Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-10-08
2002-04-09
Clark, Deborah J. R. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S009000, C800S013000, C800S014000, C536S023100, C435S320100, C435S325000
Reexamination Certificate
active
06369295
ABSTRACT:
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into :his application to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
Type X Collagen and its function Type X collagen is a homotrimer of three a1(X) chains, with a short (38aa) non-helical amino terminus (NC2), a triple helix of 463aa and a C-terminal highly conserved noncollagenous domain (NC1) of 161aa. This collagen is the major extracellular component synthesized by hypertrophic chondrocytes in growth cartilage destined to be calcified and in zones of secondary ossification (1,2). Expression of the a1(X) collagen gene is specifically associated with hypertrophic chondrocytes and precedes the onset of endochondral ossification (3). Although this collagen does not form fibrils, it has been found as fine pericellular filaments in association with cartilage collagen fibrils (4). Type X collagen molecules may also form other supramolecular structures in the matrix, since they have been shown to assemble into a hexagonal lattice in vitro (5).
Apart from association with collagen fibrils, type X collagen interacts with other matrix components, such as annexin V, chondrocalcin (6) and proteoglycans (4). Type X collagen has also been shown to be intimately associated with the calcification process by binding to Ca++ and matrix vesicles which are cell-derived microstructures found in the matrix of calcifying cartilage and bone and thought to be important in the initiation of mineral deposition (7). In addition expression of type X collagen precedes mineral deposition by cultured chondrocytes (8).
Despite the wealth of information about type X collagen, the precise function of this protein and its role in the pathogenesis of chondrodysplasia, has remained the subject of controversy. Because of its specific association with hypertrophic chondrocytes in the calcifying zone of growth plate cartilage, type X collagen has been proposed to be important for endochondral bone formation (2). Proposed functions include, providing an easily resorbed fabric for the deposition of bone matrix during endochondral growth of long bones; providing support as the cartilage matrix is degraded during endochondral ossification (9,10); or regulating the calcification process during endochondral ossification (11-14). Reconciling these opposing views has also been difficult because the consequences of gene mutations which result in type X collagen deficiency in human and mouse differ.
Mutations in the NC1 encoding domain of the human a1(X) collagen gene (COL10A1) have been found to be associated with the autosomal dominant inherited skeletal disorder, Schmid metaphyseal chondrodysplasia (SMCD) (15-18). SMCD is a relatively mild form of metaphyseal chondrodysplasia, resulting from growth plate abnormalities. The SMCD phenotype is variable in severity and characterized by short to normal stature, with genu varum (bow legs), coxa vara (a reduced angle between the femoral neck and shaft) and flaring of the metaphyses of long bone (19,20). Transgenic mice expressing truncated chicken type X collagen, display much more severe skeletal abnormalities, similar to human spondylometaphyseal dysplasia(SMD) (21) in which there is compression of the hypertrophic zone of the growth plate and a decrease in newly formed bony trabeculae.
The phenotypes of SMCD patients and the SMD-like transgenic mice favour a supportive role for type X collagen (15,16,22). Therefore it was surprising to find that mice carrying a null mutation in the a1(X) collagen gene (Col10a1) have been reported to show no abnormality and no signs of SMCD (23). K. Cheah in collaboration with others had also created a null mutation in mouse Col10a1 by homologous recombination in ES cells, to gain insight into the function of type X collagen (24). To resolve the apparently contradictory consequences of mutations in the gene in human and mouse and gain better insight into the pathogenesis of SMCD, we focused our study on the consequences of type X collagen deficiency on the structure of the growth plate and trabecular bone, and on the organization of matrix components within cartilage.
This disclosure showed that type X collagen deficiency in mice does have phenotypic consequences which partly resemble SMCD, reducing the apparent discrepancy in phenotype between human and mouse (24). Intriguingly, the major impact of type X collagen deficiency does not lie in its site of expression, the hypertrophic zone, but rather affects other zones of the growth plate and in bone. It is also found that the consequence of loss of type X collagen in mutant mice is a major change in the distribution of matrix materials such as proteoglycans and matrix vesicles, within the epiphyseal cartilage. Other features of type X collagen deficiency are a significant compression of the resting zone and articular cartilage.
Phenotypic features which partly resemble SMCD were found, such as persistence of cartilage in trabecular bone, alterations in bone mineralization and trabecular structure. In particular, type X collagen deficient mice develop coxa vara, one of phenotypic changes common in human SMCD. These findings have led us to propose a function for type X collagen. Based on these findings we propose that type X collagen plays a role in the normal compartmentalization of the cartilage matrix. Type X collagen deficiency alters the distribution of cartilage matrix components thereby impacting on the supporting properties of the growth plate and the mineralization process, resulting in abnormal trabecular bone. This hypothesis would accommodate the previously conflicting views of the function of type X collagen and of the molecular pathogenesis of SMCD.
SUMMARY OF THE INVENTION
This invention provides an isolated DNA comprising the sequence which codes for a mutated collagen X or a portion thereof wherein the expression of said DNA regulates bone growth. In an embodiment, the DNA comprises the secuence of Col10-13del as set forth in FIG.
2
. This invention also provides a vector which comprises the above-described DNA.
This invention also provides a method for production of the a polypeptide which regulates bone growth comprising a host-vector system which comprise the above vector and an appropriate host.
This invention provides a polypeptide encoded by the isolated DNA comprising the sequence which codes for a mutated collagen X or a portion thereof wherein the expression of said DNA regulates bone growth. This invention also provides a polypeptide which comprise the portion of the mutated collagen X capable of regulating bone growth.
This invention provides a composition comprising the above polypeptide and a suitable carrier. This invention provides a pharmaceutical composition for increasing bone growth comprising the above polypeptide and a pharmaceutically acceptable carrier.
This invention also provides a method of treating a subject afflicted with dwarfism comprising administering to the subject an amount of the polypeptide comprising a portion of the mutated collagen X capable of regulating bone growth or the isolated DNA comprising the sequence which codes for a mutated collagen X or a portion thereof wherein the expression of said DNA regulates bone growth effective to reverse the dwarfism.
This invention provides a method of treating a subject afflicted with low bone mass comprising administering to the subject an amount of the polypeptide comprising a portion of the mutated collagen X capable of regulating bone growth or the isolated DNA comprising the sequence which codes for a mutated collagen X or a portion thereof wherein the expression of said DNA regulates bone growth effective to treat low bone mass in the subject.
This invention provides a method of improving the quality and speed of bone union after fracture in a subject comprising administering to the subject an amount of the polypeptide comprising a portion of the mut
Cheah Kathryn S. E.
Cheung Kenneth M. C.
Clark Deborah J. R.
Cooper & Dunham LLP
Katz Robert D.
Paras, Jr. Peters
University of Hong Kong
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