WO1993022431A1 - Systemes de vecteurs epidermiques constitutifs et inductifs - Google Patents

Systemes de vecteurs epidermiques constitutifs et inductifs Download PDF

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WO1993022431A1
WO1993022431A1 PCT/US1993/003993 US9303993W WO9322431A1 WO 1993022431 A1 WO1993022431 A1 WO 1993022431A1 US 9303993 W US9303993 W US 9303993W WO 9322431 A1 WO9322431 A1 WO 9322431A1
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vector
nucleic acid
cassette
acid sequence
loricrin
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PCT/US1993/003993
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Dennis R. Roop
Joseph A. Rothnagel
David A. Greenhalgh
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Baylor College Of Medicine
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Priority to JP5519487A priority Critical patent/JPH08501202A/ja
Priority to AU42213/93A priority patent/AU669713B2/en
Priority to EP93910875A priority patent/EP0644933A4/fr
Publication of WO1993022431A1 publication Critical patent/WO1993022431A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/32—Vector systems having a special element relevant for transcription being an silencer not forming part of the promoter region
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates generally to expression vectors for use in expressing proteins and polypeptides in epidermal cells. More
  • a constitutive vector consisting of the loricrin gene promoter, its 5' flanking region, its 5' transcribed but untranslated region, its intron, its 3' transcribed but untranslated region, its contiguous non-coding DNA containing the gene's natural transcriptional termination region and its 3' flanking region. It further relates to an
  • inducible vector consisting of the K6 keratin gene promoter, its 5' flanking region, its 5' transcribed but untranslated region, its first intron, its 3' transcribed but untranslated region, its contiguous non-coding DNA containing the gene's natural transcriptional termination region and its 3' flanking region. Additionally it relates to the treatment of disease
  • the skin is the largest organ in the human body and, due to its accessibility, it is an attractive target for gene therapy.
  • the outer layer is the largest organ in the human body and, due to its accessibility, it is an attractive target for gene therapy.
  • epidermis 30 of the skin is called the epidermis, and it is particularly attractive since epidermal cells can be grown in vitro from normal and affected patients, are easily transformed genetically by vectors, and can be readily reintroduced by autografting.
  • Previous studies investigating the feasibility of using epidermal cells for gene therapy have only considered this ex vivo approach. These investigations utilized retroviral vectors and their promoters to introduce and express foreign genetic material in epidermal cells. Even though the epidermis is avascular, these studies demonstrated that proteins expressed in the epidermis were able to traverse the epidermal-dermal barrier and achieve systemic distribution (Morgan et al., Science, Vol. 237, pp. 1476-1479, (1987)? Feiyves et al., PNAS USA, Vol. 86, pp. 8803-8807, (1989)fGarlick et al., J. Invest. Dermatol., Vol. 97, pp.
  • the epidermis is a continuously regenerating stratified squamous epithelium.
  • Differentiated epidermal cells are the progeny of proliferative cells located in the basal cell layer and there is substantial evidence suggesting that the regeneration process occurs in proliferative units composed of slowly cycling, self-renewing stem cells, proliferative but non- renewing transit amplifying cells, and post-mitotic maturing epidermal cells (Iversen, et al., Cell Tissue Kinet., Vol. 1, pp. 351-367, (1968); MacKenzie, et al., Nature, Vol. 226, pp. 653-655, (1970); Christophers, et al., J. Invest. Dermatol, Vol. 56, pp.
  • the maturation process (terminal differentiation) is initiated when epidermal cells withdraw from the cell cycle and migrate from the basal layer into the spinous layer. Maturation continues as spinous cells migrate into the granular layer and terminates with the formation of the stratum corneum. Morphological and biochemical studies have shown that terminal differentiation occurs in stages. (Matoltsy, J. Invest. Dermatol., Vol. 65, pp. 127-142, (1975)).
  • Keratins K5 and K14 are m ⁇ products of basal epidermal cells (Woodcock-Mitchell, et al., J. Cell Biol., Vol. 95, pp. 580- 588, (1982)). These proteins assemble into 10 nm filaments (intermediate filaments [IF]) and, together with microtubules (tubulin) and microfilaments (actin), comprise the cytoskeleton of epidermal cells (Steinert, P.M., et al., Cell, Vol. 42, pp. 411-419, (1985)).
  • Kl and K10 Another differentiation-specific pair of keratins
  • IF containing Kl and K10 replace those containing K5 and K14 as the major products of cells in the spinous layer (Woodcock-Mitchell, et al., J. Cell Biol., Vol. 95, pp. 580-588, (1982); Roop, et al., Proc. Natl. Acad. Sci., USA, Vol. 80, pp. 716-720, (1983); Schweizer, et al, Cell, Vol. 37, pp. 159-170, (1984)).
  • the keratin IF formed by these proteins assemble into bundles.
  • transglutaminase catalyzes the crosslinking of involucrin and loricrin, by the formation of ( ⁇ -glutamyl) lysine isopeptides, into a highly insoluble comified envelope which is located just beneath the plasma membrane (Rice and Green, Cell, Vol. 11, pp. 417-422, (1977); Mehrel, et al., Cell, Vol. 61, pp. 1103-1112, (1990)).
  • K5 Lersch, et al., Mol. and Cell Biol., Vol.
  • loricrin a major keratinocyte cell envelope protein (Mehrel et al., Cell, Vol. 61, pp. 1103-1112, (1990)). Although this gene is normally only expressed in the most differentiated layers of the epidermis, the present invention demonstrates that it possible to remove sequences that normally restrict expression of the loricrin gene in undifferentiated cells and achieve high levels of expression in undifferentiated epidermal cells (greater than the viral promoter of
  • this vector is constitutively expressed in epidermal cells at all differentiation states.
  • the present invention takes advantage of the expression characteristics of another gene encoding the K6 keratin to construct an inducible vector.
  • the K6 gene is normally never expressed in the epidermis, but it can be induced under hyperproliferative conditions such as wound healing (Weiss, et al., J. Cell Biol, Vol. 98, pp. 1397-1406, (1984); Nakazawa, et al., J. Cell Biol, Vol. 103, pp. 561a, (1986); Stoler, et al., J. Cell Biol, Vol. 107, pp. 427-446, (1988) and topical application of retinoic acid (Rosenthal et al., J. Invest.
  • An object of the present invention is a loricrin constitutive vector for efficient expression of nucleic acid sequences in epidermal cells.
  • An additional object of the present invention is a keratin K6 inducible vector for regulated expression of nucleic acid sequences in epidermal cells.
  • Another object of the present invention is an in vivo method of transducing epidermal cells with a constitutive or inducible vector.
  • a further object of the present invention is a bioreactor for producing proteins and polypeptides.
  • An additional object of the present invention is an enhanced method of wound healing or healing of surgical incisions.
  • Another object of the present invention is a method of treating skin ulcers.
  • An additional object of the present invention is a method of treating psoriasis.
  • a further object of the present invention is a method of treating cancer.
  • a loricrin constitutive vector for efficient expression of nucleic acid sequences in epidermal cells comprising a 5' flanking region of the loricrin gene, said flanking region including a TATA box, a cap site and a first intron and an intron/exon boundary, all in appropriate sequential and positional relationship for expression of a nucleic acid cassette; a 3' flanking sequence of the loricrin gene; and a linker having a unique restriction endonuclease site at the location of the start and stop codon, said linker connecting the 5' flanking region to the 3' flanking sequence and said linker further providing a position for inserting the nucleic acid cassette which includes the specific nucleic acid sequence to be expressed.
  • the loricrin constitutive vector has a 5' flanking region of approximately 1.5 kb, an intron of approximately 1.1 kb and a 3' flanking sequence of approximately 2.1 kb.
  • the loricrin constitutive vector also includes a poly-linker.
  • An alternative embodiment of the present invention is a keratin K6 inducible vector for regulated expression of a nucleic acid sequence in epidermal cells, comprising a 5' flanking region of the keratin K6 gene, said flanking region including a TATA box, a cap site, a first intron and an intron/exon boundary, all in sequential and positional relationship for expression of a nucleic acid cassette; a 3' flanking sequence of the keratin K6 gene; and a poly-linker having a pluraHty of restriction endonuclease sites, said poly-linker connecting the 5' flanking region to the 3' flanking sequence and further providing a position for insertion of the nucleic acid cassette which includes the specific nucleic acid sequence to be expressed.
  • the keratin K6 inducible vector 5' flanking region of approximately 8.0 kb, an intron and intron/exon boundary of approximately 0.56 kb and the 3' flanking sequence of approximately 1.2 kb.
  • restriction endonuclease sites in the linker or poly-linker are selected from the group consisting of Cla I, Not I, Xma I, Bgl II, Pac I, Xho I, Nhe I and Sfi I.
  • the nucleic acid cassette of the constitutive or inducible vectors, contains a sequence coding for a protein, polypeptide or antisense RNA.
  • bioreactor comprising transduced epidermal cells including either the loricrin constitutive or keratin K6 inducible vectors.
  • the bioreactor can produce a variety of compounds selected from proteins, polypeptides, antisense RNA.
  • the loricrin constitutive or keratin K6 inducible vectors are used for the treatment of wounds, surgical incisions, psoriasis, skin ulcers and cancer.
  • the method of the present invention can also be used for vaccination by transducing epidermal cells with a loricrin constitutive or keratin K6 inducible vector having proteins or polypeptides which induce an immunological response.
  • Another embodiment of the present invention is the nucleotide sequences for the loricrin gene and loricrin constitutive vector.
  • Another embodiment of the present invention is the nucleotide sequences for the keratin K6 gene and keratin K6 inducible vector.
  • Figure 1 is a schematic drawing of the mouse loricrin gene and the constitutive epidermal vector derived from its regulatory sequences.
  • Figure 2 shows the expression characteristics of the constitutive epidermal vector in undifferentiated and differentiated epidermal cells utilizing a reporter gene encoding chloramphenicol acetyl transferase (CAT).
  • CAT chloramphenicol acetyl transferase
  • Figure 3 shows the expression characteristics of the constitutive epidermal vector in vivo utilizing a reporter gene encoding E. coli ⁇ -galactosidase.
  • Figure 4 demonstrates the suppression by Vitamin D 3 of a novel negative regulatory element from the human Kl keratin gene (HK1.NRE).
  • Figure 5 is a schematic representative of the constitutive epidermal vector which can be suppressed by Vitamin D s via insertion of the HK1.NRE.
  • Figure 6 is a schematic drawing of a derivative of the mouse K6 keratin gene (BCM-MK6(A)-HK1).
  • Figure 7 shows the expression characteristics of BCM-MK6(A)-HK1 in transgenic animals.
  • Figure 8 is a schematic drawing of the mouse K6 keratin gene and the proposed construction of an inducible epidermal vector from its regulatory sequences.
  • Figure 9 is a schematic representative of the inducible epidermal vector which can be suppressed by Vitamin D s via insertion of HK1.NRE.
  • the drawings are not necessarily to scale, and certain features of the invention may be exaggerated in scale and shown in schematic form in the interest of clarity and conciseness.
  • transformed refers to the process or mechanism of inducing changes in the characteristics (expressed phenotype) of a cell by the mechanism of gene transfer whereby DNA is introduced into a cell in a form where it expresses a specific gene product or alters expression of endogenous gene products.
  • transduction refers to the process of introducing a DNA expression vector into a cell.
  • Various methods of transduction are possible, including microi ⁇ jection, CaPO ⁇ , lipofection (lysosome fusion), use of a gene gun and DNA vector transporter.
  • the loricrin constitutive vector and the keratin K6 inducible vector can be transduced into the squamous epithelia cells by any of the variety of ways described above.
  • the types of epithelia cells include epidermis, oral, esophageal, vaginal, tracheal, coraeal and other squamous epithelia. They are transduced by contacting the vector with the cells. In the preferred embodiment this includes using a gene gun or DNA vector transporter.
  • DNA vector transporter refers to those molecules which bind to DNA vectors and are capable of being taken up by epidermal cells. DNA transporter is a molecular complex capable of non-covalent binding to DNA and efficiently transporting the DNA through the cell membrane.
  • nucleic acid cassette refers to the genetic material of interest which can express a protein polypeptide or RNA and which is capable of being incorporated into the epidermal cells.
  • the nucleic acid cassette is positionally and sequentially oriented within the keratin K6 inducible vector or the loricrin constitutive vector such that the nucleic acid in the cassette can be transcribed into RNA or antisense RNA and, when necessary, translated into proteins or polypeptides in the transformed epidermal cells.
  • proteins and polypeptides can be expressed by the sequence in the nucleic acid cassette in the transformed epidermal cells.
  • proteins or polypeptides which can be expressed include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, tumor antigens, viral antigens, parasitic antigens and bacterial antigens.
  • specific examples of these compounds include proinsulin, insulin, growth hormone, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding protein, epidermal growth factor TGF- ⁇ , dermal growth factor PDGF, angiogenesis factors, e.g., acid fibroblast growth factor, basic fibroblast growth factor and angiogenin for instance, matrix proteins such as Type IV collagen, Type VII collagen, laminin and proteins from viral, bacterial and parasitic organisms which can be used to induce immunologic response.
  • the genetic material which is incorporated into the epidermal cells using the loricrin constitutive vector or the keratin K6 inducible vector includes DNA not normally found in epidermal cells, DNA which is normally found in epidermal cells but not expressed at physiological significant levels, DNA normally found in epidermal cells and normally expressed at physiological desired levels, any other DNA which can be modified for expression in epidermal cells, and any combination of the above.
  • loricrin constitutive vector refers to a vector which can be inserted into epidermal cells and which once inserted, will express a constitutive (i.e.- a constant level) of protein, polypeptide or antisense RNA from the nucleic acid cassette which is part of the loricrin constitutive vector.
  • the loricrin constitutive vector is used for efficient expression of a nucleic acid sequence in epidermal cells and is comprised of a 5' flanking region of the loricrin gene, said flanking region including a TATA box, a cap site and a first intron and an intron/exon boundary all in appropriate sequential and positional relationship for expression of a nucleic acid cassette; a 3' flanking sequence of the loricrin gene; and a linker having a unique restriction endonuclease site at the location of the start and stop codon, said linker connecting the 5' flanking region to the
  • the sequence for the loricrin gene which is used for preparing the loricrin constitutive vector is shown in SEQ. ID No. 1.
  • the loricrin constitutive vector has a 5' flanking region comprising nucleotides 1 to
  • the loricrin constitutive vector has a 5' flanking region of approximately 1.5 kb, an intron of approximately 1.1 kb and a 3' flanking sequence of approximately 2.1 kb.
  • the linker of the loricrin constitutive vector can be a poly-linker.
  • the poly-linker includes a plurality of restriction endonuclease sites.
  • the term "keratin K6 inducible vector” as used herein is a vector which is useful for regulated expression of a nucleic acid sequence in epidermal cells.
  • the keratin K6 inducible vector comprises a 5' flanking region of the keratin K6 gene, said flanking region including a TATA box, a cap site, a first intron and an intron/exon boundary all in sequential and positional relationship for the expression of a nucleic acid cassette; a 3' flanking sequence of a keratin K6 gene; and a poly-linker.
  • the poly-linker includes a plurality of restriction endonuclease sites, connects the 5' flanking region to the 3' flanking sequence and further provides a position for insertion of the nucleic acid cassette.
  • the partial sequence for the keratin K6 gene which is used for preparing the keratin K6 inducible vector is shown in schematic form in Figure 8 and the sequence is shown in SEQ. ID No. 3.
  • the keratin inducible vector has a 5' flanking region which extends from a unique 5' Xho I site up to nucleotide 360 of SEQ. ID. No. 3; an intron and intron/exon boundary comprising nucleotides 928 to 1494 of SEQ. ID. No.
  • the keratin K6 inducible vector has a 5' flanking region of approximately 8.0 kb, an intron and intron/exon boundary of approximately 0.56 kb and a 3' flanking sequence of approximately 1.2 kb.
  • the restriction endonuclease sites found in the linker and poly-linker of the loricrin and keratin K6 vectors can be any restriction endonucleases which will allow insertion of the nucleic acid cassette. In the preferred embodiment they are usually selected from the group consisting of Cla I,
  • the vectors can be inserted either in vivo or ex vivo.
  • the mode of insertion will, to a certain degree, determine the available methods for the insertion.
  • a bioreactor is comprised of transformed epidermal cells which contain the loricrin constitutive vector or contain the keratin K6 inducible vector. Once the vector is inserted in the epidermal cells, the epidermal cells will express the nucleic cassette and produce the protein, polypeptide or antisense RNA of interest. This can be done either in vivo or ex vivo. Any compound which can be encoded in, and expressed by, the nucleic acid cassette can be produced by the bioreactor.
  • One method for ex vivo introduction of the loricrin constitutive vector or the keratin K6 inducible vector into epidermal cells includes a cotransfection of the vector with a selectable marker. The selectable marker is used to select those cells which have become transformed. The cells can then be used in any of the methods described in the present invention.
  • One specific embodiment of the present invention is a method for the enhanced healing of a wound or surgical incision.
  • This method comprises the in vivo transduction of epidermal cells with a loricrin constitutive vector or a keratin K6 indu le vector.
  • the nucleic acid cassette of said vector contains a nucleic acid sequence for a growth factor.
  • a plurality of vectors are introduced into the epidermal cells.
  • the cassette of at least one vector contains a nucleic acid sequence for an epidermal growth factor (TGF- ⁇ )
  • the cassette of at least one vector contains a dermal growth factor (PDGF)
  • PDGF dermal growth factor
  • a cassette of at least one vector contains a nucleic acid sequence for a matrix protein to anchor the epidermis to the dermis
  • a cassette of at least one vector contains a nucleic acid sequence for an angiogenesis factor.
  • the sequence for matrix proteins can be selected from any sequences useful for the anchoring of the epidermis to the dermis but are usually selected from the group consisting of Type IV collagen, laminin, nidogen, and Type VII collagen.
  • the angiogenesis factor is usually selected from the group consisting of acid fibroblast growth factor, basic fibroblast growth factor and angiogenin.
  • the vectors are first transduced into the epidermal cells ex vivo.
  • the transformed epidermal cells are transplanted onto the animal or human to be treated.
  • Another embodiment of the present invention is a method for treating psoriasis.
  • epidermal cells are transduced in vivo with a loricrin constitutive vector or a keratin K6 inducible vector.
  • a nucleic acid cassette in said vector contains a nucleic acid sequence for a protein or polypeptide selected from the group consisting of TGF- ⁇ , a soluble form of cytokine receptor, and an antisense RNA.
  • the cytokine receptor can be selected from the group consisting of IL-1, IL-6 and IL-8.
  • the antisense RNA sequence is selected from the group consisting of TGF- ⁇ , IL-1, IL-6 and IL-8.
  • a method of treating cancer comprises the steps of in vivo transduction of epidermal cells with a loricrin constitutive vector or a keratin K6 inducible vector into epidermal cells.
  • the nucleic acid cassette of either vector contains the nucleic acid sequence coding for antisense RNA for the E6 or E7 gene of the human papilloma virus or coding for the normal p53 protein.
  • the example given is for skin cancer, this same approach is used for cancers occurring in other squamous epithelial, since the constitutive and inducible vectors will also function in these tissue types.
  • Vitamin D regulatory element is usually introduced into the 3' flanking sequence.
  • the Vitamin D regulatory element is from the human
  • Vitamin D a commonly used substance in animals and humans.
  • An additional embodiment of the present invention is a method for vaccination comprising the step of in vivo introduction of a loricrin constitutive vector into epidermal cells.
  • the nucleic acid cassette in the vectors usually codes for a polypeptide which induces an immunological response.
  • An example of this is the viral capsid protein from the human papilloma virus.
  • any other variety of proteins can be used to generate a immunologic response and thus produce antibodies for vaccination.
  • loricrin was not identified until 1990 (Mehrel, et al., Cell, Vol. 61, pp. 1103-1112, (1990)).
  • the primary sequence of the loricrin protein was deduced from the overlapping cDNA clones described in Mehrel, id.
  • the cDNA clones were used to screen an EMBL-3 Balb/c mouse genomic library.
  • the gene encoding loricrin was located within two Bam HI fragments of 3.4 and 3.1 kb. The coding sequence within this genomic fragment is identical to the cDNA sequences and is not interrupted by introns.
  • intron in the 5' non-coding region that is approximately 1.1 kb in length.
  • intron and coding sequence there is approximately 1.5 kb of 5' flanking sequence and 2.1 kb of 3' flanking sequence.
  • a functional loricrin constitutive expression construct was designed as follows. Briefly, polymerase chain reaction (PCR) technology was used to delete the loricrin coding region, leaving the 5' and 3' flanking regions, 5' and 3' non-coding regions and the intron ( Figure 1). A unique Cla I restriction site was engineered at the start (ATG) and stop (TAA) codons to allow easy insertion of exogenous gene cassettes. To assess the expression characteristics of this vector, a reporter gene, the bacterial gene encoding chloramphenicol acetyl transferase (CAT), was inserted into the Cla I site.
  • CAT chloramphenicol acetyl transferase
  • the expression vector was analyzed by transient transfection into primary mouse epidermal cells. Positive ( ⁇ SV2.CAT, lane 1) and negative (pAlO.CAT, lane 2) control vectors were included in the assay ( Figure 2).
  • the loricrin expression vector had high activity in undifferentiated (low Ca" medium, lane 3) and differentiated (high Ca” medium, lane 4) epidermal cells, surpassing levels obtained with the strong promoter of the virus SV40. This result was unexpected, since previous in vivo studies had demonstrated that the loricrin gene was only expressed at a late stage of epidermal differentiation (Mehrel, et al., Cell, Vol. 61, pp. 1103-1112, (1990)), and indicates that additional flanking sequences are required to suppress loricrin expression in undifferentiated epidermal cells.
  • the bacterial gene encoding ⁇ -galactosidase was inserted into the Cla I site.
  • the ⁇ -galactosidase gene has frequently been used as a reporter gene to assess targeting specificity (MacGregor, et al., In: Methods in Molecular Biology, Vol. 7, pp. 217-235, (1991)).
  • This construct was designated pML- ⁇ -gal and was used in the production of transgenic mice.
  • This construct was digested with Apa I and subjected to preparative agarose gel electrophoresis to purify the pML- ⁇ -gal expression construct away from plasmid sequences (pGEM72) which might interfere with expression.
  • the separated expression construct sequences were purified and recovered using NA 45 DEAE membrane (Schleicher & Schuell). DNA was precipitated and resuspended at 1-3 ng ul. ICR outbred female mice (Sasco) were given PMS and HCG to stimulate superovulation, mated to FVB males (Taconie) and resulting one-cell fertilized embryos were collected from the oviducts. DNA was micro-i ⁇ jected into the pronuclei and the embryos were surgically transferred to pseudopregnant recipient females (the result of mating ICR females with vasectomized B e D 2 F. males
  • PCR analysis was performed on the extracted tail using oligo primers specific for ⁇ -galactosidase. Animals positive for the transgene were further analyzed to assess the expression characteristics of pML- ⁇ - gal. This was done by removing part of the ear and incubating the tissue in a staining solution containing X-gal. This was done by removing part of the ear and incubating the tissue in a staining solution containing X- gal.
  • the bacterial gene encoding ⁇ -galactosidase was inserted into the Cla I site. This data is shown in Figure 3. This observation indicates that the loricrin expression vector is useful as a constitutive vector to direct the efficient expression of exogenous DNA in both the undifferentiated and differentiated compartments of the epidermis.
  • HKl.NRE novel negative regulatory element from the human Kl keratin gene
  • the HKl.NRE is 70 nucleotides in length (see Figure 4).
  • PCR technology was used to generate Bam HI and Bgl II sites at opposite ends of this fragment. This facilitates generating multiple copies of this fragment since ligation and digestion with Bam HI and Bgl II will select for oligomers which have ligated head to tail.
  • Four tandem copies of the HKl.NRE were inserted into the Bgl II cloning site of pAlO.CAT.
  • FIG. 4 shows a schematic representative of a derivative of the loricrin constitutive epidermal vector which contains the HKl.NRE in its 3' flanking region. The activity of this vector within epidermal cells can be suppressed by topical application of Vitamin D 3 , or an analogue, to the skin.
  • keratin K6 is not expressed in normal epidermis, but is expressed under hyperproliferative conditions such as wounding (Weiss, et al., J. Cell Biol, Vol. 98, pp. 1397-1406, (1984); Nakazawa, et al., J. Cell Biol, Vol. 103, pp. 561a (1986); Stoler, et al., J. Cell Biol, Vol. 107, pp. 427-446, (1988)) or topical application of retinoic acid (Rosenthal, et al., J. Invest. Dermatol, Vol. 95, pp. 510-515, (1990).
  • K6 expression does not occur in interfollicular epidermis, it does occur in hair follicles (Nakazawa, et al., J. Cell Biol, Vol. 103, pp. 561a, (1986)).
  • K6 cDNAs that differ in sequence in only a few nucleotides.
  • These cDNA clones have been used to differentially screen a EMBL 3 Balb/c mouse genomic library and isolate two distinct K6 genes. These genes are closely linked within genomic DNA, i.e., arranged in tandem. They have almost identical 3' halves, including identical 3' non-coding and flanking regions. Interestingly, the 5' halves of the 2 genes differ greatly in their restriction fragment patterns.
  • BCM-MK6(A) The sequence of one of these genes, designated BCM-MK6(A), is shown in SEQ. ID. No. 3.
  • SEQ. ID. No. 3 The sequence of one of these genes, designated BCM-MK6(A), is shown in SEQ. ID. No. 3.
  • Nucleotides encoding the C-terminal region of the K6 protein were deleted and nucleotides encoding the amino acid sequence SEQ. ID. No. 4 were inserted. These amino acids are at the C- terminal of human keratin Kl (Johnson, et al., PNAS, USA, Vol. 82, pp. 1896-1900, (1985)).
  • FIG. 6 A schematic representative of this derivative of the ouse K6 gene (BCM-MK6(A)-HK1) is shown in Figure 6.
  • Antisera have previously been generated against the HK1 C-terminal peptide (Rosenthal, et al., J. Invest. Dermatol, Vol. 95, pp.510-515, (1990)). These antibodies are monospecific for this human Kl peptide and allow expression of the derivatized BCM-MK6(A)-HK1 transgene to be followed against the expression pattern of the endogenous mouse K6 genes.
  • the derivatized mouse K6 transgene shown in Figure 6 was used in the production of transgenic mice as outlined in Example 2.
  • Mice resulting from the initial iiyections were screened by PCR analysis for presence of the BCM-MK6(A)-HK1 transgene. Positive founders were initially analyzed for transgene expression as follows. A small ear biopsy was taken and after 48 hours a second biopsy was taken at the same site to score for expression during wound healing. Transgene expression was limited to hair follicles in the initial biopsy and was not present in interfollicular epidermis. Transgene expression was observed in the epidermis in the 48 hour biopsies, but only at the site of wounding.
  • Results obtained with the derivative of BCM-MK6(A) indicate that all of the regulatory sequences required to suppress expression of this gene in normal epidermis and activate its expression under hyperproliferative conditions, such as in wounding healing or experimentally induced hyperplasia, are located within the 13.5 kb Xho I fragment ( Figure 6). Therefore, an inducible vector was developed from this fragment.
  • This vector is very useful in gene therapy applications where dosage of pharmaceuticals needs to be regulated.
  • this vector is ideally suited for wound healing applications since it is induced during the wound healing process but suppressed after healing has occurred.
  • Figure 8 illustrates how a vector is constructed from the BCM- MK6(A) gene.
  • the vector is derived from the 13.5 kb Xho I fragment which contains the entire K6 gene.
  • the same general strategy used in construction of the constitutive epidermal vector ( Figure 1) is followed.
  • the expression vector retains all of the 5' flanking sequences, the 5' non- coding sequences up to but not including the ATG, the first intron including the splice-sites of the intron-exon boundary and all of the 3' non-coding and flanking sequences after the TAA codon.
  • a polylinker is engineered 3' of the first intron to allow easy insertion of exogenous DNA cassettes. These manipulations are performed through the use of PCR technology.
  • FIG. 8 shows a schematic representative of a derivative of the K6 inducible epidermal vector which contains the HKl.NRE in its 3' flanking region. The activity of this vector within epidermal cells is suppressed by topical appHcation of Vitamin D 3 , or an analogue, to the skin.
  • the inducible epidermal vector Utilization of the inducible epidermal vector in wound healing Greater than 3.5 milHon individuals develop skin ulcers. During normal healing, epidermal cells produce growth factors which affect not only epidermal cells but also cells within the dermis. In addition, epidermal cells synthesize several matrix proteins which provide an anchor to the underlying dermis. Many skin ulcers occur in patients with disorders such as circulatoiy problems and diabetes, and the normal healing process in impaired.
  • the inducible epidermal vector is used to target the combined expression of growth factors, to accelerate growth of cells in both the epidermal and dermal compartments; matrix proteins, to increase tensile strength; and angiogenesis factors, to improve circulation, in an attempt to improve healing these patients. EXAMPLE 8
  • Skin cancer is by far the most common form of cancer with greater than 600,000 new cases reported each year.
  • Several genes have been implicated in causing skin cancer, including loss or mutation of the host cell tumor suppressor gene, p 53 and expression of the E6 and E7 transforming genes of human papilloma virus (HPV).
  • HPV human papilloma virus
  • the constitutive epidermal vector is used to target expression of the normal p53 gene to cause reversion to a non-maHgnant phenotype or induction of programmed death in vivo.
  • the constitutive vector is used to target expression of antisense RNA specific for the E6 and E7 genes of HPV.
  • Psoriasis Utilization of the epidermal vector systems in gene therapy apprpacheg tp pspriagis Psoriasis is a common inherited skin disease which affects approximately 4 milHon individuals in the U.S., 20 million world-wide. It is characterized by the presence of inflamed scaly skin. Although the specific defect for psoriasis is not known, inappropriate expression of growth factors, and cytokines appears to be responsible for its pathogenesis. Epidermal vectors are used to inhibit the mitogenic effects of positive growth factors produced in psoriatic lesions by expressing negative growth factors which induce growth arrest of epidermal cells.
  • AU patents and pubHcations mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. AU patents and pubHcations which are incorporated herein by reference are incorporated to the same extent as if each individual pubHcation was specifically and individuaUy indicated to be incorporated by reference.
  • MOLECULE TYPE DNA (genomic)
  • GGCCAGTCAT ACACCAAACT GCCTATCCCT ATCCCAAGAA TTTGAAATCT TCATGAATGG 1440
  • CAATCCTTAC CTTCCCTACA ATTCCCTTTA CCATGTTTTT GTTAGTTTTG TTGGTTTGTT 5640
  • MOLECULE TYPE DNA (genomic)
  • GGCCAGTCAT ACACCAAACT GCCTATCCCT ATCCCAAGAA TTTGAAATCT TCATGAATGG 1440
  • ATCTCACTAC CAATAACCCT TTTCTAAACT GGTAGCCTAC AACTTTAGTT CCAGTACTTG 4860
  • AATTGACCCC ACCATCCAGC GGGTCAGGAC TGAGGAGAGG GAGCAGATCA AGACCCTCAA 900 TAACAAGTTT GCCTCCTTCA TCGACAAGGT GAGACATGGT CCTCCCTAGA GCACCCTGTG 960
  • CAAGCCATGG CTAACCTGCC AGTGCCCTAC ATGAGTTCTCTC TGCCCTCCTT AGAGAGGTGG 1320
  • ATGTACACTC CACGATTATT TTTGTTGCTC TCTCTGCCCA GTGTGCCAAC CTGCAAGCTG 3180
  • GAGTTACTGA GTTTAATTCC CAGCAACCAC CTGGTGATTC ACAATCATCT CTATTGAGAT 3600
  • CTGTGAGTTT CTAATGGCCT GAGAAACCCC ATCTCTCAAC ATCATAACCC TCCCTGTCAG 4620 TAACTGTGAC TGCCCCGTCA CTGGTCCTGT GATGTAAGTT TCTGCTCATG TGATGTCTTT 4680
  • AAAATTTTCC ACGTGCATTT TTTGTTGCAA TGTTTTTAAT ATAGAAATTC TGTGGCCTTG 4800
  • CTAGACAAGG CATCATTACA GTTCCCTCTC CCAGGTCTAT ATGTCTTCAT CTGTTAGTAT 4860

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Abstract

Un vecteur constitutif de loricrine permet l'expression efficace d'une séquence d'acide nucléique dans des cellules épidermiques comprenant la région de flanquement 5' du gène de loricrine, ladite région de flanquement contenant une boîte TATA, un site de coiffe et un premier intron et une frontière intron/exon, tous en relation appropriée de séquence et de position pour l'expression d'une cassette d'acide nucléique, une séquence de flanquement 3' du gène de loricrine et un lieur contenant un site d'endonucléase de restriction unique à l'emplacement du codon de départ et d'arrêt. Ce lieur relie la région de flanquement 5' à la séquence de flanquement 3' et procure en outre une position d'insertion de la cassette. La cassette contient la séquence d'acide nucléique spécifique à exprimer. De même, l'invention concerne un vecteur inductif de la kératine K6 pour réguler l'expression d'une séquence d'acide nucléique dans des cellules épidermiques comprenant la région de flanquement 5' du gène de kératine K6, ladite région de flanquement comprenant la boîte TATA, un site de coiffe et le premier intron et la frontière intron/exon, tous en relation de séquence et de position appropriée pour l'expression d'une cassette d'acide nucléique, une séquence de flanquement 3' du gène de la kératine K6, et un polylieur ayant une pluralité de site d'endonuclease de restriction. Le polylieur relie la région de flanquement 5' à la séquence de flanquement 3' et fournit également une position d'insertion de la cassette. Les vecteurs de la kératine K6 et de loricrine peuvent en outre être régulés par addition d'un élément régulateur de vitamine D. Les vecteurs peuvent être utilisés dans un bioréacteur afin de générer une variété de produits y compris des protéines, des polypeptides ou des ARN antisens. Les vecteurs peuvent être également utilisés en thérapie génétique pour le traitement de plusieurs maladies chez les animaux et les êtres humains y compris la cicatrisation de blessures, les incisions chirurgicales, les ulcères de la peau, le psoriasis et le cancer de la peau, ainsi qu'en vaccination.
PCT/US1993/003993 1992-04-30 1993-04-28 Systemes de vecteurs epidermiques constitutifs et inductifs WO1993022431A1 (fr)

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JP5519487A JPH08501202A (ja) 1992-04-30 1993-04-28 構成および誘導的表皮ベクター系
AU42213/93A AU669713B2 (en) 1992-04-30 1993-04-28 Constitutive and inducible epidermal vector systems
EP93910875A EP0644933A4 (fr) 1992-04-30 1993-04-28 Systemes de vecteurs epidermiques constitutifs et inductifs.

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EP0633315A2 (fr) * 1993-03-29 1995-01-11 Centro De Investigaciones Energeticas Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Animaux transgéniques pour la détermination d'agents stimulant ou réprimant l'hyperprolifération épidermique et la croissance des cheveux
WO1996027019A1 (fr) * 1995-02-25 1996-09-06 Imperial Cancer Research Technology Limited Animaux transgeniques utilises comme modele du psoriasis
EP0777730A1 (fr) * 1994-08-15 1997-06-11 Garvan Institute Of Medical Research Vecteur d'adn destine a l'expression d'un gene specifique des os
GB2324960A (en) * 1997-05-09 1998-11-11 Univ Manchester Delivery of naked DNA for wound healing
US5955059A (en) * 1995-06-06 1999-09-21 Trustees Of Boston University Use of locally applied DNA fragments
US6147056A (en) * 1995-06-06 2000-11-14 Trustees Of Boston University Use of locally applied DNA fragments
US7033829B2 (en) 2000-03-31 2006-04-25 Trustees Of Boston University Method to inhibit cell growth using oligonucleotides
US7094766B1 (en) 1995-06-06 2006-08-22 Trustees Of Boston University Use of locally applied DNA fragments
US8183222B2 (en) 1995-06-06 2012-05-22 Trustees Of Boston University Method to inhibit cell growth using oligonucleotides
WO2015089321A2 (fr) 2013-12-11 2015-06-18 The General Hospital Corporation Utilisation de protéines d'hormone anti-mullerienne (amh) pour la contraception et la préservation de la réserve ovarienne

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0633315A2 (fr) * 1993-03-29 1995-01-11 Centro De Investigaciones Energeticas Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Animaux transgéniques pour la détermination d'agents stimulant ou réprimant l'hyperprolifération épidermique et la croissance des cheveux
ES2152753A1 (es) * 1993-03-29 2001-02-01 Ct Investig Energeticas Ciemat Animales transgenicos para la determinacion de agentes que estimulan o reprimen la hiperproliferacion epidermica y el crecimiento del pelo
EP0633315A3 (fr) * 1993-03-29 1996-12-04 Invest Energet Medioambient Animaux transgéniques pour la détermination d'agents stimulant ou réprimant l'hyperprolifération épidermique et la croissance des cheveux.
EP0777730A4 (fr) * 1994-08-15 1999-10-13 Garvan Inst Med Res Vecteur d'adn destine a l'expression d'un gene specifique des os
EP0777730A1 (fr) * 1994-08-15 1997-06-11 Garvan Institute Of Medical Research Vecteur d'adn destine a l'expression d'un gene specifique des os
WO1996027019A1 (fr) * 1995-02-25 1996-09-06 Imperial Cancer Research Technology Limited Animaux transgeniques utilises comme modele du psoriasis
US5955059A (en) * 1995-06-06 1999-09-21 Trustees Of Boston University Use of locally applied DNA fragments
US6147056A (en) * 1995-06-06 2000-11-14 Trustees Of Boston University Use of locally applied DNA fragments
US7094766B1 (en) 1995-06-06 2006-08-22 Trustees Of Boston University Use of locally applied DNA fragments
US8183222B2 (en) 1995-06-06 2012-05-22 Trustees Of Boston University Method to inhibit cell growth using oligonucleotides
GB2324960A (en) * 1997-05-09 1998-11-11 Univ Manchester Delivery of naked DNA for wound healing
US7033829B2 (en) 2000-03-31 2006-04-25 Trustees Of Boston University Method to inhibit cell growth using oligonucleotides
WO2015089321A2 (fr) 2013-12-11 2015-06-18 The General Hospital Corporation Utilisation de protéines d'hormone anti-mullerienne (amh) pour la contraception et la préservation de la réserve ovarienne
EP4008339A1 (fr) 2013-12-11 2022-06-08 The General Hospital Corporation Utilisation de protéines d'hormone anti-mullerienne (amh) pour la contraception

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CA2134670A1 (fr) 1993-11-11
AU669713B2 (en) 1996-06-20

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