Patent Application: US-79833801-A

Abstract:
disclosed are materials that may be used in the design of improved devices and wound treatment platforms though covalent and / or non - covalent attachment of bioactive proteins . the proteins comprise any variety of cell growth and / or healing promoting proteins , such as growth factor . the incorporation of these whole proteins may be designed to provide controlled release thereof in a biological system through further use of enzyme degradation sites . heparin - binding protein or fusion proteins synthesized to contain a heparin - binding domain are two mechanisms that may be used in providing these properties to a matrix , such as a fibrinogen matrix . the proteins will be used to provide enhanced healing in various tissues including vasculature , skin , nerve , and liver . the materials disclosed will be used to enhance would ?? healing and other generative processes by engineering the fibrin gel to contain appropriate proteins with specifically designed release and / or degradation characteristics .

Description:
following long - standing patent law convention , the terms “ a ” and “ an ” mean “ one or more ” when used in this application , including the claims . the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . all of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the composition , methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . indirect coupling of heparin via a heparin - binding peptide to attach growth factor a peptide chimera containing both a factor xiiia substrate and a heparin - binding domain is synthesized by standard solid phase synthesis . a sample peptide is one containing the following sequence , dlnqeqvspk ( βa ) faklaarlyrka ( seq id no : 12 ), where the n - terminus of the peptide contains the factor xiiia substrate and the sequence in italics contains a modified peptide from the heparin - binding domain of atiii ( dl denotes dansyl leucine , which is used to allow detection of the peptide by fluorescence ). size exclusion chromatography was used to determine the amount of peptide cross - linked to fibrin gels using the previously developed incorporation method . a bi - domain peptide containing the heparin - binding domain from antithrombin iii and a fluorescent label was incorporated into fibrin gels during polymerization . the free peptide was washed from the gels , and the fibrin network was degraded with plasmin . the degradation products were analyzed by high performance liquid chromatography ( size exclusion chromatography ) to determine the amount of peptide ( by fluorescence ) present per mole of fibrinogen ( by uv absorbance ). the fluorescence signal from peptide - modified gels appeared at an earlier elution time than did the signal from free peptide alone , indicating that all peptide present in the modified gels was cross - linked to fibrin ( fig1 ). quantification based on standards of known concentration for both peptide and fibrin networks degraded with plasmin showed incorporation of 8 . 7 ± 0 . 2 moles of peptide per mole of fibrinogen ( n = 10 ), which is in close agreement with previously published results for a peptide containing the same factor xiiia substrate domain but a vastly different carboxy terminal sequence . a heparin - peptide chimera is synthesized by coupling a peptide , containing the factor xiiia substrate on the n - terminus and a poly - lysine on the c - terminus , to a heparin oligosaccharide , with a unique aldehyde group on one end , via reductive amination . a peptide with the following sequence , dlnqeqvsplkkkg ( seq id no : 13 ), is synthesized by standard solid phase peptide chemistry . the heparin oligosaccharides are made by standard nitrous acid degradation of heparin , resulting in the formation of an aldehyde on the reducing terminal of the cleaved oligosaccharide . during coupling , the ε - amino group of the lysine side chain attacks the aldehyde on the reducing end of the heparin oligosaccharide to form a schiff base . the schiff base is then reduced to form a stable product . a sample coupling protocol is given below . 1 ) dissolve 1 . 8 mm of peptide and 1 . 8 mm of nitrous acid degraded heparin in 50 mm borate buffer , ph 9 . react for 30 minutes . 2 ) add 160 mm nacnbh 3 and react for 12 hours . 3 ) add 240 mm nacnbh 3 and react for 12 hours . 9 ) purification of desired product is accomplished using anion exchange chromatography . 1 ) dialyze fibrinogen ( 8 mg / ml ) versus 4 l of tris buffered saline ( 33 mm tris ), ph 7 . 4 for 24 hours . 6 ) make gels : add 20 μl of tbs + 50 mm cacl 2 , 40 μl of thrombin solution ( 20 units / ml ), and 340 μl of chimera solution + fibrinogen . ( above solutions make 6 gels ). 8 ) wash 5 times in 24 hours . use 1 ml of tbs the first 4 times and neuronal media the last time . 10 ) place one ganglia in each gel and place at 37 ° c . for 1 hr . fusion proteins or peptide chimeras , which are cross - linked to fibrin gels , may be further modified to contain a degradable site between the attachment site ( i . e ., factor xiiia substrate or heparin - binding domain ) and the bioactive protein ( i . e ., growth factor or enzyme ). these sites may be degradable either by non - specific hydrolysis ( i . e ., an ester bond ) or they may be substrates for specific enzymatic ( either proteolytic or polysaccharide degrading ) degradation . these degradable sites allow the engineering of more specific release of bioactive factor from fibrin gels . for example , degradation based on enzymatic activity allows for the release of bioactive factors to be controlled by a cellular process rather than by diffusion of the factor through the gel . the degradation sites allow the bioactive factor to be released with little or no modification to the primary protein sequence , which may result in higher activity of the factor . in addition , it allows the release of the factor to be controlled by cell specific processes , such as localized proteolysis , rather than diffusion from some porous materials . this allows factors to be released at different rates within the same material depending on the location of cells within the material . cell specific proteolytic activity is vital in applications such as nerve regeneration , which occur over long periods of time . this also reduces the amount of total growth factor needed , since its release is controlled by cellular processes . conservation of growth factor and its bioavailability are distinct advantages of exploiting cell specific proteolytic activity over the use of diffusion controlled release devices which characteristically result in the loss of a significant amount of bioactive factor in an initial burst release . enzymes that could be used for proteolytic degradation are numerous . proteolytically degradable sites could include substrates for collagenase , plasmin , elastase , stromelysin , or plasminogen activators . exemplary substrates are listed below . p1 - p5 denote amino acids 1 - 5 positions toward the amino terminus of the protein from the site where proteolysis occurs . p1 ′- p4 ′ denote amino acids 1 - 4 positions toward the carboxy terminus of the protein from the site where proteolysis occurs . enzymatic degradation can occur with polysaccharide substrates for enzymes such as heparinase , heparitinase , and chondroitinase abc . each of these enzymes have polysaccharide substrates . by virtue of the presence of heparin in all of the heparin - binding , systems , the substrate for heparinase is already built into these systems . non - enzymatic degradation substrate can consist of any linkage which undergoes hydrolysis by an acid or base catalyzed mechanism . these substrates can include oligo - esters such as oligomers of lactic or glycolic acid . the rate of degradation of these materials can be controlled through the choice of oligomer . polysaccharide degradation substrate can be included in either of the embodiments , approach 1 or 2 , through the use of heparin in either system . this provides a substrate for heparinase to degrade . it could degrade either the heparin present in the heparin - peptide chimera ( approach 2 ). or , it could degrade the heparin in the non - covalent heparin - peptide complex ( approach 1 ). proteolytic substrate could be added during the peptide synthesis of either the peptide chimera or the heparin - peptide chimera . the heparin - binding peptide chimera could be modified to contain a proteolytic degradation sequence by inserting a protease substrate , such as one of the ones for plasmin described above , between the factor xiiia substrate and the heparin - binding domain . the heparin - peptide chimera could be modified to contain a proteolytic degradation sequence by inserting a protease substrate , such as one of the ones for plasmin described above , between the factor xiiia substrate and the heparin domain . a substrate with a high k m and a low k cat could be used to slow cleavage while occupying active sites of the protease . the cleavage substrates other than those for plasmin could be used to allow release of the bioactive factors to be independent of matrix degradation . an oligo - ester substrate could be inserted between the factor xiiia substrate and either the heparin - binding domain or the heparin domain of the chimera during the peptide synthesis step as well . this could be accomplished using a oligo - ester such as oligomers of lactic acid . fusion proteins together with growth factors that do not bind heparin spontaneously in order to sequester growth factors which do not spontaneously bind heparin , it is necessary to modify the protein through the addition of a functionality capable of attaching to fibrin . this can be accomplished in several ways . by way of example , this may be achieved through the addition of a factor xiiia substrate or by adding a heparin - binding domain to the resulting fusion protein . the addition of a synthetic factor xiiia substrate can be accomplished by expressing a fusion protein containing the native growth factor sequence and a factor xiiia substrate at either the amino or carboxyl terminus of the fusion protein . this modification is done at the dna level . whole proteins present difficulty in that they are synthesized by solid phase chemical synthesis . the dna sequence encoding the growth factor is adapted to optimal codon usage for bacterial expression . the dna sequence is then determined for the desired factor xiiia substrate , using codons which occur frequently in bacterial dna . a series of gene fragments is designed prior to the dna synthesis . due to the error frequency of most dna synthesis , which contains an error approximately every 50 bp , genes are constructed to be approximately 100 bp in length . this reduces the number of colonies that must be screened in order to find one containing the proper dna sequence . the location at which one gene ends and the next begins is selected based on the natural occurrence of unique restriction enzyme cut sites within the gene , resulting in fragments ( or oligonucleotides ) of variable length . the process is greatly assisted by the use of software which identifies the location and frequency of restriction enzyme sites within a given dna sequence . once the gene fragments have been successfully designed , common restriction enzyme sites are included on the ends of each fragment to allow ligation of each fragment into a cloning plasmid . for example , adding tcori and hindiii sites to each gene fragment allows it to be inserted into the polylinker cloning region of puc 19 ( yanish - perron , c ., et al , ( 1985 ). gene . 33 : 103 - 119 ). the 3 ′ and 5 ′ single strands of each gene fragment are then synthesized using standard solid phase synthesis with the proper sticky ends for insertion into the cloning vector . following cleavage and desalting , the single stranded fragments are then purified by page and annealed . after phosphorylation , the annealed fragments are ligated into a cloning vector , such as puc 19 . following ligation , the plasmids are transformed into dh5 - f ′ competent cells and plated on isopropyl - d - thiogalactopyranoside ( iptg )/ 5 - bromo - 4 - chloro - 3 - indolyl - d - galactopyranoside ( x - gal ) plates to screen for the insertion of the gene fragments . the resulting colonies which contain gene fragment are then screened for insertion of the proper length . this is accomplished by purifying plasmid from colonies of transformed cells by alkaline lysis miniprep protocol and digesting the plasmid with the restriction enzyme sites present at either end of the gene fragment . upon detection of the fragments of the proper length by agarose gel electrophoresis , the plasmids are sequenced . when a plasmid containing a gene fragment with the proper sequence is identified , the fragment is then cut out and used to assemble the full gene . each time one plasmid is cut with the enzymes at the insertion points and purified from an agarose gel after dephosphorylation of the plasmid . meanwhile , a second plasmid containing the fragment to be inserted is also cut and the fragment to be inserted is purified from an agarose gel . the insert dna is then ligated into the dephosphorylated plasmid . this process is continued until the full gene is assembled . the gene is then moved into an expression vector , such as pet 14b ( studier , f ., et al , ( 1990 ). methods in enzymology . 185 : 60 - 89 ) and transformed into bacteria for expression . after this final ligation , the full gene is sequenced to confirm that it is correct . expression of the fusion protein is accomplished by growing the bacteria until they reach mid - log phase growth and then inducing expression of the fusion protein . expression is continued for approximately 3 hours and the cells are then harvested . after obtaining a bacterial cell pellet , the cells are lysed . the cell membranes and debris are removed by washing the cell lysate pellet with triton x100 , leaving the inclusion bodies in relatively pure form . the fusion protein is solubilized using high urea concentrations and purified by histidine affinity chromatography . the resulting protein is then renatured gradually by dialysis against a slowly decreasing amount of urea and lyophilized . the addition of a synthetic heparin - binding domain can be accomplished by expressing a fusion protein containing the native growth factor sequence and heparin - binding domain at either the amino or carboxyl terminus of the fusion protein . this modification is done at the dna level . the dna sequence encoding the growth factor is adapted to optimal codon usage for bacterial expression . the dna sequence is then determined for the desired heparin - binding domain , using bacterial codons . a series of gene fragments is then constructed and assembly of the full gene is performed as described above for a factor xiiia substrate . once the full gene is assembled , it is moved to an expression plasmid and the fusion protein is synthesized as described above . this protein is purified as described above for the factor xiiia substrate fusion protein . a fusion protein containing either the factor xiiia substrate or the heparin - binding domain can be synthesized with a degradable site between the native growth factor sequence and “ cross - linking ” functionality . this may be accomplished by modification of the dna sequence . the gene is designed and assembled as described above . ngf can be expressed as fusion protein in e . coli , which contains a factor xiiia substrate at the n - terminus and the human β - ngf sequence at the c - terminus of the protein . this is accomplished by constructing a synthetic gene containing the dna which codes for the desired fusion protein . the protein sequence to be expressed is as follows : where the region in italics is the histidine tag derived from the expression vector , and the underlined region is the thrombin cleavage site . the residues are the cross - linking substrate sequence for factor xiiia . the cloning plasmid used for gene assembly was puc 18 , which is the same as puc 19 except that the sequence of the polylinker cloning region is reversed . a map of puc 19 follows , which was obtained from new england biolabs . the dna sequence of the gene is as follows from 5 ′ to 3 ′: this gene is inserted between the ecori and hindiii sites in the polylinker cloning region of puc 18 , as shown in the map . after gene assembly , this gene is inserted into the expression vector pet 14b between the nde 1 and bamhi sites . a map of the pet 14b vector follows , which was obtained from novagen . after insertion of the gene into the expression vector , the plasmid is transformed into bl21 ( de3 ) plyss competent cells . the cells are grown until they reach an o . d . of about 0 . 6 , then they are induced to express the fusion protein with iptg ( final concentration in solution 0 . 4 mm ). expression is continued for 2 - 3 hours . the cells are placed on ice for 5 minutes and then harvested by centrifugation at 5000 × g for 5 min . at 4 ° c . they are resuspended in 0 . 25 culture volume of cold 50 mm tris - hcl ph 8 . 0 at 25 ° c . the cells are centrifuged as before and the pellet is frozen . cells are lysed upon thawing . the cell lysate is centrifuged and the supernatant discarded . the pellet is resuspended in triton x100 . the solution is then centrifuged and the supernatant is discarded . the pellet is resuspended in 6m urea and the fusion protein is purified by histidine affinity chromatography . the histidine tag can be cleaved by thrombin during polymerization and washed from the gels during the standard washing procedure . ngf can be expressed as fusion protein in e . coli , which contains a heparin - binding domain at the n - terminus and the ngf sequence at the c - terminus of the protein . this is accomplished by constructing a synthetic gene containing the dna which codes for the desired fusion protein . the protein sequence to be expressed is as follows : where the region in italics is the histidine tag derived from the expression vector , and the underlined region is the thrombin cleavage site . the region underlined with a dotted underline is the heparin - binding sequence . the cloning plasmid used for gene assembly was puc 18 . the dna sequence of the gene is as follows from 5 ′ to 3 ′: this gene is inserted between the ecori and hindiii sites in the polylinker - cloning region of puc 18 , as shown in the map . after assembly this gene is inserted into the expression vector . expression and purification are then performed as described above . ngf can be expressed as fusion protein in e . coli , which contains a factor xiiia substrate at the n - terminus , a plasmin substrate in the middle , and the ngf sequence at the c - terminus of the protein . this is accomplished by constructing a synthetic gene containing the dna which codes for the desired fusion protein . the protein sequence to be expressed is as follows : where the region in italics is the histidine tag derived from the expression vector , and the underlined region is the thrombin cleavage site . the residues are the cross - linking substrate sequence for factor xiiia , and double underlined region denotes the plasmin substrate . the cloning plasmid used for gene assembly was puc 18 . the dna sequence of the gene is as follows from 5 ′ to 3 ′: this gene is inserted between the ecori and hindiii sites in the polylinker cloning region of puc 18 , as shown in the map . after assembly this gene is inserted into the expression vector . expression and purification are then performed as described above . ngf can be expressed as fusion protein in e . coli , which contains a heparin - binding domain at the n - terminus , a plasmin substrate in the middle and the ngf sequence at the c - terminus of the protein . this is accomplished by constructing a synthetic gene containing the dna which codes for the desired fusion protein . the protein sequence to be expressed is as follows : where the region in italics is the histidine tag derived from the expression vector , and the underlined region is the thrombin cleavage site . dotted underline denote the heparin - binding sequence , and double underline denotes the plasmin substrate . the cloning plasmid used for gene assembly was puc 18 . the dna sequence of the gene is as follows from 5 ′ to 3 ′: this gene is inserted between the ecori and hindiii sites in the polylinker cloning region of puc 18 , as shown in the map . after assembly this gene is inserted into the expression vector . expression and purification are then performed as described above . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . 1 . besson , c ., et al , ( 1996 ). analytical biochemistry . 237 : 216 - 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1107 . 14 . zucker , m , et al , ( 1991 ). experimental biology and medicine . 693 - 702 . 15 . smith , m . m ., et al , ( 1995 ). 1995 . 270 : 6440 - 6449 . 16 . spillmann , d ., et al , ( 1998 ). journal of biological chemistry . 273 : 15487 - 15493 . 17 . steffen , c ., et al , ( 1998 ). characterization of cell - associated and soluble forms of connective tissue growth factor ( ctgf ) produced by fibroblast cells in vitro . growth factors . 15 : 199 - 213 . 18 . studier , f ., et al , ( 1990 ). methods in enzymology . 185 : 60 - 89 . 19 . takagi , t ., et al , ( 1975 ). biochemistry . 14 : 5149 - 5156 . 20 . tessler , s ., et al , ( 1994 ). journal of biological chemistry . 269 : 12456 - 12461 . 21 . tyler - cross , r ., et al , ( 1994 ). protein science . 3 : 620 - 627 . 22 . yanish - perron , c ., et al , ( 1985 ). gene . 33 : 103 - 119 .