EP1021460A1 - Laminins and uses thereof - Google Patents

Laminins and uses thereof

Info

Publication number
EP1021460A1
EP1021460A1 EP98953370A EP98953370A EP1021460A1 EP 1021460 A1 EP1021460 A1 EP 1021460A1 EP 98953370 A EP98953370 A EP 98953370A EP 98953370 A EP98953370 A EP 98953370A EP 1021460 A1 EP1021460 A1 EP 1021460A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
laminin
gene
tissue
subunit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98953370A
Other languages
German (de)
French (fr)
Other versions
EP1021460A4 (en
Inventor
Robert E. Burgeson
Marie-France Champliaud
Pamela Olson
Manuel Koch
William Brunken
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
General Hospital Corp
Original Assignee
General Hospital Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by General Hospital Corp filed Critical General Hospital Corp
Publication of EP1021460A1 publication Critical patent/EP1021460A1/en
Publication of EP1021460A4 publication Critical patent/EP1021460A4/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the invention relates to the laminin 12, laminin subunit ⁇ 3, and laminin subunit ⁇ l, and methods of making and using these molecules.
  • the present invention is based, in part, on the discovery of a novel member of the laminin family, laminin 12. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of laminin 12 which includes an ⁇ 2 subunit, a ⁇ l subunit and a ⁇ 3 subunit.
  • the ⁇ 2 subunit has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human ⁇ 2 subunit, e.g., the human ⁇ 2 subunit of SEQ ID NO:7.
  • the ⁇ 2 subunit can be identical to a human ⁇ 2 sequence, e.g., that of SEQ ID NO:7.
  • the ⁇ 2 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO: 8.
  • the ⁇ 2 subunit can have substantially the same electrophoretic mobility as human ⁇ 2 subunit, e.g., it appears as a 205 kDa electrophoretic band on reducing gels.
  • Yet another preferred embodiment of the invention features an ⁇ 2 subunit which is reactive with an ⁇ 2-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 5H2.
  • ⁇ 2 specific antibodies can be made by methods known in the .art.
  • Another preferred embodiment of the invention features a ⁇ l subunit having at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human ⁇ l subunit, e.g., the human ⁇ l subunit of SEQ ID NO:9.
  • the ⁇ l subunit has the identical amino acid sequence of human ⁇ l subunit, e.g., that of SEQ ID NO:9.
  • the ⁇ l subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO: 10.
  • the ⁇ l subunit can have substantially the same electrophoretic mobility as human ⁇ l subunit, e.g., it appears as a 185 kDa electrophoretic band on reducing gels.
  • Yet another preferred embodiment of the invention features an ⁇ l subunit which is reactive with an ⁇ l-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 545.
  • ⁇ 1 -specific antibodies can be made by methods known in the art.
  • the ⁇ 3 subunit of laminin 12 has at least 60% to about 70%), more preferably at least about 80%, even more preferably at least about 90% to about 95%), and most preferably at least about 99% sequence identity with human ⁇ 3 subunit, e.g., the ⁇ 3 subunit of SEQ ID NO:3.
  • the ⁇ 3 subunit can be identical to a naturally occuring human ⁇ 3 subunit, e.g., that of SEQ ID NO:3.
  • the ⁇ 3 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:4.
  • the ⁇ 3 subunit can have substantially the same electrophoretic mobility as human ⁇ 3 subunit, e.g., it appears as a 170 kDa electrophoretic band on reducing gels.
  • Yet another preferred embodiment of the invention features an ⁇ 3 subunit which is reactive with an ⁇ 3-specific antibody.
  • ⁇ 3-specif ⁇ c antibodies can be made by methods known in the art and taught herein.
  • the laminin 12 is a trimer which can be found in, or can be isolated from human placental chorionic villi.
  • the laminin 12 is expressed by a recombinant cell, e.g., a bacterial cell, a cultured cell (e.g., a cultured eukaryotic cell) or a cell of a non-human transgenic animal.
  • Cultured cells can include CHO cells or SF8 cells.
  • Expression of laminin 12 in a transgenic animal can be general or can be under the control of a tissue specific promoter.
  • tissue specific promoter e.g., a milk specific promoter.
  • the present invention is also based, in part, on the discovery of a novel laminin subunit, ⁇ 3. Accordingly, the invention features a recombinant or substantially pure or isolated preparation of a ⁇ 3 polypeptide.
  • the ⁇ 3 polypeptide has the following biological acitivities: 1) it promotes adhesion between tissue elements; 2) provides a site for insertion of nerves into the basement membrane.
  • the ⁇ 3 polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:3;
  • the ⁇ 3 polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:3;
  • the ⁇ 3 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length;
  • the ⁇ 3 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:3;
  • the ⁇ 3 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring ⁇ 3 subunit;
  • the ⁇ 3 polypeptide is a vertebrate, e.g.,
  • the ⁇ 3 polypeptide is a polypeptide encoded by nucleotide sequences of the overlapping DNA inserts of more than one, preferably all seven of the plasmids deposited with ATCC as Accession No:209357.
  • the ⁇ 3 polypeptide is encoded by the nucleic acid in SEQ
  • the ⁇ 3 polypeptide includes a nidogen-binding domain.
  • the nidogen-binding domain is at least 5 residues in length and preferably, has about 70, 80, 90, or 95% sequence identity with the nidogen-binding domain of the protein shown in SEQ ID NO: 3 (amino acid residues 750-755).
  • the ⁇ 3 polypeptide includes at least 5, preferably 6 to 7, and most preferably 8 of the cysteins found in native ⁇ 3 protein.
  • a ⁇ 3 polypeptide which has antagonist activity can have inactivated or excluded regions which comprise at least one cystein found in native ⁇ 3 protein.
  • the ⁇ 3 polypeptide differs in amino acid sequence at up to
  • the ⁇ 3 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 % of the residues from a sequence in SEQ ID NO: 3.
  • the differences are such that: the ⁇ 3 polypeptide exhibits a ⁇ 3 biological activity, e.g., the ⁇ 3 polypeptide retains a biological activity of a naturally occurring ⁇ 3 subunit.
  • the ⁇ 3 polypeptide includes a ⁇ 3 subunit sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
  • the ⁇ 3 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO: 3, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence from SEQ ID NO: 3.
  • the ⁇ 3 polypeptide is a recombinant fusion protein having a first ⁇ 3 portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to ⁇ 3.
  • the second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain.
  • the fusion protein can be used in a two-hybrid assay.
  • the ⁇ 3 polypeptide includes amino acid residues 750-755 of SEQ ID NO:3.
  • the ⁇ 3 polypeptide encodes domains IV- VI of the ⁇ 3 subunit.
  • the ⁇ 3 polypeptide has antagonistic activity, and is capable of: inhibiting adhesion between connective tissues.
  • the ⁇ 3 polypeptide is a fragment of a naturally occurring ⁇ 3 which inhibits connective tissue adhesion.
  • Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
  • the ⁇ 3 polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed ⁇ 3 is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
  • the invention includes an immunogen which includes a ⁇ 3 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the ⁇ 3 polypeptide, e.g., a humoral response, an antibody response, or a cellular response.
  • the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO: 3.
  • the present invention also includes an antibody preparation specifically reactive with an epitop&of the ⁇ 3 immunogen or generally of a ⁇ 3 polypeptide, preferably an epitope which consists all or in part of residues from the the amino acid sequence of SEQ ID NO:3, or an epitope, which when bound to an antibody, results in the modulation of a biological activity.
  • the ⁇ 3-like polypeptide as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has a molecular weight of 170 kDa as determined by SDS-PAGE.
  • the ⁇ 3 polypeptide comprises amino acid residues 100-1761 of SEQ ID NO: 3.
  • the ⁇ 3 polypeptide has one or more of the following characteristics:
  • composition which includes a ⁇ 3 polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent.
  • additional component can be one which renders the composition useful for in vitro and in vivo pharmaceutical or veterinary use.
  • the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a ⁇ 3 polypeptide, e.g., a ⁇ 3 polypeptide described herein.
  • a preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 85% sequence identity to a nucleotide sequence of SEQ ID NO:4.
  • the ⁇ 3 polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least about 90% to about 95%, and more preferably about 98% to about 99% sequence identity to the nucleotide sequence from SEQ ID NO:4.
  • the ⁇ 3 polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:4.
  • the isolated nucleic acid molecule includes the nucleotide sequence of at least one and preferably all of the DNA inserts of the plasmids deposited with ATCC as Accession No: 209357.
  • the subject ⁇ 3 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the ⁇ 3 gene sequence (also referred to as LAMG3), e.g., to render the ⁇ 3 gene sequence suitable for use as an expression vector.
  • a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the ⁇ 3 gene sequence (also referred to as LAMG3), e.g., to render the ⁇ 3 gene sequence suitable for use as an expression vector.
  • the nucleic acid which encodes a ⁇ 3 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO:4. More preferably, the nucleic acid probe corresponds to at least 20 consecutive nucleotides from SEQ ID NO: 4.
  • the invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide.
  • the oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO: 4, or naturally occurring mutants thereof.
  • the probe or primer further includes a label group attached thereto.
  • the label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor.
  • the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
  • the invention involves nucleic acids, e.g., RNA or DNA, encoding a ⁇ 3 polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
  • the invention features a cell or purified preparation of cells which include a ⁇ 3 subunit transgene, or which otherwise misexpress a ⁇ 3 gene.
  • the cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells.
  • the cell or cells include a ⁇ 3 transgene, e.g., a heterologous form of a ⁇ 3 gene, e.g., a gene derived from humans (in the case of a non-human cell).
  • the ⁇ 3 transgene can be misexpressed, e.g., overexpressed or underexpressed.
  • the cell or cells include a gene which misexpress an endogenous ⁇ 3 gene, e.g., a gene the expression of which is disrupted, e.g., a knockout.
  • a gene which misexpress an endogenous ⁇ 3 gene e.g., a gene the expression of which is disrupted, e.g., a knockout.
  • Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed ⁇ 3 alleles or for use in drug screening.
  • the invention features a tr.ansgenic ⁇ 3 animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow.
  • the transgenic animal includes (and preferably express) a heterologous form of a ⁇ 3 gene, e.g., a gene derived from hum-ans.
  • the ⁇ 3 transgene includes a tissue specific promoter, e.g., a milk-specific promoter.
  • the animal has an endogenous ⁇ 3 gene which is misexpressed, e.g., a knockout.
  • transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed ⁇ 3 alleles or for use in drug screening.
  • the invention is also based, in part, on the discovery of a novel laminin subunit, ⁇ 4.
  • the invention features a recombinant or substantially pure preparation of a ⁇ 4 polypeptide.
  • the ⁇ 4 polypeptide has the following biological activities: 1) it promotes adhesion between tissue elements; 2) it aids in wound healing.
  • the ⁇ 4 polypeptide includes an amino acid sequence with at least 65%, 80%, 90%), 95%, 98%), or 99% sequence identity to an amino acid sequence from SEQ ID NO:l;
  • the ⁇ 4 polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO: 1;
  • the ⁇ 4 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length;
  • the ⁇ 4 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:l;
  • the ⁇ 4 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring ⁇ 4 subunit;
  • the ⁇ 4 polypeptide is a vertebrate, e.g., a mammalian, e.
  • the ⁇ 4 polypeptide is encoded by the nucleic acid in SEQ ID NO:2, or by a nucleic acid having at least about 65% to about 70%, more preferably at least 80%, even more preferably at least about 90% to about 95%, and most preferably about 99% sequence identity with the nucleic acid from SEQ ID NO: 2.
  • the ⁇ 4 polypeptide includes domains VI and V found in native ⁇ 4 subunits.
  • Amino acid residues from about 221-262 and 263-535 of SEQ ID NO: 1 are exemplary of domains VI and V, respectively, of ⁇ 4.
  • domain VI is at least 33 residues in length and has preferably at least about 60%, more preferably about 70% to about 80%), and most preferably about 90% to about 95% sequence identity with the amino acid residues 221-262 of the ⁇ 4 protein shown in SEQ ID NO: 1.
  • Domain V is at least 272 residues in length and has preferably at least about 60%, more preferably about 70% to about 80%), and most preferably about 90% to about 95% sequence identity with the amino acid residues 263-535 of the ⁇ 4 protein shown in SEQ ID NO: 1.
  • the ⁇ 4 polypeptide has at least 5, preferably 6 or 7, and most preferably 8 cysteins as found in native ⁇ 4.
  • a ⁇ 4 polypeptide which has antagonist activity has inactivated or excluded regions which comprise at least one of the cysteins found in native ⁇ 4 protein.
  • the ⁇ 4 polypeptide differs in .amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 1.
  • the ⁇ 4 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 %> of the residues from a sequence in SEQ ID NO: 1.
  • the differences are such that: the ⁇ 4 polypeptide exhibits a ⁇ 4 biological activity, e.g., the ⁇ 4 polypeptide retains a biological activity of a naturally occurring ⁇ 4 subunit.
  • the ⁇ 4 polypeptide includes a ⁇ 4 sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
  • the ⁇ 4 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:l, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence from SEQ ID NO:l.
  • the ⁇ 4 polypeptide is a recombinant fusion protein having a first ⁇ 4 portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to ⁇ 4.
  • the second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain.
  • the fusion protein can be used in a two-hybrid assay.
  • the ⁇ 4 polypeptide has antagonistic activity, and is capable of: inhibiting the adhesion of connective tissues.
  • the ⁇ 4 polypeptide is a fragment of a naturally occurring ⁇ 4 which inhibits connective tissue adhesion.
  • Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, .and alternative translational and postranslational events.
  • the ⁇ 4 polypeptide is a splice variant of the ⁇ 4 subunit.
  • the ⁇ 4 splice variant is encoded by a nucleic acid molecule identical to the nucleotide sequence of SEQ ID NO:6.
  • the polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed ⁇ 4 is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
  • the invention includes an immunogen which includes a ⁇ 4 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the ⁇ 4 polypeptide, e.g., a humoral response, an antibody response, or a cellular response.
  • the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO: 1.
  • the present invention also includes an antibody preparation specifically reactive with an epitope of the ⁇ 4 immunogen or generally of a ⁇ 4 polypeptide, preferably an epitope which consists all or in part of residues from the amino acid sequence of SEQ ID NO:l, or an epitope, which when bound to an antibody, results in the modulation of a biological activity.
  • the ⁇ 4-like polypeptide as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has an estimated molecular weight of 200 kDa as determined by SDS-PAGE.
  • the ⁇ 4 polypeptide has one or more of the following characteristics:
  • ⁇ 4 polypeptide of SEQ ID NO: 1 it can be isolated from human placenta chorionic villi; (v) it can associate with 3 or ⁇ 2 subunits; (vi) it has coiled coils in domains I and II. (vii) it has at least 5, preferably 6 or 7, and most preferably 8 of the cysteins found in native ⁇ 4 sequence.
  • compositions which includes a ⁇ 4 polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent.
  • additional component can be one which renders the composition for in vitro and in vivo pharmaceutical or veterinary use. Such uses can include aiding in wound healing or promotion of the adhesion of dermal and epidermal cells.
  • the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a ⁇ 4 polypeptide, e.g., a ⁇ 4 polypeptide described herein.
  • a preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 65% sequence identity to a nucleotide sequence of SEQ ID NO:2.
  • the ⁇ 4 polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least 70%, preferably 80%, more preferably about 90% to about 95%, and even more preferably about 99% sequence identity to the nucleotide sequence from SEQ ID NO:2.
  • the ⁇ 4 polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:2.
  • the subject ⁇ 4 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enh.ancer sequence, operably linked to the ⁇ 4 gene sequence (also referred to as LAMB4), e.g., to render the ⁇ 4 gene sequence suitable for use as an expression vector.
  • a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enh.ancer sequence
  • LAMB4 transcriptional enh.ancer sequence
  • the nucleic acid which encodes a ⁇ 4 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides from SEQ ID NO:2, more preferably to at least 20 consecutive nucleotides from SEQ ID NO:2.
  • the nucleic acid differs by at least one nucleotide from a nucleotide sequence of SEQ ID NO:2, nucleotides 4686-5870.
  • the invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide.
  • the oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO: 2, or naturally occurring mutants thereof.
  • the probe or primer further includes a label group attached thereto.
  • the label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor.
  • the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
  • the invention involves nucleic acids, e.g., RNA or DNA, encoding a ⁇ 4 polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
  • the invention features a cell or purified preparation of cells which include a ⁇ 4 transgene, or which otherwise misexpress a ⁇ 4 gene.
  • the cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells.
  • the cell or cells include a ⁇ 4 transgene, e.g., a heterologous form of a ⁇ 4 gene, e.g., a gene derived from humans (in the case of a non- human cell).
  • the ⁇ 4 transgene can be misexpressed, e.g., overexpressed or underexpressed.
  • the cell or cells include a gene which misexpress an endogenous ⁇ 4 gene, e.g., a gene the expression of which is disrupted, e.g., a knockout.
  • a gene which misexpress an endogenous ⁇ 4 gene e.g., a gene the expression of which is disrupted, e.g., a knockout.
  • Such cells can serve as a model for studying disorders which are related to mutated or misexpressed ⁇ 4 alleles or for use in drug screening.
  • the invention features a transgenic ⁇ 4 animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow.
  • the transgenic animal includes (and preferably express) a heterologous form of a ⁇ 4 gene, e.g., a gene derived from humans.
  • the ⁇ 4 transgene includes a tissue specific promoter, e.g., a milk-specific promoter.
  • the animal has an endogenous ⁇ 4 gene which is misexpressed, e.g., a knockout.
  • Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed ⁇ 4 alleles or for use in drug screening.
  • the invention features, a method of promoting adhesion of a first tissue element to a second tissue element.
  • the method includes contacting one or both of the first tissue element and the second tissue element with an amount of a laminin molecule described herein, e.g., laminin 12, or ⁇ 3 (or a laminin trimer which inlcudes ⁇ 3), sufficient to promote adhesion.
  • a laminin molecule described herein e.g., laminin 12, or ⁇ 3 (or a laminin trimer which inlcudes ⁇ 3)
  • the method can be performed in vivo, or in vitro.
  • the laminin is administered to the subject.
  • the administration can be directed to the site where adhesion is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
  • a tissue element can be a cell or a multi-cellular on acellular structure.
  • tissue elements include, skin cells, e.g., epidermal or dermal cells, neuronal cells, e.g., nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, non- traumatized, or non-diseased tissue is adj ascent or adhered to a specific tissue element recited herein.
  • the molecule is exogenous (e.g., administered to a subject) or is recombinant.
  • the method is an vivo method.
  • In vivo methods can be autologous, allogeneic, or xenogeneic.
  • autologous methods adhesion between two tissue elements from the subject is promoted.
  • allogeneic methods adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted.
  • xenogeneic methods adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted.
  • one element can be a donor tissue element which is implanted into a recipient subject.
  • the first tissue is healthy tissue, e.g., skin tissue
  • the second tissue is wounded, e.g., burned, diseased, traumatized, cut, and the tissue, or is a wound bed.
  • the first tissue is skin tissue, from the subject or from a donor
  • the second tissue is wounded, e.g., burned or abraided tissue.
  • the first tissue and second tissue element are normally adhered but have become detached from one another due to trauma, burn or other physical injury, disease, or age.
  • the first tissue element is a dermal cell and the second tissue element is an epidermal cell;
  • the first tissue element is a nerve cell or nerve and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adj ascent or adhered to the nerve cell or nerve;
  • the first tissue element is a retinal cell or retina tissue and the second tissue element is a cell or structure which in normal, non- traumatized, or non-diseased tissue is adj ascent or adhered to the a retinal cell or retina tissue, the first tissue is a nerve and the second tissue is basement membrane.
  • the administration of laminin can be repeated.
  • the invention features a method of promoting wound healing in a subject.
  • the method includes administering an amount of a laminin molecule described herein, e.g., laminin 12, ⁇ 3 (or a laminin trimer which inlcudes ⁇ 3), sufficient to promote healing to the wound.
  • the administration can be directed to the site where healing is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
  • the wound can be in any tissue, but preferably ina tissue in which the laminin normally occurs.
  • tissue in which the laminin normally occurs Examples skin, central or peripheral nervous tissue, tissues of the eye, e.g., the retinal, the basement membrane, or any tissue which in normal, non-traumatized, or non- diseased tissue is adjascent or adhered thereto.
  • the molecule is exogenous (e.g., administered to a subject) or is recombinant.
  • the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g, autoimmune attack, or abraided.
  • the administration of laminin can be repeated.
  • the invention features a method of promoting nerve growth or regeneration in a subject.
  • the method includes administering an amount of a laminin molecule described herein, e.g., laminin 12, or ⁇ 3 (or a laminin trimer which inlcudes ⁇ 3), sufficient to promote nerve growth or regeneration.
  • the administration can be directed to the site where nerve growth or regeneration is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
  • the molecule is exogenous (e.g., administered to a subject) or is recombinant.
  • the nerve growth or regeneration is promoted at a wound site.
  • the administration of laminin can be repeated.
  • the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes a laminin described herein, e.g., ⁇ 3 or laminin 12.
  • Such disorders include, e.g., a disorder associated with the misexpression of a laminin, e.g., laminin 12, or misexpression of the ⁇ 3 subunit; a disorder of the central or peripheral nervous system; a disorder associated with a genetic lesion at chromosome 9, region q31-34; Fukuyama-type muscular dystrophy; muscle-eye-brain disease; Walker-Warburg Syndrome (hydrocephalus, ageria, and retinal displasia); a retinal disorder, e.g, retinitis pigmentosa-deafness syndrome (which may be a subtype of Walker- Warburg Syndrome); a disorder associated with abnormal levels, e.g., abnormally low levels, of adhesion between tissues; a disorder associated with the basement membrane; a skin disorder, e.g., an epidermal or dermal, disorder; a disorder associated with the testis, spleen, placenta, thymus, ovary, small intestine, lung
  • the method includes one or more of the following: detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the ⁇ 3 gene, or other gene which encodes a subunit of laminin 12, e.g., detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5' control region; detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the ⁇ 3 gene, or other gene which encodes a subunit of laminin 12; detecting, in a tissue of the subject, the misexpression of the ⁇ 3 gene, or other gene which encodes a subunit of laminin 12 at the mRNA level, e.g., detecting a non-wild type level of a ⁇ 3, or an other laminin 12 subunit mRNA ; detecting, in a tissue of the subject, the misexpression of the ⁇ 3 gene, or other gene which encodes a sub
  • the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the ⁇ 3 gene, or other gene which encodes a subunit of laminin 12; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
  • detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:4, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the LAMG3 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
  • detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the ⁇ 3 gene, or other gene which encodes a subunit of laminin 12; the presence of a non- wild type splicing pattern of a messenger RNA transcript of the ⁇ 3 gene, or other gene which encodes a subunit of laminin 12; or a non-wild type level of ⁇ 3, or other subunit of laminin 12.
  • Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.
  • the method includes determining the structure of a ⁇ 3 gene, or other gene which encodes a subunit of laminin 12, an abnormal structure being indicative of risk for the disorder.
  • the method includes contacting a sample form the subject with an antibody to the laminin protein or a nucleic acid which hybridizes specifically with the ⁇ 3 gene, or other gene which encodes a subunit of laminin 12.
  • the invention features, a method of promoting adhesion of a first tissue element to a second tissue element.
  • the method includes contacting one or both of the first tissue element and the second tissue element with an amount of a laminin molecule described herein, e.g., ⁇ 4, sufficient to promote adhesion.
  • the method can be performed in vivo, or in vitro.
  • the laminin is administered to the subject.
  • the administration can be directed to the site where adhesion is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
  • a tissue element can be a cell or a multi-cellular on acellular structure.
  • tissue elements include, skin cells, e.g., epidermal or dermal cells, neuronal cells, e.g., nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, non- traumatized, or non-diseased tissue is adjascent or adhered to a specific tissue element recited herein.
  • skin cells e.g., epidermal or dermal cells
  • neuronal cells e.g., nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, non- traumatized, or non-diseased tissue is adjascent or adhered to a specific tissue element recited herein.
  • the molecule is exogenous (e.g., administered to a subject) or is recombinant.
  • the method is an vivo method.
  • In vivo methods can be autologous, allogeneic, or xenogeneic.
  • autologous methods adhesion between two tissue elements from the subject is promoted.
  • allogeneic methods adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted.
  • xenogeneic methods adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted.
  • one element can be a donor tissue element which is implanted into a recipient subject.
  • the first tissue is healthy tissue, e.g., skin tissue
  • the second tissue is wounded, e.g., burned, diseased, traumatized, cut, and the tissue, or is a wound bed.
  • the first tissue is skin tissue, from the subject or from a donor
  • the second tissue is wounded, e.g., burned or abraided tissue.
  • the first tissue element is a dermal cell and the second tissue element is an epidermal cell;
  • the first tissue element is a nerve cell or nerve and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to the nerve cell or nerve;
  • the first tissue is a nerve and the second tissue is basement membrane.
  • the administration of laminin can be repeated.
  • the invention features a method of promoting wound healing in a subject.
  • the method includes administering an amount of a laminin molecule described herein, e.g., ⁇ 4, sufficient to promote healing to the wound.
  • the administration can be directed to the site where healing is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
  • the wound can be in any tissue, but preferably in a tissue in which the laminin normally occurs in fetal or adult life.
  • tissue in which the laminin normally occurs in fetal or adult life.
  • examples include skin the basement membrane.
  • the molecule is exogenous (e.g., administered to a subject) or is recombinant.
  • the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g, autoimmune attack, or abraded.
  • the administration of laminin can be repeated.
  • the invention features a method of promoting tissue growth, development, or regeneration in a subject.
  • the method includes administering an amount of a laminin molecule described herein, e.g., ⁇ 4, sufficient to promote tissue growth, development, or regeneration in a subject.
  • the administration can be directed to the site where nerve growth or regeneration is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
  • the molecule is exogenous (e.g., administered to a subject) or is recombinant.
  • the nerve growth or regeneration is promoted at a wound site.
  • the administration of laminin can be repeated.
  • the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a laminin molecule described herein, e.g., ⁇ 4.
  • disorders include, e.g., a disorder associated with the misexpression of a laminin, e.g., ⁇
  • a disorder associated with a genetic lesion at chromosome region 7q22-q31.2 a developmetnal disorder
  • a disorder associated with abnormal levels e.g., abnormally low levels, of adhesion between tissues
  • a disorder associated with the basement membrane e.g., a skin disorder, e.g., an epidermal or dermal, disorder.
  • the method includes one or more of the following: detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the ⁇ 4 gene, e.g, detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5' control region; detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the ⁇ 4 gene; detecting, in a tissue of the subject, the misexpression of the ⁇ 4 gene, e.g., detecting a non-wild type level of a ⁇ 4 mRNA ; detecting, in a tissue of the subject, the misexpression of the ⁇ 4, at the protein level, e.g., detecting a non-wild type level of a ⁇ 4 polypeptide.
  • the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the ⁇ 4; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the ⁇ 4 gene, a gross chromosomal rearrangement of the ⁇ 4 gene, e.g., a translocation, inversion, or deletion.
  • detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:2, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the LAMB4 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
  • detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the ⁇ 4; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the ⁇ 4; or a non-wild type level of ⁇ 4.
  • Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.
  • the method includes determining the structure of the a ⁇ 4, an abnormal structure being indicative of risk for the disorder.
  • the method includes contacting a sample form the subject with an antibody to the ⁇ 4 protein or a nucleic acid which hybridizes specifically with the ⁇ 4.
  • the invention features, a method of evaluating a compound for the ability to interact with, e.g., bind, a subject laminin polypeptide, e.g., laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇ 4, or a laminin trimer which includes ⁇ 4.
  • the method includes: contacting the compound with the subject laminin polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject laminin polypeptide.
  • This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay.
  • This method can be used to identify naturally occurring molecules which interact with subject laminin polypeptide. It can also be used to find natural or synthetic inhibitors of subject laminin polypeptide.
  • the invention features, a method of evaluating a compound, e.g., a polypeptide, e.g., a naturally occurring ligand of or a naturally occuring substrate to which binds a subject laminin polypeptide, e.g., of laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇
  • a compound e.g., a polypeptide, e.g., a naturally occurring ligand of or a naturally occuring substrate to which binds a subject laminin polypeptide, e.g., of laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇
  • the method includes: contacting the compound with the subject laminin polypeptide; and evaluating the ability of the compound to interact with, e.g., to bind or form a complex with the subject laminin polypeptide, e.g., the ability of the compound to inhibit a subject laminin polypeptide/ligand interaction.
  • This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay.
  • This method can be used to identify compounds, e.g., fragments or analogs of a subject laminin polypeptide, which are agonists or antagonists of a subject laminin polypeptide.
  • the invention features, a method of evaluating a first compound, e.g., a subject laminin polypeptide, e.g., laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇
  • a first compound e.g., a subject laminin polypeptide, e.g., laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇
  • a laminin trimer which includes ⁇ 4 for the ability to bind a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or substrate to which binds a subject laminin polypeptide.
  • the method includes: contacting the first compound with the second compound; and evaluating the ability of the first compound to form a complex with the second compound.
  • This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject laminin polypeptide, which are agonists or antagonists of a subject laminin polypeptide.
  • the invention features a method for evaluating a compound, e.g., for the ability to modulate an interaction, e.g., the ability to inhibit an interaction of a subject laminin polypeptide, e.g., of laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇ 4, or a laminin trimer which includes ⁇ 4, with a second polypeptide, e.g., a polypeptide, e.g., a natural ligand of the of or a substrate wo which binds a subject laminin polypeptide, or a fragment thereof.
  • a subject laminin polypeptide e.g., of laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇ 4, or a laminin trimer which includes ⁇ 4
  • a second polypeptide e.g., a polypeptide, e.g., a natural ligand of the of or a substrate wo which
  • the method includes the steps of (i) combining the second polypeptide (or preferably a purified preparation thereof), a subject laminin polypeptide, (or preferably a purified preparation thereof), and a compound, e.g., under conditions wherein in the absence of the compound, the second polypeptide, and the subject laminin polypeptide, are able to interact, e.g., to bind or form a complex; and (ii) detecting the interaction, e.g., detecting the formation (or dissolution) of a complex which includes the second polypeptide, and the subject laminin polypeptide.
  • a change e.g., a decrease or increase, in the formation of the complex in the presence of a compound (relative to what is seen in the absence of the compound) is indicative of a modulation, e.g., an inhibition or promotion, of the interaction between the second polypeptide, and the subject laminin polypeptide.
  • the second polypeptide, and the subject laminin polypeptide are combined in a cell-free system and contacted with the compound; the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture; the subject laminin polypeptide, and the second polypeptide are simultaneously expressed in a cell, and the cell is contacted with the compound, e.g.
  • the invention features a two-phase method (e.g., a method having an in vitro, e.g., in a cell free system, and an in vivo phase) for evaluating a compound, e.g., for the ability to modulate, e.g., to inhibit or promote, an interaction of a subject laminin polypeptide subject laminin polypeptide, e.g., of laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇ 4, or a laminin trimer which includes ⁇ 4, with a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or a substrate to which binds a subject laminin polypeptide, or a fragment thereof.
  • a two-phase method e.g., a method having an in vitro, e.g., in a cell free system, and an in vivo phase
  • a compound e.g., for
  • the method includes steps (i) and (ii) of the method described immediately above performed in vitro, and further includes: (iii) determining if the compound modulates the interaction in vitro, e.g., in a cell free system, and if so; (iv) administering the compound to a cell or animal; and (v) evaluating the in vivo effect of the compound on an interaction, e.g., inhibition, of a subject laminin polypeptide, with a second polypeptide.
  • the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a subject laminin polypeptide, e.g., a laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇ 4, or a laminin trimer which includes ⁇ 4 polypeptide regulatory sequence.
  • the method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid.
  • the invention features a method of making a ⁇ 3 or ⁇ 4 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring ⁇ 3 or ⁇ 4 polypeptide, e.g., a naturally occurring ⁇ 3 or ⁇ 4 polypeptide.
  • the method includes: altering the sequence of a ⁇ 3 or ⁇ 4 polypeptide, e.g., altering the sequence , e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.
  • the invention features a method of making a fragment or analog of a ⁇ 3 or ⁇ 4 polypeptide having a biological activity of a naturally occurring ⁇ 3 or ⁇ 4 polypeptide.
  • the method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a ⁇ 3 or ⁇ 4 polypeptide, e.g., altering the sequence of a non- conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
  • the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid which encodes a subject laminin polypeptide, e.g., a laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇ 4, or a laminin trimer which includes ⁇ 4.
  • a subject laminin polypeptide e.g., a laminin 12, ⁇ 3, a laminin trimer which inlcudes ⁇ 3, ⁇ 4, or a laminin trimer which includes ⁇ 4.
  • the invention includes: a ⁇ 3, ⁇ 4 nucleic acid, e.g., a ⁇ 3, ⁇ 4 nucleic acid inserted into a vector; a cell transformed with a ⁇ 3, ⁇ 4 nucleic acid; a ⁇ 3, ⁇ 4 made by culturing a cell transformed with a ⁇ 3, ⁇ 4 nucleic acid; and a method of making a ⁇ 3, ⁇ 4 polypeptide including culturing a a cell transformed with a ⁇ 3, ⁇ 4 nucleic acid.
  • a ⁇ 3, ⁇ 4 nucleic acid e.g., a ⁇ 3, ⁇ 4 nucleic acid inserted into a vector
  • a cell transformed with a ⁇ 3, ⁇ 4 nucleic acid e.g., a ⁇ 3, ⁇ 4 nucleic acid inserted into a vector
  • a cell transformed with a ⁇ 3, ⁇ 4 nucleic acid e.g., a ⁇ 3, ⁇ 4 nucleic acid inserted
  • ⁇ 3 forms laminin 12 in association with ⁇ 2 and ⁇ l .
  • ⁇ 3 and ⁇ 4 polypetides of the invention can be expressed with, assembled with, or administered with other laminin subunits in any of the methods described herein.
  • ⁇ 3 can be assembled with an ⁇ and a ⁇ subunit to form a laminin trimer.
  • ⁇ 4 can be assembled with an ⁇ and a ⁇ subunit to form a laminin trimer.
  • any treatment or therapeutic application which administers ⁇ 3, a ⁇ 2 subunit can also be administered.
  • heterologous promoter is a promoter which is not naturally associated with a gene or a purified nucleic acid.
  • a “purified'Or “substantially pure” or isolated “preparation” of a polypeptide means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs.
  • the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which .are used to purify it.
  • the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation.
  • the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 ⁇ g of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
  • a “purified preparation of cells”, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
  • a “treatment”, as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
  • An "isolated” or "pur nucleic acid”, e.g., a substantially pure DNA, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived.
  • the term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences.
  • Substantially pure DNA can also includes a recombinant DNA which is part of a hybrid gene encoding sequence.
  • Sequence identity or homology refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position.
  • the percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity.
  • the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • transgene means a nucleic acid sequence (encoding, e.g., one or more subject laminin polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an
  • transgenic cell refers to a cell containing a transgene.
  • a transgenic animal is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue.
  • the term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
  • Unrelated to a ⁇ 3 or ⁇ 4 amino acid or nucleic acid sequence means having less than 30%) sequence identity, less than 20% sequence identity, or, preferably, less than 10% homology with a naturally occuring ⁇ 3 or ⁇ 4 sequence disclosed herein.
  • a polypeptide has ⁇ 3 biological activity if it has one or more of the properties of ⁇ 3 disclosed herein.
  • a polypeptide has biological activity if it is an antagonist, agonist, or super- agonist of a polypeptide having one of the properties of ⁇ 3 disclosed herein.
  • a polypeptide has ⁇ 4 biological activity if it has one or more of the properties of ⁇ 4 disclosed herein.
  • a polypeptide has biological activity if it is an antagonist, agonist, or super- agonist of a polypeptide having one of the properties of ⁇ 4 disclosed herein.
  • “Misexpression”, as used herein, refers to a non- wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
  • Subject can refer to a mammal, e.g., a human, or to an experimental or animal or disease model.
  • the subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.
  • nucleic acid as used herein can include fragments and equivalents.
  • equivalent refers to nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and include sequences that differ from the nucleotide sequences disclosed herein by degeneracy of the genetic code.
  • Figure I depicts the cDNA sequence for human ⁇ 2 subunit.
  • Figure 2 depicts the predicted amino acid sequence for human ⁇ 2 subunit.
  • Figure 3 depicts the cDNA sequence for human ⁇ 4 subunit.
  • Figure 4 depicts the predicted amino acid sequence for human ⁇ 4 subunit.
  • Figure 5 depicts an alignment of the amino acid sequence of human ⁇ 4 of SEQ ID NO: 1 and ⁇ 4 splice varient of SEQ ID NO:5 and laminin ⁇ l, ⁇ 2, and ⁇ 3 subunits.
  • Figure 6 provides a comparision of the similarities of laminin ⁇ 4 domains with the domains of other known laminin ⁇ subunits. Isolation of laminin 12
  • the soluble fraction was collected following centrifugation (30000 x g, 60 min) and precipitated by 300g/l of Ammonium Sulfate.
  • the sample was then dialyzed against the same buffer. Following dialysis, 0.5 volumes of buffer equilibrated DEAE-cellulose (DE-52, Whatman) was added and the mixture shaken overnight.
  • the final fraction of interest resulting from the above protocol contains multiple laminins.
  • the laminin 12 was resolved from this mixture by SDS-PAGE (3-5% polyacrylamide) under non-reducing conditions. Six b.and were resolved. Only the bands at approximately 560 kDa and at the top of the gel were shown to be reactive with poly clonal anti-laminin antiserum (Sigma, St. Louis, MO).
  • Protein sequencing was done according to Aebersold et al. (1987).
  • the complex laminin 5-laminin 7 was run on a polyacrylamide gel in the presence of 2-mercaptoethanol and blotted onto a nitrocellulose membrane (Biorad).
  • the 190 kDa band of ⁇ 2 and the 165 kDa ⁇ 3 band were separately excised and digested by protease trypsin.
  • the digested product was separated by HPLC and one fragment was sequenced on an Applied Biosystems sequenator (Applied Biosystems, Foster City, CA).
  • the 205 kDa chain contained a sequence identical to human laminin ct2, and was thus identified as human laminin ⁇ 2 subunit.
  • the 185 kDa produced two peptides identical to human ⁇ l, and was thus identified as human laminin ⁇ l subunit.
  • the band at 170 kDa contained three sequences not contained in any known laminin chain.
  • a N-terminal sequence of the 170 kDa chain was also determined.
  • the N-terminal sequence was not identical to any known laminin sequence.
  • the cDNA sequences of human ⁇ l and ⁇ 2 were used to probe the National Center for Biomedical Information (NCBI) dBestTM data base by BLAST search and a clone was isolated that was homologous, but not identical to ⁇ l and ⁇ 2. This clone was extended by PCR at the 5' end using Marathon cDNA from human placenta from Clonetech (Palo Alto, CA). The resulting sequence was determined to be 100% identical to all three of the 170 kDa band peptide sequences.
  • NCBI National Center for Biomedical Information
  • the human ⁇ 3 protein contains a nidogen-binding domain, which can be found, for example, from about amino acids 750-755 of SEQ ID NO:3.
  • the ⁇ 3 amino acid sequence and the nucleotide sequence encoding human laminin ⁇ 3 is shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
  • SEQ ID NO:3 and SEQ ID NO:4 By Northern analysis the size of the ⁇ 3 mRNA is approximately 5 kb, which is consistent with other laminin ⁇ subunits.
  • the ⁇ 3 mRNA transcript is expressed in human tissues including spleen, testis, brain, placenta, lung, and possibly liver. Chromosomal mapping using the ⁇ 3 cDNA sequence indicates that the human ⁇ 3 gene is located on chromosome 9q31-34. The location of ⁇ 3 on chromosome 9 was confirmed by FISH analysis using a 1.3 kb ⁇ 3 cDNA probe within the predicted domains I and II, which are the regions of the least sequence identity among ⁇ subunits.
  • Four human genes associated with Walker- Walburg syndrome, Fukuyama muscular dystrophy, retinitis pigmentosa-deafness syndrome and Eye, Muscle, Brain disease have also been mapped to chromosome 9q31-34.
  • the 170 kDa ( ⁇ 3) chain was excised from the reducing SDS-PAGE gel described above and injected into a rabbit for antibody production.
  • the resulting serum (rabbit 16) was evaluated by Western analysis and shown to react with the 170 kDa ⁇ 3 chain, and showed minor crossreactivity with other laminin chains.
  • this antiserum shows localization of ⁇ 3 to the following tissue areas: 1) sites of insertions of nerves into the dermal-epidermal junction basement membrane of human skin; 2) the inner nuclear layers, outer nuclear layers, and outer limiting membranes of human, mouse and rat neural retina; 3) the Purkinje cells, and molecular layers, and (perhaps) the glial cells of the mouse and rat cerebellum; 4) the neuromuscular junctions of skeletal muscle; and, 5) the taste buds of the cow tongue.
  • the ⁇ 3 was also shown to colocalize with protein ubiquitin carboxy terminal hydrolase I using antibody pGp 9.5.
  • the ⁇ 3 subunit also appears to colocalize with the ⁇ 2 subunit in the same tissue sections.
  • the initial 350 bp fragment of human laminin ⁇ 4 cDNA was amplified by touchdown RT-PCR from cultured human keratinocyte total RNA using nested primers made from the published chicken laminin ⁇ x 503 bp cDNA sequence (as described in Ybot- Gonzalez et al. (1995)).
  • Subsequent cDNA clones were isolated by nested PCR directly from a human placenta cDNA library packaged in lambda-gtl 1 (Clontech, Palo Alto, CA) or by nested PCR directly from human placenta Marathon-Ready cDNA (Clontech, Palo Alto, CA).
  • the 5' end of the cDNA was cloned using the 5'-RACE technique from human placenta total RNA.
  • the Expanded Long Template PCR System (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used for all PCR reactions.
  • the PCR products were ligated into the pCR2.1 vector (Invitrogen, San Diego, CA) and recombinant plasmids purified for sequencing using the QIAprepTM kit (Qiagen).
  • the DNA sequence was determined using either the Sequenase version 2.0 DNA Sequencing Kit (Amersham) and 35s-dATP or the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham) and 33p. ddNTPs.
  • PCR product bands were sequenced directly by cycle sequencing after excision from a TAE-EtBr agarose gel and purification using QIAquick Gel Extraction kit (Qiagen). Structural Analysis of DNA encoding ⁇ 4
  • the human ⁇ 4 protein retains the highest amino acid sequence identity with domains VI and V, which can be found, for example, from about amino acids 221-262 and about 263-535 of SEQ ID NO:l.
  • a short form, splice variant of ⁇ 4, which is approximately 3.84 kb and an estimated molecular weight of 120 kDa was also isolated.
  • the splice variant has 132 nucleotide sequence identical to the long form of ⁇ 4, with the sequence diverging at nucleotide 3375 and spliced into a unique 3' untranslated region.
  • the short form cDNA encodes a truncated ⁇ 4 subunit which contains only the short arm of the ⁇ 4 subunit and is missing the domains necessary for heterodimerization.
  • the ⁇ 4 amino acid sequence and the nucleotide sequence encoding human laminin ⁇ 4 is shown in SEQ ID NO:l and SEQ ID NO:2, respectively.
  • Northern analysis was performed using total RNA prepared from JAR cell, cultured human keratinocytes and human placenta using either Trizol (Gibco BRL, Bethesda, MD) or RNeasyTM (Qiagen) which was denatured, separated on a formaldehyde agarose gel and blotted onto nitrocellulose according to standard protocols (Sambrook, et al., 1989).
  • Trizol Gibco BRL, Bethesda, MD
  • RNeasyTM Qiagen
  • 32P-dCTP-labelled probes were generated from gel-purified restriction fragments using RediprimeTM random primer labeling kit (Amersham).
  • 32P-UTP-labelled antisense RNA probes were generated using the RNA transcription kit (Stratagene, La Jolla, CA) from cDNAs subcloned into Bluescript II KS+ (Stratagene, La Jolla, CA).
  • Chromosomal mapping with a ⁇ 4 cDNA probe indicates that the human ⁇ 4 subunit is located at locus 7q22-q31.2.
  • the gene encoding ⁇ l is located near, but not on, this position of chromosome 7.
  • Statistical analysis of the mapping data using markers for ⁇ l and ⁇ 4 suggest that the gene encoding ⁇ l is linked to both ends of the gene encoding ⁇ 4.
  • neonatal cutis laxa with manifold phenotype has been mapped near, but not in the same position, as the gene encoding ⁇ 4.
  • In situ hybridization to wounded human skin grafted into nude mice suggests that laminin ⁇ x is expressed in the dermis underneath the migrating epidermal tongues during wound closure.
  • ⁇ 4 specific antibody and tissue localization of ⁇ 4 Antibodies were raised in rabbits against a 26 kDa bacterial fusion protein which corresponds to the 175 amino acid residues of domain VI (e.g., from about amino acid residues 221-262) of SEQ ID NO: 1. Briefly the fusion protein was made by PCR amplification of nucleotides 302-785 of the cDNA encoding ⁇ 4 using adapter primers and cloned in- frame into the Ndel and SacII sites of pET-15b (Novagen). The fusion protein construct was confirmed by restriction mapping and DNA sequencing. Expression of the fusion protein was induced and separated from E. coli proteins using reducing SDS-PAG ⁇ .
  • mice against the fusion protein reacted well with ⁇ 4, as well as, ⁇ l and ⁇ 2 polypeptides.
  • the ⁇ 4 subunit contains six domains, and ⁇ interruption and a signal peptide.
  • the signal peptide and domain VI can be found, for example, at about amino acid residues 1-262 of S ⁇ Q ID NO:l.
  • Domain V can be found, for example, at about amino acid residues 263- 535 of S ⁇ Q ID NO:l.
  • Domains IV and III can be found, for example, at about amino acid residues 536-767 and 768-1178 of S ⁇ Q ID NO:l, respectively.
  • Domain I can be found, for example, at about amino acid residues 1409-1761 of S ⁇ Q ID NO:l.
  • the ⁇ 4 subunit (long form) is most similar in size and domain structure to laminin ⁇ l with an amino acid sequence identity of 42.5%. ⁇ 4 retains the highest levels of amino acid identity with the other laminin ⁇ subunits in domains VI and V, and the lowest levels in domains I and II, as shown in Figure 6. Using the MulticoilTM program, it was determined that only domains I and II of ⁇ 4 have a high probability of forming coiled coil structures.
  • Domains I and II of ⁇ 4 look most similar to human ⁇ 3. Both ⁇ 4 and ⁇ 3 are epithelial and the coiled coil structures in domains I and II dictate the ⁇ and ⁇ subunits with which the ⁇ subunits are associated. Thus, it is likely that ⁇ 4 associates with ⁇ 3 and ⁇ 2, as does the laminin ⁇ 3 subunit.
  • the cDNA encoding the splice variant of ⁇ 4 contains only the short arm of the ⁇ 4 subunit, and is missing the EGF repeat of domain III, as shown in Figure 5.
  • the ⁇ 4 polypeptide encoded by the ⁇ 4 c DNA splice variant is missing the coiled coil structures in domains I and II, rendering the short subunit unable to associate into a laminin heterotrimer.
  • PCR amplification of human genomic DNA suggest that the exon which encodes the alternative short form 3' untr-anslated region is located downstream from the carboxyl-most common exon, exon 23, and is splices out of the ⁇ 4 subunit, long form, by exon skipping.
  • Analogs can differ from naturally occurring ⁇ 3 or ⁇ 4 in amino acid sequence or in ways that do not involve sequence, or both.
  • Non-sequence modifications include in vivo or in vitro chemical derivatization of ⁇ 3 or ⁇ 4.
  • Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.
  • Preferred analogs include ⁇ 3 or ⁇ 4 (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the ⁇ 3 or ⁇ 4 biological activity.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be taken from the table below.
  • analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids; and cyclic analogs.
  • the gene constructs of the invention can also be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of a ⁇ 3 or ⁇ 4 polypeptide.
  • the invention features expression vectors for in vivo transfection and expression of a ⁇ 3 or ⁇ 4 polypeptide in particular cell types so as to reconstitute the function of, or alternatively, antagonize the function of ⁇ 3 or ⁇ 4 polypeptide in a cell in which that polypeptide is misexpressed.
  • Expression constructs of ⁇ 3 or ⁇ 4 polypeptides may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the ⁇ 3 or ⁇ 4 gene to cells in vivo.
  • Approaches include insertion of the subject gene in viral vectors including recombinant refroviruses, adeno virus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO 4 precipitation carried out in vivo.
  • a preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding a ⁇ 3 or ⁇ 4 polypeptide.
  • a viral vector containing nucleic acid e.g. a cDNA, encoding a ⁇ 3 or ⁇ 4 polypeptide.
  • Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid.
  • molecules encoded within the viral vector e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
  • Retroviras vectors .and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
  • the development of specialized cell lines (termed "packaging cells") which produce only replication-defective refroviruses has increased the utility of refroviruses for gene therapy, and defective refroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271).
  • a replication defective retroviras can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant refroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology. Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable refroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art.
  • Suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am. Refroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro .and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460- 6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci.
  • Another viral gene delivery system useful in the present invention utilizes adenovirus- derived vectors.
  • the genome of an adenoviras can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
  • Suitable adenoviral vectors derived from the adenoviras strain Ad type 5 dl324 or other strains of adenoviras are known to those skilled in the art.
  • adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al. (1992) cited supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral
  • DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenoviras or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32- 39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).
  • non-viral methods can also be employed to cause expression of a ⁇ 3 or ⁇ 4 polypeptide in the tissue of an animal.
  • Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non- viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject ⁇ 3 or ⁇ 4 gene by the targeted cell.
  • Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
  • a gene encoding a ⁇ 3 or ⁇ 4 polypeptide can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551 ; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
  • the gene delivery systems for the therapeutic ⁇ 3 or ⁇ 4 gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by Stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3054-3057).
  • the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • the invention includes transgenic animals which include cells (of that animal) which contain a ⁇ 3 or ⁇ 4 transgene and which preferably (though optionally) express (or misexpress) an endogenous or exogenous ⁇ 3 or ⁇ 4 gene in one or more cells in the animal.
  • the ⁇ 3 or ⁇ 4 transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs.
  • the expression of the transgene is restricted to specific subsets of cells, or tissues utilizing, for example, cis-acting sequences that control expression in the desired pattern.
  • Tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns, e.g., to restrict production to the milk or other secreted product of the animal.
  • Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.
  • Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.
  • DNA which encodes a protein or a particular domain or region of a protein.
  • Useful methods include PCR mutagenesis and saturation mutagenesis.
  • a library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein.)
  • PCR mutagenesis reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11-15). This is a very powerful and relatively rapid method of introducing random mutations.
  • the DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn 2+ to the PCR reaction.
  • the pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.
  • Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242).
  • This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, a . nd synthesis of a complimentary DNA strand.
  • the mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.
  • a library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev.
  • Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
  • Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989).
  • a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine).
  • Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
  • Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al, (DNA 2:183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA.
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single- stranded DNA template molecule.
  • the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
  • the starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated.
  • the codon(s) in the protein subunit DNA to be mutated are identified.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques.
  • This double-stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
  • This plasmid now contains the mutated desired protein subunit DNA sequence.
  • Combinatorial Mutagenesis can also be used to generate mutants.
  • the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences.
  • the variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.
  • Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to other laminin subunits, assembly into a trimeric laminin molecules, binding to natural ligands or substrates, facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • a desired activity e.g., in this case, binding to other laminin subunits, assembly into a trimeric laminin molecules, binding to natural ligands or substrates.
  • Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify fragments or analogs. These may include agonists, superagonists, and antagonists. (The subject protein and a protein it interacts with are used as the bait protein and fish proteins.) .
  • the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay".
  • the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).
  • a detectably labeled ligand can be used to score for potentially functional peptide homologs.
  • Fluorescently labeled ligands e.g., receptors, can be used to detect homolog which retain ligand-binding activity.
  • the use of fluorescently labeled ligands allows cells to be visually inspected and separated under a fluorescence microscope, or, where the mo ⁇ hology of the cell permits, to be separated by a fluorescence-activated cell sorter.
  • a gene library can be expressed as a fusion protein on the surface of a viral particle.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • coli filamentous phages Ml 3, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle.
  • Foreign epitopes can be expressed at the NH2-terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; .and Barbas et al. (1992) PNAS 89:4457-4461).
  • E. coli the outer membrane protein, LamB
  • LamB the outer membrane protein
  • Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands, e.g., to antibodies, and can elicit an immune response when the cells are administered to animals.
  • Other cell surface proteins e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al.
  • Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment.
  • Another large surface stracture used for peptide display is the bacterial motive organ, the flagellum.
  • Fusion of peptides to the subunit protein flagellin offers a dense array of may peptides copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083).
  • Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane protease IgA of Neisseria (Hansson et al. (1992) J. Bacteriol. 174, 4239-4245 .and Klauser et al. (1990) EMBOJ. 9, 1991-1999).
  • the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within.
  • An alternative scheme uses the DNA-binding protein Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3'- end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced.
  • This fusion retains the natural ability of Lad to bind to a short DNA sequence known as LacO operator (LacO).
  • LacO operator By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide- DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides.
  • the associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands.
  • a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dyno ⁇ hin B.
  • a cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dyno ⁇ hin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89- 1869)
  • peptides-on-plasmids differs in two important ways from the phage display methods.
  • the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini.
  • Both of the filamentous phage coat proteins, pill and pVIII are anchored to the phage through their C-termini, and the guest peptides are placed into the outward- extending N-terminal domains.
  • the phage-displayed peptides are presented right at the amino terminus of the fusion protein.
  • a second difference is the set of biological biases affecting the population of peptides actually present in the libraries.
  • the Lad fusion molecules are confined to the cytoplasm of the host cells.
  • the phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles.
  • the peptides in the Lad and phage libraries may differ significantly as a result of their exposure to different proteolytic activities.
  • phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to inco ⁇ oration into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). These particular biases are not a factor in the Lad display system.
  • RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening.
  • the polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification.
  • polysome-derived peptides on phage By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ⁇ LISA, or for binding specificity in a completion phage ⁇ LISA (Barret, et al. (1992) Anal. Biochem 204,357-364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host.
  • Secondary Screens The high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists.
  • the type of a secondary screen used will depend on the desired activity that needs to be tested.
  • an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.
  • the invention also provides for reduction of the protein binding domains of the subject ⁇ 3 or ⁇ 4 polypeptides to generate mimetics, e.g. peptide or non-peptide agents.
  • mimetics e.g. peptide or non-peptide agents.
  • Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein European patent applications EP-412,762A and EP-B31 ,080A.
  • Non-hydrolyzable peptide analogs of critical residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,
  • the invention also includes antibodies specifically reactive with a subject ⁇ 3 or ⁇ 4 polypeptides.
  • Anti-protein anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • Antibodies which specifically bind ⁇ 3 or ⁇ 4 epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of ⁇ 3 or ⁇ 4.
  • Anti ⁇ 3 or ⁇ 4 antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate ⁇ 3 or ⁇ 4 levels in tissue or bodily fluid as part of a clinical testing procedure.
  • antibodies of the present invention is in the immuno logical screening of cDNA libraries constructed in expression vectors such as ⁇ gtl 1, ⁇ gtl 8-23, ⁇ ZAP, and ⁇ ORF8.
  • Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
  • ⁇ gtl 1 will produce fusion proteins whose amino termini consist of ⁇ -galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
  • Antigenic epitopes of a subject polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with antibodies of the invention.
  • Phage, scored by this assay, can then be isolated from the infected plate.
  • the presence of homologs can be detected and cloned from other animals, and alternate isoforms (including splicing variants) can be detected and cloned from human sources.
  • allelic variations include allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide of SEQ ID NO: 1 or SEQ ID NO:3 (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby inco ⁇ orated by reference); and, polypeptides specifically bound by antisera to ⁇ 3 or ⁇ 4.
  • Nucleic acids and polypeptides of the invention includes those that differ from the sequences discolosed herein by virtue of sequencing errors in the disclosed sequences.
  • the invention also includes fragments, preferably biologically active fragments, or analogs of ⁇ 3 or ⁇ 4.
  • a biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of the ⁇ 3 or ⁇ 4 shown in SEQ ID NO:3 and SEQ ID NO:l, respectively, or of other naturally occurring ⁇ 3 or ⁇ 4, e.g., one or more of the biological activities described above.
  • fragments which exist in vivo e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's.
  • Fragments include those expressed in native or endogenous cells, e.g., as a result of post-translational processing, e.g., as the result of the removal of an amino-terminal signal sequence, as well as those made in expression systems, e.g., in CHO cells.
  • Particularly preferred fragments are fragments, e.g., active fragments, which are generated by proteolytic cleavage or alternative splicing events.
  • Other embodiments are within the following claims. What is claimed is:

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Abstract

The invention is drawn to a purified laminin 12 polypeptide that includes the α2 subunit, the β1 subunit and the η3 subunit. The invention is also drawn to isolated laminin β4 subunit and to isolated laminin η3 subunit. The invention is also drawn to polynucleotides encoding purified laminin 12 polypeptide as well as to polynucleotides encoding laminin β4 subunit and laminin η3 subunit.

Description

LAMININS AND USES THEREOF
BACKGROUND OF THE INVENTION The invention relates to the laminin 12, laminin subunit γ3, and laminin subunit βl, and methods of making and using these molecules.
SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery of a novel member of the laminin family, laminin 12. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of laminin 12 which includes an α2 subunit, a βl subunit and a γ3 subunit.
In a preferred embodiment, the α2 subunit has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human α2 subunit, e.g., the human α2 subunit of SEQ ID NO:7. The α2 subunit can be identical to a human α2 sequence, e.g., that of SEQ ID NO:7. In another embodiment, the α2 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO: 8. In addition, the α2 subunit can have substantially the same electrophoretic mobility as human α2 subunit, e.g., it appears as a 205 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an α2 subunit which is reactive with an α2-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 5H2. α2 specific antibodies can be made by methods known in the .art.
Another preferred embodiment of the invention features a βl subunit having at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human βl subunit, e.g., the human βl subunit of SEQ ID NO:9. Preferably, the βl subunit has the identical amino acid sequence of human βl subunit, e.g., that of SEQ ID NO:9. In another embodiment, the βl subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO: 10. In addition, the βl subunit can have substantially the same electrophoretic mobility as human βl subunit, e.g., it appears as a 185 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an βl subunit which is reactive with an βl-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 545. β 1 -specific antibodies can be made by methods known in the art.
In yet another preferred embodiment, the γ3 subunit of laminin 12 has at least 60% to about 70%), more preferably at least about 80%, even more preferably at least about 90% to about 95%), and most preferably at least about 99% sequence identity with human γ3 subunit, e.g., the γ3 subunit of SEQ ID NO:3. The γ3 subunit can be identical to a naturally occuring human γ3 subunit, e.g., that of SEQ ID NO:3. In another embodiment, the γ3 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:4. In addition, the γ3 subunit can have substantially the same electrophoretic mobility as human γ3 subunit, e.g., it appears as a 170 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an γ3 subunit which is reactive with an γ3-specific antibody. γ3-specifιc antibodies can be made by methods known in the art and taught herein. In a preferred embodiment, the laminin 12 is a trimer which can be found in, or can be isolated from human placental chorionic villi. In another embodiment, the laminin 12 is expressed by a recombinant cell, e.g., a bacterial cell, a cultured cell (e.g., a cultured eukaryotic cell) or a cell of a non-human transgenic animal. Cultured cells can include CHO cells or SF8 cells. Expression of laminin 12 in a transgenic animal can be general or can be under the control of a tissue specific promoter. Preferably, one or more sequences which encode subunits of the laminin 12 trimer are expressed in a preferred cell-type by a tissue specific promoter, e.g., a milk specific promoter.
The present invention is also based, in part, on the discovery of a novel laminin subunit, γ3. Accordingly, the invention features a recombinant or substantially pure or isolated preparation of a γ3 polypeptide.
In a preferred embodiment, the γ3 polypeptide has the following biological acitivities: 1) it promotes adhesion between tissue elements; 2) provides a site for insertion of nerves into the basement membrane. In other preferred embodiments: the γ3 polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:3; the γ3 polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:3; the γ3 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the γ3 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:3; the γ3 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring γ3 subunit; the γ3 polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, γ3 polypeptide. In a preferred embodiment, the invention includes a γ3 polypeptide encoded by a
DNA insert of a plasmid deposited with ATCC as Accession No: 209357. In another embodiment, the γ3 polypeptide is a polypeptide encoded by nucleotide sequences of the overlapping DNA inserts of more than one, preferably all seven of the plasmids deposited with ATCC as Accession No:209357. In preferred embodiments: the γ3 polypeptide is encoded by the nucleic acid in SEQ
ID NO:4, or by a nucleic acid having at least about 85%, more preferably at least about 90% to about 95%), and most preferably at least about 99% sequence identity with the nucleic acid from SEQ ID NO: 4. In preferred embodiments, the γ3 polypeptide includes a nidogen-binding domain.
Generally, the nidogen-binding domain is at least 5 residues in length and preferably, has about 70, 80, 90, or 95% sequence identity with the nidogen-binding domain of the protein shown in SEQ ID NO: 3 (amino acid residues 750-755). In another embodiment, the γ3 polypeptide includes at least 5, preferably 6 to 7, and most preferably 8 of the cysteins found in native γ3 protein. In yet another embodiment of the invention features a γ3 polypeptide that does not include or has an inactivated nidogen-binding domain which serves as an antagonist to γ3 biological activities. Furthermore, a γ3 polypeptide which has antagonist activity can have inactivated or excluded regions which comprise at least one cystein found in native γ3 protein. In a preferred embodiment, the γ3 polypeptide differs in amino acid sequence at up to
1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 3. In other preferred embodiments, the γ3 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 % of the residues from a sequence in SEQ ID NO: 3. Preferably, the differences are such that: the γ3 polypeptide exhibits a γ3 biological activity, e.g., the γ3 polypeptide retains a biological activity of a naturally occurring γ3 subunit.
In preferred embodiments the γ3 polypeptide includes a γ3 subunit sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In preferred embodiments, the γ3 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO: 3, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence from SEQ ID NO: 3.
In yet other preferred embodiments, the γ3 polypeptide is a recombinant fusion protein having a first γ3 portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to γ3. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In a preferred embodiment the γ3 polypeptide includes amino acid residues 750-755 of SEQ ID NO:3. In another embodiment, the γ3 polypeptide encodes domains IV- VI of the γ3 subunit. In preferred embodiments the γ3 polypeptide has antagonistic activity, and is capable of: inhibiting adhesion between connective tissues.
In a preferred embodiment, the γ3 polypeptide is a fragment of a naturally occurring γ 3 which inhibits connective tissue adhesion.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. The γ3 polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed γ3 is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
The invention includes an immunogen which includes a γ3 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the γ3 polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO: 3.
The present invention also includes an antibody preparation specifically reactive with an epitop&of the γ3 immunogen or generally of a γ3 polypeptide, preferably an epitope which consists all or in part of residues from the the amino acid sequence of SEQ ID NO:3, or an epitope, which when bound to an antibody, results in the modulation of a biological activity. In preferred embodiments the γ3-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has a molecular weight of 170 kDa as determined by SDS-PAGE.
In another embodiment, the γ3 polypeptide comprises amino acid residues 100-1761 of SEQ ID NO: 3.
In a preferred embodiment, the γ3 polypeptide has one or more of the following characteristics:
(i) it has the ability to promote adhesion between connective tissues; (ii) it has a molecular weight, amino acid composition or other physical characteristic of γ3 subunit of SEQ ID NO:3;
(iii) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with a γ3 polypeptide of SEQ ID NO:3; (iv) it can be isolated from human placenta chorionic villi; (v) it has a nidogen-binding domain which is preferably about 70%, 80%, 90% or 95% with amino acid residues 750-755 of SEQ ID NO:3;
(vi) it can colocalize with protein ubiquitin carboxy terminal hydroxylase I; (vii) it has at least 5, preferably 6 or 7, and most preferably 8 of the cysteins found amino acid sequence of native γ3.
Also included in the invention is a composition which includes a γ3 polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent. The additional component can be one which renders the composition useful for in vitro and in vivo pharmaceutical or veterinary use.
In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a γ3 polypeptide, e.g., a γ3 polypeptide described herein.
A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 85% sequence identity to a nucleotide sequence of SEQ ID NO:4. In other preferred embodiments, the γ3 polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least about 90% to about 95%, and more preferably about 98% to about 99% sequence identity to the nucleotide sequence from SEQ ID NO:4. In another preferred embodiment, the γ3 polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:4.
In prefered embodiments, the isolated nucleic acid molecule includes the nucleotide sequence of at least one and preferably all of the DNA inserts of the plasmids deposited with ATCC as Accession No: 209357.
In preferred embodiments, the subject γ3 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the γ3 gene sequence (also referred to as LAMG3), e.g., to render the γ3 gene sequence suitable for use as an expression vector.
In yet a further preferred embodiment, the nucleic acid which encodes a γ3 polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO:4. More preferably, the nucleic acid probe corresponds to at least 20 consecutive nucleotides from SEQ ID NO: 4. The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO: 4, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a γ3 polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention features a cell or purified preparation of cells which include a γ3 subunit transgene, or which otherwise misexpress a γ3 gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a γ3 transgene, e.g., a heterologous form of a γ3 gene, e.g., a gene derived from humans (in the case of a non-human cell). The γ3 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous γ3 gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed γ3 alleles or for use in drug screening.
In another aspect, the invention features a tr.ansgenic γ3 animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of a γ3 gene, e.g., a gene derived from hum-ans. In a further embodiment, the γ3 transgene includes a tissue specific promoter, e.g., a milk-specific promoter. In other preferred embodiments, the animal has an endogenous γ3 gene which is misexpressed, e.g., a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed γ3 alleles or for use in drug screening. The invention is also based, in part, on the discovery of a novel laminin subunit, β4.
Accordingly, the invention features a recombinant or substantially pure preparation of a β4 polypeptide.
In preferred embodiment, the β4 polypeptide has the following biological activities: 1) it promotes adhesion between tissue elements; 2) it aids in wound healing. In other preferred embodiments: the β4 polypeptide includes an amino acid sequence with at least 65%, 80%, 90%), 95%, 98%), or 99% sequence identity to an amino acid sequence from SEQ ID NO:l; the β4 polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO: 1; the β4 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the β4 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:l; the β4 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring β4 subunit; the β4 polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, β4 polypeptide.
In preferred embodiments: the β4 polypeptide is encoded by the nucleic acid in SEQ ID NO:2, or by a nucleic acid having at least about 65% to about 70%, more preferably at least 80%, even more preferably at least about 90% to about 95%, and most preferably about 99% sequence identity with the nucleic acid from SEQ ID NO: 2.
In preferred embodiments, the β4 polypeptide includes domains VI and V found in native β4 subunits. Amino acid residues from about 221-262 and 263-535 of SEQ ID NO: 1 are exemplary of domains VI and V, respectively, of β4. Generally, domain VI is at least 33 residues in length and has preferably at least about 60%, more preferably about 70% to about 80%), and most preferably about 90% to about 95% sequence identity with the amino acid residues 221-262 of the β4 protein shown in SEQ ID NO: 1. Domain V is at least 272 residues in length and has preferably at least about 60%, more preferably about 70% to about 80%), and most preferably about 90% to about 95% sequence identity with the amino acid residues 263-535 of the β4 protein shown in SEQ ID NO: 1. In another embodiment, the β4 polypeptide has at least 5, preferably 6 or 7, and most preferably 8 cysteins as found in native β4. In yet another embodiment, a β4 polypeptide which has antagonist activity has inactivated or excluded regions which comprise at least one of the cysteins found in native β4 protein.
In a preferred embodiment, the β4 polypeptide differs in .amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 1. In other preferred embodiments, the β4 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 %> of the residues from a sequence in SEQ ID NO: 1. Preferably, the differences are such that: the β4 polypeptide exhibits a β4 biological activity, e.g., the β4 polypeptide retains a biological activity of a naturally occurring β4 subunit.
In preferred embodiments the β4 polypeptide includes a β4 sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence. In preferred embodiments, the β4 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:l, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence from SEQ ID NO:l.
In yet other preferred embodiments, the β4 polypeptide is a recombinant fusion protein having a first β4 portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to β4. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In preferred embodiments the β4 polypeptide has antagonistic activity, and is capable of: inhibiting the adhesion of connective tissues.
Preferably, the β4 polypeptide is a fragment of a naturally occurring β4 which inhibits connective tissue adhesion.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, .and alternative translational and postranslational events. In one aspect of the invention, the β4 polypeptide is a splice variant of the β4 subunit. In another preferred embodiment, the β4 splice variant is encoded by a nucleic acid molecule identical to the nucleotide sequence of SEQ ID NO:6. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed β4 is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
The invention includes an immunogen which includes a β4 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the β4 polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO: 1.
The present invention also includes an antibody preparation specifically reactive with an epitope of the β4 immunogen or generally of a β4 polypeptide, preferably an epitope which consists all or in part of residues from the amino acid sequence of SEQ ID NO:l, or an epitope, which when bound to an antibody, results in the modulation of a biological activity. In preferred embodiments the β4-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has an estimated molecular weight of 200 kDa as determined by SDS-PAGE. In a preferred embodiment, the β4 polypeptide has one or more of the following characteristics:
(i) it has the ability to promote adhesion between connective tissues; (ii) it has a molecular weight, amino acid composition or other physical characteristic of β4 subunit of SEQ ID NO: 1 ; (iii) it has an overall sequence similarity of at least 50%, preferably at least
65%, more preferably at least 70, 80, 90, or 95%, with a β4 polypeptide of SEQ ID NO: 1 ; (iv) it can be isolated from human placenta chorionic villi; (v) it can associate with 3 or γ2 subunits; (vi) it has coiled coils in domains I and II. (vii) it has at least 5, preferably 6 or 7, and most preferably 8 of the cysteins found in native β4 sequence.
Also included in the invention is a composition which includes a β4 polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent. The additional component can be one which renders the composition for in vitro and in vivo pharmaceutical or veterinary use. Such uses can include aiding in wound healing or promotion of the adhesion of dermal and epidermal cells.
In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a β4 polypeptide, e.g., a β4 polypeptide described herein. A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 65% sequence identity to a nucleotide sequence of SEQ ID NO:2. In other preferred embodiments, the β4 polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least 70%, preferably 80%, more preferably about 90% to about 95%, and even more preferably about 99% sequence identity to the nucleotide sequence from SEQ ID NO:2. In another preferred embodiment, the β4 polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:2.
In preferred embodiments, the subject β4 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enh.ancer sequence, operably linked to the β4 gene sequence (also referred to as LAMB4), e.g., to render the β4 gene sequence suitable for use as an expression vector.
In yet a further preferred embodiment, the nucleic acid which encodes a β4 polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides from SEQ ID NO:2, more preferably to at least 20 consecutive nucleotides from SEQ ID NO:2. In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence of SEQ ID NO:2, nucleotides 4686-5870.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO: 2, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a β4 polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention features a cell or purified preparation of cells which include a β4 transgene, or which otherwise misexpress a β4 gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a β4 transgene, e.g., a heterologous form of a β4 gene, e.g., a gene derived from humans (in the case of a non- human cell). The β4 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous β4 gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or misexpressed β4 alleles or for use in drug screening.
In another aspect, the invention features a transgenic β4 animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of a β4 gene, e.g., a gene derived from humans. In a further embodiment, the β4 transgene includes a tissue specific promoter, e.g., a milk-specific promoter. In other preferred embodiments, the animal has an endogenous β4 gene which is misexpressed, e.g., a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed β4 alleles or for use in drug screening.
In another aspect, the invention features, a method of promoting adhesion of a first tissue element to a second tissue element. The method includes contacting one or both of the first tissue element and the second tissue element with an amount of a laminin molecule described herein, e.g., laminin 12, or γ3 (or a laminin trimer which inlcudes γ3), sufficient to promote adhesion. The method can be performed in vivo, or in vitro. In in vivo methods the laminin is administered to the subject. The administration can be directed to the site where adhesion is desired, e.g., by topical appication or by injection, or administered in a systemic fashion. A tissue element can be a cell or a multi-cellular on acellular structure. Examples of tissue elements include, skin cells, e.g., epidermal or dermal cells, neuronal cells, e.g., nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, non- traumatized, or non-diseased tissue is adj ascent or adhered to a specific tissue element recited herein.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the method is an vivo method. In vivo methods can be autologous, allogeneic, or xenogeneic. In autologous methods, adhesion between two tissue elements from the subject is promoted. In allogeneic methods, adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted. In xenogeneic methods, adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted. Thus, one element can be a donor tissue element which is implanted into a recipient subject.
In preferred embodiments the first tissue is healthy tissue, e.g., skin tissue, and the second tissue is wounded, e.g., burned, diseased, traumatized, cut, and the tissue, or is a wound bed. For example, the first tissue is skin tissue, from the subject or from a donor, and the second tissue is wounded, e.g., burned or abraided tissue. In preferred embodiments the first tissue and second tissue element are normally adhered but have become detached from one another due to trauma, burn or other physical injury, disease, or age.
In preferred embodiments: the first tissue element is a dermal cell and the second tissue element is an epidermal cell; the first tissue element is a nerve cell or nerve and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adj ascent or adhered to the nerve cell or nerve; the first tissue element is a retinal cell or retina tissue and the second tissue element is a cell or structure which in normal, non- traumatized, or non-diseased tissue is adj ascent or adhered to the a retinal cell or retina tissue, the first tissue is a nerve and the second tissue is basement membrane. The administration of laminin can be repeated.
In another aspect, the invention features a method of promoting wound healing in a subject. The method includes administering an amount of a laminin molecule described herein, e.g., laminin 12, γ3 (or a laminin trimer which inlcudes γ3), sufficient to promote healing to the wound. The administration can be directed to the site where healing is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
The wound can be in any tissue, but preferably ina tissue in which the laminin normally occurs. Examples skin, central or peripheral nervous tissue, tissues of the eye, e.g., the retinal, the basement membrane, or any tissue which in normal, non-traumatized, or non- diseased tissue is adjascent or adhered thereto. In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g, autoimmune attack, or abraided. The administration of laminin can be repeated.
In another aspect, the invention features a method of promoting nerve growth or regeneration in a subject. The method includes administering an amount of a laminin molecule described herein, e.g., laminin 12, or γ3 (or a laminin trimer which inlcudes γ3), sufficient to promote nerve growth or regeneration. The administration can be directed to the site where nerve growth or regeneration is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the nerve growth or regeneration is promoted at a wound site.
The administration of laminin can be repeated.
In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes a laminin described herein, e.g., γ3 or laminin 12. Such disorders include, e.g., a disorder associated with the misexpression of a laminin, e.g., laminin 12, or misexpression of the γ3 subunit; a disorder of the central or peripheral nervous system; a disorder associated with a genetic lesion at chromosome 9, region q31-34; Fukuyama-type muscular dystrophy; muscle-eye-brain disease; Walker-Warburg Syndrome (hydrocephalus, ageria, and retinal displasia); a retinal disorder, e.g, retinitis pigmentosa-deafness syndrome (which may be a subtype of Walker- Warburg Syndrome); a disorder associated with abnormal levels, e.g., abnormally low levels, of adhesion between tissues; a disorder associated with the basement membrane; a skin disorder, e.g., an epidermal or dermal, disorder; a disorder associated with the testis, spleen, placenta, thymus, ovary, small intestine, lung, or liver. The method includes one or more of the following: detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the γ3 gene, or other gene which encodes a subunit of laminin 12, e.g., detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5' control region; detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the γ3 gene, or other gene which encodes a subunit of laminin 12; detecting, in a tissue of the subject, the misexpression of theγ3 gene, or other gene which encodes a subunit of laminin 12 at the mRNA level, e.g., detecting a non-wild type level of a γ3, or an other laminin 12 subunit mRNA ; detecting, in a tissue of the subject, the misexpression of the γ3 gene, or other gene which encodes a subunit of laminin 12, at the protein level, e.g., detecting a non-wild type level of a γ3, or an other laminin 12 subunit polypeptide.
In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the γ3 gene, or other gene which encodes a subunit of laminin 12; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:4, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the LAMG3 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion. In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the γ3 gene, or other gene which encodes a subunit of laminin 12; the presence of a non- wild type splicing pattern of a messenger RNA transcript of the γ3 gene, or other gene which encodes a subunit of laminin 12; or a non-wild type level of γ3, or other subunit of laminin 12. Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.
In preferred embodiments the method includes determining the structure of a γ3 gene, or other gene which encodes a subunit of laminin 12, an abnormal structure being indicative of risk for the disorder. In preferred embodiments the method includes contacting a sample form the subject with an antibody to the laminin protein or a nucleic acid which hybridizes specifically with the γ3 gene, or other gene which encodes a subunit of laminin 12.
In another aspect, the invention features, a method of promoting adhesion of a first tissue element to a second tissue element. The method includes contacting one or both of the first tissue element and the second tissue element with an amount of a laminin molecule described herein, e.g., β4, sufficient to promote adhesion. The method can be performed in vivo, or in vitro. In in vivo methods the laminin is administered to the subject. The administration can be directed to the site where adhesion is desired, e.g., by topical application or by injection, or administered in a systemic fashion. A tissue element can be a cell or a multi-cellular on acellular structure. Examples of tissue elements include, skin cells, e.g., epidermal or dermal cells, neuronal cells, e.g., nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, non- traumatized, or non-diseased tissue is adjascent or adhered to a specific tissue element recited herein.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant. In preferred embodiments the method is an vivo method. In vivo methods can be autologous, allogeneic, or xenogeneic. In autologous methods, adhesion between two tissue elements from the subject is promoted. In allogeneic methods, adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted. In xenogeneic methods, adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted. Thus, one element can be a donor tissue element which is implanted into a recipient subject.
In preferred embodiments the first tissue is healthy tissue, e.g., skin tissue, and the second tissue is wounded, e.g., burned, diseased, traumatized, cut, and the tissue, or is a wound bed. For example, the first tissue is skin tissue, from the subject or from a donor, and the second tissue is wounded, e.g., burned or abraided tissue.
In preferred embodiments: the first tissue element is a dermal cell and the second tissue element is an epidermal cell; the first tissue element is a nerve cell or nerve and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to the nerve cell or nerve; the first tissue is a nerve and the second tissue is basement membrane.
The administration of laminin can be repeated.
In another aspect, the invention features a method of promoting wound healing in a subject. The method includes administering an amount of a laminin molecule described herein, e.g., β4, sufficient to promote healing to the wound. The administration can be directed to the site where healing is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
The wound can be in any tissue, but preferably in a tissue in which the laminin normally occurs in fetal or adult life. Examples examples include skin the basement membrane. In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g, autoimmune attack, or abraded. The administration of laminin can be repeated. In another aspect, the invention features a method of promoting tissue growth, development, or regeneration in a subject. The method includes administering an amount of a laminin molecule described herein, e.g., β4, sufficient to promote tissue growth, development, or regeneration in a subject. The administration can be directed to the site where nerve growth or regeneration is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant. In preferred embodiments the nerve growth or regeneration is promoted at a wound site.
The administration of laminin can be repeated.
In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a laminin molecule described herein, e.g., β4. Such disorders include, e.g., a disorder associated with the misexpression of a laminin, e.g., β
4; a disorder associated with a genetic lesion at chromosome region 7q22-q31.2; a developmetnal disorder; a disorder associated with abnormal levels, e.g., abnormally low levels, of adhesion between tissues; a disorder associated with the basement membrane; a skin disorder, e.g., an epidermal or dermal, disorder. The method includes one or more of the following: detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the β4 gene, e.g, detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5' control region; detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the β4 gene; detecting, in a tissue of the subject, the misexpression of the β4 gene, e.g., detecting a non-wild type level of a β4 mRNA ; detecting, in a tissue of the subject, the misexpression of the β4, at the protein level, e.g., detecting a non-wild type level of a β4 polypeptide. In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the β4; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the β4 gene, a gross chromosomal rearrangement of the β4 gene, e.g., a translocation, inversion, or deletion. For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:2, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the LAMB4 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
In preferred embodiments: detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the β 4; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the β4; or a non-wild type level of β4.
Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder. In preferred embodiments the method includes determining the structure of the a β4, an abnormal structure being indicative of risk for the disorder.
In preferred embodiments the method includes contacting a sample form the subject with an antibody to the β4 protein or a nucleic acid which hybridizes specifically with the β4. In another aspect, the invention features, a method of evaluating a compound for the ability to interact with, e.g., bind, a subject laminin polypeptide, e.g., laminin 12, γ3, a laminin trimer which inlcudes γ3, β4, or a laminin trimer which includes β4. The method includes: contacting the compound with the subject laminin polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject laminin polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with subject laminin polypeptide. It can also be used to find natural or synthetic inhibitors of subject laminin polypeptide.
In another aspect, the invention features, a method of evaluating a compound, e.g., a polypeptide, e.g., a naturally occurring ligand of or a naturally occuring substrate to which binds a subject laminin polypeptide, e.g., of laminin 12, γ3, a laminin trimer which inlcudes γ
3, β4, or a laminin trimer which includes β4, for the ability to bind a subject laminin polypeptide. The method includes: contacting the compound with the subject laminin polypeptide; and evaluating the ability of the compound to interact with, e.g., to bind or form a complex with the subject laminin polypeptide, e.g., the ability of the compound to inhibit a subject laminin polypeptide/ligand interaction. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject laminin polypeptide, which are agonists or antagonists of a subject laminin polypeptide.
In another aspect, the invention features, a method of evaluating a first compound, e.g., a subject laminin polypeptide, e.g., laminin 12, γ3, a laminin trimer which inlcudes γ3, β
4, or a laminin trimer which includes β4, for the ability to bind a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or substrate to which binds a subject laminin polypeptide. The method includes: contacting the first compound with the second compound; and evaluating the ability of the first compound to form a complex with the second compound. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject laminin polypeptide, which are agonists or antagonists of a subject laminin polypeptide.
In yet another aspect, the invention features a method for evaluating a compound, e.g., for the ability to modulate an interaction, e.g., the ability to inhibit an interaction of a subject laminin polypeptide, e.g., of laminin 12, γ3, a laminin trimer which inlcudes γ3, β4, or a laminin trimer which includes β4, with a second polypeptide, e.g., a polypeptide, e.g., a natural ligand of the of or a substrate wo which binds a subject laminin polypeptide, or a fragment thereof. The method includes the steps of (i) combining the second polypeptide (or preferably a purified preparation thereof), a subject laminin polypeptide, (or preferably a purified preparation thereof), and a compound, e.g., under conditions wherein in the absence of the compound, the second polypeptide, and the subject laminin polypeptide, are able to interact, e.g., to bind or form a complex; and (ii) detecting the interaction, e.g., detecting the formation (or dissolution) of a complex which includes the second polypeptide, and the subject laminin polypeptide. A change, e.g., a decrease or increase, in the formation of the complex in the presence of a compound (relative to what is seen in the absence of the compound) is indicative of a modulation, e.g., an inhibition or promotion, of the interaction between the second polypeptide, and the subject laminin polypeptide. In preferred embodiments: the second polypeptide, and the subject laminin polypeptide, are combined in a cell-free system and contacted with the compound; the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture; the subject laminin polypeptide, and the second polypeptide are simultaneously expressed in a cell, and the cell is contacted with the compound, e.g. in an interaction trap assay (e.g., a two-hybrid assay). In yet another aspect, the invention features a two-phase method (e.g., a method having an in vitro, e.g., in a cell free system, and an in vivo phase) for evaluating a compound, e.g., for the ability to modulate, e.g., to inhibit or promote, an interaction of a subject laminin polypeptide subject laminin polypeptide, e.g., of laminin 12, γ3, a laminin trimer which inlcudes γ3, β4, or a laminin trimer which includes β4, with a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or a substrate to which binds a subject laminin polypeptide, or a fragment thereof. The method includes steps (i) and (ii) of the method described immediately above performed in vitro, and further includes: (iii) determining if the compound modulates the interaction in vitro, e.g., in a cell free system, and if so; (iv) administering the compound to a cell or animal; and (v) evaluating the in vivo effect of the compound on an interaction, e.g., inhibition, of a subject laminin polypeptide, with a second polypeptide.
In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a subject laminin polypeptide, e.g., a laminin 12, γ3, a laminin trimer which inlcudes γ3, β4, or a laminin trimer which includes β4 polypeptide regulatory sequence. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid.
In another aspect, the invention features a method of making a γ3 or β4 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring γ3 or β4 polypeptide, e.g., a naturally occurring γ3 or β4 polypeptide. The method includes: altering the sequence of a γ3 or β4 polypeptide, e.g., altering the sequence , e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity. In another aspect, the invention features a method of making a fragment or analog of a γ3 or β4 polypeptide having a biological activity of a naturally occurring γ3 or β4 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a γ3 or β4 polypeptide, e.g., altering the sequence of a non- conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid which encodes a subject laminin polypeptide, e.g., a laminin 12, γ3, a laminin trimer which inlcudes γ3, β4, or a laminin trimer which includes β4.
In another aspect, the invention includes: a γ3, β4 nucleic acid, e.g., a γ3, β4 nucleic acid inserted into a vector; a cell transformed with a γ3, β4 nucleic acid; a γ3, β4 made by culturing a cell transformed with a γ3, β4 nucleic acid; and a method of making a γ3, β4 polypeptide including culturing a a cell transformed with a γ3, β4 nucleic acid.
The inventors have shown that γ3 forms laminin 12 in association with α2 and βl . However, we are unsure of the chain associations of γ3 within other tissues. It is very likely that γ3 can also associate with γ3, α3, α4, and α5; with β2, β3, β4 and β5. Therefore, our results predict 25 new laminins: laminins 12-37. γ3 and β4 polypetides of the invention can be expressed with, assembled with, or administered with other laminin subunits in any of the methods described herein. E.g., γ3 can be assembled with an α and a β subunit to form a laminin trimer. β4 can be assembled with an α and a β subunit to form a laminin trimer. In any treatment or therapeutic application which administers γ3, a β2 subunit can also be administered.
A "heterologous promoter", as used herein is a promoter which is not naturally associated with a gene or a purified nucleic acid.
A "purified'Or "substantially pure" or isolated "preparation" of a polypeptide, as used herein, means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which .are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 μg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A "purified preparation of cells", as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A "treatment", as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug. An "isolated" or " pure nucleic acid", e.g., a substantially pure DNA, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA can also includes a recombinant DNA which is part of a hybrid gene encoding sequence.
"Sequence identity or homology", as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology.
The terms "peptides", "proteins", and "polypeptides" are used interchangeably herein. As used herein, the term "transgene" means a nucleic acid sequence (encoding, e.g., one or more subject laminin polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term "transgenic cell" refers to a cell containing a transgene. As used herein, a "transgenic animal" is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term "tissue-specific promoter" means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue. The term also covers so-called "leaky" promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
"Unrelated to a γ3 or β4 amino acid or nucleic acid sequence" means having less than 30%) sequence identity, less than 20% sequence identity, or, preferably, less than 10% homology with a naturally occuring γ3 or β4 sequence disclosed herein.
A polypeptide has γ3 biological activity if it has one or more of the properties of γ3 disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or super- agonist of a polypeptide having one of the properties of γ3 disclosed herein.
A polypeptide has β4 biological activity if it has one or more of the properties of β4 disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or super- agonist of a polypeptide having one of the properties of β4 disclosed herein.
"Misexpression", as used herein, refers to a non- wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
Subject, as used herein, can refer to a mammal, e.g., a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.
As described herein, one aspect of the invention features a substantially pure (or recombinant) nucleic acid which includes a nucleotide sequence encoding a γ3 or β4 polypeptide and/or equivalents of such nucleic acids. The term nucleic acid as used herein can include fragments and equivalents. The term equivalent refers to nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and include sequences that differ from the nucleotide sequences disclosed herein by degeneracy of the genetic code.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.
Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
DETAILED DESCRIPTION
The drawings are briefly described.
Figure I depicts the cDNA sequence for human α2 subunit.
Figure 2 depicts the predicted amino acid sequence for human α2 subunit.
Figure 3 depicts the cDNA sequence for human β4 subunit.
Figure 4 depicts the predicted amino acid sequence for human β4 subunit.
Figure 5 depicts an alignment of the amino acid sequence of human β4 of SEQ ID NO: 1 and β4 splice varient of SEQ ID NO:5 and laminin βl, β2, and β3 subunits.
Figure 6 provides a comparision of the similarities of laminin β4 domains with the domains of other known laminin β subunits. Isolation of laminin 12
Laminin 12 was isolated from human placental chor ionic villi. Briefly, human chorionic placental villi were frozen in liquid nitrogen, ground in a Waring blender and washed in 1 M NaCl. The final tissue pellet (200g, wet weight) was suspended in 1 L of extraction buffer (50 mM Tris-HCl 50 mM, pH=7.8; NaCl 0.5M, EDTA lOmM, 625 mg/1 of N-ethylmaleimide, 150 mg/1 of phenylmethylsulphonyl fluoride. The suspension was incubated at 4°C with stirring for 48 h. Unless otherwise noted, all subsequent steps were performed at 4°C. The soluble fraction was collected following centrifugation (30000 x g, 60 min) and precipitated by 300g/l of Ammonium Sulfate. The precipitated proteins were collected by centrifugation (30000 x g, 60 min) and redissolved into chromatography buffer (2M Urea, 25 mM NaCl, 5 mM EDTA, and 50 mM Tris-HCl, pH=7.8). The sample was then dialyzed against the same buffer. Following dialysis, 0.5 volumes of buffer equilibrated DEAE-cellulose (DE-52, Whatman) was added and the mixture shaken overnight. Material not bound to DEAE-cellulose was collected by filtration on a Buchner funnel (Whatman filter 4) and precipitated by addition of 300g/l of ammonium sulfate. The proteins were collected by centrifugation (30000 x g, 60 min), redissolved in the Concanavalin-A buffer (0.5 M NaCl, 5 mM CaCl2, 5 mM MgCl2, and Tris-HCl 50 mM, pH=7.8) and dialyzed against the same buffer overnight. The fraction was applied to a 2.5 x 5 cm Concanavalin-A sepharose column (Pharmacia), and unbound material was removed by extensive washing. Bound proteins were first eluted with 10 mM α-D- Mannopyrannoside (Sigma, St. Louis, MO) and secondly with 1 M α-D-Glucopyrannoside (Sigma, St. Louis, MO). A third elution with 1M α-D-Manno-pyrannoside (Sigma, St. Louis, MO) allowed the recovery of the proteins of interest. Each fraction was independently concentrated to 10 ml on a Amicon™ concentrator (30 kDa membrane) and applied to a 2.5 x 100 cm Sephacryl S-500 column in a 0.5 M NaCl, 50 mM Tris-HCl, pH=7.8 buffer. The fractions of interest were pooled, dialyzed against
Mono-Q buffer (0.1 M NaCl, 25 mM Tris-HCl, ρH=7.8) and applied to the 1 x 5 cm Mono-Q column (Pharmacia). Elution was achieved with a 60 ml 0.1-0.5 M NaCl gradient.
The final fraction of interest resulting from the above protocol contains multiple laminins. The laminin 12 was resolved from this mixture by SDS-PAGE (3-5% polyacrylamide) under non-reducing conditions. Six b.and were resolved. Only the bands at approximately 560 kDa and at the top of the gel were shown to be reactive with poly clonal anti-laminin antiserum (Sigma, St. Louis, MO).
Isolation of α2. βl.γ3 subunits from laminin 12 Laminin 12 was excised, equilibrated and reduced in 10% 2-me SDS-PAGE sample buffer, and resolved by 5% SDS-PAGE. Three bands were resolved, which were approximately 205 kDa, 185 kDa, and 170 kDa. The band at 185 kDa reacted with monoclonal antibody 545, specific to the laminin βl subunit. Each of the three bands were digested with trypsin and the peptides were resolved by HPLC. The selected resolves were subject to peptide sequencing.
Sequencing of the α2. βl subunits of laminin 12
Protein sequencing was done according to Aebersold et al. (1987). The complex laminin 5-laminin 7 was run on a polyacrylamide gel in the presence of 2-mercaptoethanol and blotted onto a nitrocellulose membrane (Biorad). The 190 kDa band of β2 and the 165 kDa α3 band were separately excised and digested by protease trypsin. The digested product was separated by HPLC and one fragment was sequenced on an Applied Biosystems sequenator (Applied Biosystems, Foster City, CA). The 205 kDa chain contained a sequence identical to human laminin ct2, and was thus identified as human laminin α2 subunit. The 185 kDa produced two peptides identical to human βl, and was thus identified as human laminin βl subunit. The band at 170 kDa contained three sequences not contained in any known laminin chain. A N-terminal sequence of the 170 kDa chain was also determined. In addition, the N-terminal sequence was not identical to any known laminin sequence.
Identification of the γ3 subunit
The cDNA sequences of human γl and γ2 were used to probe the National Center for Biomedical Information (NCBI) dBest™ data base by BLAST search and a clone was isolated that was homologous, but not identical to γl and γ2. This clone was extended by PCR at the 5' end using Marathon cDNA from human placenta from Clonetech (Palo Alto, CA). The resulting sequence was determined to be 100% identical to all three of the 170 kDa band peptide sequences.
Comparison of the nucleotide sequence of the isolated γ3 subunit to γl, demonstrated about 80% sequence identity.
Structural Analysis of γ3 encoding DNA
The human cDNA encoding γ3, which is approximately 4710 nucleotides in length, encodes a protein having an estimated molecular weight of approximately 146 kDa (including post-translational modifications) and which is approximately 1570 amino acid residues in length. The human γ3 protein contains a nidogen-binding domain, which can be found, for example, from about amino acids 750-755 of SEQ ID NO:3. The γ3 amino acid sequence and the nucleotide sequence encoding human laminin γ3 is shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. By Northern analysis the size of the γ3 mRNA is approximately 5 kb, which is consistent with other laminin γ subunits. The γ3 mRNA transcript is expressed in human tissues including spleen, testis, brain, placenta, lung, and possibly liver. Chromosomal mapping using the γ3 cDNA sequence indicates that the human γ3 gene is located on chromosome 9q31-34. The location of γ3 on chromosome 9 was confirmed by FISH analysis using a 1.3 kb γ3 cDNA probe within the predicted domains I and II, which are the regions of the least sequence identity among γ subunits. Four human genes associated with Walker- Walburg syndrome, Fukuyama muscular dystrophy, retinitis pigmentosa-deafness syndrome and Eye, Muscle, Brain disease have also been mapped to chromosome 9q31-34.
Production of a γ3 specific antibody and tissue localization of γ3
The 170 kDa (γ3) chain was excised from the reducing SDS-PAGE gel described above and injected into a rabbit for antibody production. The resulting serum (rabbit 16) was evaluated by Western analysis and shown to react with the 170 kDa γ3 chain, and showed minor crossreactivity with other laminin chains.
Using immunofluorescence, this antiserum shows localization of γ3 to the following tissue areas: 1) sites of insertions of nerves into the dermal-epidermal junction basement membrane of human skin; 2) the inner nuclear layers, outer nuclear layers, and outer limiting membranes of human, mouse and rat neural retina; 3) the Purkinje cells, and molecular layers, and (perhaps) the glial cells of the mouse and rat cerebellum; 4) the neuromuscular junctions of skeletal muscle; and, 5) the taste buds of the cow tongue.
The γ3 was also shown to colocalize with protein ubiquitin carboxy terminal hydrolase I using antibody pGp 9.5. The γ3 subunit also appears to colocalize with the α2 subunit in the same tissue sections.
Isolation and Sequencing of cDNA encoding β4
The initial 350 bp fragment of human laminin β4 cDNA was amplified by touchdown RT-PCR from cultured human keratinocyte total RNA using nested primers made from the published chicken laminin β x 503 bp cDNA sequence (as described in Ybot- Gonzalez et al. (1995)). Subsequent cDNA clones were isolated by nested PCR directly from a human placenta cDNA library packaged in lambda-gtl 1 (Clontech, Palo Alto, CA) or by nested PCR directly from human placenta Marathon-Ready cDNA (Clontech, Palo Alto, CA). The 5' end of the cDNA was cloned using the 5'-RACE technique from human placenta total RNA. The Expanded Long Template PCR System (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used for all PCR reactions. The PCR products were ligated into the pCR2.1 vector (Invitrogen, San Diego, CA) and recombinant plasmids purified for sequencing using the QIAprep™ kit (Qiagen). The DNA sequence was determined using either the Sequenase version 2.0 DNA Sequencing Kit (Amersham) and 35s-dATP or the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham) and 33p. ddNTPs. At least two independent cDNA subclones were sequenced to rule out Taq polymerase-generated nucleotide substitutions. In some cases, PCR product bands were sequenced directly by cycle sequencing after excision from a TAE-EtBr agarose gel and purification using QIAquick Gel Extraction kit (Qiagen). Structural Analysis of DNA encoding β4
The human cDNA encoding a long form β4, which is approximately 5.87 kb, encodes a protein having an estimated molecular weight of approximately 200 kDa and which is approximately 1761 amino acid residues in length. The human β4 protein retains the highest amino acid sequence identity with domains VI and V, which can be found, for example, from about amino acids 221-262 and about 263-535 of SEQ ID NO:l. In addition, a short form, splice variant of β4, which is approximately 3.84 kb and an estimated molecular weight of 120 kDa, was also isolated. The splice variant has 132 nucleotide sequence identical to the long form of β4, with the sequence diverging at nucleotide 3375 and spliced into a unique 3' untranslated region. The short form cDNA encodes a truncated β4 subunit which contains only the short arm of the β4 subunit and is missing the domains necessary for heterodimerization. The β4 amino acid sequence and the nucleotide sequence encoding human laminin β4 is shown in SEQ ID NO:l and SEQ ID NO:2, respectively.
Northern analysis was performed using total RNA prepared from JAR cell, cultured human keratinocytes and human placenta using either Trizol (Gibco BRL, Bethesda, MD) or RNeasy™ (Qiagen) which was denatured, separated on a formaldehyde agarose gel and blotted onto nitrocellulose according to standard protocols (Sambrook, et al., 1989). In addition, A human multiple tissue northern blot (Clontech, Palo Alto, CA) and Human Northern Territory normal tissue blots and custom fetal skin northern blot (Invitrogen, San Diego, CA) were used. Hybridization and washing were performed using NorthernMAX™ buffer system (Ambion) by manufacturer's recommended protocols. 32P-dCTP-labelled probes were generated from gel-purified restriction fragments using Rediprime™ random primer labeling kit (Amersham). 32P-UTP-labelled antisense RNA probes were generated using the RNA transcription kit (Stratagene, La Jolla, CA) from cDNAs subcloned into Bluescript II KS+ (Stratagene, La Jolla, CA).
Northern blotting showed that human laminin β4 is expressed in JAR cells, derived from undeveloped chronic villi and in placenta. By RT-PCR, it is also expressed in cultured keratinocytes. Using a northern blot of human fetal skin developmental progression, β4 subunit (long form) demonstrates strong expression at week twelve of fetal development and persists until birth, but expression is barely detectable in adult skin. The β4 splice variant, however, is expressed in various tissues including adult heart, brain, lung, liver, skeletal muscle, kidney, spleen, stomach, esophagus, intestine, colon, uterus, bladder, adipose tissue and pancreas. Chromosomal mapping with a β4 cDNA probe indicates that the human β4 subunit is located at locus 7q22-q31.2. The gene encoding βl is located near, but not on, this position of chromosome 7. Statistical analysis of the mapping data using markers for βl and β4 suggest that the gene encoding βl is linked to both ends of the gene encoding β4. In addition, neonatal cutis laxa with manifold phenotype has been mapped near, but not in the same position, as the gene encoding β4. In situ hybridization to wounded human skin grafted into nude mice suggests that laminin β x is expressed in the dermis underneath the migrating epidermal tongues during wound closure.
A GenBank™ search using the human nucleotide sequence encoding β4 as shown in SEQ ID NO: 3 revealed an EST, which corresponds to nucleotides 4686-5870 of the human nucleotide sequence encoding β4 depicted in SEQ ID NO:3. Alignment of cDNA encoding β4 with the genes encoding human laminin βl and laminin β2 shows 61% and 59% sequence identity, respectively, as shown in Figure 5.
Production of a β4 specific antibody and tissue localization of β4 Antibodies were raised in rabbits against a 26 kDa bacterial fusion protein which corresponds to the 175 amino acid residues of domain VI (e.g., from about amino acid residues 221-262) of SEQ ID NO: 1. Briefly the fusion protein was made by PCR amplification of nucleotides 302-785 of the cDNA encoding β4 using adapter primers and cloned in- frame into the Ndel and SacII sites of pET-15b (Novagen). The fusion protein construct was confirmed by restriction mapping and DNA sequencing. Expression of the fusion protein was induced and separated from E. coli proteins using reducing SDS-PAGΕ. Bands corresponding to the fusion protein were excised from the gel, equilibrated and homogenized using Freud's adjuvant. The same fusion protein was also western blotted on nitrocellulose, dissolved in DMSO and used to immunize mice for monoclonal antibody production.
The polyclonal antisera raised in mice against the fusion protein reacted well with β4, as well as, βl and β2 polypeptides.
Structural Analysis of the β4 subunit and the β4 splice variant The β4 subunit contains six domains, and α interruption and a signal peptide. The signal peptide and domain VI can be found, for example, at about amino acid residues 1-262 of SΕQ ID NO:l. Domain V can be found, for example, at about amino acid residues 263- 535 of SΕQ ID NO:l. Domains IV and III can be found, for example, at about amino acid residues 536-767 and 768-1178 of SΕQ ID NO:l, respectively. Domain I can be found, for example, at about amino acid residues 1409-1761 of SΕQ ID NO:l.
The β4 subunit (long form) is most similar in size and domain structure to laminin βl with an amino acid sequence identity of 42.5%. β4 retains the highest levels of amino acid identity with the other laminin β subunits in domains VI and V, and the lowest levels in domains I and II, as shown in Figure 6. Using the Multicoil™ program, it was determined that only domains I and II of β4 have a high probability of forming coiled coil structures.
Domains I and II of β4 look most similar to human β3. Both β4 and β3 are epithelial and the coiled coil structures in domains I and II dictate the α and γ subunits with which the β subunits are associated. Thus, it is likely that β4 associates with α3 and γ2, as does the laminin β3 subunit.
The cDNA encoding the splice variant of β4 contains only the short arm of the β4 subunit, and is missing the EGF repeat of domain III, as shown in Figure 5. Thus, the β4 polypeptide encoded by the β4 c DNA splice variant is missing the coiled coil structures in domains I and II, rendering the short subunit unable to associate into a laminin heterotrimer. PCR amplification of human genomic DNA suggest that the exon which encodes the alternative short form 3' untr-anslated region is located downstream from the carboxyl-most common exon, exon 23, and is splices out of the β4 subunit, long form, by exon skipping.
Analogs of γ3 and β4
Analogs can differ from naturally occurring γ3 or β4 in amino acid sequence or in ways that do not involve sequence, or both. Non-sequence modifications include in vivo or in vitro chemical derivatization of γ3 or β4. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation. Preferred analogs include γ3 or β4 (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the γ3 or β4 biological activity. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be taken from the table below.
TABLE 1
CONSERVATIVE AMINO ACID REPLACEMENTS
Other analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., β or γ amino acids; and cyclic analogs.
Gene Therapy The gene constructs of the invention can also be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of a γ3 or β 4 polypeptide. The invention features expression vectors for in vivo transfection and expression of a γ3 or β4 polypeptide in particular cell types so as to reconstitute the function of, or alternatively, antagonize the function of γ3 or β4 polypeptide in a cell in which that polypeptide is misexpressed. Expression constructs of γ3 or β4 polypeptides, may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the γ3 or β4 gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant refroviruses, adeno virus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation carried out in vivo. A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding a γ3 or β4 polypeptide. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
Retroviras vectors .and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective refroviruses has increased the utility of refroviruses for gene therapy, and defective refroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). A replication defective retroviras can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant refroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology. Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable refroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 and ψ Am. Refroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro .and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460- 6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).
Another viral gene delivery system useful in the present invention utilizes adenovirus- derived vectors. The genome of an adenoviras can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenoviras strain Ad type 5 dl324 or other strains of adenoviras (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art.
Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al. (1992) cited supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral
DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).
Yet another viral vector system useful for delivery of the subject gene is the adeno- associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenoviras or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in
Micro, and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32- 39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).
In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a γ3 or β4 polypeptide in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non- viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject γ3 or β4 gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
In a representative embodiment, a gene encoding a γ3 or β4 polypeptide can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551 ; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
In clinical settings, the gene delivery systems for the therapeutic γ3 or β4 gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by Stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3054-3057).
The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced in tact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
Transgenic Animals
The invention includes transgenic animals which include cells (of that animal) which contain a γ3 or β4 transgene and which preferably (though optionally) express (or misexpress) an endogenous or exogenous γ3 or β4 gene in one or more cells in the animal. The γ3 or β4 transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs. In preferred embodiments, the expression of the transgene is restricted to specific subsets of cells, or tissues utilizing, for example, cis-acting sequences that control expression in the desired pattern. Tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns, e.g., to restrict production to the milk or other secreted product of the animal.
Production of Fragments and Analogs Generation of Fragments Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.
Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.
Generation of Analogs: Production of Altered DNA and Peptide Sequences by Random Methods Amino acid sequence variants of a protein can be prepared by random mutagenesis of
DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein.)
PCR Mutagenesis
In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11-15). This is a very powerful and relatively rapid method of introducing random mutations. The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn2+ to the PCR reaction. The pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.
Saturation Mutagenesis
Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, a . nd synthesis of a complimentary DNA strand. The mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.
Degenerate Oligonucleotides A library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815).
Generation of Analogs: Production of Altered DNA and Peptide Sequences by Directed Mutagenesis
Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
Alanine Scanning Mutagenesis Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989). In alanine scanning, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
Oligonucleotide-Mediated Mutagenesis
Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al, (DNA 2:183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA. Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single- stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
Cassette Mutagenesis
Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. (Gene, 34:315[ 1985]). The starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the desired protein subunit DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated desired protein subunit DNA sequence.
Combinatorial Mutagenesis Combinatorial mutagenesis can also be used to generate mutants. E.g., the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences. The variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.
Primary High-Through-Put Methods for Screening Libraries of Peptide Fragments or
Homologs
Various techniques are known in the art for screening generated mutant gene products. Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to other laminin subunits, assembly into a trimeric laminin molecules, binding to natural ligands or substrates, facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques.
Two Hybrid Systems
Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify fragments or analogs. These may include agonists, superagonists, and antagonists. (The subject protein and a protein it interacts with are used as the bait protein and fish proteins.) .
Display Libraries In one approach to screening assays, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay". For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140). In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, e.g., receptors, can be used to detect homolog which retain ligand-binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the moφhology of the cell permits, to be separated by a fluorescence-activated cell sorter.
A gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at concentrations well over 1013 phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages Ml 3, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle. Foreign epitopes can be expressed at the NH2-terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; .and Barbas et al. (1992) PNAS 89:4457-4461).
A common approach uses the maltose receptor of E. coli (the outer membrane protein, LamB) as a peptide fusion partner (Charbit et al. (1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands, e.g., to antibodies, and can elicit an immune response when the cells are administered to animals. Other cell surface proteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al. (1991) Bio/Tech 9, 1369-1372), as well as large bacterial surface structures have served as vehicles for peptide display. Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment. Another large surface stracture used for peptide display is the bacterial motive organ, the flagellum. Fusion of peptides to the subunit protein flagellin offers a dense array of may peptides copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane protease IgA of Neisseria (Hansson et al. (1992) J. Bacteriol. 174, 4239-4245 .and Klauser et al. (1990) EMBOJ. 9, 1991-1999).
In the filamentous phage systems and the LamB system described above, the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within. An alternative scheme uses the DNA-binding protein Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3'- end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced. This fusion retains the natural ability of Lad to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide- DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynoφhin B. A cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynoφhin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89- 1869)
This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pill and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outward- extending N-terminal domains. In some designs, the phage-displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382) A second difference is the set of biological biases affecting the population of peptides actually present in the libraries. The Lad fusion molecules are confined to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the Lad and phage libraries may differ significantly as a result of their exposure to different proteolytic activities. The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incoφoration into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). These particular biases are not a factor in the Lad display system.
The number of small peptides available in recombinant random libraries is enormous. Libraries of l07-109 independent clones are routinely prepared. Libraries as large as 1011 recombinants have been created, but this size approaches the practical limit for clone libraries. This limitation in library size occurs at the step of transforming the DNA containing randomized segments into the host bacterial cells. To circumvent this limitation, an in vitro system based on the display of nascent peptides in polysome complexes has recently been developed. This display library method has the potential of producing libraries 3-6 orders of magnitude larger than the currently available phage/phagemid or plasmid libraries. Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.
In one application of this method (Gallop et al. (1994) J. Med. Chem. 37(9): 1233- 1251), a molecular DNA library encoding 1012 decapeptides was constracted and the library expressed in an E. coli S30 in vitro coupled transcription translation system. Conditions were chosen to stall the ribosomes on the mRNA, causing the accumulation of a substantial proportion of the RNA in polysomes and yielding complexes containing nascent peptides still linked to their encoding RNA. The polysomes are sufficiently robust to be affinity purified on immobilized receptors in much the same way as the more conventional recombinant peptide display libraries are screened. RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening. The polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification. By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ΕLISA, or for binding specificity in a completion phage ΕLISA (Barret, et al. (1992) Anal. Biochem 204,357-364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host.
Secondary Screens The high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.
Therefore, methods for generating fragments and analogs and testing them for activity are known in the art. Once the core sequence of interest is identified, it is routine to perform for one skilled in the art to obtain analogs and fragments.
Peptide Mimetics
The invention also provides for reduction of the protein binding domains of the subject γ3 or β4 polypeptides to generate mimetics, e.g. peptide or non-peptide agents. See, for example, "Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein" European patent applications EP-412,762A and EP-B31 ,080A.
Non-hydrolyzable peptide analogs of critical residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem SocPerkin Trans 1:1231), and β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Communl26:4l9; and Dann et al. (1986) Biochem Biophys Res Commun 134:71).
Antibodies
The invention also includes antibodies specifically reactive with a subject γ3 or β4 polypeptides. Anti-protein anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). Antibodies which specifically bind γ3 or β4 epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of γ3 or β4. Anti γ3 or β4 antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate γ3 or β4 levels in tissue or bodily fluid as part of a clinical testing procedure.
Another application of antibodies of the present invention is in the immuno logical screening of cDNA libraries constructed in expression vectors such as λgtl 1, λgtl 8-23, λZAP, and λORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, λgtl 1 will produce fusion proteins whose amino termini consist of β-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a subject polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with antibodies of the invention. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of homologs can be detected and cloned from other animals, and alternate isoforms (including splicing variants) can be detected and cloned from human sources.
Other Embodiments
Included in the invention are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide of SEQ ID NO: 1 or SEQ ID NO:3 (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby incoφorated by reference); and, polypeptides specifically bound by antisera to γ3 or β4.
Nucleic acids and polypeptides of the invention includes those that differ from the sequences discolosed herein by virtue of sequencing errors in the disclosed sequences. The invention also includes fragments, preferably biologically active fragments, or analogs of γ3 or β4. A biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of theγ3 or β4 shown in SEQ ID NO:3 and SEQ ID NO:l, respectively, or of other naturally occurring γ3 or β4, e.g., one or more of the biological activities described above. Especially preferred are fragments which exist in vivo, e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells, e.g., as a result of post-translational processing, e.g., as the result of the removal of an amino-terminal signal sequence, as well as those made in expression systems, e.g., in CHO cells. Particularly preferred fragments are fragments, e.g., active fragments, which are generated by proteolytic cleavage or alternative splicing events. Other embodiments are within the following claims. What is claimed is:

Claims

1. An isolated laminin 12 which includes an ╬▒2 subunit, a ╬▓l subunit and a ╬│3 subunit.
2. An isolated ╬│3 subunit.
3. An isolated ╬▓4 subunit.
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Title
BURGESON R E ET AL: "A NEW NOMENCLATURE FOR THE LAMININS" MATRIX BIOLOGY, XX, XX, vol. 14, no. 3, 1994, pages 209-211, XP001070885 *
DATABASE EMBL 18 April 1997 (1997-04-18), "EST113184 Gall bladder II Homo sapiens cDNA 5' end similar to laminin, B2 chain" XP002294054 Database accession no. AA297625 *
DATABASE EMBL 6 September 1996 (1996-09-06), "NIB8 Normalized infant brain, Bento Soares Homo sapiens cDNA 3'end similar to H. sapiens putatively transcribed partial sequence; UK-HGMP sequence ID AAACUKW" XP002294055 Database accession no. T17365 *
IIVANAINEN A ET AL: "Molecular cloning and tissue-specific expression of a novel murine laminin gamma3 chain." THE JOURNAL OF BIOLOGICAL CHEMISTRY. 14 MAY 1999, vol. 274, no. 20, 14 May 1999 (1999-05-14), pages 14107-14111, XP002294053 ISSN: 0021-9258 *
KOCH M ET AL: "Characterization and expression of the laminin gamma3 chain: a novel, non-basement membrane-associated, laminin chain." THE JOURNAL OF CELL BIOLOGY. 3 MAY 1999, vol. 145, no. 3, 3 May 1999 (1999-05-03), pages 605-617, XP002294052 ISSN: 0021-9525 *
LINDBLOM A ET AL: "Characterization of native laminin from bovine kidney and comparison with other laminin variants." EUROPEAN JOURNAL OF BIOCHEMISTRY / FEBS. 15 JAN 1994, vol. 219, no. 1-2, 15 January 1994 (1994-01-15), pages 383-392, XP008034636 ISSN: 0014-2956 *
See also references of WO9919348A1 *

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