP-QMlO-knob virions present in the same vesicles containing electron- dense heterogeneous material (Fig. 11, a) , amorphous inclusions or both (Fig. 11, b) , or in large vesicles containing multiple vesicles of smaller diameter reminiscent of multivesicular bodies (Fig. 11, c) . The morphological aspect of these vesicular contents was characteristic of the lysosomal compartment . Higher magnifications of the vector-vesicular membrane junctions showed fuzzy material interrupting the dark line of the membrane leaflet (Fig.11, d, e) . These gaps might represent the first stages of endosomolysis. A similar pattern of cellular compartmentalization was observed in A549 cells with knobless AdGFP-QMlO vector, whose most virions were found associated with multivesicular bodies (Fig. 12, a, b) , or with multilamellar inclusions of phospholipids already described in A549 cells (Fig. 12, c) . These observations suggested that AdGFP-QMlO-knob and AdGFP-QMlO could follow the same endocytic pathway as Ad5WT through early endosomes leading to their endosomal escape, but that late endosomes and lysosomes represented the preferred endocytic compartment whereto QMlO-liganded vectors were rapidly redirected and accumulated. The cytoplasm of CF-KM4 cells has been shown to contain a very rich vesicular network, and this made difficult to distinguish between early and late endosomal compartments in these particular cell line by using conventional EM. However, virions of control vectors AdGFP-R7-knob and AdGFP-WTFi were more frequently found within vesicles with characteristics of clathrin-coated pits (Fig. 13, a,b,d,e), compared to QMlO-liganded vectors, whose majority was found within large vesicles reminiscent of late endosomes or lysosomes (Fig. 13, f, g) . No obvious difference was observed between AdGFP- QMlO-knob and its knobless version AdGFP-QMlO in terms of preferred vesicular residence (Fig. 13, compare panels f and g) . Careful examination of intravesicular virions under the EM showed that most of the particles of AdGFP- QMlO-knob and AdGFP-QMlO presented a blurred aspect and/or irregular morphology, whereas AdGFP-WTFi and AdGFP-R7-knob virions had retained their sharp contour and regular, icosahedral shape (Fig . 13, compare panels a-e to f-g) . This suggested that at 1 h post-infection at 37°C in CF-KM4 cells, endocytosed virions of AdGFP-QMlO- knob or AdGFP-QMlO were at a more advanced stage of uncoating than those of non-liganded vectors, a process which might contribute to the higher efficiency of transduction of CF-KM4 cells by QMlO-liganded vectors.
EXAMPLE 2
Ligand Insertion into the pIX protein and transduction of melanoma cells
MATERIALS AND METHODS
Cells Melanoma cells derived from human patient explants referred to as Ml, M2, M3, M4 (a gift from the Hospices Civils de Lyon) , and M8 (a gift from Dr. A. Blondel, Institut Cochin, Paris) and human melanoma cell line MeWo (from ATCC) were grown at 37°C in RPMI supplemented with 10% Fetal Calf Serum.
Ligand insertion Insertion of SY12 or QM10 peptide ligands into the gene encoding the human adenovirus serotype 5 pIX protein was performed as follows. The pIX gene was mutated using the QuickChange site-directed mutagenesis system (Stratagene) , to introduce mutations and additional sequence (s) into the pIX coding sequence, into a novel Bam H I site inserted between the base codons encoding Leu-131 and Lys-132 residues within the C-terminal moiety of pIX. Introduction of the coding sequence of QM10 and SY12 peptides between codons encoding Leul31 and Lysl32 residues within C terminal part of pIX The QuickChange site-directed mutagenesis system (Stratagene) was used to introduce a Bam HI restriction site between the base codons encoding Leu-131 and Lys-132 within the pIX coding sequence. The following sense and antisense oligonucleotides were used : 5'-cgc cag cag gtt tct gcc ctg gga tec aag get tec tec cct ccc aat gcg g3 ' and 5'-c cgc att ggg agg gga gga age ctt gga tec cag ggc aga aac ctg ctg gcg-3'. For insertion of the QM10 ligand, the oligonucleotides 5 '-cgc gga tec gga cac ccc cga cag atg tea cac gtc tac tag gga tec gcg-3' and 5 ' -gcg gga tec eta gta cac gtg tga cat ctg teg ggg gtg tec gga tec gcg-3 ' were hybridised to each other and cloned into the Bam HI restriction site to directly introduce the nucleotide coding sequence of QM10 peptide within the C-terminal portion of pIX. Alternatively, for insertion of the SY12 ligand, the oligonucleotides 5 '-cgc gga tec aca gca tac tec tec tac atg aag gga gga ggc aaa ttt tag gga tec gcg-3' and 5 ' -gcg gga tec eta aaa ttt gcc tec tec ctt cat gta gga gga gtc tgc tgt gga tec gcg-3 ' were hybridised to each other and cloned into the Bam HI restriction site to directly introduce the nucleotide coding sequence of SY12 peptide within the C-terminal part of pIX. The resulting amino acid sequence of the C-terminal domain of the modified pIX protein is as follows, starting from the Leu residue at position 100: 100-LTALLAQLDSLTRELNWSQQLLDLRQQVSALGHPRQMSHVY-C02H for the pIX-QMlO peptide, and 100-LTALLAQLDSLTRELNWSQQLLDLRQQVSALTAYSSYMKGGKF-C02H for the pIX-SY12 peptide. As a control, an adenoviral vector carrying pIX proteins liganded with the model peptide ligand 7K was used. Insertion of an oligo-lysine peptide ligand (Lys- Lys-Lys-Lys-Lys-Lys-Lys, abbreviated 7K) in the pIX protein between Leu-131 and Lys-132 residues within the C-terminal domain of pIX was performed using the same protocol as for pIX-QMlO and pIX-SY12. The oligonucleotides 5 ' -gcg gga tec aag aag aag aag aag aag aag taa gga tec gcg -3' , and 5'- gcg gga tec tta ctt ctt ctt ctt ctt ctt ctt gga tec gcg-3 ' were hybridised to each other and cloned into the Bam HI restriction site to directly introduce the nucleotide coding sequence of 7K peptide within the C-terminal portion of pIX. The 7K- liganded pIX sequence is: 100-LTALLAQLDSLTRELNWSQQLLDLRQQVSALKKKKKKK-C02H .
Vectors and reporter gene
All adenoviral vectors contained, in addition to alterations of the pIX-encoding gene, a deletion in El and in E3, and carry the LacZ gene (encoding the bacterial beta-galactosidase) driven by the CMV promoter, in lieu of the deleted El gene-encoding region. The vectors were referred to as AdβGal-pIX-QMlO, AdβGal-pIX-
SY12 and AdβGal-pIX-7K, respectively, and AdβGal-pIX-WT for the control, unmodified pIX vector. Vectors were generated and vector stocks produced in the conventional El-trans-complementing cell line HEK-293 cells.
RESULTS
Efficiency of gene transduction
The gene transduction efficiency of the QM10- or SY12-liganded pIX constructs, AdβGal-pIX-QMlO and AdβGal- pIX-SY12, was evaluated in a number of melanoma cell explants issued from various patients, or in a human melanoma cell line. Efficiency of gene transduction was compared with (i) AdβGal-pIX-WT, an adenoviral vector carrying unmodified capsid proteins pIX, and (ii) AdβGal- pIX-7K, an adenoviral vector carrying pIX proteins liganded with the model peptide ligand 7K. The oligo- lysine ligand is supposed to bind nonspecifically to sialic acid residues of most sialylated cell surface glycoproteins, or/and to acidic glycosamino-glycans such as heparan sulfate, also present on plasma membrane of most cell types.
Conditions of adenovirus infection and reporter gene transduction Adenoviral vectors in the range of concentrations of 100, 200, 500 and 2,000 virus particles/cell were added to target cell monolayers for 1 h at 4°C. The inoculum was removed and the cells were washed twice with cold medium, then further incubated for 48 h at 37°C. Cells were then fixed and stained to determine beta- galactosidase activity, using a beta-galactosidase staining kit (Chemicon) . Infected cells, visible as blue cells (adenovirus-positive cells) were then counted under the light microscope. Alternatively, the beta- galactosidase activity was determined on the whole cell lysate, and monitored using a chemiluminescent substrate (luminescent beta-galactosidase detection kit, Clontech, Palo Alto) .
Adenoviral vector infection and gene transduction efficiency
For melanoma cells Ml, M2, M3 and M4, the infectivity and transducing capacity of AdβGal-pIX-QMlO was significantly higher than those of other vectors : a factor of 10- to 100-fold was obtained compared to the model vector AdβGal-pIX-7K, and a factor of 1, 000-fold, compared to AdβGal-pIX-WT carrying unmodified capsid protein pIX (Figure 14) . For melanoma cells M8, the infectivity and transducing capacity of AdβGal-pIX-QMlO and AdβGal-pIX- SY12 were equivalent, but significantly higher (by a factor of 10- to 100-fold) than those of AdβGal-pIX-WT (Figure 15) . However, AdβGal-pIX-SY12 infects and transduces melanoma cell line MeWo with a 100-fold higher efficiency than of AdβGal-pIX-QMlO, and 1, 000-fold higher than AdβGal-pIX-WT (Figure 16) .
CONCLUSION
This study demonstrates that : (i) QM10 and SY12 peptide ligands can be inserted at the C-terminus of the Ad5 pIX protein, and generate a viable virus progeny in trans-complementing cell line HEK-293 cells. (ii) Both AdβGal-pIX-QMlO and AdβGal-pIX-SY12 show a similar level of infectivity and gene transduction capacity in a variety of human melanoma cells, a cell type which is known to be non permissive or poorly permissive to adenovirus, compared to a non-liganded vector (AdβGal-pIX-WT) or a vector liganded by the nonspecific oligo-lysine peptide (AdβGal-pIX-7K) . (iii) The level of infectivity of AdβGal-pIX-QMlO and AdβGal-pIX-SY12 in a variety of melanoma cells from human melanoma explants is 2-log to 3-log higher than that of AdβGal vector with non-liganded pIX. (iv) QMIO and SY12 using the pIX protein as the insertion platform will allow retargeting of Ad5 vectors liganded with these tumour-specific and/or selective ligands. This is an advantageous alternative to the fibre platform more generally used. The use of QMIO and SY12 ligands therefore expands the applicability of adenoviral vectors to cancer gene therapy. (v) Based on our knowledge of the pIX structure and its localisation in the capsid of Ad5 (Ref. Rosa-Catarava et al., J. Virol. , 2001; Fabry et al., 2005, and our unpublished data) , we can predict that the nucleotide sequences coding for the QM10 or SY12 peptides could be inserted at other sites than those cited above (between residues 131 and 132) , e.g. between codons coding for other residues within the C-terminal portion of the pIX protein, from amino acid position 100 to the terminal amino acids 139 and 140. (vi) On the basis of knowledge of pIX structure and its localisation in the capsid of Ad5 (Rosa-Catarava et al., 2001, 75 : 7131-41; Fabry et al . EMBO J. 2005 April 21, in press, it is predicted that nucleotide sequences coding for the QM10 or SY12 peptides could be inserted within the C-terminal portion of a pIX protein different from the WT pIX, i.e. containing other mutations/modifications in its C-terminal domain, to permit more accessibility and/or functionality of the ligands. By way of example, pIX could contain an extra amino acid sequence, a ^linker', at its C-terminus, which would allow the QMIO or SY12 to protrude further at the surface of viral capsid. This linker could have different lengths in terms of amino acid number.

Claims

1. A vector comprising a nucleic acid molecule and a peptide or peptide derivative, said peptide or peptide derivative comprising a peptide of general Formula (I) :
R1-R2-R3-R4-R5-R6 (I) wherein:
Rx is arginine or lysine; R2 is glutamine or asparagine; R3 is methionine; R4 is serine or threonine; Rs is histidine, tryptophan, arginine or lysine; and R6 is valine, leucine or isoleucine; or a functionally active fragment thereof.
2. The vector of claim 1 wherein the peptide of general Formula (I) is selected from
R-Q-M-S-H-V R-Q-M-T-H-I R-Q-M-S-H-L R-Q-M-T-W-V R-Q-M-S-H-I R-Q-M-T-R-L R-Q-M-S-W-V R-Q-M-T-R-V R-Q-M-S-R-L R-Q-M-T-W-L R-Q-M-S-R-V R-Q-M-T-W-I R-Q-M-S-W-L R-Q-M-T-R-I R-Q-M-S-W-I R-Q-M-T-K-V R-Q-M-S-R-I R-Q-M-T-K-L R-Q-M-S-K-V R-Q-M-T-K-I R-Q-M-S-K-L R-N-M-S-H-V R-Q-M-S-K-I R-N-M-S-H-L R-Q-M-T-H-V R-N-M-S-H-I R-Q-M-T-H-L R-N-M-S-W-V R-N-M- S-R-L K-Q-M-T-R-V R-N-M- S-R-V K-Q-M-T-W-L R-N-M- S-W-L K-Q-M-T-W-I R-N-M- S-W-I K-Q-M-T-R-I R-N-M- S-R-I K-Q-M-T-K-V R-N-M- S-K-V K-Q-M-T-K-L R-N-M- S-K-L K-Q-M-T-K-I R-N-M- S-K-I K-N-M-S-H-V R-N-M- T-H-V K-N-M-S-H-L R-N-M- T-H-L K-N-M-S-H-I R-N-M- T-H-I K-N-M-S-W-V R-N-M- T-W-V K-N-M-S-R-L R-N-M- T-R-L K-N-M-S-R-V R-N-M- T-R-V K-N-M-S-W-L R-N-M- T-W-L K-N-M-S-W-I R-N-M- T-W-I K-N-M-S-R-I R-N-M- T-R-I K-N-M-S-K-V R-N-M- T-K-V K-N-M-S-K-L R-N-M- T-K-L K-N-M-S-K-I R-N-M- T-K-I K-N-M-T-H-V K-Q-M- S-H-V K-N-M-T-H-L K-Q-M- S-H-L K-N-M-T-H-I K-Q-M- S-H-I K-N-M-T-W-V K-Q-M- S-W-V K-N-M-T-R-L K-Q-M- S-R-L K-N-M-T-R-V K-Q-M- S-R-V K-N-M-T-W-L K-Q-M- S-W-L K-N-M-T-W-I K-Q-M- S-W-I K-N-M-T-R-I K-Q-M- S-R-I K-N-M-T-K-V K-Q-M- S-K-V K-N-M-T-K-L K-Q-M- S-K-L K-N-M-T-K-I K-Q-M- -S-K-I K-Q-M- T-H-V K-Q-M- T-H-L K-Q-M- -T-H-I K-Q-M- T-W-V K-Q-M- -T-R-L or functionally active fragments thereof.
3. The vector of claim 1 or claim 2 wherein the peptide of general Formula (I) has the sequence RQMSHV.
4. The vector of claim 1 wherein the peptide or peptide derivative comprises a peptide of general Formula (II) :
R7-R8-R9-(I)-R10 (II) wherein:
(I) is a peptide of general Formula (I) as defined in claim 1; R7 is glycine or alanine; R8 is histidine, tryptophan, arginine or lysine; R9 is proline; R10 is tyrosine or phenylalanine or a functionally active fragment thereof.
5. The vector of claim 4 wherein the peptide of Formula (II) has the sequence GHPRQMSHVY.
6. A vector comprising a nucleic acid molecule and a peptide or peptide derivative, said peptide or peptide derivative comprising a peptide of general Formula (III) l ~ R_ ~ ^3 ~ R- - R5 ~ &6 ~ &7 ~ ^8 ~ ^-9 ~ Ε-l ( I I I )
wherein :
Ri is threonine; R2 is alanine; R3 is tyrosine; R4 is serine; Rs is serine R6 is tyrosine R7 is any amino acid, R8 is any amino acid; R9 is glycine; and R10 is glycine
or a functionally active fragment thereof .
7. The vector of claim 6 wherein R7 is methionine, leucine, isoleucine, valine, threonine, tryptophan or phenylalanine; and
Rs is lysine or arginine
8. The vector of claim 7 wherein the peptide of Formula III has the sequence TAYSSYMKGGKF .
9. The vector of claim 5 or 6 wherein the peptide or peptide derivative comprise a peptide of general Formula (IV) :
(IID-Rn-Ru (IV) wherein: (III) is a peptide of general Formula (III) as defined in claim 6; Rlx is lysine or arginine R12 is phenylalanine or tyrosine or a functionally active fragment thereof, wherein said peptide or peptide derivative does not have the sequence TAYSSYMKGGKF.
10. A modified adenovirus incorporating in one or more of its capsid proteins a peptide or peptide derivative as defined in any one of claims 1 to 9 or a functionally active fragment thereof .
11. The modified adenovirus of claim 10 wherein the capsid protein is selected from the group consisting of penton fiber, hexon, or protein IX.
12. A peptide or peptide derivative incorporating a region having the sequence of Formula (I), (II), (III) or (IV) as defined in any one of claims 1 to 9.
13. The peptide or peptide derivative of claim 12 wherein the peptide or peptide derivative incorporates a region having the sequence GHPRQMSHVY or TAYSSMKGGKF or a functionally active fragment of either sequence.
14. A method of gene delivery which method comprises contacting a cell population ex vivo with the vector of any one of claims 1 to 9 or the modified adenovirus of either of claims 10 or 11 or the peptide of claims 12 or 13.
15. The method of claim 14 wherein the cell population is of mammalian origin.
16. The method of claim 15 wherein the cell population is a human cell line.
17. The vector of any one of claims 1 to 9 or the modified adenovirus of either claim 10 or 11 or the peptide of either claim 12 or 13 for use in therapy.
18. The vector of claim 17 wherein the therapy is an anti-melanoma therapy.
19. The use of the vector of any one of claims 1 to 9 or the modified adenovirus of either claim 10 or 11 or the peptide of either claim 12 or 13 in the manufacture of a medicament for the treatment of cancer or cystic fibrosis.
20. The use of claim 19 wherein the cancer is a melanoma .
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