EP1251888A1 - Chirurgisches nahtmaterial mit spinnenseide - Google Patents
Chirurgisches nahtmaterial mit spinnenseideInfo
- Publication number
- EP1251888A1 EP1251888A1 EP01903537A EP01903537A EP1251888A1 EP 1251888 A1 EP1251888 A1 EP 1251888A1 EP 01903537 A EP01903537 A EP 01903537A EP 01903537 A EP01903537 A EP 01903537A EP 1251888 A1 EP1251888 A1 EP 1251888A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- suture
- silk
- spider silk
- gene
- film
- 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- Withdrawn
Links
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/44—Medicaments
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
- A61L17/005—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters containing a biologically active substance, e.g. a medicament or a biocide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
- A61L17/06—At least partially resorbable materials
- A61L17/08—At least partially resorbable materials of animal origin, e.g. catgut, collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
- A61L17/06—At least partially resorbable materials
- A61L17/10—At least partially resorbable materials containing macromolecular materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
Definitions
- Surgical sutures, sealants, and surgical adhesion barriers play a key role in many aspects of medicine, from major surgery to repair of minor wounds.
- the requirements of sutures for particular applications vary depending on the application. For example, eye surgery requires extremely fine sutures, while thoracic surgery generally requires stronger, larger-diameter sutures with high tensile strength.
- Surgical sealants or films are used in preventing, for example, leakage following surgery or trauma. In geriatric cases where skin is weak and prone to tearing, adhesives are preferable.
- the sutures can be used in combination with the sealants, for example, in cardiac or vascular procedures.
- many available sutures are composed of materials that provide desired properties, there is a need for improved sutures with superior properties, such as tensile strength, elasticity, and biodegradability.
- the invention features surgical sutures having superior properties by virtue of their having, either as the only structural element, or as one of two or more structural elements, spider silk, a term which encompasses one of a number of proteins made by spiders for, e.g., the construction of webs.
- spider silk a term which encompasses one of a number of proteins made by spiders for, e.g., the construction of webs.
- spider silk proteins have been made in recombinant systems, including mammalian cell culture and in the urine and milk of transgenic mammals, as will be described in greater detail below.
- the suture is a spider silk monofilament that is spun from a solution of spider silk monomer.
- the sutures because they are used in wound closing, are preferably sterile.
- the sutures may comprise spider silk only, or a combination of spider silk and other non-spider silk structural elements, for example, collagen or elastin.
- the spider silk used in the sutures of the invention can have the native amino acid sequence, or it can be engineered to contain sites that are cleavable in the body of a living human patient, e.g., by proteolytic enzymes of the patient. These biodegradable sutures are used in circumstances in which the sutures are allowed to be reabsorbed, rather than removed.
- the suture contains multiple monofilaments that are braided or twisted together.
- Silk sutures from the silk worm have been used as surgical material. Silk is superior to other braided suture materials because of its good “knot security” and relatively low “tie down resistance.”(Tomita et al., J. Appl. Biomater., 4: 61-65, 1993).
- the silkworm based sutures appear to have inferior physical properties when compared to synthetic materials such as polytefra fluoroethylene, and polylactic acid/polyglycolic acid, in terms of strain to failure, tensile strength, knot tensile strength etc.(Charbit et al., Biomed. Lustrum. TechnoL, 33: 71-75, 1999). Therefore, we propose recombinant (re) spider silks as an alternative superior material.
- Some of the filaments in these multiple filament sutures can be filaments of materials other than spider silk, e.g., conventional suture materials (collagen or elastin).
- the sutures of the invention can be impregnated or coated with a therapeutic substance, such as an antibacterial agent. This is particularly advantageous where the suture is composed of multiple filaments that are twisted or braided together, because such sutures provide a potential route for infection.
- the multiple filament sutures of the invention can also be treated with a biocompatible material such as a thermoplastic elastomer, to fill in the interstices created by the multiple filament nature of the suture; this treatment can minimize infection, and render the suture smoother and more easily used by the surgeon.
- the filaments can also be coated with other proteins such as serum albumin or fibrinogen.
- the invention features a solid film, adhesive, or sealant for human medical use, composed of spider silk monomer, either by itself or in combination with a biocompatible polymer such as hyaluronic acid, a polyester, collagen, or elastin.
- a biocompatible polymer such as hyaluronic acid, a polyester, collagen, or elastin.
- These films, adhesives, or sealants can be used, e.g., in surgery to prevent adhesions between organs following surgery, and can further comprise an antibacterial agent.
- the invention also features solid surgical dressing, e.g., adhesives or sealants and non-adhesive wound-covering pads or bandages.
- the invention also features biocompatible gels for human therapeutic use, containing spider silk monomer.
- gels can contain other components as well, including, for example, hyaluronic acid, collagen, or elastin.
- the gels of the invention can be used as coatings for surgical instruments and organs to prevent adhesions, and as replacement material for aqueous humor in eye surgery.
- the invention further features a synthetic ligament or tendon comprising multiple fibers of a spider silk and fibers of a biocompatible polymer, for example, collagen or elastin.
- sutures and other items of the invention can be adapted for a wide range of applications; elasticity, tensile strength, and biodegradability, as well as other properties, can be controlled by choosing the appropriate spider silk, altering the thickness of the filaments, choosing appropriate additional components, and varying the conditions under which the spider silk monomer is spun or woven or formed into a gel or sealant.
- silk is meant a fibrous protein that is normally produced and secreted by any one of a variety of insects and arachnids.
- Silks are composed of alternating crystalline and amorphous regions.
- Exemplary silks include spider silk, an externally spun fibrous protein secretion found in a variety of arachnids (e.g., Nephila clavipes), and fibroin, an externally spun fibrous protein secretion found in a variety of insects (e.g., Bombyx mori).
- Preferable silks when secreted such that the secretion is subjected to shear forces and mechanical extension, have a poly-alanine segment, forming a crystal-forming domain, that undergoes a helix to ⁇ -sheet transition, forming a ⁇ -crystal that stabilizes its structure.
- the amorphous domain of a silk forms a ⁇ -pleated sheet such that inter- ⁇ sheet spacings are between 3 angstroms and 8 angstroms in size, preferably, between 3.5 angstroms and 7.5 angstroms in size.
- a silk has a C-terminal portion with an amino acid repeat motif.
- a silk has an amino acid repeat motif (creating both the amorphous domain and the crystal-forming domain) having a sequence that is at least 50% identical to the sequence of SEQ ID NO: 25, more preferably, at least 70% identical, and most preferably, at least 90% identical to SEQ ID NO: 25.
- a silk has a consensus sequence that is at least 50% identical to the sequence of SEQ ID NO: 26, more preferably, at least 70% identical, and most preferably, at least 90% identical to SEQ ID NO: 26.
- promoter is meant a nucleic acid sequence sufficient to direct transcription.
- promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the native gene.
- Preferred promoters of the invention direct transcription of a protein in a milk-producing cell; such promoters include, without limitation, promoters from the following genes: whey acidic protein, ⁇ Sl -casein, ⁇ S2-casein, ⁇ -casein, ⁇ -casein, ⁇ -lactoglobulin, and ⁇ - lactalbumin.
- promoters of the invention direct transcription of a protein in a urine-producing cell (e.g., a uroepithelial cell or a kidney cell); such promoters include, without limitation, the promoter from the uroplakin gene or the uromodulin gene. Yet another preferred promoter of the invention directs transcription of a protein in an embryonal cell.
- leader sequence or a “signal sequence” is meant a nucleic acid sequence which, when operably linked to a nucleic acid molecule, allows for the secretion of the product of the nucleic acid molecule.
- the leader sequence is preferably located 5' to the nucleic acid molecule.
- the leader sequence is obtain from same gene as the promoter that is used to direct the transcription of the nucleic acid molecule, or is obtained from the gene from which the nucleic acid molecule is derived.
- culture medium is meant the medium surrounding a cell, and in which the cell has been living. If the cell is secreting a protein (e.g., a silk monomer), the culture medium of the cell will contain the protein secreted by that cell.
- a protein e.g., a silk monomer
- a transfected cell or a “transformed cell” is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid molecule encoding a polypeptide of the invention.
- the cell is a eukaryotic cell from a multicellular animal (e.g., a mammal).
- embryonal cell By an “embryonal cell” is meant a cell that is capable of being a progenitor to all the somatic and germ-line cells of an organism.
- exemplary embryonal cells are embryonic stem cells (ES cells) and fertilized oocytes.
- ES cells embryonic stem cells
- fertilized oocytes Preferably, the embryonal cells of the invention are mammalian embryonal cells.
- operably linked is meant that a nucleic acid molecule and one or more regulatory sequences (e.g., a promoter) are connected in such a way as to permit expression and/or secretion of the product (i.e., a polypeptide) of the nucleic acid molecule when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
- regulatory sequences e.g., a promoter
- transgene any piece of nucleic acid that is inserted by artifice into a cell, or an ancestor thereof, and becomes part of the genome of the animal which develops from that cell.
- Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic animal, or may represent a gene homologous to an endogenous gene of the animal.
- transgenic any cell which includes a nucleic acid sequence that has been inserted by artifice into a cell, or an ancestor thereof, and becomes part of the genome of the animal which develops from that cell.
- the transgenic animals are transgenic mammals (e.g., rodents or ruminants).
- the nucleic acid (transgene) is inserted by artifice into the nuclear genome.
- Fig. 1A is a schematic representation of the goat ⁇ -casein/NcDS-1 construct containing the ⁇ -casein promoter and signal sequence, the 1.5 kb NcDS-1 cDNA, and the 3' UTR from ⁇ -casein.
- Fig. IB is a schematic representation of the Whey Acidic Protein
- WAP WAP/NcDS-l construct containing the WAP gene promoter, the WAP signal sequence, a 1.5 kb cDNA encoding dragline silk (NcDS-1), and the 3' end of the WAP gene.
- Fig. IC is a schematic representation of the uroplakin II/NcDS-1 construct containing the promoter and signal sequence from the uroplakin II gene, the NcDS-1 cDNA insert, and the 3' UTR region from the mouse protamine-1 (Mp-1) gene.
- Biofilaments such as spider silk, have a number of high performance mechanical properties that make them comparable to the "super-filaments” SpectraTM (commercially available from AlliedSignal) or KevlarTM (commercially available from DuPont).
- SpectraTM commercially available from AlliedSignal
- KevlarTM commercially available from DuPont
- biofilaments are resistant to digestion by proteolytic enzymes, and are insoluble in dilute acids and bases.
- Biofilaments are a natural source of material that is renewable.
- Transgenic animals expressing biofilaments such as the animals described herein, may be managed as normal livestock using available feedstuffs.
- the manufacture of this commodity is domestic and renewable.
- the truly natural source, the spiders themselves, are, unfortunately, too small to produce significant amounts of this material.
- spiders manifest territorial behavior, they cannot be raised in close quarters for mass production.
- Spiders manufacture up to seven different silks, each one of them tailored for a specific application.
- Each spider silk has a unique amino acid composition, consisting of repetitive sequences with polyalanine regions (crystalline domains) sandwiched between sequences (amorphous or elastic domains) which, depending on the silk, for example, dragline silk, are rich in glycine.
- the biofilaments of the present invention deal with spider silks secreted from the major ampullate gland (producing dragline silk), the minor ampullate gland (producing minor ampullate silk which exhibits lower tensile strength and elasticity, but which also demonstrates a lack of super contraction upon wetting), the flagelliform gland (producing silk with very high elasticity, but with a tenacity of only approximately 25% that of the silks produced by the major or minor ampullate gland), or the cylindrical gland (producing cocoon silk).
- the properties of a suture may be varied, depending on the application for which the suture is to be used.
- ultra- fine spun silk fibers may be generated for uses in eye surgery, cosmetic surgery, or nerve reconstruction.
- Such an ultra-fine spun fiber would ideally have a diameter of 0.1 ⁇ M to 100 ⁇ M; 5% to 300% elasticity; and tensile strength of 0.1 g/denier to 30 g/denier.
- a denier is defined as the weight of a fiber 9,000 meters in length and is often used instead of diameter measurement.
- Sutures are categorized by size, design, material and behavior. Absorbable and non-absorbable materials are further divided into natural versus synthetics that can be in braided and/or monofilament form (Greenwald et al., J. Surg. Res., 56: 372-373, 1994).
- Silks have evolved in certain insects and arachnids having very specialized anatomical adaptations and gene evolution. In spiders, the silk is produced by a series of abdominal silk glands. The formation and size of the monomer may depend upon, among other things, the primary amino acid sequence composition. The production and secretion of the major silks occurs in the major ampullate gland, the minor ampullate gland, the flagelliform gland, and the cylindrical gland, which produce dragline silk, minor ampullate, flag silk, and cocoon silk, respectively. It will be understood that the transgenic animals described herein, and the constructs used to generate such animals, may produce any of these silks, or any variations thereof, such that a silk is produced having inter- ⁇ -sheet spacings of between 3.5 to 7.5 angstroms. Examples of various silks and each of their properties relevant to the present invention are presented in Table 1.
- Dragline silk is a high stiffness fiber and has properties similar to nylon. Dragline silk contains 20-30% crystal, by volume, and has the following characteristics: stiff (initial Young's modulus is 10 GPa), strong (tensile strength is 1.5 GPa), and tough (energy required to break is 150 Mjm "3 ).
- the flag silk produced by the flagelliform gland forms the core fiber of the sticky spiral of the web. Similar to other silks, the flagelliform protein is composed of iterated repeats with the dominant one being: Gly-Pro-Gly- Gly-X (X: Ala or Ser or Tyr or Val; SEQ. ID NO: 34) (Hayashi and Lewis, J. Molecular Biology, 275: 773-784, 1998). Viscid silk contains less than 5% crystal by volume, is elastomeric in its native state, and has properties similar to Lycra. It has the following characteristics: mechanically similar to lightly cross-linked rubber (e.g., spandex), low stiffness (initial Young's modulus is 3 MPa), and highly extensible.
- lightly cross-linked rubber e.g., spandex
- low stiffness initial Young's modulus is 3 MPa
- the cylindrical gland produces cocoon silk that is similar to the cocoon silk produced by silkworms (B. mori).
- the size of the native dragline silk protein has not been determined conclusively, but has been observed to range from 274 kDa to 750 kDa.
- Partial cDNA clones (spidroin 1 and 2) from Nephilia clavipes (the golden orb weaver found in Brazil and southern Florida) and from Araneus diadematus (Guerette et al., Science 272: 112-115, 1996) have been isolated.
- a summary of the clones isolated, for example, from Araneus, and the glands from which they are derived, is shown below in Table 2. Table 2
- Araneus diadematus genes (libraries screened using spidroin-1 3 2 from N clavipes) Distribution-Expression of specific silk type (as determined by Northern Blot)
- ADF-1 (Araneus diadematus fibroin) is 68% pofy(A) 5 (i.e., AAAAA; SEQ ID NO: 1) or (GA) 2.7 (SEQ ID NOS: 2-7), and 32% GGYGQGY (SEQ ID NO: 8);
- ADF-2 is 19% poly(A) 8 (SEQ ID NO: 9) 5 and 81% [GGAGQGGY (SEQ ID NO: 10) and
- Nephila and Araneus Any of a variety of procedures well known in the art may be utilized to clone additional silk-encoding genes using the known nucleic acid sequences from Nephila and Araneus, and one so skilled will routinely adapt one of these methods in order to obtain the desired gene.
- the full 5 length clones of Nephila and Araneus genes may be likewise isolated.
- One such method for obtaining a silk-encoding gene sequence is to use an oligonucleotide probe generated by the Nephila clavipes spidroin 1 gene sequence (Arcidiacono et al, Appl. Microbiol. Biotechnol. 49: 31-38, 1998 to screen an arachnid or insect cDNA or genomic DNA library for sequences which hybridize to the probe.
- Hybridization techniques are well known to the skilled artisan, and are described, for example, in Ausubel et al, supra, and Sambrook et al, supra.
- cDNA or genomic DNA library preparation is also well known in the art. A large number of prepared nucleic acid libraries from a variety of species are also commercially available.
- the oligonucleotide probes are readily designed using the sequences described herein and standard techniques.
- the oligonucleotide probes may be based upon the sequence of either strand of DNA encoding the spidroin 1 gene product.
- Exemplary oligonucleotide probes are degenerate probes (i.e., a mixture of all possible coding sequences for the N. clavipes spidroin 1 protein).
- Spider silk genes can be cloned and expressed, using a variety of techniques. Spider silk cD ⁇ A sequences cloned to date share similarities in overall organization and in regions of sequence conservation. The consensus repeats are rich in glycine and glutamine, with poly (Ala) regions integrated into larger repeating units. Examples of silk genes sequences which may be expressed are found, for example, in EPO
- the major ampullae gland produces the dragline silk (spidroin 1 and spidroin 2) with the following representative sequences: a 34 amino acid long repeat motif (forming both the amorphous domain and crystal-forming domain); and a 47 amino acid long consensus sequence (Xu and Lewis, Proc. Natl. Acad. Sci. U.S.A. 87: 7120-7124, 1990; Beckwitt and Arcidiacono, J. Biol. Chem. 269: 6661-6663, 1994).
- the 34 amino acid long repeat motif of Nephila spidroin 1 has the following sequence:
- SEQ ID NO: 25 AGQ GGY GGL GSQ GAG RGG LGG QGA GAA AAA AAG G (SEQ ID NO: 25). Note the poly-alanine region (AAAAAAA) at the C-terminal end of SEQ ID NO: 2, as well as numerous glycine blocks forming amorphous domains (two glycine residues separated by 3 amino acids; e.g., GG LGG). These sequences being part of the native gene may be used to produce silk protein according to the methods of the invention.
- the 47 amino acid long consensus sequence of Nephila spidroin 2 has the following sequence:
- AAA AA SEQ ID NO: 26. These sequences being part of the native gene may also be used to produce silk protein according to the methods of the invention.
- the candidate gene will be represented by one of the major dragline genes from N. clavipes, spidroin 1 (Arcidiacono et al, Appl. Microbiol. Biotechnol. 49: 31-38, 1998).
- the highly repetitive nature of these genes raises the concerns over the stability of the genes and the possibility of recombination.
- the repeats can be avoided based on suggestions offered, for example, by Arcidiacono et al., Appl. Microbiol. Biotechnol. 49: 31-38, 1998; Fahnestock and Irwin (Fahnestock and Irwin, Appl. Microbiol. Biotechnol. 47: 23-32, 1997).
- a series of constructs can be generated using a similar strategy (supra), generating 4- 20 (or more) consecutive repeats, and can be tested in cell lines prior to the generation of transgenic animals.
- Blocks of synthetic repeats are constructed so they have different sizes and contain non-coding sequences (e.g., introns from casein or immunoglobulin genes) in order to facilitate transcription of the encoded silks and enhance expression.
- the blocks can be alternating using head-to-tail construct strategy (McGrath, K. P., Ph.D. Dissertation, University of Massachusetts at Amherst, 1991; Ferrari et al, U.S. Pat. No. 5,243,038).
- Codon selection may also be designed to maximize expression, since premature termination may occur if the gene contains a greater number of codons recognized by tRNA species present in lower abundance in the cell (Rosenberg et al, J. Bacteriol. 175: 716-722, 1993, Manley, J. Mol. Biol. 125:407-432, 1978).
- the genes to be expressed are designed and synthesized using codons favored in the tissue being expressed (i.e., casein genes in the mammary gland; uroplakins in the bladder).
- Eukaryotic expression vectors may be generated which drive the synthesis and secretion of proteins (e.g., silk proteins) in the milk or urine ) of an animal transgenic for a nucleic acid molecule encoding such a 5 protein. These vectors are prepared according to standard molecular biology techniques.
- the synthesized nucleic acid molecule(s) may have a sequence encoding an epitope tag attached for easy identification and/or purification of the encoded polypeptide. Such purification may be accomplished, for 10 example, by affinity chromatography for the epitope tag.
- a site-specific proteolytic or chemical agent recognition site may be added to the sequence to facilitate removal of the epitope tag following purification of epitope-tagged spider silk monomers (Saito et al., J. Biochem. 101: 123- 134, 1987).
- the sites and site-specific proteolytic agents cleave 15 at or near the junction of the epitope tag and the silk protein monomer.
- a variety of chemical cleavage agents and their recognition sites are known in the art, and include the following: Hydroxylamine (N or G); formic acid (D or P), cyanogen bromide (M); or acetic acid (D or P).
- N or G Hydroxylamine
- D or P formic acid
- M cyanogen bromide
- D or P acetic acid
- a cyanogen bromide (CNBr) may be 20 used to cleave a Met (M) residue introduced between the epitope tag and the silk protein.
- natural or synthetic proteases may be used. Examples of these (and their recognition sites) include enterokinase (DDDK; SEQ ID NO: 27); Factor Xa (IEGR; SEQ ID NO: 28); chymotrypsin (W and Y and 25 F); renin (YIHPFHLL; SEQ ID NO: 29); trypsin ® and K); and thrombin (RGPR; SEQ ID NO: 30).
- the epitope tag may be attached to the silk via a thrombin-recognition site. Following affinity purification of epitope tag-containing proteins, since silks are generally resistant to proteolysis, the epitope tag may be easily removed upon proteolytic cleavage with thrombin.
- the expression cassette consists of elements necessary for proper transcription, translation, and secretion in the desired eukaryotic cell (i.e., a promoter, signal sequence for secretion, intron sequences, and a polyadenylation signal).
- a promoter for secretion, intron sequences, and a polyadenylation signal.
- Many milk or urine specific promoters can be used with their signal sequences or with the silk and/or fibroin gene signal sequence.
- the silk-encoding nucleic acid molecule should not contain its own translation initiation codon but, rather, should be in frame with the 3' end of the signal sequence.
- the 3' end of the silk- encoding nucleic acid molecule may contain its own polyadenylation signal, or may contain the 3 ! untranslated sequence normally found on the gene used for the promoter and/or signal sequence.
- a silk- encoding nucleic acid molecule may be in an expression vector cassette with a promoter, signal sequence, and 3' untranslated sequence
- the eukaryotic expression constructs to be used may include one or more of the following basic components.
- promoter of transcriptional initiation regulatory region may be heterologous to the cell to be modified and may include synthetic and natural viral sequences (e.g., human cytomegalovirus immediate early promoter (CMV); simian virus 40 early promoter (SV40); Rous sarcoma virus (RSV); or adenovirus major late promoter) which confers a strong level of transcription of the nucleic acid molecule to which they are operably linked.
- the promoter can also be modified by the deletion of nonimportant sequences and/or addition of sequences, such as enhancers (e.g., an enhancer element CMV, SV40, or RSV), or tandem repeats of such sequences.
- enhancers e.g., an enhancer element CMV, SV40, or RSV
- the addition of strong enhancer elements may increase transcription by 10-100 fold.
- the promoter region may be native to a ruminant mammary-specific gene.
- examples include: ⁇ Sl-casein (PCT Application Nos.: WO91/08216 and W093/25567), ⁇ S2-casein, ⁇ -casein (Rosen, U.S. Pat. No. 5,304,489; Lee et al, Nucleic Acids Res. 16: 1027-1041, 1988), ⁇ -casein, ⁇ -lactoglobin, and ⁇ -lactalbumin (Vilotte et al, Eur. J. Biochem. 186: 43-48, 1989; PCT Application No.: WO88/01648).
- These promoters can drive a high level of expression of a variety of proteins in a tissue and lactation specific manner.
- Intron Inclusion Genes containing an intron are expressed at higher levels than infron-less genes. Hence, inclusion of an intron placed between the transcription initiation site and the translational start codon, 3' to the translational stop codon, or inside the coding region of the silk- encoding gene may result in a higher level of expression.
- the intron sequence includes a 5' splice site (donor site) and a 3' splice site (acceptor site). Sequences of at least 100 base pairs are found between these two sites. The origin of these intronic sequences will be derived from the promoter being used, or from the native gene (Ichimura and Mita, J. Mol. Evol.
- Each expression vector will contain a signal sequence which directs the expressed gene product to be secreted from the mammary or uroepithelial cells.
- This signal sequence is present in any gene which is secreted naturally.
- a signal sequence from a relative fibroin gene e.g., B. mori heavy and light fibroin gene, P25 and ss l60
- a gene specific to the tissue of expression e.g., casein or uroplakin gene
- a general signal sequence e.g., from human alkaline phosphatase, mellitin, and CD33 signal peptides
- signal sequences for secretion may be interchanged between mammalian/insect genes; for example, signal sequences from mellitin, casein, or the sequences from native silk (from arachnids) or fibroin (from insects) genes may be used.
- the transcription termination region of the nucleic acid constructs may involve the 3'-end and polyadenylation signal from which the 5'-promoter region is derived.
- the 3'-end of the nucleic acid construct will contain transcription termination and polyadenylation signals which are known to regulate post-transcriptional mRNA stability such as those derived from bovine growth hormone, ⁇ -globin genes, or the SV40 early region.
- the expression vectors designed for gene transfer also contain an origin or replication for propagation E. coli, SV40 origin of replication, ampicillin resistance gene, neomycin resistance gene for selection in 5 eukaryotic and/or genes (i.e., dihydrofolate reductase gene) that amplify the dominant selectable marker plus the gene of interest.
- the expression vectors may contain appropriate flanking sequences at their 5 ! and 3' ends that will allow for enhanced integration rates in the transduced cells (ITR sequences; Lebkowski et al, Mol. Cell. Biol. 8: 3988-3996,
- cosmids are the vectors of choice. Although plasmid vectors can, in theory, carry large inserts, the resulting recombinants transform Escherichia coli very inefficiently. Cosmids have the capacity to propagate large pieces of foreign DNA (Royal et al, Nature 279: 125, 1979). 0
- the expression vectors used for the generation of transgenic animals may be linearized by restriction endonuclease digestion prior to transformation of a cell.
- a digestion fragment that includes the coding, 5'-end regulatory sequences (e.g., the promoter), and 3'-end regulatory sequences (e.g., the 3' untranslated 5 region) from, for example, bovine casein or growth hormone sequences will be used to transform cells.
- a cell transformed with such a fragment will not, consequently, contain any sequences that are necessary solely for plasmid propagation in bacteria (e.g., the cell will not contain the E. coli origin or replication or a nucleic acid molecule encoding an antibiotic- resistance protein (e.g., an ampicillin-resistance protein) that is useful for selecting prokaryotic cells).
- an antibiotic- resistance protein e.g., an ampicillin-resistance protein
- the digestion fragment used to transform a cell will include the coding region, the 5' and 3' regulatory sequences, and a nucleic acid molecule (including a promoter and 3' untranslated region) encoding a protein capable of conferring resistance to a antibiotic useful for selecting eukaryotic cells (e.g., neomycin or puromycin).
- the silk gene of interest may be modified in its 5' untranslated region (UTR), its 3' UTR, and/or its region coding for the N-terminus in order to preferentially improve expression.
- sequences within the coding sequence of the silk encoding-nucleic acid molecule may be deleted or mutated in order to increase secretion and/or avoid retention of the gene product within the cell due, for example, to the presence of endoplasmic reticulum (ER) retention signals or other sorting inhibitory signals.
- the transgenic construct may contain sequences that possess chromatin opening domain activity such that they confer reproducible activation of tissue-specific expression of a linked transgene (Ellis et al, PCT Application No.: W095/33841; Chung and Felsenfield, PCT Application No.: WO96/04390).
- the assembled constructs are partially characterized by sequencing the areas where two pieces of DNA have been fused together by ligation.
- a partial restriction endonuclease map will provide information on the correct orientation of the ligated regulatory sequences with respect to the silk-encoding nucleic acid molecule.
- the silk gene used in the following examples is the 1.5 kb NcDS-1 insert described and cloned by Arcidiacono et al, Appl. Microbiol. Biotechnol. 49: 31-38, 1998.
- the above-described silk protein monomer expression constructs may be introduced into the pronuclei of fertilized oocytes, and transgenic animals which produce the desired silk monomer may be generated.
- transgenic animals which produce the desired silk monomer may be generated.
- animals such as mice
- fertilization is performed in vivo and fertilized ova are surgically removed.
- the ova can be removed from live, or from newly-dead (e.g., slaughterhouse) animals and fertilized in vitro.
- Transgenes are usually introduced by microinjection (Ogata et al, U.S. Pat. No. 4,873,292).
- the microinjected zygotes are fransferrred to an appropriate female resulting in the birth of a transgenic or chimeric animal, depending upon the stage of development when the transgene is integrated.
- Chimeric animals can be bred to form true germline transgenic animals.
- transgenes can be introduced into embryonic stem cells (e.g., mouse ES cells) or somatic or fetal cells.
- Transgenes can be introduced into such cells by lipofection, electroporation, microinjection, or any other techniques used for the transfection of cells which are known to the skilled artisan.
- Transformed cells are combined with blastocysts from the animal from which they originate, for example, in the case of mice, using nuclear transfer methods. The cells colonize the embryo, and in some embryos these cells form the germline of the resulting chimeric animal (Jaenisch, R., Science 240: 1468-1474, 1988).
- ES cells can be used as a source of nuclei for transplantation into an enucleated fertilized oocyte, thus giving rise to a transgenic animal.
- the silk protein transgene is under the transcriptional control of tissue specific regulatory elements, which direct the expression of the desired protein.
- the protein is produced by a cell which secretes the protein into a bodily fluid, for example milk or urine.
- expression of the silk gene may be directed by the uroplakin promoter, which would result in the silk protein being produced in the urine.
- the transgenic animals produce and secrete the desired silk protein monomer.
- the silk protein may be expressed under the control of the mammary specific whey acidic protein (WAP) promoter, in which case the silk protein would be produced in the milk.
- WAP mammary specific whey acidic protein
- silk protein monomers may be produced using cell culture systems.
- the protein monomers are produced in biologically relevant cell cultures.
- mammary epithelial cells or other cell lines BHK, COS-7, etc.
- the expression of the protein is driven preferably by appropriate regulatory elements (e.g., viral promoters as previously described).
- the desired silk protein monomer is secreted into the tissue culture medium and can then be purified and formed into fibers/filaments that can be used as sutures. Purification of the silk monomers can be performed using standard chromatographic techniques.
- the fibers are spun from solutions in which the silk monomers (resembling either dragline, minor, or flag silk monomers) form a liquid crystal phase.
- the protein concentration in solution is between .1% and 50%, and more preferably between 4% and 30%.
- the pH of the solution is preferably between 4 and 5.
- fibers are spun from monomer solutions in aqueous buffers. The fibers are extruded by dropping the pH of the buffers to between 4 and 5.
- the fibers can be extruded from the silk monomer solution through a small diameter needle (for example, a 22/ gauge needle) into a methanol or acetone bath or acetic acid/methanol or aceti acid/acetone bath (called the coagulation bath).
- the formed fiber can be either aged into the solvent or aged for a period of time before it is drawn and extended in order to produce a stronger fiber.
- Silk monomers may be extruded by wet spinning, dry spinning, melt spinning, or dry-jet wet spinning, and formed into fibers which may be used as sutures using any of several method known in the art, for example, as described in EPO 0488687 A2, and U.S. Pat. No. 5,252,285, hereby incorporated by reference.
- Parameters which may be altered in the spinning process include temperature, pH, protein concentration, buffer composition, denaturing agents such as those cited above, coagulation bath, the silk monomer, monomer that is already partly fibrous to act as the "nucleation effector" for fiber formation, drawing speed, and aging in the bath. Alteration of such parameters will affect the diameter, elasticity and, tensile strength.
- the monomer solutions can be dried to a powder, and solubilized into solvents, for example, HFIP and then extruded in a manner similar to that described above.
- the fibers or filaments formed have a diameter between 0.1 ⁇ M and 100 ⁇ M, and more preferably the diameter is between 0.5 ⁇ M and 50 ⁇ M.
- the elasticity of the fibers or filaments is between 5% and 300%, more preferably between 5% and 200%.
- the fibers or filaments possess tensile strengths of 0.1 to 30 grams/denier.
- the fibers or filaments of the present invention may be used as monofilament sutures.
- a very strong monofilament suture may be desirable because other commonly used twisted or braided yarns may be a route of infection
- the monofilaments of the present invention can be made into a stronger suture by forming it into yarns, using a twisting and entangling process.
- the yarns are then interlaced into fabrics by weaving, knitting, or braiding.
- Such yarns may be comprised of the same, or two or more different, monofilaments, each one derived from a different silk protein monomer (see Table 1).
- the yarn can comprise one filament having high tenacity, and another filament having high elasticity.
- the fibers of the present invention can be used alone in a suture or they can be used in combination with other biomaterials that can take the form of fibers, for example, collagen or elastin.
- the suture can be a yarn comprising a silk filament and an elastin filament.
- engineered silk monomers can be produced which are sensitive to cleavage by enzymes.
- enzyme-sensitive sites can be engineered into the silk protein monomers by forming expression vectors containing nucleic acid sequences encoding the silk monomers and enzyme-sensitive sites.
- the sequences that can be incorporated into the spider silk sequences can be recognized by enzymes of the body, thereby allowing for the selective attack of the silk.
- These sites can be incorporated within the monomer spider silk proteins and in specific sites, such as the amorphous domain or the crystalline domain.
- the recognition site is a peptide bond that separates two or more monomers, said monomers linked to each other in tandem repeats (head to tail) from each other. The number and specificity of such sites may determine the rate by which resorption occurs.
- a large number of site- specific enzymes and their recognition motifs are known and can be used to carry out the present invention and.
- site specific sites include, but are not limited to: chymotrypsin: W1Y1FI collagenase: PX1 GP endoproteinase: Kl enterokinase: DDDKI factor X a : IEGR1 kallikrein: PFRI renin: YIHPFHLIL thrombin: RGPRI trypsin: Rl Kl (SEQ ID NOS: 27-31) Since the efficiency of site recognition is enhanced by the presence of flanking poly-Gly peptides, silks being rich in Gly residues would be good substrates for the suggested modification.
- Silk proteins are composed of an amino terminal portion that contains the repetitive interspaced crystalline (poly- Ala) and amorphous domains (Gly), and a COOH non-repetitive domain.
- the amino terminal position is not sensitive to enzyme (for example, trypsin) digestion, as opposed to the COOH domain.
- enzyme for example, trypsin
- MaSp2 one of the two components of the dragline silk, in which the non-repetitive COOH terminus appears to be sensitive to proteolytic degradation.
- silk monomers can be of the same silk or different silk species (i.e., dragline MaSp2 fused to flag monomer).
- the silk fibers or filaments can also be mixed with other materials to produce a blended mixture with altered physical and biological properties, such as a mixture of silk and hyaluronic acid, or polyesters, or silk and collagen, or silk and elastin, extruded as described above.
- Silk can be used as a monofilament suture (for example in ocular surgery, constructive surgery, nerve reconstruction, vascular closure and cosmetic surgery), or as a multifilament containing strands that are braided or twisted.
- These fibers can be coated, for example, with a thermoplastic elastomer in order to fill the interstices between the silk filaments (as taught in U.S. Pat. No. 4,461,298).
- Such treated sutures may elicit reduced tissue reaction and may retard cellular infiltration.
- the interstices of the multifilament strands can also be filled with germicidal compounds, as taught in U.S. Pat. No. 3,642,003 using methods such as those described in U.S. Pat. No.
- Films of spider rc-silk monomers can be formed for example by using a 5% to 50% monomer solution (w/v) by, for example, a solvent evaporation method.
- Solvents such as HFIP in a film casting apparatus and/or with a combination of vacuum and/or high temperature (50°C- 60°C in an air or nitrogen atmosphere) to remove the solvent (see U.S. Pat. No. 5,252,285) can be used.
- the tensile strength of films so produced can be measured using standard methods. For example, tensile strength of, as low as, 15 MPa to 30 Mpa of the resulting films would be sufficient for use in various wound healing or surgical applications.
- these films or adhesives may be a useful alternative to sutures for wound closures, allowing wound sealing without trauma on the wound from the needle and suture itself.
- the silk monomers can be mixed with hyaluronate in various proportions to prepare a film which, when compared with 100% hyaluronate gels, would exhibit decreased resorption rates and increased mechanical properties (strength, stiffness).
- silk monomers can be mixed with elastin, collagen, or fibrinogen and formed into films.
- Example 1 Construction of a Silk Protein Expression Vector Using the Casein Promoter
- the design of the construct includes the use of the goat ⁇ -casein promoter (Ebert et al, Bio/Technology 12: 699-701, 1993), followed by its own signal sequence for expression, followed by a 1.5 kb insert containing the silk clone (Arcidiacono et al, supra and Xu and Lewis, Proc. Nail. Acad. Sci., 87: 7120-7124, 1990) in frame with the 5' and 3' ends of the casein gene.
- a schematic diagram of this construct is shown in Fig. 1 A.
- the nucleic acid molecule encoding a silk (e.g., a silk or fibroin gene, or fragment thereof) is fused to the casein promoter and secretion of the silk protein is driven by signal sequences from the gene from which the promoter is derived, or from the silk nucleic acid molecule to be expressed. Termination sequences can be derived from the silk gene itself, or from the promoter gene. Furthermore, a hybrid gene can be created to increase the level of expression.
- the silk or fibroin gene (or fragment thereof) can be inserted between exon 2 (just upstream of the ATG) and exon 7 (downstream of the stop codon) of the goat ⁇ -casein gene (Ebert et al, Bio/Technology 12: 699-701, 1993). Since the highly repetitive nature of the construct raises concerns over the stability of the gene and the possibility of recombination due to the repetitive sequences, they can be disrupted by inserting the introns from the casein gene (introns 3 to 7).
- Construction of the vector can be performed in a cosmid vector (supercos), due to the large size of the final construct or the backbone of the plasmid could consist of any known bacterial vector (preferably ones that accept large DNA sizes) which contains sequences necessary for its amplification in an E. coli host (Sambrook et al, supra).
- Example 2 Construction of a Silk Protein Expression Vector Using the Whey Acidic Protein (WAP) Promoter
- WAP Whey Acidic Protein
- the present example demonstrates the generation of a hybrid gene composed of the WAP gene promoter, its signal sequence, a 1.5 kb cDNA encoding dragline silk (Arcidiacono et al, supra), followed by the 3' end of the WAP gene (Velander et al, Proc. Natl. Acad. Sci U.S.A. 89: 12003-12007, 1992). The details of its construction are described below and diagramed in Fig. IB.
- Whey acidic protein (WAP), the major whey protein in rodents, is expressed at high levels exclusively in the mammary gland during late pregnancy and lactation (Hobbes et al, J. Biol. Chem. 257: 3598-3605, 1982).
- the genomic marine WAP gene consists of 2.6 K.P. (kilo base pairs) of 5' flanking promoter sequence, 3.0 K.P. of coding sequence (exon and introns), and 16 K.P. of 3' flanking DNA (see Fig. 1A) (Velander et al, Proc. Natl. Acad. Sci U.S.A. 89: 12003-12007, 1992; Velander et al, Ann. N. Y. Acad Sci. 665: 391-403, 1992).
- a hybrid gene composed of the WAP gene promoter and a cDNA encoding dragline silk may be constructed.
- the hybrid dragline- silk encoding gene is created by inserting the gene(s) or nucleic acid molecule(s) of interest (in this case, a cDNA encoding dragline silk) between the marine WAP promoter and 5' sequence (nucleotide position - 949 to +33) at the 5' end and the WAP 3' sequence at the 3' end (843 bp; portion of exon 3, last 30 bases and all of intron C, exon 4, and 70 bp of 3' UTR) (Campbell et al, Nucleic Acids Res. 12: 8685-8697, 1984).
- the signal sequence comes from the native silk gene.
- One can also use the WAP gene signal sequence by including an additional 56 bp at the 5' end (position -949 to +89).
- the hybrid gene is created by inserting nucleotide sequence encoding part of the 5' untranslated region and a 19 amino acid signal sequence from the marine WAP gene (nucleotides +1 to +90) (Hennighausen et al, Nucl. Acids Res. 10: 3733-3744, 1982), and by amplifying the 1.5 kb (kilo base) silk gene (NsDS-1; Arcidiacono et al, supra) using 5' primers containing the 90 bp (base pair) sequence encoding the signal sequence flanked by a Kpnl restriction endonuclease recognition site.
- the amplification is performed to maintain the correct reading frame, and 3' primers creating a Kpnl restriction endonuclease recognition site at the 3' end of the gene.
- This PCR product is then insert at the Kpnl site at the first exon of WAP.
- the hybrid gene can be cut out of the vector by digestion with the EcoRI restriction endonuclease (see Fig. IB), and purified for microinjection.
- Example 3 Construction of a Silk Protein Expression Vector Using the Uroplakin Promoter
- the uroplakin II promoter (Lin et al, Proc. Natl. Acad. Sci. U.S.A. 92:679-683, 1995; Sun, T., PCT Application No.: W096/39494) is used to drive the expression of the fibroin or silk gene(s) in the urothelium of transgenic animals.
- the fibroin or silk gene(s) is inserted downstream of a 3.6 kb 5' flanking sequence of the mouse uroplakin II (UPII) gene (Lin et al, Proc. Natl. Acad. Sci. U.S.A.
- a sequence containing part of exon 1 and all of intron 1 and exon 2 of the mouse protamine-1 (Mp-1) gene (Peschon et al, Proc. Natl. Acad. Sci. U.S.A. 84: 5416, 1987) is placed at the 3' end of the gene to provide an exon/intron splicing site and a polyadenylation signal.
- Mp-1 mouse protamine-1
- Fig. IC A diagram of this construct is shown in Fig. IC.
- DNA shuffling Another method for producing a silk-encoding gene cassette is by DNA shuffling.
- DNA shuffling is a process for recombination and mutation, performed by random fragmentation of a pool of related genes, followed by reassembly of the fragments by primerless PCR (Stemmer, W. P., Nature 370: 389-391, 1994; Stemmer, W. P., Proc. Natl. Acad. Sci. U.S.A. 91: 10747-10751, 1994).
- the goal of DNA shuffling is to optimize the function of genes without first determining which gene product is rate limiting.
- Genes themselves can be "shuffled,” or can be from different species (i.e., spidroin 1 and 2 and ADF 1-4). Furthermore, different repeats (e.g., GGAGQGGY (SEQ ID NO: 32) from ADF 3) within one species can be “shuffled” (with, for example, repeats from ADF 1, 2, and 4) and expressed to determine the combination which yields favorable characteristics.
- different repeats e.g., GGAGQGGY (SEQ ID NO: 32) from ADF 3
- GGAGQGGY SEQ ID NO: 32
- the silk-encoding nucleic acid molecule may be inserted into an expression cassette for secretion in milk or urine, and used to transform mammalian cells.
- Chimeric molecules are synthesized that include a fusion of the silk (containing either crystal or amorphous domains) in frame with one or more domains of collagen or fibrillin, that may allow for increased crosslinking, stability, or elasticity. Such a chimera is especially useful as a surgical biomaterial.
- Example 7 Production of Silks in Mammary Epithelial or Uroepithelial Cell Lines
- the synthesized silk-encoding nucleic acid molecules of the invention are produced in homologous biologically relevant cell culture systems. Genetic stability of the synthetic genes, secretion ability, and attributes of the produced silk(s) can, thus, be evaluated very efficiently before transgenic animal studies are initiated.
- Mammary epithelial and urine-producing cell lines are transfected according to standard techniques (see, e.g., Ausubel et al, supra) with their respective plasmids (e.g., a milk-specific promoter containing expression construct is transfected into mammary epithelial cells).
- culture media is harvested from the stable cell lines (24-96 hours post- differentiation for mammary epithelial cells) and resolved by SDS-PAGE for Western blotting analysis using anti-spider silk antibodies (commercially available from Monsanto, St. Louis, MO), or antibodies raised against peptides having sequences homologous to SEQ ID NOS: 25, 26, 32, or GAGAGS (SEQ ID NO: 33), the crystal-forming domain which alternates with amorphous domains in Bombyx mori (silkworms).
- SEQ ID NOS anti-spider silk antibodies
- transgenes are introduced into the pronuclei of fertilized oocytes.
- animals such as mice
- fertilization is performed in vivo and fertilized ova are surgically removed.
- the ova can be removed from live, or from newly-dead (e.g., slaughterhouse) animals and fertilized in vitro.
- Transgenes are usually introduced by microinjection (Ogata et al, U.S. Pat. No. 4,873,292). The microinjected zygotes are transferred to an appropriate female resulting in the birth of a transgenic or chimeric animal, depending upon the stage of development when the transgene is integrated. Chimeric animals can be bred to form true germline transgenic animals.
- transgenes can be introduced into embryonic stem cells (ES cells).
- Transgenes can be introduced into such cells by lipofection, electroporation, microinjection, or any other techniques used for the transfection of cells which are known to the skilled artisan.
- Transformed cells are combined with blastocysts from the animal from which they originate. The cells colonize the embryo, and in some embryos these cells form the germline of the resulting chimeric animal (Jaenisch, R., Science 240: 1468-1474, 1988).
- engineered ES cells or somatic cells with a silk gene can be used as a source of nucleic for transplantation into an enucleated fertilized oocytes, thus giving rise to a transgenic animal.
- the spider silk gene is subcloned into an expression vector that contains a casein gene promoter, such that the expression of the spider silk gene is controlled by the casein gene promoter.
- a casein expression vector based on the vectors described by Rosen, J. M. (U.S. Pat. No. 5,304,489) and Meade and Lonberg (U.S. Pat. No. 4,873,316).
- the WAP/silk gene hybrid fragment (the EcoRI fragment described above in Example 2) is purified and microinjected into fertilized mouse eggs, which are then implanted into foster mothers.
- the presence of the WAP/silk gene is identified by Southern blot analysis of tail DNA (DNA isolated from tail tissue) in accordance with methods well known in the art. Positive founder animals are then back-crossed to generate hemizygous animals that are used for studying transgene expression.
- the spider silk gene may be subcloned into an expression vector such that the spider silk gene is under the transcriptional control of the uroplakin promoter (described by Lin et al, Proc. Natl. Acad. Sci. U.S.A. 92:679-683, 1995).
- Urine has the added advantage that its pH and salt composition can be modulated to allow for the incorporation of certain components that may influence the formation of the silk in a most native structure.
- a transgenic animal may be generated that produces a silk protein (e.g., spider silk) in both its urine and its milk.
- One method for constructing such an animal is to transform an embryonal cell of the animal with two constructs, one construct in which the expression of the silk-encoding nucleic acid molecule is directed by a promoter capable of expressing and secreting the silk from a milk-producing cell; and a second construct in which the expression of the silk-encoding nucleic acid molecule is directed by a promoter capable of expressing and secreting the silk from a urine- producing cell.
- the doubly-transformed cell is used to generate a transgenic animal.
- mammalian (e.g., ruminant) zygotes are microinjected (or co- microinjected) with two nucleic acid fragments: one that expresses the silk protein under the control of a milk promoter, and one that expresses the silk protein under the influence of a urine specific promoter.
- the generated transgenic animal will be secreting/producing the silk in both its milk and in its urine. This will increase the total output of silks produced per transgenic animal unit.
- a second method for producing such an animal capable of producing a silk in both its milk and its urine is to separately generate an embryonic stem cell or somatic cells carrying a construct capable of expressing and secreting the silk in a milk-producing cell and an embryonic stem cell carrying a construct capable of expressing and secreting the silk in a urine- producing cell.
- Both transformed cell types are then combined with blastocysts from the animal from which they originate to produce chimeric animals using nuclear transfer techniques.
- the chimeric animals may then be bred to homozygosity.
- This type of double-expressing animal has a number of advantages. First, animals of both genders will produce silks in the urine on a continual basis from birth, and female animals will then be able to produce additional silk protein in their milk as a lactating adult. And second, the amount of silk produced by any individual female animal may be increased (by inducing lactation) or reduced (by not inducing lactation) as the need for silk changes.
- Example 10 Generation of Transgenic Animals That Coexpress and Assemble a Silk Protein From Two Monomers
- a transgenic animal may be generated that produces two proteins which are both required for the correct assembly of a silk protein in its milk, urine, or both.
- Araneus diadematus dragline silk consists of a ratio of ADF3 and 4).
- One method for constructing such an animal is to transform an embryonal cell of the animal with two constructs, each expressing one the two proteins. These proteins may be expressed in the urine or milk, or both.
- the construct described in Example 5 which encodes a polycisfronic message may be employed. This construct may be able to secrete its products in the milk, urine, or both. Once the two proteins are present in the same fluid (i.e., both in the milk or both in the urine), they can assemble correctly and be purified.
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US20050033362A1 (en) * | 2001-09-13 | 2005-02-10 | Grafton R. Donald | High strength suture with collagen fibers |
EP1558444B1 (de) * | 2002-06-24 | 2016-09-21 | Tufts University | Seidebiomaterial und verwendungsverfahren davon |
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DE102005042455A1 (de) * | 2005-09-06 | 2007-04-12 | Medizinische Hochschule Hannover | Nervenimplantat |
CA2632871A1 (en) * | 2006-01-30 | 2007-08-09 | Angiotech Pharmaceuticals, Inc. | Sutures and fibrosing agents |
EP2097117B1 (de) | 2006-12-11 | 2013-05-22 | Medizinische Hochschule Hannover | Implantat aus vernetzten spinnen-seidenfäden |
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EP0452925B1 (de) * | 1990-04-20 | 2000-02-09 | The University Of Wyoming | Für Spinnenseideprotein kodierende DNS, DNS enthaltender Vektor, transformierte Zelle und Produkte davon |
AU8702491A (en) * | 1990-11-08 | 1992-05-14 | Matrix Pharmaceutical, Inc. | Fibrin/collagen membrane material for biomedical use |
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IL123398A0 (en) * | 1995-08-22 | 1998-09-24 | Agricola Tech Inc | Cloning methods for high strength spider silk proteins |
US7157615B2 (en) * | 1998-03-17 | 2007-01-02 | Nexia Biotechnologies, Inc. | Production of biofilaments in transgenic animals |
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2001
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- 2001-02-02 EP EP01903537A patent/EP1251888A1/de not_active Withdrawn
- 2001-02-02 CA CA002398635A patent/CA2398635A1/en not_active Abandoned
- 2001-02-02 WO PCT/CA2001/000120 patent/WO2001056626A1/en not_active Application Discontinuation
- 2001-02-02 AU AU2001231451A patent/AU2001231451A1/en not_active Abandoned
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WO2001056626A1 (en) | 2001-08-09 |
JP2003521350A (ja) | 2003-07-15 |
AU2001231451A1 (en) | 2001-08-14 |
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