Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
2000-09-29
2002-10-22
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C435S002000, C435S007100, C530S350000, C530S300000
Reexamination Certificate
active
06468731
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the fields of matrices that are modified to enclose particularly designed fusion proteins. More particularly, it concerns the use of fusion proteins that include internal degradation sites and/or enzymatic cleavage sites. Artificial matrices may be designed having desired degradation rates, as well as to include particular active biological molecules, such as growth factor or enzyme.
BACKGROUND OF THE INVENTION
It has been demonstrated that bi-domain peptides, which contain a factor XIIIa substrate sequence and a bioactive peptide sequence, can be cross-linked into fibrin gels and furthermore, that this bioactive peptide retains its cellular activity in vitro. While peptides can partially mimic the bioactivity of the whole protein from which they are derived, this bioactivity is usually lower than the bioactivity of the whole protein, and sometimes it is impossible to mimic certain proteins with only a short peptide. In order to incorporate the specific bioactivity of these type of factors, such as growth factors, it would be beneficial for the entire protein to be incorporated into the fibrin matrix.
SUMMARY OF THE INVENTION
Whole proteins can be incorporated into fibrin gels in a number of ways as taught in this invention. One method is to attach heparin to the gel by either covalent or non-covalent methods. This permits heparin-binding proteins including heparin-binding growth factors to be non-covalently bound to the fibrin gel. If the protein to be bound does not contain a native heparin-binding sequence, a fusion protein can be constructed containing the native protein sequence and a synthetic heparin-binding domain. Alternatively, a fusion protein can be constructed which contains a factor XIIIa substrate and the native protein sequence and this fusion protein can be sequestered by cross-linking it to the gel with factor XIIIa.
Fusion Protein Synthesis
Synthesis of either of the fusion proteins described above can be accomplished by utilizing molecular biology techniques. To do this, a fusion protein can be created that contains the entire protein sequence of interest with a cross-linking or binding sequence fused onto the amino or carboxyl terminus. This is done at the DNA level, as sequences encoding for either a factor XIIIa cross-linking substrate or a heparin-binding domain can be inserted at the beginning or the end of the codons for the original protein. When these modified proteins are expressed, they will then contain the additional domain of interest at the amino terminus. By using the natural machinery designed for protein synthesis, it becomes possible to synthesize and purify large proteins with high fidelity.
Incorporation of Fusion Proteins
Once the protein is purified, it can then be incorporated into the fibrin gels using several different schemes. In the first design, a factor XIIIa substrate has been directly incorporated onto the protein. When this modified protein is present during the polymerization of the fibrin, it is directly incorporated into the fibrin matrix in a manner similar to the bi-domain peptides previously demonstrated (14). A separate method involves fusion proteins that have been synthesized with a heparin-binding domain. In this example, a bi-domain peptide, heparin, and the heparin-binding fusion protein are included in the fibrin polymerization mixture. During polymerization, the bi-domain peptide is cross-linked into the fibrin gel. This bi-domain peptide would contain a factor XIIIa substrate sequence in addition to a heparin-binding sequence. The heparin binds to the bi-domain peptide that has been incorporated in the fibrin gel and is trapped in the fibrin matrix. This entrapped heparin serves to sequester the heparin-binding fusion protein within the fibrin gel by binding to the engineered heparin-binding domains. This incorporation has been shown to be stable enough to sequester the growth factor until the cross linked peptide is removed from the gel via cell controlled proteolysis.
This technique can be further modified by incorporating an enzymatic degradation site between the factor XIIIa substrate sequence and the sequence encoding the protein of interest. By careful selection of K
m
and k
cat
of this enzymatic degradation site, degradation could be controlled to occur either before or after the protein matrix and/or by utilizing similar or dissimilar enzymes to degrade the matrix, with the placement of the degradation site being tailored for each type of protein and application. This new protein could be directly cross-linked into the fibrin matrix as described above. However, incorporating an enzymatic degradation site alters the release of the protein during proteolysis. When the cell-derived proteases reach the sequestered protein, they can cleave the engineered protein at the newly formed degradation site. The resulting degradation products would include the liberated protein, which would now be free of any engineered fusion sequences, as well as any degraded fibrin. Therefore, the free protein would now be identical in primary sequence to the native growth factor and potentially more bioactive. A similar method can be used with the heparin-binding fusion proteins. These new proteins would then contain the protease degradation site, as well as the new heparin-binding domain. The heparin-binding fusion proteins will be sequestered into the matrix by the incorporation of heparin into the fibrin via the covalent immobilization of heparin-binding peptides. Once again, with the new protease degradation site added, the released protein would be identical in primary sequence to the natural protein.
2. Modification of Growth Factors to Provide Synthetic Heparin-binding Domains
Using standard molecular biology techniques, fusion proteins can be made of any growth factor for which the protein or DNA sequence is known, allowing the addition of novel domains such as heparin-binding domains or enzymatic substrates. These fusion proteins can be constructed so as to add a novel domain to either the N or C-terminus of the protein. The modifications are made at the DNA level by constructing a gene containing both the DNA sequence coding for the growth factor and the DNA sequence coding for a heparin-binding domain. This DNA is then ligated into an expression plasmid and transformed into bacteria. Upon induction of expression, the bacteria will produce large amounts of this fusion protein. Following expression, the protein must be purified from the cell lysate and refolded. Purification is often simplified due to the tendency of mammalian proteins expressed at high level to form inclusion bodies in bacteria.
The simplest way to incorporate proteins into fibrin is to attach heparin to the fibrin gels and use the heparin to sequester heparin-binding proteins, such as heparin-binding growth factors. This can be accomplished one of two ways, either by directly coupling a heparin-peptide chimera (where the heparin is chemically attached to a peptide containing a factor XIIIa substrate), or indirectly by cross-linking a heparin-binding peptide into the fibrin gel and binding heparin to this peptide non-covalently (using a bifunctional peptide containing a heparin-binding domain and a factor XIIIa substrate). This heparin can then sequester proteins, such as growth factors with heparin affinity, in the fibrin gel in a manner similar to the way that they are sequestered to the extracellular matrix in nature. Heparin can also protect these factors from proteolytic degradation and prolong their activity until they are released from the matrix.
Despite their relatively strong affinity for heparin, heparin-binding growth factors dissociate from the matrix on a short time scale. Therefore, a high excess of binding sites is essential to ensure that they do not diffuse far before they bind to the matrix again. This equilibrium also allows for the binding of free growth factor to cell surface receptors that are in close proximity to the site of dissociation. This method of control
Hubbell Jeffrey A.
Sakiyama Shelly E.
Schense Jason C.
Carlson Karen Cochrane
Eidgenossische Technische Hochschule Zurich
Holland & Knight LLP
Robinson Hope A.
LandOfFree
Enzyme-mediated modification of fibrin for tissue... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Enzyme-mediated modification of fibrin for tissue..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Enzyme-mediated modification of fibrin for tissue... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2970606