Methods and compositions for multiple gene transfer into bone cells

Disclosed are methods, compositions, kits and devices for use in transferring nucleic acids into bone cells in situ and/or for stimulating bone progenitor cells. Type II collagen and, particularly, osteotropic genes, are shown to stimulate bone progenitor cells and to promote bone growth, repair and regeneration in vivo. Gene transfer protocols are disclosed for use in transferring various nucleic acid materials into bone, as may be used in treating various bone-related diseases and defects including fractures, osteoporosis, osteogenesis imperfecta and in connection with bone implants.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to the field of bone cells and 
tissues. More particularly, certain embodiments concern the transfer of 
genetic material into bone and other embodiments concern type II collagen. 
In certain examples, the invention concerns the use of type II collagen 
and nucleic acids to stimulate bone growth, repair and regeneration. 
Methods, compositions, kits and devices are provided for transferring an 
osteotropic gene into bone progenitor cells, which is shown to stimulate 
progenitor cells and to promote increased bone formation in vivo. 
2. Description of the Related Art 
Defects in the process of bone repair and regeneration are linked to the 
development of several human diseases and disorders, e.g., osteoporosis 
and osteogenesis imperfecta. Failure of the bone repair mechanism is, of 
course, also associated with significant complications in clinical 
orthopaedic practice, for example, fibrous non-union following bone 
fracture, implant interface failures and large allograft failures. The 
lives of many individuals would be improved by the development of new 
therapies designed to stimulate and strengthen the fracture repair 
process. 
Naturally, any new technique to stimulate bone repair would be a valuable 
tool in treating bone fractures. A significant portion of fractured bones 
are still treated by casting, allowing natural mechanisms to effect wound 
repair. Although there have been advances in fracture treatment in recent 
years, including improved devices, the development of new processes to 
stimulate, or complement, the wound repair mechanisms would represent 
significant progress in this area. 
A very significant patient population that would benefit from new therapies 
designed to promote fracture repair, or even prevent or lessen fractures, 
are those patients suffering from osteoporosis. The term osteoporosis 
refers to a heterogeneous group of disorders characterized by decreased 
bone mass and fractures. Clinically, osteoporosis is segregated into type 
I and type II. Type I osteoporosis occurs predominantly in middle aged 
women and is associated with estrogen loss at the menopause, while 
osteoporosis type II is associated with advancing age. 
An estimated 20-25 million people are at increased risk for fracture 
because of site-specific bone loss. The cost of treating osteoporosis in 
the United States is currently estimated to be in the order of $10 billion 
per year. Demographic trends, i.e., the gradually increasing age of the US 
population, suggest that these costs may increase 2-3 fold by the year 
2020 if a safe and effective treatment is not found. 
The major focus of current therapies for osteoporosis is fracture 
prevention, not fracture repair. This is an important consideration, as it 
is known that significant morbidity and mortality are associated with 
prolonged bed rest in the elderly, especially those who have suffered hip 
fracture. New methods are clearly needed for stimulating fracture repair, 
thus restoring mobility in these patients before the complications arise. 
Osteogenesis imperfecta (OI) refers to a group of inherited connective 
tissue diseases characterized by bone and soft connective tissue fragility 
(Byers & Steiner, 1992; Prockop, 1990). Males and females are affected 
equally, and the overall incidence is currently estimated to be 1 in 
5,000-14,000 live births. Hearing loss, dentinogenesis imperfecta, 
respiratory insufficiency, severe scoliosis and emphysema are.just some of 
the conditions that are associated with one or more types of OI. While 
accurate estimates of the health care costs are not available, the 
morbidity and mortality associated with OI certainly result from the 
extreme propensity to fracture (OI types I-IV) and the deformation of 
abnormal bone following fracture repair (OI types II-IV) (Bonadio & 
Goldstein, 1993). The most relevant issue with OI treatment is to develop 
new methods by which to improve fracture repair and thus to improve the 
quality of life of these patients. 
The techniques of bone reconstruction, such as is used to reconstruct 
defects occurring as a result of trauma, cancer surgery or errors in 
development, would also be improved by new methods to promote bone repair. 
Reconstructive methods currently employed, such as using autologous bone 
grafts, or bone grafts with attached soft tissue and blood vessels, are 
associated with significant drawbacks of both cost and difficulty. For 
example, harvesting a useful amount of autologous bone is not easily 
achieved, and even autologous grafts often become infected or suffer from 
resorption. 
The process of bone repair and regeneration resembles the process of wound 
healing in other tissues. A typical sequence of events includes; 
hemorrhage; clot formation; dissolution of the clot with concurrent 
removal of damaged tissues; ingrowth of granulation tissue; formation of 
cartilage; capillary ingrowth and cartilage turnover; rapid bone formation 
(callus tissue); and, finally, remodeling of the callus into cortical and 
trabecular bone. Therefore, bone repair is a complex process that involves 
many cell types and regulatory molecules. The diverse cell populations 
involved in fracture repair include stem cells, macrophages, fibroblasts, 
vascular cells, osteoblasts, chondroblasts, and osteoclasts. 
Regulatory factors involved in bone repair are known to include systemic 
hormones, cytokines, growth factors, and other molecules that regulate 
growth and differentiation. Various osteoinductive agents have been 
purified and shown to be polypeptide growth-factor-like molecules. These 
stimulatory factors are referred to as bone morphogenetic or morphogenic 
proteins (BMPs), and have also been termed osteogenic bone inductive 
proteins or osteogenic proteins (OPs). Several BMP (or OP) genes have now 
been cloned, and the common designations are BMP-1 through BMP-8. Although 
the BMP terminology is widely used, it may prove to be the case that there 
is an OP counterpart term for every individual BMP (Alper, 1994). 
BMPs 2-8 are generally thought to be osteogenic, although BMP-1 is a more 
generalized morphogen (Shimell et al., 1991). BMP-3 is also called 
osteogenin (Luyten et al., 1989) and BMP-7 is also called OP-1 (Ozkaynak 
et al., 1990). BMPs are related to, or part of, the transforming growth 
factor-.beta. (TGF-.beta.) superfamily, and both TGF-.beta.1 and 
TGF-.beta.2 also regulates osteoblast function (Seitz et al., 1992). 
Several BMP (or OP) nucleotide sequences and polypeptides have been 
described in U.S. Pat. Nos. e.g., 4,795,804; 4,877,864; 4,968,590; 
5,108,753; including, specifically, BMP-1 disclosed in U.S. Pat. No. 
5,108,922; BMP-2A (currently referred to as BMP-2) in U.S. Pat. Nos. 
5,166,058 and 5,013,649; BMP-2B (currently referred to as BMP-4) disclosed 
in U.S. Pat. No. 5,013,649; BMP-3 in 5,116,738; BMP-5 in 5,106,748; BMP-6 
in 5,187,076; BMP-7 in 5,108,753 and 5,141,905; and OP-1, COP-5 and COP-7 
in 5,011,691. 
Other growth factors or hormones that have been reported to have the 
capacity to stimulate new bone formation include acidic fibroblast growth 
factor (Jingushi et al., 1990); estrogen (Boden et al., 1989); macrophage 
colony stimulating factor (Horowitz et al., 1989); and calcium regulatory 
agents such as parathyroid hormone (PTH) (Raisz & Kream, 1983). 
Several groups have investigated the possibility of using bone stimulating 
proteins and polypeptides, particularly recombinant BMPs, to influence 
bone repair in vivo. For example, recombinant BMP-2 has been employed to 
repair surgically created defects in the mandible of adult dogs (Toriumi 
et al., 1991), and high doses of this molecule have been shown to 
functionally repair segmental defects in rat femurs (Yasko et al., 1992). 
Chen and colleagues showed that a single application of 25-100 mg of 
recombinant TGF-.beta.1 adjacent to cartilage induced endochondral bone 
formation in the rabbit ear full thickness skin wounds (Chen et al., 
1991). It has also been reported that an application of TGF-.beta.1 in a 
3% methylcellulose gel was able to repair surgically induced large skull 
defects that otherwise heal by fibrous connective tissue and never form 
bone (Beck et al., 1991). 
However, there are many drawbacks associated with these type of treatment 
protocols, not least the expensive and time-consuming purification of the 
recombinant proteins from their host cells. Also, polypeptides, once 
administered to an animal are more unstable than is generally desired for 
a therapeutic agent, and they are susceptible to proteolytic attack. 
Furthermore, the administration of recombinant proteins can initiate 
various inhibitive or otherwise harmful immune responses. It is clear, 
therefore, that a new method capable of promoting bone repair and 
regeneration in vivo would represent a significant scientific and medical 
advance with immediate benefits to a large number of patients. A method 
readily adaptable for use with a variety of matrices and bone-stimulatory 
genes would be particularly advantageous. 
SUMMARY OF THE INVENTION 
The present invention overcomes one or more of these and other drawbacks 
inherent in the prior art by providing novel methods, compositions and 
devices for use in transferring nucleic acids into bone cells and tissues, 
and for promoting bone repair and regeneration. Certain embodiments of the 
invention rest, generally, with the inventors' surprising finding that 
nucleic acids can be effectively transferred to bone progenitor cells in 
vivo and that, in certain embodiments, the transfer of an osteotropic gene 
stimulates bone repair in an animal. 
The invention, in general terms, thus concerns methods, compositions and 
devices for transferring a nucleic acid segment into bone progenitor cells 
or tissues. The methods of the invention generally comprise contacting 
bone progenitor cells with a composition comprising a nucleic acid segment 
in a manner effective to transfer the nucleic acid segment into the cells. 
The cells may be cultured cells or recombinant cells maintained in vitro, 
when all that is required is to add the nucleic acid composition to the 
cells, e.g., by adding it to the culture media. 
Alternatively, the progenitor cells may be located within a bone progenitor 
tissue site of an animal, when the nucleic acid composition would be 
applied to the site in order to effect, or promote, nucleic acid transfer 
into bone progenitor cells in vivo. In transferring nucleic acids into 
bone cells within an animal, a preferred method involves first adding the 
genetic material to a bone-compatible matrix and then using the 
impregnated matrix to contact an appropriate tissue site within the 
animal. 
An extremely wide variety of genetic material can be transferred to bone 
progenitor cells or tissues using the compositions and methods of the 
invention. For example, the nucleic acid segment may be DNA (double or 
single-stranded) or RNA (e.g., mRNA, tRNA, rRNA); it may also be a "coding 
segment", i.e., one that encodes a protein or polypeptide, or it may be an 
antisense nucleic acid molecule, such as antisense RNA that may function 
to disrupt gene expression. The nucleic acid segments may thus be genomic 
sequences, including exons or introns alone or exons and introns, or 
coding cDNA regions, or in fact any construct that one desires to transfer 
to a bone progenitor cell or tissue. Suitable nucleic acid segments may 
also be in virtually any form, such as naked DNA or RNA, including linear 
nucleic acid molecules and plasmids; functional inserts within the genomes 
of various recombinant viruses, including viruses with DNA genomes and 
retroviruses; and any form of nucleic acid segment, plasmid or virus 
associated with a liposome or a gold particle, the latter of which may be 
employed in connection with the gene gun technology. 
The invention may be employed to promote expression of a desired gene in 
bone cells or tissues and to impart a particular desired phenotype to the 
cells. This expression could be increased expression of a gene that is 
normally expressed (i.e., "over-expression"), or it could be used to 
express a gene that is not normally associated with bone progenitor cells 
in their natural environment. Alternatively, the invention may be used to 
suppress the expression of a gene that is naturally expressed in such 
cells and tissues, and again, to change or alter the phenotype. Gene 
suppression may be a way of expressing a gene that encodes a protein that 
exerts a down-regulatory function, or it may utilize antisense technology. 
Bone Progenitor Cells and Tissues 
In certain embodiments, this invention provides advantageous methods for 
using genes to stimulate bone progenitor cells. As used herein, the term 
"bone progenitor cells" refers to any or all of those cells that have the 
capacity to ultimately form, or contribute to the formation of, new bone 
tissue. This includes various cells in different stages of 
differentiation, such as, for example, stem cells, macrophages, 
fibroblasts, vascular cells, osteoblasts, chondroblasts, osteoclasts, and 
the like. Bone progenitor cells also include cells that have been isolated 
and manipulated in vitro, e.g., subjected to stimulation with agents such 
as cytokines or growth factors or even genetically engineered cells. The 
particular type or types of bone progenitor cells that are stimulated 
using the methods and compositions of the invention are not important, so 
long as the cells are stimulated in such a way that they are activated 
and, in the context of in vivo embodiments, ultimately give rise to new 
bone tissue. 
The term "bone progenitor cell" is also used to particularly refer to those 
cells that are located within, are in contact with, or migrate towards 
(i.e., "home to"), bone progenitor tissue and which cells directly or 
indirectly stimulate the formation of mature bone. As such, the progenitor 
cells may be cells that ultimately differentiate into mature bone cells 
themselves, i.e., cells that "directly" form new bone tissue. Cells that, 
upon stimulation, attract further progenitor cells or promote nearby cells 
to differentiate into bone-forming cells (e.g., into osteoblasts, 
osteocytes and/or osteoclasts) are also considered to be progenitor cells 
in the context of this disclosure--as their stimulation "indirectly" leads 
to bone repair or regeneration. Cells affecting bone formation indirectly 
may do so by the elaboration of various growth factors or cytokines, or by 
their physical interaction with other cell types. Although of scientific 
interest, the direct or indirect mechanisms by which progenitor cells 
stimulate bone or wound repair is not a consideration in practicing this 
invention. 
Bone progenitor cells and bone progenitor tissues may be cells and tissues 
that, in their natural environment, arrive at an area of active bone 
growth, repair or regeneration (also referred to as a wound repair site). 
In terms of bone progenitor cells, these may also be cells that are 
attracted or recruited to such an area. These may be cells that are 
present within an artificially-created osteotomy site in an animal model, 
such as those disclosed herein. Bone progenitor cells may also be isolated 
from animal or human tissues and maintained in an in vitro environment. 
Suitable areas of the body from which to obtain bone progenitor cells are 
areas such as the bone tissue and fluid surrounding a fracture or other 
skeletal defect (whether or not this is an artificially created site), or 
indeed, from the bone marrow. Isolated cells may be stimulated using the 
methods and compositions disclosed herein and, if desired, be returned to 
an appropriate site in an animal where bone repair is to be stimulated. In 
such cases, the nucleic-acid containing cells would themselves be a form 
of therapeutic agent. Such ex vivo protocols are well known to those of 
skill in the art. 
In important embodiments of the invention, the bone progenitor cells and 
tissues will be those cells and tissues that arrive at the area of bone 
fracture or damage that one desires to treat. Accordingly, in treatment 
embodiments, there is no difficulty associated with the identification of 
suitable target progenitor cells to which the present therapeutic 
compositions should be applied. All that is required in such cases is to 
obtain an appropriate stimulatory composition, as disclosed herein, and 
contact the site of the bone fracture or defect with the composition. The 
nature of this biological environment is such that the appropriate cells 
will become activated in the absence of any further targeting or cellular 
identification by the practitioner. 
Certain methods of the invention involve, generally, contacting bone 
progenitor cells with a composition comprising one or more osteotropic 
genes (with or without additional genes, proteins or other biomolecules) 
so as to promote expression of said gene in said cells. As outlined above, 
the cells may be contacted in vitro or in vivo. This is achieved, in the 
most direct manner, by simply obtaining a functional osteotropic gene 
construct and applying the construct to the cells. The present inventors 
surprisingly found that there are no particular molecular biological 
modifications that need to be performed in order to promote effective 
expression of the gene in progenitor cells. Contacting the cells with DNA, 
e.g., a linear DNA molecule, or DNA in the form of a plasmid or other 
recombinant vector, that contains the gene of interest under the control 
of a promoter, along with the appropriate termination signals, is 
sufficient to result in uptake and expression of the DNA, with no further 
steps necessary. 
In preferred embodiments, the process of contacting the progenitor cells 
with the osteotropic gene composition is conducted in vivo. Again, a 
direct consequence of this process is that the cells take up and express 
the gene and that they, without additional steps, function to stimulate 
bone tissue growth, repair or regeneration. 
An assay of an osteoinductive gene may be conducted using the bone 
induction bioassay of Sampath & Reddi (1981; incorporated herein by 
reference). This is a rat bone formation assay that is routinely used to 
evaluate the osteogenic activity of bone inductive factors. However, for 
analyzing the effects of osteotropic genes on bone growth, one is 
generally directed to use the novel osteotomy model disclosed herein. 
Osteotropic Genes 
As used herein, the terms "osteotropic and osteogenic gene" are used to 
refer to a gene or DNA coding region that encodes a protein, polypeptide 
or peptide that is capable of promoting, or assisting in the promotion of, 
bone formation, or one that increases the rate of primary bone growth or 
healing (or even a gene that increases the rate of skeletal connective 
tissue growth or healing). The terms promoting, inducing and stimulating 
are used interchangeably throughout this text to refer to direct or 
indirect processes that ultimately result in the formation of new bone 
tissue or in an increased rate of bone repair. Thus, an osteotropic gene 
is a gene that, when expressed, causes the phenotype of a cell to change 
so that the cell either differentiates, stimulates other cells to 
differentiate, attracts bone-forming cells, or otherwise functions in a 
manner that ultimately gives rise to new bone tissue. 
In using the new osteotomy model of the invention, an osteotropic gene is 
characterized as a gene that is capable of stimulating proper bone growth 
in the osteotomy gap to any degree higher than that observed in control 
studies, e.g., parallel studies employing an irrelevant marker gene such 
as .beta.-galactosidase. This stimulation of "proper bone growth" includes 
both the type of tissue growth and the rate of bone formation. In using 
the model with a 5 mm osteotomy gap, an osteotropic gene is generally 
characterized as a gene that is capable of promoting or inducing new bone 
formation, rather than abnormal bone fracture repair, i.e. fibrous 
non-union. In using the 2 mm osteotomy gap, one may characterize 
osteotropic genes as genes that increase the rate of primary bone healing 
as compared to controls, and more preferably, genes capable of stimulating 
repair of the osteotomy defect in a time period of less than nine weeks. 
In general terms, an osteotropic gene may also be characterized as a gene 
capable of stimulating the growth or regeneration of skeletal connective 
tissues such as, e.g., tendon, cartilage, and ligament. Thus, in certain 
embodiments, the methods and compositions of the invention may be employed 
to stimulate the growth or repair of both bone tissue itself and also of 
skeletal connective tissues. 
A variety of osteotropic genes are now known, all of which are suitable for 
use in connection with the present invention. Osteotropic genes and the 
proteins that they encode include, for example, systemic hormones, such as 
parathyroid hormone (PTH) and estrogen; many different growth factors and 
cytokines; chemotactic or adhesive peptides or polypeptides; molecules 
such as activin (U.S. Pat. No. 5,208,219, incorporated herein by 
reference); specific bone morphogenetic proteins (BMPs); and even growth 
factor receptor genes. 
Examples of suitable osteotropic growth factors include those of the 
transforming growth factor (TGF) gene family, including TGFs 1-4, and 
particularly TGF-.beta.1, TGF-.beta.2 and TGF-.beta.2, (U.S. Pat. Nos. 
4,886,747 and 4,742,003, incorporated herein by reference), with 
TGF-.alpha. (U.S. Pat. No. 5,168,051, incorporated herein by reference) 
also being of possible use; and also fibroblast growth factors (FGF), such 
as acidic FGF and kFGF; granulocyte/macrophage colony stimulating factor 
(GMCSF); epidermal growth factor (EGF); platelet derived growth factor 
(PDGF); insulin-like growth factors (IGF), including IGF-I and IGF-II; and 
leukemia inhibitory factor (LIF), also known as HILDA and DIA. Any of the 
above or other related genes, or DNA segments encoding the active portions 
of such proteins, may be used in the novel methods and compositions of the 
invention. 
Certain preferred osteotropic genes and DNA segments are those of the TGF 
superfamily, such as TGF-.beta.1, TGF-.beta.2, TGF-.beta.3 and members of 
the BMP family of genes. For example, several BMP genes have been cloned 
that are ideal candidates for use in the nucleic acid transfer or delivery 
protocols of the invention. Suitable BMP genes are those designated BMP-2 
through BMP-12. BMP-1 is not considered to be particularly useful at this 
stage. 
There is considerable variation in the terminology currently employed in 
the literature in referring to these genes and polypeptides. It will be 
understood by those of skill in the art that all BMP genes that encode an 
active osteogenic protein are considered for use in this invention, 
regardless of the differing terminology that may be employed. For example, 
BMP-3 is also called osteogenin and BMP-7 is also called OP-1 (osteogenic 
protein-1). It is likely that the family of factors termed OP(s) is as 
large as that termed BMP(s), and that these terms, in fact, describe the 
same set of molecules (Alper, 1994). 
The DNA sequences for several BMP (or OP) genes have been described both in 
scientific articles and in U.S. Pat. Nos. such as 4,877,864; 4,968,590; 
5,108,753. Specifically, BMP-1 sequences are disclosed in U.S. Pat. No. 
5,108,922; BMP-2A (currently referred to as BMP-2) in U.S. Pat. Nos. 
5,166,058 and 5,013,649; BMP-2B (currently referred to as BMP-4) disclosed 
in U.S. Pat. No. 5,013,649; BMP-3 in 5,116,738; BMP-5 in 5,106,748; BMP-6 
in 5,187,076; and BMP-7 in 5,108,753 and 5,141,905; all incorporated 
herein by reference). The article by Wozney et al. (1988; incorporated 
herein by reference) is considered to be particularly useful for 
describing BMP molecular clones and their activities. DNA sequences 
encoding the osteogenic proteins designated OP-1, COP-5 and COP-7 are also 
disclosed in U.S. Pat. No. 5,011,691. 
All of the above issued U.S. Patents are incorporated herein by reference 
and are intended to be used in order to supplement the present teachings 
regarding the preparation of BMP and OP genes and DNA segments that 
express osteotropic polypeptides. As disclosed in the above patents, and 
known to those of skill in the art, the original source of a recombinant 
gene or DNA segment to be used in a therapeutic regimen need not be of the 
same species as the animal to be treated. In this regard, it is 
contemplated that any recombinant PTH, TGF or BMP gene may be employed to 
promote bone repair or regeneration in a human subject or an animal, such 
as, e.g., a horse. Particularly preferred genes are those from human, 
mouse and bovine sources, in that such genes and DNA segments are readily 
available, with the human or mouse forms of the gene being most preferred 
for use in human treatment regimens. Recombinant proteins and polypeptides 
encoded by isolated DNA segments and genes are often referred to with the 
prefix "r" for recombinant and "rh" for recombinant human. As such, DNA 
segments encoding rBMPs, such as rhBMP-2 or rhBMP-4, are contemplated to 
be particularly useful in connection with this invention. 
The definition of a "BMP gene", as used herein, is a gene that hybridizes, 
under relatively stringent hybridization conditions (see, e.g., Mainiatis 
et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor 
Laboratory), to DNA sequences presently known to include BMP gene 
sequences. 
To prepare an osteotropic gene segment or cDNA one may follow the teachings 
disclosed herein and also the teachings of any of patents or scientific 
documents specifically referenced herein. Various nucleotide sequences 
encoding active BMPs are disclosed in U.S. Pat. Nos. 5,166,058, 5,013,649, 
5,116,738, 5,106,748, 5,187,076, 5,108,753 and 5,011,691, each 
incorporated herein by reference. By way of example only, U.S. Pat. No. 
5,166,058, teaches that hBMP-2 is encoded by a nucleotide sequence from 
nucleotide #356 to nucleotide #1543 of the sequence shown in Table II of 
the patent. One may thus obtain a hBMP-2 DNA segment using molecular 
biological techniques, such as polymerase chain reaction (PCR") or 
screening a CDNA or genomic library, using primers or probes with 
sequences based on the above nucleotide sequence. The practice of such 
techniques is a routine matter for those of skill in the art, as taught in 
various scientific articles, such as Sambrook et al. (1989), incorporated 
herein by reference. Certain documents further particularly describe 
suitable mammalian expression vectors, e.g., U.S. Pat. No. 5,168,050, 
incorporated herein by reference. 
Osteotropic genes and DNA segments that are particularly preferred for use 
in certain aspects of the present compositions and methods are the TGF, 
PTH and BMP genes. TGF genes are described in U.S. Pat. Nos. 5,168,051; 
4,886,747 and 4,742,003, each incorporated herein by reference. TGF.alpha. 
may not be as widely applicable as TGF.beta., but is proposed for use 
particularly in applications involving skeletal soft tissues. The PTH 
gene, or a DNA segment encoding the active fragment thereof, such as a DNA 
segment encoding a polypeptide that includes the amino acids 1-34 
(hPTH1-34; Hendy et al., 1981; incorporated herein by reference) is 
another preferred gene; as are the BMP genes termed BMP-4 and BMP-2, such 
as the gene or CDNA encoding the mouse BMP-4 disclosed herein. 
It is also contemplated that one may clone further genes or cDNAs that 
encode an osteotropic protein or polypeptide. The techniques for cloning 
DNA molecules, i.e., obtaining a specific coding sequence from a DNA 
library that is distinct from other portions of DNA, are well known in the 
art. This can be achieved by, for example, screening an appropriate DNA 
library, as disclosed in Example XIII herein, which relates to the cloning 
of a wound healing gene. The screening procedure may be based on the 
hybridization of oligonucleotide probes, designed from a consideration of 
portions of the amino acid sequence of known DNA sequences encoding 
related osteogenic proteins. The operation of such screening protocols are 
well known to those of skill in the art and are described in detail in the 
scientific literature, for example, in Sambrook et al. (1989), 
incorporated herein by reference. 
Osteotropic genes with sequences that vary from those described in the 
literature are also encompassed by the invention, so long as the altered 
or modified gene still encodes a protein that functions to stimulate bone 
progenitor cells in any direct or indirect manner. These sequences include 
those caused by point mutations, those due to the degeneracies of the 
genetic code or naturally occurring allelic variants, and further 
modifications that have been introduced by genetic engineering, i.e., by 
the hand of man. 
Techniques for introducing changes in nucleotide sequences that are 
designed to alter the functional properties of the encoded proteins or 
polypeptides are well known in the art, e.g., U.S. Pat. No. 4,518,584, 
incorporated herein by reference, which techniques are also described in 
further detail herein. Such modifications include the deletion, insertion 
or substitution of bases, and thus, changes in the amino acid sequence. 
Changes may be made to increase the osteogenic activity of a protein, to 
increase its biological stability or half-life, to change its 
glycosylation pattern, and the like. All such modifications to the 
nucleotide sequences are encompassed by this invention. 
It will, of course, be understood that one or more than one osteotropic 
gene may be used in the methods and compositions of the invention. The 
nucleic acid delivery methods may thus entail the administration of one, 
two, three, or more, osteotropic genes. The maximum number of genes that 
may be applied is limited only by practical considerations, such as the 
effort involved in simultaneously preparing a large number of gene 
constructs or even the possibility of eliciting an adverse cytotoxic 
effect. The particular combination of genes may be two or more distinct 
BMP genes; or it may be such that a growth factor gene is combined with a 
hormone gene, e.g. a BMP gene and a PTH gene; a hormone or growth factor 
gene may even be combined with a gene encoding a cell surface receptor 
capable of interacting with the polypeptide product of the first gene. 
In using multiple genes, they may be combined on a single genetic construct 
under control of one or more promoters, or they may be prepared as 
separate constructs of the same of different types. Thus, an almost 
endless combination of different genes and genetic constructs may be 
employed. Certain gene combinations may be designed to, or their use may 
otherwise result in, achieving synergistic effects on cell stimulation and 
bone growth, any and all such combinations are intended to fall within the 
scope of the present invention. Indeed, many synergistic effects have been 
described in the scientific literature, so that one of ordinary skill in 
the art would readily be able to identify likely synergistic gene 
combinations, or even gene-protein combinations. 
It will also be understood that, if desired, the nucleic segment or gene 
could be administered in combination with further agents, such as, e.g. 
proteins or polypeptides or various pharmaceutically active agents. So 
long as genetic material forms part of the composition, there is virtually 
no limit to other components which may also be included, given that the 
additional agents do not cause a significant adverse effect upon contact 
with the target cells or tissues. The nucleic acids may thus be delivered 
along with various other agents, for example, in certain embodiments one 
may wish to administer an angiogenic factor, and/or an inhibitor of bone 
resorption, as disclosed in U.S. Pat. Nos. 5,270,300 and 5,118,667, 
respectively, each incorporated herein by reference. 
Gene Constructs and DNA Segments 
As used herein, the terms "gene" and "DNA segment" are both used to refer 
to a DNA molecule that has been isolated free of total genomic DNA of a 
particular species. Therefore, a gene or DNA segment encoding an 
osteotropic gene refers to a DNA segment that contains sequences encoding 
an osteotropic protein, but is isolated away from, or purified free from, 
total genomic DNA of the species from which the DNA is obtained. Included 
within the term "DNA segment", are DNA segments and smaller fragments of 
such segments, and also recombinant vectors, including, for example, 
plasmids, cosmids, phage, retroviruses, adenoviruses, and the like. 
The term "gene" is used for simplicity to refer to a functional protein or 
peptide encoding unit. As will be understood by those in the art, this 
functional term includes both genomic sequences and cDNA sequences. 
"Isolated substantially away from other coding sequences" means that the 
gene of interest, in this case, an osteotropic gene, forms the significant 
part of the coding region of the DNA segment, and that the DNA segment 
does not contain large portions of naturally-occurring coding DNA, such as 
large chromosomal fragments or other functional genes or cDNA coding 
regions. of course, this refers to the DNA segment as originally isolated, 
and does not exclude genes or coding regions, such as sequences encoding 
leader peptides or targeting sequences, later added to the segment by the 
hand of man. 
This invention provides novel ways in which to utilize various known 
osteotropic DNA segments and recombinant vectors. As described above, many 
such vectors are readily available, one particular detailed example of a 
suitable vector for expression in mammalian cells is that described in 
U.S. Pat. No. 5,168,050, incorporated herein by reference. However, there 
is no requirement that a highly purified vector be used, so long as the 
coding segment employed encodes a osteotropic protein and does not include 
any coding or regulatory sequences that would have an adverse effect on 
bone progenitor cells. Therefore, it will also be understood that useful 
nucleic acid sequences may include additional residues, such as additional 
non-coding sequences flanking either of the 5' or 3' portions of the 
coding region or may include various internal sequences, i.e., introns, 
which are known to occur within genes. 
After identifying an appropriate osteotropic gene or DNA molecule, it may 
be inserted into any one of the many vectors currently known in the art, 
so that it will direct the expression and production of the osteotropic 
protein when incorporated into a bone progenitor cell. In a recombinant 
expression vector, the coding portion of the DNA segment is positioned 
under the control of a promoter. The promoter may be in the form of the 
promoter which is naturally associated with an osteotropic gene, as may be 
obtained by isolating the 5' non-coding sequences located upstream of the 
coding segment or exon, for example, using recombinant cloning and/or PCR 
technology, in connection with the compositions disclosed herein. 
In other embodiments, it is contemplated that certain advantages will be 
gained by positioning the coding DNA segment under the control of a 
recombinant, or heterologous, promoter. As used herein, a recombinant or 
heterologous promoter is intended to refer to a promoter that is not 
normally associated with an osteotropic gene in its natural environment. 
Such promoters may include those normally associated with other 
osteotropic genes, and/or promoters isolated from any other bacterial, 
viral, eukaryotic, or mammalian cell. Naturally, it will be important to 
employ a promoter that effectively directs the expression of the DNA 
segment in bone progenitor cells. 
The use of recombinant promoters to achieve protein expression is generally 
known to those of skill in the art of molecular biology, for example, see 
Sambrook et al. (1989). The promoters employed may be constitutive, or 
inducible, and can be used under the appropriate conditions to direct high 
level or regulated expression of the introduced DNA segment. The currently 
preferred promoters are those such as CMV, RSV LTR, the SV40 promoter 
alone, and the SV40 promoter in combination with the SV40 enhancer. 
Osteotropic genes and DNA segments may also be in the form of a DNA insert 
which is located within the genome of a recombinant virus, such as, for 
example a recombinant adenovirus, adeno-associated virus (AAV) or 
retrovirus. In such embodiments, to place the gene in contact with a bone 
progenitor cell, one would prepare the recombinant viral particles, the 
genome of which includes the osteotropic gene insert, and simply contact 
the progenitor cells or tissues with the virus, whereby the virus infects 
the cells and transfers the genetic material. 
In preferred embodiments, one would impregnate a matrix or implant material 
with virus by soaking the material in recombinant virus stock solution, 
e.g., for 1-2 hours, and then contact the bone progenitor cells or tissues 
with the impregnated matrix. Cells then penetrate, or grow into, the 
matrix, thereby contacting the virus and allowing viral infection which 
leads to the cells taking up the desired gene or cDNA and expressing the 
encoded protein. 
Bone-Compatible Matrices 
In certain preferred embodiments, the methods of the invention involved 
preparing a composition in which the osteotropic gene, genes, DNA 
segments, or cells already incorporating such genes or segments, are 
associated with, or impregnated within, a bone-compatible matrix, to form 
a "matrix-gene composition" and the matrix-gene composition is then placed 
in contact with the bone progenitor cells or tissue. The matrix may become 
impregnated with a gene DNA segment simply by soaking the matrix in a 
solution containing the DNA, such as a plasmid solution, for a brief 
period of time of anywhere from about 5 minutes or so, up to and including 
about an hour. Matrix-gene compositions are all those in which a gene is 
adsorbed, absorbed, or otherwise maintained in contact with the matrix. 
The type of matrix that may be used in the compositions, devices and 
methods of the invention is virtually limitless, so long as it is a 
"bone-compatible matrix". This means that the matrix has all the features 
commonly associated with being "biocompatible", in that it is in a form 
that does not produce an adverse, allergic or other untoward reaction when 
administered to an animal, and it is also suitable for placing in contact 
with bone tissue. This latter requirement takes into consideration factors 
such as the capacity of the matrix to provide a structure for the 
developing bone and, preferably, its capacity to resorbed into the body 
after the bone has been repaired. 
The choice of matrix material will differ according to the particular 
circumstances and the site of the bone that is to be treated. Matrices 
such as those described in U.S. Pat. No. 5,270,300 (incorporated herein by 
reference) may be employed. Physical and chemical characteristics, such 
as, e.g., biocompatibility, biodegradability, strength, rigidity, 
interface properties and even cosmetic appearance may be considered in 
choosing a matrix, as is well known to those of skill in the art. 
Appropriate matrices will both deliver the gene composition and also 
provide a surface for new bone growth, i.e., will act as an in situ 
scaffolding through which progenitor cells may migrate. 
A particularly important aspect of the present invention is its use in 
connection with orthopaedic implants and interfaces and artificial joints, 
including implants themselves and functional parts of an implant, such as, 
e.g., surgical screws, pins, and the like. In preferred embodiments, it is 
contemplated that the metal surface or surfaces of an implant or a portion 
thereof, such as a titanium surface, will be coated with a material that 
has an affinity for nucleic acids, most preferably, with hydroxyl apatite, 
and then the coated-metal will be further coated with the gene or nucleic 
acid that one wishes to transfer. The available chemical groups of the 
absorptive material, such as hydroxyl apatite, may be readily manipulated 
to control its affinity for nucleic acids, as is known to those of skill 
in the art. 
In certain embodiments, non-biodegradable matrices may be employed, such as 
sintered hydroxyapatite, bioglass, aluminates, other bioceramic materials 
and metal materials, particularly titanium. A suitable ceramic delivery 
system is that described in U.S. Pat. No. 4,596,574, incorporated herein 
by reference. Polymeric matrices may also be employed, including acrylic 
ester polymers and lactic acid polymers, as disclosed in U.S. Pat. Nos. 
4,526,909, and 4,563,489, respectively, each incorporated herein by 
reference. 
In preferred embodiments, it is contemplated that a biodegradable matrix 
will likely be most useful. A biodegradable matrix is generally defined as 
one that is capable of being resorbed into the body. Potential 
biodegradable matrices for use in connection with the compositions, 
devices and methods of this invention include, for example, biodegradable 
and chemically defined calcium sulfate, tricalciumphosphate, 
hydroxyapatite, polylactic acid, polyanhydrides, matrices of purified 
proteins, and semi-purified extracellular matrix compositions. 
A particularly preferred group of matrices are those prepared from tendon 
or dermal collagen, as may be obtained from a variety of commercial 
sources, such as e.g., Sigma and Collagen Corporation, or collagen 
matrices prepared as described in U.S. Pat. Nos. 4,394,370 and 4,975,527, 
each incorporated herein by reference. One preferred collagenous material 
is that termed UltraFiber.TM., obtainable from Norian Corp. (Mountain 
View, Calif.). 
The various collagenous materials may also be in the form of mineralized 
collagen. U.S. Pat. No. 5,231,169, incorporated herein by reference, 
describes the preparation of mineralized collagen through the formation of 
calcium phosphate mineral under mild agitation in situ in the presence of 
dispersed collagen fibrils. Such a formulation may be employed in the 
context of delivering a nucleic acid segment to a bone tissue site. 
Certain other preferred collagenous materials are those based upon type II 
collagen. Type II collagen preparations have been discovered to have the 
surprising and advantageous property of, absent any osteotropic gene, 
being capable of stimulating bone progenitor cells. Prior to the present 
invention, it was thought that type II collagen only had a structural role 
in the extracellular matrix and the present finding that type II collagen 
is actually an osteoconductive/osteoinductive material is unexpected. The 
present invention thus contemplates the use of a variety of type II 
collagen preparations as bone cell stimulants, either with or without DNA 
segments, including native type II collagen, as prepared from cartilage, 
and recombinant type II collagen. 
Nucleic Acid Transfer Embodiments 
Once a suitable matrix-gene composition has been prepared or obtained, all 
that is required to deliver the osteotropic gene to bone progenitor cells 
within an animal is to place the matrix-gene composition in contact with 
the site in the body in which one wishes to promote bone growth. This 
could be a simple bone fracture site that one wishes to repair, an area of 
weak bone, such as in a patient with osteoporosis, or a bone cavity site 
that one wishes to fill with new bone tissue. Bone cavities may arise as a 
result of an inherited disorder, birth defect, or may result following 
dental or periodontal surgery or after the removal of an osteosarcoma. 
The amount of gene construct that is applied to the matrix and the amount 
of matrix-gene material that is applied to the bone tissue will be 
determined by the attending physician or veterinarian considering various 
biological and medical factors. For example, one would wish to consider 
the particular osteotropic gene and matrix, the amount of bone weight 
desired to be formed, the site of bone damage, the condition of the 
damaged bone, the patient's or animal's age, sex, and diet, the severity 
of any infection, the time of administration and any further clinical 
factors that may affect bone growth, such as serum levels of various 
factors and hormones. The suitable dosage regimen will therefore be 
readily determinable by one of skill in the art in light of the present 
disclosure, bearing in mind the individual circumstances. 
In treating humans and animals, progress may be monitored by periodic 
assessment of bone growth and/or repair, e.g., using x-rays. The 
therapeutic methods and compositions of the invention are contemplated for 
use in both medical and veterinary applications, due to the lack of 
species specificity in bone inductive factors. In particular, it is 
contemplated that domestic, farm and zoological animals, as well as 
thoroughbred horses, would be treatable using the nucleic acid transfer 
protocols disclosed herein. 
The present methods and compositions may also have prophylactic uses in 
closed and open fracture reduction and also in the improved fixation of 
artificial joints. The invention is applicable to stimulating bone repair 
in congenital, trauma-induced, or oncologic resection-induced craniofacial 
defects, and also is useful in the treatment of periodontal disease and 
other tooth repair processes and even in cosmetic plastic surgery. The 
matrix-gene compositions and devices of this invention may also be used in 
wound healing and related tissue repair, including, but not limited to 
healing of burns, incisions and ulcers. 
The present invention also encompasses DNA-based compositions for use in 
cellular transfer to treat bone defects and disorders. The compositions of 
the invention generally comprise an osteotropic gene in association with a 
bone-compatible matrix, such as type II collagen, wherein the composition 
is capable of stimulating bone growth, repair or regeneration upon 
administration to, or implantation within, a bone progenitor tissue site 
of an animal. The osteotropic gene or genes may be any of those described 
above, with TGF-.alpha. (for soft skeletal tissues), TGF-.beta.1, 
TGF-.beta.2, TGF-.beta.2, PTH, BMP-2 and BMP-4 genes being generally 
preferred. Likewise, irrespective of the choice of gene, the 
bone-compatible matrix may be any of those described above, with 
biodegradable matrices such as collagen and, more particularly, type II 
collagen, being preferred. 
In still further embodiments, the present invention concerns osteotropic 
devices, which devices may be generally considered as molded or designed 
matrix-gene compositions. The devices of the invention naturally comprise 
a bone-compatible matrix in which an osteotropic gene is associated with 
the matrix. The combination of genes and matrix components is such that 
the device is capable of stimulating bone growth or healing when implanted 
in an animal. The devices may be of virtually any size or shape, so that 
their dimensions are adapted to fit a bone fracture or bone cavity site in 
the animal that is to be treated, allowing the fracture join and/or bone 
regrowth to be more uniform. Other particularly contemplated devices are 
those that are designed to act as an artificial joint. Titanium devices 
and hydroxylapatite-coated titanium devices will be preferred in certain 
embodiments. Parts of devices in combination with an osteotropic nucleic 
acid segment, such as a DNA-coated screw for an artificial joint, and the 
like, also fall within the scope of the invention. 
Therapeutic kits comprising, in suitable container means, a bone compatible 
matrix, such as type II collagen, and an osteotropic gene form another 
aspect of the invention. Such kits will generally contain a 
pharmaceutically acceptable formulation of the matrix and a 
pharmaceutically acceptable formulation of an osteotropic gene, such as 
PTH, BMP, TGF-.beta., FGF, GMCSF, EGF, PDGF, IGF or a LIF gene. Currently 
preferred genes include PTH, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, and 
BMP-4 genes. 
The kits may comprise a single container means that contains both the 
biocompatible matrix and the osteotropic gene. In this sense, the 
container means may contain a pharmaceutically acceptable sterile 
gelatinous matrix having associated with it, the osteotropic gene 
composition. The gelatinous matrix-DNA formulation may be in the form of a 
syringeable composition, e.g., a type II collagen-DNA composition. In 
which cases, the container means may itself be a syringe, pipette, or 
other such like apparatus, from which the matrix-DNA material may be 
applied to a bone tissue site or wound area. However, the single container 
means may contain a dry, or lyophilized, mixture of a matrix and 
osteotropic gene composition, which may or may not require pre-wetting 
before use. 
Alternatively, the kits of the invention may comprise distinct container 
means for each component. In such cases, one container would contain the 
osteotropic gene, either as a sterile DNA solution or in a lyophilised 
form, and the other container would include the matrix, which may or may 
not itself be prewetted with a sterile solution. 
The kits may also comprise a second or third container means for containing 
a sterile, pharmaceutically acceptable buffer or other diluent. Such a 
solution may be required to formulate either the DNA component, the matrix 
component, both components separately, or a pre-mixed combination of the 
components, into a more gelatinous form for application to the body. It 
should be noted, however, that all components of a kit could be supplied 
in a dry form, which would allow for "wetting" upon contact with body 
fluids. Thus, the presence of any type of pharmaceutically acceptable 
buffer is not a requirement for the kits of the invention. 
The container means will generally be a container such as a vial, test 
tube, flask, bottle, syringe or other container means, into which the 
components of the kit may placed. The matrix and gene components may also 
be aliquoted into smaller containers, should this be desired. The kits of 
the present invention may also include a means for containing the 
individual containers in close confinement for commercial sale, such as, 
e.g., injection or blow-molded plastic containers into which the desired 
vials or syringes are retained. 
Irrespective of the number of containers, the kits of the invention may 
also comprise, or be packaged with, an instrument for assisting with the 
placement of the ultimate matrix-gene composition within the body of an 
animal. Such an instrument may be a syringe, pipette, forceps, or any such 
medically approved delivery vehicle. 
Type II Collagen as an Osteoconductive/inductive Material 
The present invention also provides methods for stimulating bone progenitor 
cells, as may be applied, in certain circumstances, to promote new bone 
formation, or to stimulate wound-healing. As such, the bone progenitor 
cells that are the targets of the invention may also be termed "wound 
healing bone progenitor cells", although the function of wound healing 
itself is not required to practice all aspects of the invention. 
To stimulate a bone progenitor cell in accordance with these aspects of the 
invention, generally one would contact a bone progenitor cell with a 
composition comprising a biologically effective amount of type II 
collagen. Although preparations of crushed bone and mineralized collagen 
have been shown to be osteoconductive, this property has not previously 
been ascribed to type II collagen. The present inventors have found that 
type II collagen alone is surprisingly effective at promoting new bone 
formation, it being able to bridge a 5 mm osteotomy gap in only three 
weeks in all animals tested. 
The forms of type II collagen that may be employed in this invention are 
virtually limitless. For example, type II collagen may be purified from 
hyaline cartilage, as isolated from diarthrodial joints or growth plates. 
Purified type II collagen is commercially available and may be purchased 
from, e.g., Sigma Chemical Company, St. Louis. Any form of recombinant 
type II collagen may also be employed, as may be obtained from a type II 
collagen-expressing recombinant host cell, including bacterial, yeast, 
mammalian, and insect cells. One particular example of a recombinant type 
II collagen expression system is a yeast cell that includes an expression 
vector that encodes type II collagen, as disclosed herein in Example VI. 
The type II collagen used in the invention may, if desired, be supplemented 
with additional minerals, such as calcium, e.g., in the form of calcium 
phosphate. Both native and recombinant type II collagen may be 
supplemented by admixing, adsorbing, or otherwise associating with, 
additional minerals in this manner. Such type II collagen preparations are 
clearly distinguishable from the types of "mineralized collagen" 
previously described, e.g., in U.S. Pat. No. 5,231,169 that describes the 
preparation of mineralized total collagen fibrils. 
An object of this aspect of the invention is to provide a source of 
osteoconductive matrix material, that may be reproducibly prepared in a 
straightforward and cost-effective manner, and that may be employed, with 
or without an osteotropic gene segment, to stimulate bone progenitor 
cells. Recombinant type II collagen was surprisingly found to satisfy 
these criteria. Although clearly not required for effective results, the 
combination of native or recombinant type II collagen with mineral 
supplements, such as calcium, is encompassed by this invention. 
A biologically effective amount of type II collagen is an amount of type II 
collagen that functions to stimulate a bone progenitor cell, as described 
herein. By way of example, one measure of a biologically effective amount 
is an amount effective to stimulate bone progenitor cells to the extent 
that new bone formation is evident. In this regard, the inventors have 
shown that 10 mg of lyophilized collagen functions effectively to close a 
5 mm osteotomy gap in three weeks. This information may be used by those 
of skill in the art to optimize the amount of type II collagen needed for 
any given situation. 
Depending on the individual case, the artisan would, in light of this 
disclosure, readily be able to calculate an appropriate amount, or dose, 
of type II collagen for stimulating bone cells and promoting bone growth. 
Suitable effective amounts of collagen include between about 1 mg and 
about 100 mgs of lyophilized type II collagen per bone tissue site. Of 
course, it is likely that there will be variations due to, e.g., 
individual responses, particular tissue conditions, and the speed with 
which bone formation is required. While 10 mg were demonstrated to be 
useful in the illustrative example, the inventors contemplate that 1, 5, 
10, 15, 20, 30, 40, 50, 75, 100, 125 mg, and the like, may be usefully 
employed. Of course any values within these contemplated ranges may be 
useful in any particular case. Exceptional cases where even greater 
amounts of type II collagen need to be employed are not excluded from the 
methods of the invention. 
In contacting or applying type II collagen, with or without a DNA segment, 
to bone progenitor cells located within a bone progenitor tissue site of 
an animal, bone tissue growth will be stimulated. Thus, bone cavity sites 
and bone fractures may be filled and repaired. 
The use of type II collagen in combination with a nucleic acid segment that 
encodes a polypeptide or protein that stimulates bone progenitor cells 
when expressed in said cells is preferred, as described above. Nucleic 
acid segments that comprise an isolated PTH gene, BMP gene, growth factor 
gene, growth factor receptor gene, cytokine gene or a chemotactic factor 
gene are preferred, with PTH, TGF-.beta. and BMP genes being most 
preferred. The genes function subsequent to their transfer into, and 
expression in, bone progenitor cells of the treated animal, thus promoting 
bone growth. 
Although type II collagen alone is effective, its combined use with an 
osteotropic gene segment may prove to give synergistic and particularly 
advantageous effects. Type II collagen, whether native or recombinant, may 
thus also be formulated into a therapeutic kit with an osteotropic gene 
segment, in accordance with those kits described herein above. This 
includes the use of single or multiple container means; any medically 
approved delivery vehicle, including, but not limited to, syringes, 
pipettes, forceps, additional diluents, and the like.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
1. Applications of Bone Repair Technology to Human Treatment 
The following is a brief discussion of four human conditions to exemplify 
the variety of diseases and disorders that would benefit from the 
development of new technology to improve bone repair and healing 
processes. In addition to the following, several other conditions, such 
as, for example, vitamin D deficiency; wound healing in general; soft 
skeletal tissue repair; and cartilage and tendon repair and regeneration, 
may also benefit from technology concerning the stimulation of bone 
progenitor cells. 
The first example is the otherwise healthy individual who suffers a 
fracture. Often, clinical bone fracture is treated by casting to alleviate 
pain and allow natural repair mechanisms to repair the wound. There has 
been progress in the treatment of fracture in recent times, however, even 
without considering the various complications that may arise in treating 
fractured bones, any new procedures to increase bone healing in normal 
circumstances would be represent a great advance. 
A second example which may benefit from new treatment methods is 
osteogenesis imperfecta (OI). OI encompasses various inherited connective 
tissue diseases that involve bone and soft connective tissue fragility in 
humans (Byers & Steiner, 1992; Prockop, 1990). About one child per 
5,000-14,000 born is affected with OI and the disease is associated with 
significant morbidity throughout life. A certain number of deaths also 
occur, resulting from the high propensity for bone fracture and the 
deformation of abnormal bone after fracture repair (OI types II-IV; 
Donadio & Goldstein, 1993). The relevant issue here is quality of life; 
clearly, the lives of affected individuals would be improved by the 
development of new therapies designed to stimulate and strengthen the 
fracture repair process. 
OI type I is a mild disorder characterized by bone fracture without 
deformity, blue sclerae, normal or near normal stature, and autosomal 
dominant inheritance (Bonadio & Goldstein). Osteopenia is associated with 
an increased rate of lone bone fracture upon ambulation (the fracture 
frequency decreases dramatically at puberty and during young adult life, 
but increases once again in late middle age). Hearing loss, which often 
begins in the second or third decade, is a feature of this disease in 
about half the families and can progress despite the general decline in 
fracture frequency. Dentinogenesis imperfecta is observed in a subset of 
individuals. 
In contrast, OI types II-VI represent a spectrum of more severe disorders 
associated with a shortened life-span. OI type II, the perinatal lethal 
form, is characterized by short stature, a soft calvarium, blue sclerae, 
fragile skin, a small chest, floppy appearing lower extremities (due to 
external rotation and abduction of the femurs), fragile tendons and 
ligaments, bone fracture with severe deformity, and death in the perinatal 
period due to respiratory insufficiency. Radiographic signs of bone 
weakness include compression of the femurs, bowing of the tibiae, broad 
and beaded ribs, and calvarial thinning. 
OI type III is characterized by short stature, a triangular facies, severe 
scoliosis, and bone fracture with moderate deformity. Scoliosis can lead 
to emphysema and a shortened life-span due to respiratory insufficiency. 
OI type IV is characterized by normal sclerae, bone fracture with mild to 
moderate deformity, tooth defects, and a natural history that essentially 
is intermediate between OI type II and OI type I. 
More than 150 OI mutations have been characterized since 1989 (reviewed in 
Byers & Steiner, 1992; Prockop, 1990). The vast majority occur in the 
COL1A1 and COL1A2 genes of type I collagen. Most cases of OI type I appear 
to result from heterozygous mutations in the COL1A1 gene that decrease 
collagen production but do not alter primary structure, i.e. heterozygous 
null mutations affecting COL1A1 expression. 
A third, important example is osteoporosis. The term osteoporosis refers to 
a heterogeneous group of disorders characterized by decreased bone mass 
and fractures. An estimated 20-25 million people are at increased risk for 
fracture because of site-specific bone loss. Risk factors for osteoporosis 
include increasing age, gender (more females), low bone mass, early 
menopause, race (Caucasians), low calcium intake, reduced physical 
activity, genetic factors, environmental factors (including cigarette 
smoking and abuse of alcohol or caffeine), and deficiencies in 
neuromuscular control that create a propensity to fall. 
More than a million fractures in the USA each year can be attributed to 
osteoporosis, and in 1986 alone the treatment of osteoporosis cost an 
estimated 7-10 billion health care dollars. Demographic trends (i.e., the 
gradually increasing age of the US population) suggest that these costs 
may increase 2-3 fold by the year 2020 if a safe and effective treatment 
is not found. Clearly, osteoporosis is a significant health care problem. 
Clinically, osteoporosis is segregated into type I and type II. Type I 
osteoporosis occurs predominantly in middle aged women and is associated 
with estrogen loss at the menopause, while osteoporosis type II is 
associated with advancing age. Much of the morbidity and mortality 
associated with osteoporosis results from immobilization of elderly 
patients following fracture. 
Current therapies for osteoporosis patients focus on fracture prevention, 
not fracture repair. This remains an important consideration because of 
the literature, which clearly states that significant morbidity and 
mortality are associated with prolonged bed rest in the elderly, 
particularly those who have suffered hip fractures. Complications of bed 
rest include blood clots and pneumonia. These complications are recognized 
and measures are usually taken to avoid them, but these is hardly the best 
approach to therapy. thus, the osteoporotic patient population would 
benefit from new therapies designed to strengthen bone and speed up the 
fracture repair process, thus getting these people on their feet before 
the complications arise. 
A fourth example is related to bone reconstruction and, specifically, the 
ability to reconstruct defects in bone tissue that result from traumatic 
injury; as a consequence of cancer or cancer surgery; as a result of a 
birth defect, an error in development, or a heritable disorder; or as a 
result of aging. There is a significant orthopaedic need for more frequent 
implants, and cranial and facial bone are particular targets for this type 
of reconstructive need. The availability of new implant materials, e.g., 
titanium, has permitted the repair of relatively large defects. Titanium 
implants provide excellent temporary stability across bony defects. 
However, experience has shown that a lack of viable bone bridging the 
defect can result in exposure of the appliance, infection, structural 
instability and, ultimately, failure to repair the defect. 
Autologous bone grafts are another possibility, but they have several 
demonstrated disadvantages in that they must be harvested from a donor 
site such as iliac crest or rib, they usually provide insufficient bone to 
completely fill the defect, and the bone that does form is sometimes prone 
to infection and resorption. Partially purified xenogeneic preparations 
are not practical for clinical use because microgram quantities are 
purified from kilograms of bovine bone, making large scale commercial 
production both costly and impractical. Allografts and demineralized bone 
preparations are therefore often employed. 
Microsurgical transfers of free bone grafts with attached soft tissue and 
blood vessels can close bony defects with an immediate source of blood 
supply to the graft. However, these techniques are time consuming, have 
been shown to produce a great deal of morbidity, and can only be used by 
specially trained individuals. Furthermore, the bone implant is often 
limited in quantity and is not readily contoured. In the mandible, for 
example, the majority of patients cannot wear dental appliances using 
presently accepted techniques (even after continuity is established), and 
thus gain little improvement in the ability to masticate. Toriumi et al. 
have written the "Reconstructive surgeons should have at their disposal a 
bone substitute that would be reliable, biocompatible, easy to use, and 
long lasting and that would restore mandibular continuity with little 
associated morbidity." 
In connection with bone reconstruction, specific problem areas for 
improvement are those concerned with treating large defects, such as 
created by trauma, birth defects, or particularly, following tumor 
resection; and also the area of artificial joints. The success of 
orthopaedic implants, interfaces and artificial joints could conceivably 
be improved if the surface of the implant, or a functional part of an 
implant, were to be coated with a bone stimulatory agent. The surface of 
implants could be coated with one or more appropriate materials in order 
to promote a more effective interaction with the biological site 
surrounding the implant and, ideally, to promote tissue repair. 
2. Bone Repair 
Bone tissue is known to have the capacity for repair and regeneration and 
there is a certain understanding of the cellular and molecular basis of 
these processes. The initiation of new bone formation involves the 
commitment, clonal expansion, and differentiation of progenitor cells. 
Once initiated, bone formation is promoted by a variety of polypeptide 
growth factors, Newly formed bone is then maintained by a series of local 
and systemic growth and differentiation factors. 
The concept of specific bone growth-promoting agents is derived from the 
work of Huggins and Urist. Huggins et al. demonstrated that autologous 
transplantation of canine incisor tooth to skeletal muscle resulted in 
local new bone formation (Huggins et al., 1936). Urist and colleagues 
reported that demineralized lyophilized bone segments induced bone 
formation (Urist, 1965; Urist et al., 1983), a process that involved 
macrophage chemotaxis; the recruitment of progenitor cells; the formation 
of granulation tissue, cartilage, and bone; bone remodeling; and marrow 
differentiation. The initiation of cartilage and bone formation in an 
extraskeletal site, a process referred to as osteoinduction, has permitted 
the unequivocal identification of initiators of bone morphogenesis (Urist, 
1965; Urist et al., 1983; Sampath et al., 1984; Wang et al., 1990; 
Cunningham et al., 1992). 
Significant progress has now been made in characterizing the biological 
agents elaborated by active bone tissue during growth and natural bone 
healing. Demineralized bone matrix is highly insoluble; Sampath and Reddi 
(1981) showed that only 3% of the proteins can be extracted using strong 
combinations of denaturants and detergents. They also showed that the 
unfractionated demineralized bone extract will initiate bone 
morphogenesis, a critical observation that led to the purification of 
"osteoinductive" molecules. Families of proteinaceous osteoinductive 
factors have now been purified and characterized. They have been variously 
referred to in the literature as bone morphogenetic or morphogenic 
proteins (BMPs), osteogenic bone inductive proteins or osteogenic proteins 
(OPs). 
3. Bone Repair and Bone Morphogenetic Proteins (BMPs) 
Following their initial purification, several bone morphogenetic protein 
genes have now been cloned using molecular techniques (Wozney et al., 
1988; Rosen et al., 1989; summarized in Alper, 1994). This work has 
established BMPs as members of the transforming growth factor-.beta. 
(TGF-.beta.) superfamily based on DNA sequence homologies. Other TGF 
molecules have also been shown to participate in new bone formation, and 
TGF-.beta. is regarded as a complex multifunctional regulator of 
osteoblast function (Centrella et al., 1988; Carrington et al., 1969-175; 
Seitz et al., 1992). Indeed, the family of transforming growth factors 
(TGF-.alpha. and TGF-.beta.) has been proposed as potentially useful in 
the treatment of bone disease (U.S. Pat. No. 5,125,978, incorporated 
herein by reference). 
The cloning of distinct EMP genes has led to the designation of individual 
BMP genes and proteins as BMP-1 through BMP-8. BMPs 2-8 are generally 
thought to be osteogenic (BMP-1 may be a more generalized morphogen; 
Shimell et al., 1991). BMP-3 is also called osteogenin (Luyten et al., 
1989) and BMP-7 is also called OP-1 (Ozkaynak et al., 1990). TGFs and BMPs 
each act on cells via complex, tissue-specific interactions with families 
of cell surface receptors (Roberts & Sporn, 1989; Paralkar et al., 1991). 
Several BMP (or OP) nucleotide sequences and vectors, cultured host cells 
and polypeptides have been described in the patent literature. For 
example, U.S. Pat. Nos., e.g., 4,877,864, 4,968,590 and 5,108,753 all 
concern osteogenic factors. More specifically, BMP-1 is disclosed in U.S. 
Pat. No. 5,108,922; BMP-2 species, including BMP-2A and BMP-2B, are 
disclosed in U.S. Pat. Nos. 5,166,058, 5,013,649, and 5,013,649; BMP-3 in 
5,116,738; BMP-5 in 5,106,748; BMP-6 in 5,187,076; and BMP-7 in 5,108,753 
and 5,141,905; all incorporated herein by reference. Various BMP clones 
and their activities are particularly described by Wozney et al. (1988; 
incorporated herein by reference). DNA sequences encoding the osteogenic 
proteins designated OP-1, COP-5 and COP-7 are also disclosed in U.S. Pat. 
No. 5,011,691. Although the BMP terminology is widely used, it may prove 
to be the case that there is an OP counterpart term for every individual 
BMP (Alper, 1994). 
4. Bone Repair and Growth Factors and Cytokines 
Transforming growth factors (TGFs) have a central role in regulating tissue 
healing by affecting cell proliferation, gene expression, and matrix 
protein synthesis (Roberts & Sporn, 1989). While not a direct effect, 
Bolander and colleagues have provided evidence that TGF-.beta.1 and 
TGF-.beta.2 can initiate both chondrogenesis and osteogenesis (Joyce et 
al., 1990; Izumi et al., 1992; Jingushi et al., 1992). In these studies 
new cartilage and bone formation appeared to be dose dependent (i.e. 
dependent on the local growth factor concentration). The data also 
suggested that TGF-.beta.1 and TGF-.beta.2 stimulated cell differentiation 
by a similar mechanism, even though they differed in terms of the ultimate 
amount of new cartilage and bone that was formed. 
Other growth factors/hormones besides TGF and BMP may influence new bone 
formation following fracture. Bolander and colleagues injected recombinant 
acidic fibroblast growth factor into a rat fracture site (Jingushi et al., 
1990). The major effect of multiple high doses (1.0 mg/50 ml) was a 
significant increase in cartilage tissue in the fracture gap, while lower 
doses had no effect. These investigators also used the reverse 
transcriptase-polymerase chain reaction (PCR) technique to demonstrate 
expression of estrogen receptor transcripts in callus tissue (Boden et 
al., 1989). These results suggested a role for estrogen in normal fracture 
repair. 
Horowitz and colleagues have shown that activated osteoblasts will 
synthesize the cytokine, macrophage colony stimulating factor (Horowitz et 
al., 1989). The osteotropic agents used in this study included 
lipopolysaccharide, PTH1-84, PTH1-34, vitamin D and all-trans retinoic 
acid. This observation has led to the suggestion that osteoblast 
activation following fracture may lead to the production of cytokines that 
regulate both hematopoiesis and new bone formation. Various other proteins 
and polypeptides that have been found to be expressed at high levels in 
osteogenic cells, such as, e.g., the polypeptide designated Vgr-1 (Lyons 
et al. 1989), also have potential for use in connection with the present 
invention. 
5. Bone Repair and Calcium Regulating Hormones 
Calcium regulating hormones such as parathyroid hormone (PTH) participate 
in new bone formation and bone remodeling (Raisz & Kream, 1983). PTH is an 
84 amino acid calcium-regulating hormone whose principle function is to 
raise the Ca.sup.+2 concentration in plasma and extracellular fluid. 
Studies with the native hormone and with synthetic peptides have 
demonstrated that the amino-terminus of the molecule (aa 1-34) contains 
the structural requirements for biological activity (Tregear et al., 1973; 
Hermann-Erlee et al., 1976; Riond, 1993). PTH functions by binding to a 
specific cell surface receptor that belongs to the G protein-coupled 
receptor superfamily (Silve et al., 1982; Rizzoli et al., 1983; Juppner et 
al., 1991). 
Using a retroviral approach, a human full-length PTH gene construct has 
been introduced into cultured rat fibroblasts to create recombinant 
PTH-secreting cells. These cells were then transplanted into syngeneic rat 
recipients that were observed to develop hypercalcemia mediated by the 
increased serum concentrations of PTH (Wilson et al., 1992). The object of 
these studies was to create an animal model of primary 
hyperparathyroidism. 
PTH has a dual effect on new bone formation, a somewhat confusing aspect of 
hormone function despite intensive investigation. PTH has been shown to be 
a potent direct inhibitor of type I collagen production by osteoblasts 
(Kream et al., 1993). Intact PTH was also shown to stimulate bone 
resorption in organ culture over 30 years ago, and the hormone is known to 
increase the number and activity of osteoclasts. Recent studies by Gay and 
colleagues have demonstrated binding of .sup.125 I!PTH(1-84) to 
osteoclasts in tissue sections and that osteoclasts bind intact PTH in a 
manner that is both saturable and time- and temperature dependent 
(Agarwala & Gay, 1992). While these properties are consistent with the 
presence of PTH/PTHrP receptors on the osteoclast cell surface, this 
hypothesis is still considered controversial. A more accepted view, 
perhaps, is that osteoclast activation occurs via an osteoblast signaling 
mechanism. 
On the other hand, osteosclerosis may occur in human patients with primary 
hyperparathyroidism (Seyle, 1932). It is well known that individuals with 
hyperparathyroidism do not inexorably lose bone mass, but eventually 
achieve a new bone remodeling steady state after an initial period of net 
bone loss. Chronic, low dose administration of the amino-terminal fragment 
of PTH (aa 1-34) also can induce new bone formation according to a time- 
and dose-dependent schedule (Seyle, 1932; Parsons & Reit, 1974). 
Human PTH1-34 has recently been shown to: stimulate DNA synthesis in chick 
osteoblasts and chondrocytes in culture (van der Plas, 1985; Schluter et 
al., 1989; Somjen et al., 1990); increase bone cell number in vivo 
(Malluche et al., 1986); enhance the in vitro growth of chick embryonic 
cartilage and bone (Kawashima, 1980; Burch & Lebovitz, 1983; Lewinson & 
Silbermann, 1986; Endo et al., 1980; Klein-Nulend et al., 1990); enhance 
surface bone formation (both cortical and trabecular bone) in normal and 
osteogenic animals and in humans with osteoporosis (Reeve et al., 1976; 
Reeve et al., 1980; Tam et al., 1982; Hefti et al., 1982; Podbesek et al., 
1983; Stevenson & Parsons, 1983; Slovik et al., 1986; Gunness-Hey & Hock, 
1984; Tada et al., 1988; Spencer et al., 1989; Hock & Fonseca, 1990; Liu & 
Kalu, 1990; Hock & Gera, 1992; Mitlak et al., 1992; Ejersted et al., 
1993); and delay and reverse the catabolic effects of estrogen deprivation 
on bone mass (Hock et al., 1988; Hori et al., 1988; Gunness-Hey & Hock, 
1989) Liu et al., 1991). Evidence of synergistic interactions between 
hPTH-1-34 and other anabolic molecules has been presented, including 
insulin-like growth factor, BMP-2, growth hormone, vitamin D, and 
TGF-.beta. (Slovik et al., 1986; Spencer et al., 1989; Mitlak et al., 
1992; Canalis et al., 1989; Linkhart & Mohan, 1989; Seitz et al., 1992; 
Vukicevic et al., 1989). 
Anecdotal observation has shown that serum PTH levels may be elevated 
following bone fracture (Meller et al., 1984; Johnston et al., 1985; 
Compston et al., 1989; Hardy et al., 1993), but the significance of this 
observation is not understood. There are apparently no reports in the 
literature concerning attempts to localize either PTH or the PTH/PTHrP 
receptor in situ in human fracture sites or in experimental models. 
Furthermore, no attempt has been made to augment bone repair by the 
exogenous addition of PTH peptides. Although hPTH1-34 has known to 
function as an anabolic agent for bone, prior to the present invention, 
much remained to be learned about the role (if any) of PTH during bone 
regeneration and repair. 
6. Protein Administration and Bone Repair 
Several studies have been conducted in which preparations of protein growth 
factors, including BMPs, have been administered to animals in an effort to 
stimulate bone growth. The results of four such exemplary studies are 
described blow. 
Toriumi et al. studied the effect of recombinant BMP-2 on the repair of 
surgically created defects in the mandible of adult dogs (Toriumi et al., 
1991). Twenty-six adult hounds were segregated into three groups following 
the creation of a 3 cm full thickness mandibular defect: 12 animals 
received test implants composed of inactive dog bone matrix carrier and 
human BMP-2, 10 animals received control implants composed of carrier 
without BMP-2, and BMP-4 animals received no implant. The dogs were 
euthanized at 2.5-6 months, and the reconstructed segments were analyzed 
by radiography, histology, histomorphometry, and biomechanical testing. 
Animals that received test implants were euthanized after 2.5 months 
because of the presence of well mineralized bone bridging the defect. The 
new bone allowed these animals to chew a solid diet, and the average 
bending strength of reconstructed mandibles was 27% of normal (`normal` in 
this case represents the unoperated, contralateral hemimandible). In 
contrast, the implants in the other two groups were non-functional even 
after 6 months and showed minimal bone formation. 
Yasko et al. published a related study in which the effect of BMP-2 on the 
repair of segmental defects in the rat femur was examined (Yasko et al., 
1992). The study design included a group that received a dose of 1.4 mg of 
BMP-2, another group that received 11.0 mg of BMP-2, and a control group 
that received carrier matrix alone. Endochondral bone formation was 
observed in both groups of animals that received BMP-2. As demonstrated by 
radiography, histology, and whole bone (torsion) tests of mechanical 
integrity, the larger dose resulted in functional repair of the 5 mm 
defect beginning 4.5 weeks after surgery. The lower dose resulted in 
radiographic and histological evidence of new bone formation, but 
functional union was not observed even after 9 weeks post surgery. There 
was also no evidence of bone formation in control animals at this time. 
Chen et al. showed that a single application of 25-100 mg of recombinant 
TGF-.beta.1 adjacent to cartilage induced endochondral bone formation in 
the rabbit ear full thickness skin wounds (Chen et al., 1991). Bone 
formation began 21 days following the creation of the wound and reached a 
peak at day 42, as demonstrated by morphological methods. Active bone 
remodeling was observed beyond this point. 
In a related study, Beck et al. demonstrated that a single application of 
TGF-.beta.1 in a 3% methylcellulose gel was able to repair surgically 
induced large skull defects that otherwise heal by fibrous connective 
tissue and never form bone (Beck et al., 1991). Bony closure was achieved 
within 28 days of the application of 200 mg of TGF-.beta.1 and the rate of 
healing was shown to be dose dependent. 
Studies such as those described above have thus established that exogenous 
growth factors can be used to stimulate new bone 
formation/repair/regeneration in vivo. Certain U.S. Patents also concern 
methods for treating bone defects or inducing bone formation. For example, 
U.S. Pat. No. 4,877,864 relates to the administration of a therapeutic 
composition of bone inductive protein to treat cartilage and/or bone 
defects; U.S. Pat. No. 5,108,753 concerns the use of a device containing a 
pure osteogenic protein to induce endochondral bone formation and for use 
in periodontal, dental or craniofacial reconstructive procedures. 
However, nowhere in this extensive literature does there appear to be any 
suggestion that osteogenic genes themselves may be applied to an animal in 
order to promote bone repair or regeneration. Indeed, even throughout the 
patent literature that concerns genes encoding various bone stimulatory 
factors and their in vitro expression in host cells to produce recombinant 
proteins, there seems to be no mention of the possibility of using nucleic 
acid transfer in an effort to express an osteogenic gene in bone 
progenitor cells in vivo or to promote new bone formation in an animal or 
human subject. 
7. Biocompatible Matrices for use in Bone Repair 
There is a considerable amount of work that has been directed to the 
development of biocompatible matrices for use in medical implants, 
including those specifically for bone implantation work. In context of the 
present invention, a matrix may be employed in association with the gene 
or DNA coding region encoding the osteotropic polypeptide in order to 
easily deliver the gene to the site of bone damage. The matrix is thus a 
"biofiller" that provides a structure for the developing bone and 
cartilage. Such matrices may be formed from a variety of materials 
presently in use for implanted medical applications. 
Matrices that may be used in certain embodiments include non-biodegradable 
and chemically defined matrices, such as sintered hydroxyapatite, 
bioglass, aluminates, and other ceramics. The bioceramics may be altered 
in composition, such as in calcium-aluminate-phosphate; and they may be 
processed to modify particular physical and chemical characteristics, such 
as pore size, particle size, particle shape, and biodegradability. Certain 
polymeric matrices may also be employed if desired, these include acrylic 
ester polymers and lactic acid polymers, as disclosed in U.S. Pat. Nos. 
4,526,909, and 4,563,489, respectively, each incorporated herein by 
reference. Particular examples of useful polymers are those of 
orthoesters, anhydrides, propylene-cofumarates, or a polymer of one or 
more .alpha.-hydroxy carboxylic acid monomers, (e.g. .alpha.-hydroxy 
acetic acid (glycolic acid) and/or .alpha.-hydroxy propionic acid (lactic 
acid). 
Optimally, the best matrices for present purposes are those that are 
capable of being resorbed into the body. Potential biodegradable matrices 
for use in bone gene transfer include, for example, biodegradable and 
chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, 
polylactic acid and polyanhydrides. Furthermore, biomatrices comprised of 
pure proteins and/or extracellular matrix components may be employed. 
The inventors currently prefer to use bone or dermal collagenous materials 
as matrices, as may be prepared from various commercially-available 
lyophilized collagen preparations, such as those from bovine or rat skin. 
Collagen matrices may also be formulated as described in U.S. Pat. No. 
4,394,370, incorporated herein by reference, which concerns the use of 
collagenous matrices as delivery vehicles for osteogenic protein. 
UltraFiber.TM., as may be obtained from Norian Corp. (Mountain View, 
Calif.), is a preferred matrix. Preferred matrices are those formulated 
with type II collagen, and most preferably, recombinant type II collagen 
and mineralized type II collagen. 
Further suitable matrices may also be prepared from combinations of 
materials, such as polylactic acid and hydroxyapatite or collagen and 
tricalciumphosphate. Although sufficient sequestration and subsequent 
delivery of an osteotropic gene is in no way a limitation of the present 
invention, should it be desired, a porous matrix and gene combination may 
also be administered to the bone tissue site in combination with an 
autologous blood clot. The basis for this is that blood clots have 
previously been employed to increase sequestration of osteogenic proteins 
for use in bone treatment (U.S. Pat. No. 5,171,579, incorporated herein by 
reference) and their use in connection with the present invention is by no 
means excluded (they may even attract growth factors for cytokines). 
8. Collagen 
Although not previously proposed for use with a nucleic acid molecule, the 
use of collagen as a pharmaceutical delivery vehicle has been described. 
The biocompatibility of collagen matrices is well known in the art. U.S. 
Pat. Nos. 5,206,028, 5,128,136, 5,081,106, 4,585,797, 4,390,519, and 
5,197,977 (all incorporated herein by reference) describe the 
biocompatibility of collagen-containing matrices in the treatment of skin 
lesions, use as a wound dressing, and as a means of controlling bleeding. 
In light of these documents, therefore, there is no question concerning 
the suitability of applying a collagen preparation to a tissue site of an 
animal. 
U.S. Pat. No. 5,197,977 describes the preparation of a collagen-impregnated 
vascular graft including drug materials complexed with the collagen to be 
released slowly from the graft following implant. U.S. Pat. No. 4,538,603 
is directed to an occlusive dressing useful for treating skin lesions and 
a granular material capable of interacting with wound exudate. U.S. Pat. 
No. 5,162,430 describes a pharmaceutically acceptable, non-immunogenic 
composition comprising atelopeptide collagen chemically conjugated to a 
synthetic hydrophilic polymer. 
Further documents that one of skill in the art may find useful include U.S. 
Pat. Nos. 4,837,285, 4,703,108, 4,409,332, and 4,347,234, each 
incorporated herein by reference. These references describe the uses of 
collagen as a non-immunogenic, biodegradable, and bioresorbable binding 
agent. 
The inventors contemplate that collagen from many sources will be useful in 
the present invention. Particularly useful are the amino acid sequences of 
type II collagen. Examples of type II collagen are well known in the art. 
For example, the amino acid sequences of human (Lee et al., 1989), rat 
(Michaelson et al., 1994), and mouse (Ortman et al., 1994) have been 
determined (SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14, respectively). 
Although not previously known to be capable of stimulating bone progentor 
cells itself, type II collagen is herein surprisingly shown to possess 
this property, which thus gives rise to new possibilities for clinical 
use. 
9. Nucleic Acid Delivery 
The transfer of nucleic acids to mammalian cells has been proposed a method 
for treating certain diseases or disorders. Nucleic acid transfer or 
delivery is often referred to as "gene therapy". Initial efforts toward 
postnatal (somatic) gene therapy relied on indirect means of introducing 
genes into tissues, e.g., target cells were removed from the body, 
infected with viral vectors carrying recombinant genes, and implanted into 
the body. These type of techniques are generally referred to as ex vivo 
treatment protocols. Direct in vivo gene transfer has recently been 
achieved with formulations of DNA trapped in liposomes (Ledley et al., 
1987); or in proteoliposomes that contain viral envelope receptor proteins 
(Nicolau et al., 1983); calcium phosphate-coprecipitated DNA (Benvenisty & 
Reshef, 1986); and DNA coupled to a polylysine-glycoprotein carrier 
complex (Wu & Wu, 1988). The use of recombinant replication-defective 
viral vectors to infect target cells in vivo has also been described 
(e.g., Seeger et al., 1984). 
In recent years, Wolff et al. demonstrated that direct injection of 
purified preparations of DNA and RNA into mouse skeletal muscle resulted 
in significant reporter gene expression (Wolfe et al., 1990). This was an 
unexpected finding, and the mechanism of gene transfer could not be 
defined. The authors speculated that muscle cells may be particularly 
suited to take up and express polynucleotides in vivo or that damage 
associated with DNA injection may allow transfection to occur. 
Wolff et al. suggested several potential applications of the direct 
injection method, including (a) the treatment of heritable disorders of 
muscle, (b) the modification of non-muscle disorders through muscle tissue 
expression of therapeutic transgenes, (c) vaccine development, and (d) a 
reversible type of gene transfer, in which DNA is administered much like a 
conventional pharmaceutical treatment. In an elegant study Liu and 
coworkers recently showed that the direct injection method can be 
successfully applied to the problem of influenza vaccine development 
(Ulmer et al., 1993). 
The use of gene transfer to synoviocytes as a means of treating arthritis 
has also been discussed (Bandara et al., 1992; Roessler et al., 1993). The 
protocols considered have included both the ex vivo treatment of isolated 
synoviocytes and their re-introduction into the animal and also direct 
gene transfer in which suitable vectors are injected into the joint. The 
transfer of marker genes into synoviocytes has already been demonstrated 
using both retroviral and adenoviral technology (Bandara et al., 1992; 
Roessler et al., 1993). 
Despite the exclusive emphasis on protein treatment by those working in 
this field, the present inventors saw that there was great potential for 
using nucleic acids themselves to promote bone regeneration/repair in 
vivo. In addition to the ease and cost, it was reasoned that using DNA 
transfer rather than peptide transfer would provide many further 
advantages. For example, DNA transfer allows for the expression or 
over-expression of integral membrane receptors on the surface of bone 
regeneration/repair cells, whereas this cannot be done using peptide 
transfer because the latter (a priori) is an extracellular manipulation. 
Importantly, DNA transfer also allows for the expression of polypeptides 
modified in a site-directed fashion with the very minimum of additional 
work (i.e., straightforward molecular biological manipulation without 
protein purification). 
The inventors contemplated that both naked DNA and viral-mediate DNA could 
be employed in an effort to transfer genes to bone progenitor cells. In 
beginning to study this, the most appropriate animal model had to be 
employed, that is, one in which the possibilities of using nucleic acids 
to promote bone repair could be adequately tested in controlled studies. 
10. Osteotomy Model 
Prior to the present invention, three model systems were available for 
study in this area, including Mov13 mice, an animal model of OI. 
Unfortunately, each of the models suffers from significant drawbacks. With 
the Mov13 mice, first, these mice typically die in young adulthood because 
of retrovirus-induced leukemia (Schnieke et al., 1983); second, gene 
transfer studies in Mov13 mice conducted between postnatal weeks 8-16 
(i.e. prior to the development of leukemia) may be complicated by a 
natural adaptation in which a significant amount of new bone is deposited 
on the periosteal surface (Bonadio et al., 1993); and third, an 
osteotropic gene transferred into an osteotomy site may synergize with the 
active retrovirus and make it even more virulent. 
Another system is the in vivo bone fracture model created by Einhorn and 
colleagues (Bonnarens & Einhorn, 1984). However, this model is a closed 
system that would not easily permit gene transfer in vivo. The organ 
culture model developed by Bolander and colleagues (Joyce et al., 1991) 
was also available, but again, this model is not suitable for studying 
gene transfer in vivo. Due to the unsuitability of the above models for 
studying the effects of gene transfer on bone repair and regeneration, the 
inventors employed a rat osteotomy system, as described below. 
The important features of the rat osteotomy model are as follows: Under 
general anesthesia, four 1.2 mm diameter pins are screwed into the femoral 
diaphysis of normal adult Sprague-Dawley rats. A surgical template ensures 
parallel placement of the pins. An external fixator is then secured on the 
pins, and a 2 mm, or 5 mm, segmental defect is created in the central 
diaphysis with a Hall micro 100 oscillating saw. A biodegradable implant 
material, soaked in a solution of plasmid DNA, other genetic construct or 
recombinant virus preparation, is then placed in the intramedullary canal 
and the defect is closed (FIG. 1). 
New bone formation can be detected as early as three weeks later in the 2 
mm gap, although up to 9 weeks is generally allowed for new bone formation 
to occur. The fixator provided the necessary stability, and there were no 
limitations on animal ambulation. The surgical protocol has been 
successfully performed on 21/21 animals to date. None of these animals 
have died. Assays of new bone formation are performed after sacrifice, 
except plain film radiography, which is performed weekly from the time of 
surgery to sacrifice. 
Previous studies in Sprague-Dawley rats have shown that the 5 mm osteotomy 
gap will heal as a fibrous non-union, whereas a gap of less than 3 mm, 
(such as the 2 mm gap routinely employed in the studies described herein) 
will heal by primary bone formation. Studies using the 5 mmgap thus allow 
a determination of whether transgene expression can stimulate new bone 
formation when fibrous tissue healing normally is expected. On the other 
hand, studies with the 2 mm gap allow a determination of whether transgene 
expression can speed up natural primary bone healing. 
11. Gene Transfer Promotes Bone Repair In Vivo 
The present inventors surprisingly found that gene transfer into bone 
progenitor cells in vivo (i.e., cells in the regenerating tissue in the 
osteotomy gap) could be readily achieved. Currently, the preferred methods 
for achieving gene transfer generally involve using a fibrous collagen 
implant material soaked in a solution of DNA shortly before being placed 
in the site in which one desires to promote bone growth. As the studies 
presented herein show, the implant material facilitates the uptake of 
exogenous plasmid constructs by cells (in the osteotomy gap) which clearly 
participate in bone regeneration/repair. The transgenes, following 
cellular uptake, direct the expression of recombinant polypeptides, as 
evidenced by the in vivo expression of functional marker gene products. 
Further studies are presented herein demonstrating that the transfer of an 
osteotropic gene results in cellular expression of a recombinant 
osteotropic molecule, which expression is directly associated with 
stimulation of new bone formation. After considering a relatively large 
number of candidate genes, a gene transfer vector coding for a fragment of 
human parathyroid hormone (hPTH1-34) was chosen for the inventors' initial 
studies. Several factors were considered in making this choice: (a), 
recombinant hPTH1-34 peptides can be discriminated from any endogenous rat 
hormone present in osteotomy tissues; (b), hPTH1-34 peptides will 
stimulate new bone formation in Sprague-Dawley rats, indicating that the 
human peptide can efficiently bind the PTH/PTHrP receptor on the rat 
osteoblast cell surface; and (c), there is only one PTH/PTHrP receptor, 
the gene for this receptor has been cloned, and cDNA probes to the 
receptor are available. 
Thus, in terms of understanding the mechanism of action of the transgene on 
new bone formation in vivo, the inventors reasoned it most straightforward 
to correlate the expression of recombinant hPTH1-34 peptide and its 
receptor with new bone formation in the rat osteotomy model. Of course, 
following these initial studies, it is contemplated that any one of a wide 
variety of genes may be employed in connection with the bone gene transfer 
embodiments of the present invention. 
Previous studies have indicated that hPTH1-34 is a more powerful anabolic 
agent when given intermittently as opposed to continuously. Despite the 
fact that an anabolic effect would still be expected with continuous 
dosing, as documented by the studies of Parsons and co-workers (Tam et 
al., 1982) and Spencer et al. (1989), there was a concern that the 
PLJ-hPTH1-34 transgene may not function very effectively as transfected 
cells would be expected to express recombinant hPTH1-34 molecules in a 
constitutive manner. The finding that transfection and expression of the 
LPH-hPTH1-34 transgene did effectively stimulate bone formation in the rat 
osteotomy model was therefore an important result. 
As the osteotomy site in this model is highly vascularized, one possible 
complication of the studies with the PLJ-hPTH1-34 transgene is the 
secretion of recombinant human PTH from the osteotomy site with consequent 
hypercalcemia and (potentially) animal death. Weekly serum calcium levels 
should therefore be determined when using this transgene. the fact that no 
evidence of disturbed serum calcium levels has been found in this work is 
therefore a further encouraging finding. 
These studies complement others by the inventors in which direct gene 
transfer was employed to introduce genes into Achilles' tendon and 
cruciate ligament, as described in Example XI. 
Various immediate applications for using nucleic acid delivery in 
connection with bone disorders became apparent to the inventors following 
these surprising findings. The direct transfer of an osteotropic gene to 
promote fracture repair in clinical orthopaedic practice is just one use. 
Other important aspects of this technology include the use of gene 
transfer to treat patients with "weak bones", such as in diseases like 
osteoporosis; to improve poor healing which may arise fro unknown reasons, 
e.g., fibrous non-union; to promote implant integration and the function 
of artificial joints; to stimulate healing of other skeletal tissues such 
as Achilles' tendon; and as an adjuvant to repair large defects. In all 
such embodiments, DNA is being used as a direct pharmaceutical agent. 
The use of the methods and compositions of the present invention in 
stimulating vascular graft survival is also contemplated. The invention 
may thus be employed in connection with the technology described by 
Sandusky et al. (1992; incorporate herein by reference). In this case, the 
matrix part of the composition would be the biological graft, preferably 
made from acellular collagen, and more preferably type II collagen, and 
most preferably recombinant type II collagen, such as a small intestine 
submucosa (SIS) graft. To practice these aspects of the invention one 
would simply impregnate the biological graft with the nucleic acid that 
one desired to transfer to the tissue surrounding the graft site. 
12. Biological Functional Equivalents 
As mentioned above, modification and changes may be made in the structure 
of an osteotropic gene and still obtain a functional molecule that encodes 
a protein or polypeptide with desirable characteristics. The following is 
a discussion based upon changing the amino acids of a protein to create an 
equivalent, or even an improved, second-generation molecule. The amino 
acid changes may be achieved by change the codons of the DNA sequence, 
according to the following codon table: 
TABLE 1 
______________________________________ 
Amino Acids Codons 
______________________________________ 
Alanine Ala A GCA GCC GCG GCU 
Cysteine Cys C UGC UGU 
Aspartic acid 
Asp D GAC GAU 
Glutamic acid 
Glu E GAA GAG 
Phenylalanine 
Phe F UUC UUU 
Glycine Gly G GGA GGC GGG GGU 
Histidine 
His H CAC CAU 
Isoleucine 
Ile I AUA AUC AUU 
Lysine Lys K AAA AAG 
Leucine Leu L UUA UUG CUA CUC CUG CUU 
Methionine 
Met M AUG 
Asparagine 
Asn N AAC AAU 
Proline Pro P CCA CCC CCG CCU 
Glutamine 
Gln Q CAA CAG 
Arginine Arg R AGA AGG CGA CGC CGG CGU 
Serine Ser S AGC AGU UCA UCC UCG UCU 
Threonine 
Thr T ACA ACC ACG ACU 
Valine Val V GUA GUC GUG GUU 
Tryptophan 
Trp W UGG 
Tyrosine Tyr Y UAC UAU 
______________________________________ 
For example, certain amino acids may be substituted for other amino acids 
in a protein structure without appreciable loss of interactive binding 
capacity with structures such as, for example, antigen-binding regions of 
antibodies or binding sites on substrate molecules. Since it is the 
interactive capacity and nature of a protein that defines that protein's 
biological functional activity, certain amino acid sequence substitutions 
can be made in a protein sequence, and, of course, its underlying DNA 
coding sequence, and nevertheless obtain a protein with like properties. 
It is thus contemplated by the inventors that various changes may be made 
in the DNA sequences of osteotropic genes without appreciable loss of 
their biological utility or activity. 
In making such changes, the hydropathic index of amino acids may be 
considered. The importance of the hydropathic amino acid index in 
conferring interactive biologic function on a protein is generally 
understood in the art (Kyte & Doolittle, 1982, incorporate herein by 
reference). It is accepted that the relative hydropathic character of the 
amino acid contributes to the secondary structure of the resultant 
protein, which in turn defines the interaction of the protein with other 
molecules, for example, enzymes, substrates, receptors, DNA, antibodies, 
antigens, and the like. 
Each amino acid has been assigned a hydropathic index on the basis of their 
hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these 
are: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine 
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); 
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); 
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); 
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and 
arginine (-4.5). 
It is known in the art that certain amino acids may be substituted by other 
amino acids having a similar hydropathic index or score and still result 
in a protein with similar biological activity, i.e., still obtain a 
biological functionally equivalent protein. In making such changes, the 
substitution of amino acids whose hydropathic indices are within .+-.2 is 
preferred, those which are within .+-.1 are particularly preferred, and 
those within .+-.0.5 are even more particularly preferred. 
It is also understood in the art that the substitution of like amino acids 
can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 
4,554,101, incorporated herein by reference, states that the greatest 
local average hydrophilicity of a protein, as governed by the 
hydrophilicity of its adjacent amino acids, correlates with a biological 
property of the protein. 
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values 
have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); 
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine 
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline 
(-0.5.+-.1); alanine (-0.5); histidine *-0.5); cysteine (-1.0); methionine 
(-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); 
phenylalanine (-2.5); tryptophan (-3.4). 
It is understood that an amino acid can be substituted for another having a 
similar hydrophilicity value and still obtain a biologically equivalent, 
and in particular, an immunologically equivalent protein. In such changes, 
the substitution of amino acids whose hydrophilicity values are within 
.+-.2 is preferred, those which are within .+-.1 are particularly 
preferred, and those within .+-.0.5 are even more particularly preferred. 
As outline above, amino acid substitutions are generally therefore based on 
the relative similarity of the amino acid side-chain substituents, for 
example, their hydrophobicity, hydrophilicity, charge, size, and the like. 
Exemplary substitutions which take various of the foregoing 
characteristics into consideration are well known to those of skill in the 
art and include: arginine and lysine; glutamate and aspartate; serine and 
threonine; glutamine and asparagine; and valine, leucine and isoleucine. 
13. Site-Specific Mutagenesis 
Site-specific mutagenesis is a technique useful in the preparation of 
individual peptides, or biologically functional equivalent proteins or 
peptides, through specific mutagenesis of the underlying DNA. The 
technique further provides a ready ability to prepare and test sequence 
variants, for example, incorporating one or more of the foregoing 
considerations, by introducing one or more nucleotide sequence changes 
into the DNA. Site-specific mutagenesis allows the production of mutants 
through the use of specific oligonucleotide sequences which encode the DNA 
sequence of the desired mutation, as well as a sufficient number of 
adjacent nucleotides, to provide a primer sequence of sufficient size and 
sequence complexity to form a stable duplex on both sides of the deletion 
junction being traversed. Typically, a primer of about 17 to 25 
nucleotides in length is preferred, with about 5 to 10 residues on both 
sides of the junction of the sequence being altered. 
In general, the technique of site-specific mutagenesis is well known in the 
art, as exemplified by various publications. As will be appreciated, the 
technique typically employs a phage vector which exists in both a single 
stranded and double stranded form. Typical vectors useful in site-directed 
mutagenesis include vectors such as the M13 phage. These phage are readily 
commercially available and their use is generally well known to those 
skilled in the art. Double stranded plasmids are also routinely employed 
in site directed mutagenesis which eliminates the step of transferring the 
gene of interest from a plasmid to a phage. 
In general, site-directed mutagenesis in accordance herewith is performed 
by first obtaining a single-stranded vector or melting apart of two 
strands of a double stranded vector which includes within its sequence a 
DNA sequence which encodes the desired osteotropic protein. An 
oligonucleotide primer bearing the desired mutated sequence is prepared, 
generally synthetically. This primer is then annealed with the 
single-stranded vector, and subjected to DNA polymerizing enzymes such as 
E. coli polymerase I Klenow fragment, in order to complete the synthesis 
of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one 
strand encodes the original non-mutated sequence and the second strand 
bears the desired mutation. This heteroduplex vector is then used to 
transform appropriate cells, such as E. coli cells, and clones are 
selected which include recombinant vectors bearing the mutated sequence 
arrangement. 
The preparation of sequence variants of the selected osteotropic gene using 
site-directed mutagenesis is provided as a means of producing potentially 
useful species and is not meant to be limiting as there are other ways in 
which sequence variants of osteotropic genes may be obtained. For example, 
recombinant vectors encoding the desired osteotropic gene may be treated 
with mutagenic agents, such as hydroxylamine, to obtain sequence variants. 
14. LTBP-2 
Other aspects of the present invention concern isolated DNA segments and 
recombinant vectors encoding LTBP-2, and the creation and use of 
recombinant host cells through the application of DNA technology, that 
express LTBP-2 gene products. As such, the invention concerns DNA segment 
comprising an isolated gene that encodes a protein or peptide that 
includes an amino acid sequence essentially as set forth by a contiguous 
sequence from SEQ ID NO:3. These DNA segments are represented by those 
that include a nucleic acid sequence essentially as set forth by a 
contiguous sequence from SEQ ID NO:2 (FIG. 17). Compositions that include 
a purified protein that has an amino acid sequence essentially as set 
forth by the amino acid sequence of SEQ ID NO:3 (FIG. 18) are also 
encompassed by the invention. 
Transforming growth factor-.beta. (TGF-.beta.) is a family of structurally 
related molecules with diverse effects on cell shape, growth, and 
differentiation (Roberts and Sporn, 1990; Lyons and Moses, 1990). 
TGF-.beta. is initially synthesized as a precursor molecule with an 
amino-terminal propeptide followed by the mature or active form of 
TGF-.beta.. Two chains of newly synthesized pro-TGF-.beta. associate to 
form a M.sub.r =.about.106,000 disulfide bonded dimer; homodimers are most 
common but heterodimers have also been described (Cheifetz et al., 1987; 
Ogawa et al. 1992). Cultured cells secrete all three TGF-.beta. isoforms 
as latent complexes. The carboxy-termini of TGF-.beta. precursors are 
cleaved from their amino-terminal propeptides during secretion, and 
latency results in part from the non-covalent association of the 
propeptide dimer and the mature TGF-.beta. dimer. Consequently, the 
propeptide dimer is also known as the latency associated protein (LAP). 
(For additional discussion of the mechanism of latency, see Pircher et 
al., 1984; Pircher et al., 1986; Lyons et al., 1988; Miyazono et al., 
1988; Miyazono and Heldin, 1989; Kovacina et al., 1989; Brown et al., 
1990.) LAP plus the 25 kDa disulfide-bonded TGF-.beta. dimer is also known 
as the small latent complex. 
Latent complexes produced by cultured cells may also contain additional 
high molecular weight proteins to form large latent complexes (Miyazono et 
al., 1988; Wakefiled et al., 1988; Olofsson et al., 1992; Pircher et al., 
1984, 1986; Wakefield et al., 1987). The best characterized of these 
proteins is a molecule known as the latent TGF-.beta. binding protein 
(LTBP), which covalently binds LAP by a disulfide bond (Miyazono et al., 
1988). Molecular cloning of human LTBP has shown that the molecule 
contains 17 epidermal growth factor-like repeats and 3 copies of a unique 
motif containing 8 cysteine residues (Kanzaki et al., 1990). LTBP has been 
shown to bind calcium, probably via its EGF-like repeats, which in turn 
induces a structural change that protects the molecule against proteolysis 
(Colosetti et al., 1993). The EGF-like repeats of LTBP may also be 
modified to contain hydroxyaspartic acid and hydroxyasparagine, but the 
significance of this finding is unknown (Kanzaki et al., 1990). LTBP 
contains several structurally distinct domains and it is heterogeneous, 
ranging in size from 125-205 kDa in different cell types because of 
alternative splicing of the LTBP transcript and cell specific proteolysis 
(Tsuji et al., 1990). 
LTBP may have an important role in the assembly and secretion of latent 
TGF-.beta. from cells, as evidenced by the fact that the small latent 
complex is secreted slowly from cultured cells and may contain anomalous 
disulfide bonds. The large latent TGF-.beta. complex, on the other hand, 
is efficiently secreted (Miyazono et al., 1991; Miyazono et al., 1992). 
LTBP appears to be covalently bound to the extracellular matrix following 
its secretion and may therefore have the additional function of targeting 
latent TGF-.beta. to specific types of connective tissue. (Taipale et al., 
1994). Recent studies have shown that the subsequent release of mature 
TGF-.beta. and LAP from the extracellular matrix of cultured cells occurs 
as a secondary consequence of the cleavage of LTBP by proteases such as 
plasmin and thrombin (Taipale et al., 1992; Falcone et al., 1993; Benezra 
et al., 1993). 
Regarding the novel protein LTBP-2, the present invention concerns DNA 
segments, that can be isolated from virtually any mammalian source, that 
are free from total genomic DNA and that encode proteins having 
LTBP-2-like activity. DNA segments encoding LTBP-2-like species may prove 
to encode proteins, polypeptides, subunits, functional domains, and the 
like. 
As used herein, the term "DNA segment" refers to a DNA molecule that has 
been isolated free of total genomic DNA of a particular species. 
Therefore, a DNA segment encoding LTBP-2 refers to a DNA segment that 
contains LTBP-2 coding sequences yet is isolated away from, or purified 
free from, total genomic DNA of the species from which the DNA segment is 
obtained. Included within the term "DNA segment", are DNA segments and 
smaller fragments of such segments, and also recombinant vectors, 
including, for example, plasmids, cosmids, phagemids, phage, viruses, and 
the like. 
Similarly, a DNA segment comprising an isolated or purified LTBP-2 gene 
refers to a DNA segment including LTBP-2 coding sequences and, in certain 
aspects, regulatory sequences, isolated substantially away from other 
naturally occurring genes or protein encoding sequences. In this respect, 
the term "gene" is used for simplicity to refer to a functional protein, 
polypeptide or peptide encoding unit. As will be understood by those in 
the art, this functional term includes both genomic sequences, cDNA 
sequences and smaller engineered gene segments that express, or may be 
adapted to express, proteins, polypeptides or peptides. 
"Isolated substantially away from other coding sequences" means that the 
gene of interest, in this case, a gene encoding LTBP-2, forms the 
significant part of the coding region of the DNA segment, and that the DNA 
segment does not contain large portions of naturally-occurring coding DNA, 
such as large chromosomal fragments or other functional genes or cDNA 
coding regions. Of course, this refers to the DNA segment as originally 
isolated, and does not exclude genes or coding regions later added to the 
segment by the hand of man. 
In particular embodiments, the invention concerns isolated DNA segments and 
recombinant vectors incorporating DNA sequences that encode an LTBP-2 
species that includes within its amino acid sequence an amino acid 
sequence essentially as set forth in SEQ ID NO:3. In other particular 
embodiments, the invention concerns isolated DNA segments and recombinant 
vectors incorporating DNA sequences that include within their sequence a 
nucleotide sequence essentially as set forth in SEQ ID NO:2. 
The term "a sequence essentially as set forth in SEQ ID NO:3" means that 
the sequence substantially corresponds to a portion of SEQ ID NO:3 and has 
relatively few amino acids that are not identical to, or a biologically 
functional equivalent of, the amino acids of SEQ ID NO:3. The term 
"biologically functional equivalent" is well understood in the art and is 
further defined in detail herein (for example, see section 7, preferred 
embodiments). Accordingly, sequences that have between about 70% and about 
80%; or more preferably, between about 81% and about 90%; or even more 
preferably, between about 91% and about 99%; of amino acids that are 
identical or functionally equivalent to the amino acids of SEQ ID NO:3 
will be sequences that are "essentially as set forth in SEQ ID NO:3". 
In certain other embodiments, the invention concerns isolated DNA segments 
and recombinant vectors that include within their sequence a nucleic acid 
sequence essentially as set forth in SEQ ID NO:2. The term "essentially as 
set forth in SEQ ID NO:2" is used in the same sense as described above and 
means that the nucleic acid sequence substantially corresponds to a 
portion of SEQ ID NO:2 and has relatively few codons that are not 
identical, or functionally equivalent, to the codons of SEQ ID NO:2. 
Again, DNA segments that encode proteins exhibiting LTBP-2-like activity 
will be most preferred. 
It will also be understood that amino acid and nucleic acid sequences may 
include additional residues, such as additional N- or C-terminal amino 
acids or 5' or 3' sequences, and yet still be essentially as set forth in 
one of the sequences disclosed herein, so long as the sequence meets the 
criteria set forth above, including the maintenance of biological protein 
activity where protein expression is concerned. The addition of terminal 
sequences particularly applies to nucleic acid sequences that may, for 
example, include various non-coding sequences flanking either of the 5' or 
3' portions of the coding region or may include various internal 
sequences, i.e., introns, which are known to occur within genes. 
Naturally, the present invention also encompasses DNA segments that are 
complementary, or essentially complementary, to the sequence set forth in 
SEQ ID NO:2. Nucleic acid sequences that are "complementary" are those 
that are capable of basepairing according to the standard Watson-Crick 
complementarity rules. As used herein, the term "complementary sequences" 
means nucleic acid sequences that are substantially complementary, as may 
be assessed by the same nucleotide comparison set forth above, or as 
defined as being capable of hybridizing to the nucleic acid segment of SEQ 
ID NO:2, under relatively stringent conditions such as those described 
herein. 
The nucleic acid segments of the present invention, regardless of the 
length of the coding sequence itself, may be combined with other DNA 
sequences, such as promoters, polyadenylation signals, additional 
restriction enzyme sites, multiple cloning sites, other coding segments, 
and the like, such that their overall length may vary considerably. It is 
therefore contemplated that a nucleic acid fragment of almost any length 
may be employed, with the total length preferably being limited by the 
ease of preparation and use in the intended recombinant DNA protocol. For 
example, nucleic acid fragments may be prepared that include a short 
contiguous stretch identical to or complementary to SEQ ID NO:2, such as 
about 14 nucleotides, and that are up to about 10,000 or about 5,000 base 
pairs in length, with segments of about 3,000 being preferred in certain 
cases. DNA segments with total lengths of about 1,000, about 500, about 
200, about 100 and about 50 base pairs in length (including all 
intermediate lengths) are also contemplated to be useful. 
It will be readily understood that "intermediate lengths", in these 
contexts, means any length between the quoted ranges, such as 14, 15, 16, 
17, 18, 19, 20, etc.; 21, 22, 23, etc; 30, 31, 32, etc.; 50, 51, 52, 53, 
etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all 
integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 
3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 
12,001, 12,002, 13,001, 13,002 and the like. 
It will also be understood that this invention is not limited to the 
particular nucleic acid and amino acid sequences of SEQ ID NO:2 and SEQ ID 
NO:3. Recombinant vectors and isolated DNA segments may therefore 
variously include the LTBP-2 coding regions themselves, coding regions 
bearing selected alterations or modifications in the basic coding region, 
or they may encode larger polypeptides that nevertheless include 
LTEP-2-coding regions or may encode biologically functional equivalent 
proteins or peptides that have variant amino acids sequences. 
The DNA segments of the present invention encompass biologically functional 
equivalent LTBP-2 proteins and peptides. Such sequences may arise as a 
consequence of codon redundancy and functional equivalency that are known 
to occur naturally within nucleic acid sequences and the proteins thus 
encoded. Alternatively, functionally equivalent proteins or peptides may 
be created via the application of recombinant DNA technology, in which 
changes in the protein structure may be engineered, based on 
considerations of the properties of the amino acids being exchanged. 
Changes designed by man may be introduced through the application of 
site-directed mutagenesis techniques, e.g., to introduce improvements to 
the antigenicity of the protein or to test mutants in order to examine 
activity at the molecular level. 
If desired, one may also prepare fusion proteins and peptides, e.g., where 
the LTBP-2 coding regions are aligned within the same expression unit with 
other proteins or peptides having desired functions, such as for 
purification or immunodetection purposes (e.g., proteins that may be 
purified by affinity chromatography and enzyme label coding regions, 
respectively). 
Recombinant vectors form further aspects of the present invention. 
Particularly useful vectors are contemplated to be those vectors in which 
the coding portion of the DNA segment, whether encoding a full length 
protein or smaller peptide, is positioned under the control of a promoter. 
The promoter may be in the form of the promoter that is naturally 
associated with a LTBP-2 gene, as may be obtained by isolating the 5' 
non-coding sequences located upstream of the coding segment or exon, for 
example, using recombinant cloning and/or PCR technology, in connection 
with the compositions disclosed herein. 
In other embodiments, it is contemplated that certain advantages will be 
gained by positioning the coding DNA segment under the control of a 
recombinant, or heterologous, promoter. As used herein, a recombinant or 
heterologous promoter is intended to refer to a promoter that is not 
normally associated with an LTBP-2 gene in its natural environment. Such 
promoters may include LTBP-2 promoters normally associated with other 
genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or 
mammalian cell. Naturally, it will be important to employ a promoter that 
effectively directs the expression of the DNA segment in the cell type, 
organism, or even animal, chosen for expression. The use of promoter and 
cell type combinations for protein expression is generally known to those 
of skill in the art of molecular biology, for example, see Sambrook et al. 
(1989). The promoters employed may be constitutive, or inducible, and can 
be used under the appropriate conditions to direct high level expression 
of the introduced DNA segment, such as is advantageous in the large-scale 
production of recombinant proteins or peptides. Appropriate promoter 
systems contemplated for use in high-level expression include, but are not 
limited to, the Pichia expression vector system (Pharmacia LKB 
Biotechnology) (see Example XVI herein). 
In connection with expression embodiments to prepare recombinant LTBP-2 
proteins and peptides, it is contemplated that longer DNA segments will 
most often be used, with DNA segments encoding the entire LTBP-2 protein 
or functional domains, subunits, etc. being most preferred. However, it 
will be appreciated that the use of shorter DNA segments to direct the 
expression of LTBP-2 peptides or epitopic core regions, such as may be 
used to generate anti-LTBP-2 antibodies, also falls within the scope of 
the invention. DNA segments that encode peptide antigens from about 15 to 
about 50 amino acids in length, or more preferably, from about 15 to about 
30 amino acids in length are contemplated to be particularly useful. 
The LTBP-2 gene and DNA segments may also be used in connection with 
somatic expression in an animal or in the creation of a transgenic animal. 
Again, in such embodiments, the use of a recombinant vector that directs 
the expression of the full length or active LTBP-2 protein is particularly 
contemplated. 
In addition to their use in directing the expression of the LTBP-2 protein, 
the nucleic acid sequences disclosed herein also have a variety of other 
uses. For example, they also have utility as probes or primers in nucleic 
acid hybridization embodiments. As such, it is contemplated that nucleic 
acid segments that comprise a sequence region that consists of at least a 
14 nucleotide long contiguous sequence that has the same sequence as, or 
is complementary to, a 14 nucleotide long contiguous sequence of SEQ ID 
NO:2 will find particular utility. Longer contiguous identical or 
complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 
500, 1000 (including all intermediate lengths) and even up to full length 
sequences will also be of use in certain embodiments. 
The ability of such nucleic acid probes to specifically hybridize to 
LTBP-2-encoding sequences will enable them to be of use in detecting the 
presence of complementary sequences in a given sample. However, other uses 
are envisioned, including the use of the sequence information for the 
preparation of mutant species primers, or primers for use in preparing 
other genetic constructions. 
Nucleic acid molecules having sequence regions consisting of contiguous 
nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 
nucleotides or so, identical or complementary to SEQ ID NO:2, are 
particularly contemplated as hybridization probes for use in, e.g., 
Southern and Northern blotting. This would allow LTBP-2 structural or 
regulatory genes to be analyzed, both in diverse cell types and also in 
various mammalian cells. The total size of fragment, as well as the size 
of the complementary stretch(es), will ultimately depend on the intended 
use or application of the particular nucleic acid segment. Smaller 
fragments will generally find use in hybridization embodiments, wherein 
the length of the contiguous complementary region may be varied, such as 
between about 10-14 and about 100 nucleotides, but larger contiguous 
complementary stretches may be used, according to the length complementary 
sequences one wishes to detect. 
The use of a hybridization probe of about 10-14 nucleotides in length 
allows the formation of a duplex molecule that is both stable and 
selective. Molecules having contiguous complementary sequences over 
stretches greater than 10 bases in length are generally preferred, though, 
in order to increase stability and selectivity of the hybrid, and thereby 
improve the quality and degree of specific hybrid molecules obtained. One 
will generally prefer to design nucleic acid molecules having 
gene-complementary stretches of 15 to 20 contiguous nucleotides, or even 
longer where desired. 
Hybridization probes may be selected from any portion of any of the 
sequences disclosed herein. All that is required is to review the sequence 
set forth in SEQ ID NO:2 and to select any continuous portion of the 
sequence, from about 10-14 nucleotides in length up to and including the 
full length sequence, that one wishes to utilize as a probe or primer. The 
choice of probe and primer sequences may be governed by various factors, 
such as, by way of example only, one may wish to employ primers from 
towards the termini of the total sequence. 
The process of selecting and preparing a nucleic acid segment that includes 
a contiguous sequence from within SEQ ID NO:2 may alternatively be 
described as preparing a nucleic acid fragment. Of course, fragments may 
also be obtained by other techniques such as, e.g., by mechanical shearing 
or by restriction enzyme digestion. Small nucleic acid segments or 
fragments may be readily prepared by, for example, directly synthesizing 
the fragment by chemical means, as is commonly practiced using an 
automated oligonucleotide synthesizer. Also, fragments may be obtained by 
application of nucleic acid reproduction technology, such as the PCR 
technology of U.S. Pat. No. 4,603,102 (incorporated herein by reference), 
by introducing selected sequences into recombinant vectors for recombinant 
production, and by other recombinant DNA techniques generally known to 
those of skill in the art of molecular biology. 
Accordingly, the nucleotide sequences of the invention may be used for 
their ability to selectively form duplex molecules with complementary 
stretches of LTBP-2 genes or cDNAs. Depending on the application 
envisioned, one will desire to employ varying conditions of hybridization 
to achieve varying degrees of selectivity of probe towards target 
sequence. For applications requiring high selectivity, one will typically 
desire to employ relatively stringent conditions to form the hybrids, 
e.g., one will select relatively low salt and/or high temperature 
conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 
50.degree. C. to 70.degree. C. Such selective conditions tolerate little, 
if any, mismatch between the probe and the template or target strand, and 
would be particularly suitable for isolating LTBP-2 genes. 
Of course, for some applications, for example, where one desires to prepare 
mutants employing a mutant primer strand hybridized to an underlying 
template or where one seeks to isolate LTBP-2-encoding sequences from 
related species, functional equivalents, or the like, less stringent 
hybridization conditions will typically be needed in order to allow 
formation of the heteroduplex. In these circumstances, one may desire to 
employ conditions such as 0.15M-0.9M salt, at temperatures ranging from 
20.degree. C. to 55.degree. C. Cross-hybridizing species can thereby be 
readily identified as positively hybridizing signals with respect to 
control hybridizations. In any case, it is generally appreciated that 
conditions can be rendered more stringent by the addition of increasing 
amounts of formamide, which serves to destabilize the hybrid duplex in the 
same manner as increased temperature. Thus, hybridization conditions can 
be readily manipulated, and thus will generally be a method of choice 
depending on the desired results. 
In certain embodiments, it will be advantageous to employ nucleic acid 
sequences of the present invention in combination with an appropriate 
means, such as a label, for determining hybridization. A wide variety of 
appropriate indicator means are known in the art, including fluorescent, 
radioactive, enzymatic or other ligands, such as avidin/biotin, which are 
capable of giving a detectable signal. In preferred embodiments, one will 
likely desire to employ a fluorescent label or an enzyme tag, such as 
urease, alkaline phosphatase or peroxidase, instead of radioactive or 
other environmental undesirable reagents. In the case of enzyme tags, 
calorimetric indicator substrates are known that can be employed to 
provide a means visible to the human eye or spectrophotometrically, to 
identify specific hybridization with complementary nucleic acid-containing 
samples. 
In general, it is envisioned that the hybridization probes described herein 
will be useful both as reagents in solution hybridization as well as in 
embodiments employing a solid phase. In embodiments involving a solid 
phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a 
selected matrix or surface. This fixed, single-stranded nucleic acid is 
then subjected to specific hybridization with selected probes under 
desired conditions. The selected conditions will depend on the particular 
circumstances based on the particular criteria required (depending, for 
example, on the G+C contents, type of target nucleic acid, source of 
nucleic acid, size of hybridization probe, etc.). Following washing of the 
hybridized surface so as to remove nonspecifically bound probe molecules, 
specific hybridization is detected, or even quantified, by means of the 
label. 
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. 
EXAMPLE I 
Animal Model for Assessing New Bone Formation 
As various animal models were not suitable for studying the effects of 
nucleic acid transfer on bone formation, the inventors employed the 
following model system. The important features of the rat osteotomy model 
are as described in the following protocol (which is generally completed 
in 25-35 minutes). 
The osteotomy was performed on one femur per animal. Right to left 
differences have not been apparent, but such differences are monitored in 
these studies, since the limb receiving the osteotomy is randomized. 
After pre-operative preparation (i.e. shaving and betadine scrub), adult 
male Sprague Dawley rats (.about.500 gm, retired male breeders) were 
anesthetized using a 3%. halothane 97% oxygen mixture (700 ml/min. flow 
rate). A lateral approach to the femur was made on one limb. Utilizing 
specially designed surgical guides, four 1.2 mm diameter pins were screwed 
into the diaphysis after pre-drilling with a high speed precision bit. A 
surgical template ensured precise and parallel placement of the pins. The 
order of pin placement was always the same: outer proximal first and then 
outer distal, inner proximal and inner distal (with "outer" and "inner" 
referring to the distance from the hip joint). Pin placement in the center 
of the femur was ensured by fluoroscopic imaging during pin placement. The 
external fixator was secured on the pins and a t mm or 2 mm segmental 
defect was created in the central diaphysis through an incision using a 
Hall Micro 100 Oscillating saw (#5053-60 Hall surgical blades) under 
constant irrigation. Other than the size of the segmental defect, there is 
no difference between the 5 mm and 2 mm osteotomy protocols (FIG. 1). 
The contents of the osteotomy site were irrigated with sterile saline and 
the fibrous collagen implant material, previously soaked in a solution of 
plasmid DNA or other DNA construct, if appropriate, was placed in situ. 
The wound was then closed in layers. Since the fixator provided the 
necessary stability no limitations on animal ambulation existed, and other 
supports were not required. The surgical protocol has been successfully 
performed on 21/21 animals to date. None of these animals have died and no 
significant adverse effects have been observed, other than complications 
that might be associated with surgical fracture repair. Minor 
complications that were experienced include 1 animal that developed a 
post-operative osteomyelitis and 1 animal in which 2/4 pins loosened as a 
consequence of bone fracture. 
EXAMPLE II 
Implant Material for use in Bone Gene Transfer 
Various implant materials may be used for transferring genes into the site 
of bone repair and/or regeneration in vivo. These materials are soaked in 
a solution containing the DNA or gene that is to be transferred to the 
bone regrowth site. 
One particular example of a suitable material is fibrous collagen, which 
may be lyophilized following extraction and partial purification from 
tissue and then sterilized. A particularly preferred collagen is the 
fibrous collagen implant material termed UltraFiber.TM., as may be 
obtained from Norian Corp., (1025 Terra Bella Ave., Mountain View, Calif., 
94043). Detailed descriptions of the composition and use of UltraFiber.TM. 
are provided in Gunasekaran et al. (1993a,b; each incorporated herein by 
reference). 
A more particularly preferred collagen is type II collagen, with most 
particularly preferred collagen being either recombinant type II collagen, 
or mineralized type II collagen. 
Prior to placement in osteotomy sites, implant materials are soaked in 
solutions of DNA (or virus) under sterile conditions. The soaking may be 
for any appropriate and convenient period, e.g., from 6 minutes to 
over-night. The DNA (e.g., plasmid) solution will be a sterile aqueous 
solution, such as sterile water or an acceptable buffer, with the 
concentration generally being about 0.5-1.0 mg/ml. Currently preferred 
plasmids are those such as pGL2 (Promega), pSV40.beta.-gal, pAd.CMVlacZ, 
and pLJ. 
EXAMPLE III 
Parathyroid Hormone Gene Constructs 
The active fragment of the human parathyroid hormone gene (hPTH1-34) was 
chosen as the first of the osteotropic genes to be incorporated into an 
expression vector for use in gene transfer to promote new bone formation 
in the rat osteotomy model. 
The inventors chose to construct the hPTH1-34 transgene in the PLJ 
expression vector (FIG. 2), since this vector was appropriate for studies 
of transgene function both in vitro and in vivo. A schematic of the 
PLJ-hPTH1-34 transgene is shown in FIG. 2. The DNA and amino acid 
sequences of the hPTH1-34 are well known, e.g., see Hendy et al. (1981, 
incorporated herein by reference). To insert the transgene into the PLJ 
expression vector PCR of a full-length PTH recombinant clone was employed, 
followed by standard molecular biological manipulation. 
A retroviral stock was then generated following CaPO.sub.4 -mediated 
transfection of psi crip cells with the PLJ-hPTH1-34 construct, all 
according to standard protocols (Sambrook et al., 1989). Independent 
transduced Rat-1 clones were obtained by standard infection and selection 
procedures (Sambrook et al., 1989). 
One clone (YZ-15) was analyzed by Southern analysis, demonstrating that the 
PLJ-hPTH1-34 transgene had stably integrated into the Rat-1 genome (FIG. 
3). A Northern analysis was next performed to show that the YZ-15 clone 
expressed the PLJ-hPTH1-34 transgene, as evidenced by the presence of 
specific PLJ-hPTH1-34 transcripts (FIG. 4A). 
EXAMPLE IV 
Parathyroid Hormone Polypeptide Expression and Activity 
A sensitive and specific radioimmunoassay was performed to demonstrate that 
the YZ-15 cells expressed and secreted a recombinant hPTH1-34 molecule 
(Table 2). The radioimmunoassay was performed on media from transduced 
Rat-1 clones. To quantify secretion of the recombinant hPTH-1-34 peptide 
produced by YZ-15 cells, the culture medium from one 100 mm confluent dish 
was collected over a 24 hour period and assayed with the NH2-terminal hPTH 
RIA kit (Nichols Institute Diagnostics) according to the manufacturer's 
protocol. PLJ-hPTH1-87 cells and BAG cells served as positive and negative 
controls, respectively. 
Protein concentrations in Table 2 are expressed as the average of three 
assays plus the standard deviation (in parenthesis). The concentration of 
the 1-34 and full length (1-84) peptides was determined relative to a 
standard curve generated with commercially available reagents (Nichols 
Institute Diagnostics). 
TABLE 2 
______________________________________ 
CELL LINES PTH (pg/ml) 
______________________________________ 
YZ-15 247 (.+-.38) 
PLJ-hPTH1-84 2616 (.+-.372) 
BAG 13 (.+-.3) 
______________________________________ 
As shown in Table 2, PTH expression was detected in both YZ-15 cells and 
PLJ-hPTH1-84 cells. BAG cells produced no detectable PTH and served as a 
baseline for the RIA. These results demonstrate that YZ-15 cells expressed 
recombinant hPTH1-34 protein. 
The recombinant hPTH1-34 molecule was added to rat osteosarcoma cells and a 
cAMP response assay conducted in order to determine whether the secreted 
molecule had biological activity. Unconcentrated media was collected from 
YZ-15 cells, PLJ-hPTH1-84 cells, and BAG cells and was used to treat 
ROS17/2.8 cells for 10 minutes, as described (Majmudar et al., 1991). cAMP 
was then extracted from treated cells and quantified by RIA (Table 3). The 
amount of cAMP shown is the average of three assays. The standard 
deviation of the mean is shown in parenthesis. 
TABLE 3 
______________________________________ 
CELL LINES cAMP (pmol) 
______________________________________ 
YZ-15 20.3 (.+-.0.25) 
PLJ-hPTH184 88.5 (.+-.4.50) 
BAG 7.6 (.+-.0.30) 
______________________________________ 
A cAMP response was induced by the recombinant PTH secreted by the YZ-15 
cells and by PLJ-hPTH1-84 cells. BAG cells produced no PTH and served as 
the baseline for the cAMP assay. These results provide direct in vitro 
evidence that the PLJ-hPTH1-34 transgene directs the expression and 
secretion of a functional osteotropic agent. 
EXAMPLE V 
Bone Morphogenetic Protein (BMP) Gene Constructs 
The mouse bone morphogenetic protein-4 (BMP-4) was chosen as the next of 
the osteotropic genes to be incorporated into an expression vector for use 
in promoting bone repair and regeneration. 
A full length mouse BMP-4 cDNA was generated by screening a mouse 3%3 cell 
CDNA library (Stratagene). The human sequence for BMP-4 is well known to 
those of skill in the art and has been deposited in Genbank. Degenerate 
oligos were prepared and employed in standard PCR to obtain a murine cDNA 
sequence. 
The ends of the CDNA clone were further modified using the polymerase chain 
reaction so that the full length cDNA (5'.fwdarw.3' direction) codes for: 
the natural mouse initiator Met codon, the full length mouse coding 
sequence, a 9 amino acid tag (known as the HA epitope), and the natural 
mouse stop codon. The amino acid sequence encoded by the mouse BMP-4 
transgene is shown in FIG. 9; this entire sequence, including the tag, is 
represented by SEQ ID NO:1. As of the filing of this application, the 
precise nucleic acid sequence has not yet been determined, and various 
"wobble position" bases remain unknown. 
Placement of the HA epitope at the extreme carboxy terminus should not 
interfere with the function of the recombinant molecule sequence in vitro 
or in vivo. The advantage of the epitope is for utilization in 
immunohistochemical methods to specifically identify the recombinant mouse 
BMP-4 molecule in osteotomy tissues in vivo, e.g., the epitope can be 
identified using a commercially available monoclonal antibody 
(Boehringer-Mannheim), as described herein. 
Studies to demonstrate that the mouse BMP-4 transgene codes for a 
functional osteotropic agent include, for example, (a) transfection of COS 
cells and immunoprecipitation of a protein band of the correct size using 
a monoclonal anti-HA antibody (Boehringer-Mannheim); and (b) a 
quantitative in vivo bone induction bioassay (Sampath & Reddi, 1981) that 
involves implanting proteins from the medium of transfected COS cells 
beneath the skin of male rats and scoring for new bone formation in the 
ectopic site. 
EXAMPLE VI 
Detection of mRNA by Tissue in situ Hybridization 
The following technique describes the detection of mRNA in tissue obtained 
from the site of bone regeneration. This may be useful for detecting 
expression of the transgene mRNA itself, and also in detecting expression 
of hormone or growth factor receptors or other molecules. This method may 
be used in place of, or in addition to, Northern analyses, such as those 
described in FIG. 7. 
DNA from a plasmid containing the gene for which mRNA is to be detected is 
linearized, extracted, and precipitated with ethanol. Sense and antisense 
transcripts are generated from 1 mg template with T3 and T7 polymerases, 
e.g., in the presence of .sup.35 S! UTP at &gt;6 mCi/ml (Amersham Corp., 
&gt;1200 Ci/mmol) and 1.6 U/ml RNasin (Promega), with the remaining in vitro 
transcription reagents provided in a kit (SureSite, Novagen Inc.). After 
transcription at 37.degree. C. for 1 hour, DNA templates are removed by a 
15 minute digestion at 37.degree. C. with 0.5 U/ml RNase-free DNase I, 
extracted, and precipitated with ethanol. Riboprobes are hydrolyzed to an 
average final length of 150 bp by incubating in 40 mM NaHCO.sub.3, 60 mM 
Na.sub.2 CO.sub.3, 80 mM DTT at 60.degree. C., according to previously 
determined formula. Hydrolysis is terminated by addition of sodium 
acetate, pH 6.0, and glacial acetic acid to 0.09M and 0.005% (v/v), 
respectively, and the probes are then ethanol precipitated, dissolved in 
0.1M DTT, counted, and stored at -20.degree. C. until use. 
RNase precautions are taken in all stages of slide preparation. Bouins 
fixed, paraffin embedded tissue sections are heated to 65.degree. C. for 
10 minutes, deparaffinized in 3 changes of xylene for 5 minutes, and 
rehydrated in a descending ethanol series, ending in phosphate-buffered 
saline (PBS). Slides will be soaked in 0.2 N HCl for 5 min., rinsed in 
PBS, digested with 0.0002% proteinase K in PBS for 30 minutes at 
37.degree. C. and rinsed briefly with DEPC-treated water. After 
equilibrating for 3 minutes in 0.1M triethanolamine-HCl (TEA-HCl), pH 8.0, 
sections are acetylated in 0.25% (v/v) acetic anhydride in 0.1M TEA-HCl 
for 10 minutes at room temperature, rinsed in PBS, and dehydrated in an 
ascending ethanol series. Each section receives 100-200 ml 
prehybridization solution (0.5 mg/ml denatured RNase-free tRNA 
(Boehringer-Mannheim), 10 mM DTT, 5 mg/ml denatured, sulfurylated salmon 
sperm DNA, 50% formamide, 10% dextran sulfate, 300 mM NaCl, 1.times. 
RNase-free Denhardt's solution (made with RNase-free bovine serum albumin, 
Sigma), 10 mM Tris-HCl, pH 7.4, 1 mM EDTA) and then incubate on a 
50.degree. C. slide warmer in a humidified enclosure for 2 hours. The 
sulfurylated salmon-sperm DNA blocking reagent is used in both 
prehybridization and hybridization solutions to help reduce nonspecific 
binding to tissue by .sup.35 SH groups on the probe. It is prepared by 
labeling RNase-free salmon sperm DNA (Sigma) with non-radioactive 
.alpha.-thio-dCTP and .alpha.-thio-dATP (Amersham) in a standard random 
oligonucleotide-primed DNA labeling reaction. Excess prehybridization 
solution is removed with a brief rinse in 4.times. SSC before application 
of probe. 
Riboprobes, fresh tRNA and sulfurylated salmon sperm DNA will be denatured 
for 10 minutes at 70.degree. C., and chilled on ice. Hybridization 
solution, identical to prehybridization solution except with denatured 
probe added to 5.times.10.sup.6 CPM/ml, is applied and slides incubated at 
50.degree. C. overnight in sealed humidified chambers on a slide warmer. 
Sense and antisense probes are applied to serial sections. Slides are 
rinsed 3 times in 4.times. SSC, washed with 2.times. SSC, 1 mM DTT for 30 
min. at 50.degree. C., digested with RNase A (20 mg/ml RNase A, 0.5M NaCl, 
10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0) for 30 min. at 37.degree. C., and 
rinsed briefly with 2.times. SSC, 1 mM DTT. Three additional washes are 
performed, each at 50.degree. C. for 30 minutes: once in 2.times. SSC, 50% 
formamide, 1 mM DTT, and twice in 1.times. SSC, 0.13% (w/v) sodium 
pyrophosphate, 1 mM DTT. 
Slides are dehydrated in an ascending ethanol series (with supplementation 
of the dilute ethanols (50% and 70%) with SSC and DTT to 0.1.times. and 1 
mM, respectively). Slides are exposed to X-ray film for 20-60 hours to 
visualize overall hybridization patterns, dipped in autoradiographic 
emulsion (Kodak NTB-2, diluted to 50% with 0.3M ammonium acetate), slowly 
dried for 2 hours, and exposed (4.degree. C.) for periods ranging from 8 
days to 8 weeks. After developing emulsion, sections are counter strained 
with hematoxylin and eosin, dehydrated, and mounted with xylene-based 
medium. The hybridization signal is visualized under darkfield microscopy. 
The above in situ hybridization protocol may be used, for example, in 
detecting the temporal and spatial pattern of PTH/PTHrP receptor 
expression. A suitable rat PTH/PTHrP receptor cDNA probe (R15B) is one 
that consists of a 1810 bp region encoding the full length rat bone 
PTH/PTHrP receptor (Abou-Samra et al., 1992). The cDNA fragment is 
subcloned into pcDNA 1 (Invitrogen Corp., San Diego, Calif.) and is cut 
out using XbaI and BamHI. This probe has provided positive signals for 
northern blot analysis of rat, murine, and human osteoblastic cell lines, 
rat primary calvarial cells, and murine bone tissue. The pcDNA I plasmid 
contains a T7 and SP6 promoter that facilitate the generation of cRNA 
probes for in situ hybridization. The full length transcript has been used 
to detect PTH/PTHrP receptor in sections of bone (Lee et al., 1994). The 
PTHrP cDNA probe (Yasuda et al., 1989) is a 400 bp subcloned fragment in 
pBluescript 1 KS (Stratagene). This probe has been used for in situ 
hybridization, generating an antisense cRNA probe using BamHI cleavage and 
the T3 primer and a sense cRNA probe using EcoRI cleavage and the T7 
primer. 
EXAMPLE VII 
In vivo Protein Detection following Transgene Expression 
1. .beta.-galactosidase Transgene 
Bacterial .beta.-galactosidase is detected immunohistochemically and by 
substrate utilization assays. Osteotomy tissue specimens are fixed in 
Bouins fixative, demineralized, and then split in half along the 
longitudinal plane. One-half of each specimen is embedded in paraffin for 
subsequent immunohistochemical identification of the bacterial 
.beta.-galactosidase protein. 
For immunohistochemistry, cross-Sections (2-3 mm thick) were transferred to 
poly-L-Lysine coated microscope slides and fixed in acetone at 0.degree. 
C. for at least 20 min. Sections were rehydrated in PBS. Endogenous 
peroxidase activity was quenched by immersion of tissue sections in 0.1% 
hydrogen peroxide (in 95% methanol) at room temperature for 10 min, and 
quenched sections were washed 3.times. in PBS. In some cases, sectioned 
calvariae were demineralized by immersion in 4% EDTA, 5% polyvinyl 
pyrrolidone, and 7% sucrose, pH 7.4, for 24 h at 4.degree. C. 
Demineralized sections were washed 3.times. before application for 
antibodies. Primary antibodies were used without dilution in the form of 
hybridoma supernatant. Purified antibodies were applied to tissue sections 
at a concentration of 5 mg/ml. Primary antibodies were detected with 
biotinylated rabbit antimouse IgG and peroxidase conjugated streptavidin 
(Zymed Histostain-SPkit). After peroxidase staining, sections were 
counterstained with hematoxylin. 
Substrate utilization assays (for both .beta.-gal and luciferase) are 
conducted using commercially available kits (e.g., Promega) according to 
the manufacturers' instructions. 
2. PTH Transgenes 
Recombinant PTH, such as hPTH1-34 peptide, is assayed in homogenates of 
osteotomy gap tissue, for example, using two commercially available 
radioimmunoassay kits according to the manufacturer's protocols (Nichols 
Institute Diagnostics, San Juan Capistrano, Calif.). 
One kit is the Intact PTH-Parathyroid Hormone 100T Kit. This 
radioimmunoassay utilizes an antibody to the carboxy terminus of the 
intact hormone, and thus is used to measure endogenous levels of hormone 
in gap osteotomy tissue. This assay may be used to establish a baseline 
value PTH expression in the rat osteotomy model. 
The second kit is a two-site immunoradiometric kit for the measurement of 
rat PTH. This kit uses affinity purified antibodies specific for the amino 
terminus of the intact rat hormone (PTH1-34) and thus will measure 
endogenous PTH production as well as the recombinant protein. Previous 
studies have shown that these antibodies cross-react with human PTH and 
thus are able to recognize recombinant molecules in vivo. 
Values obtained with kit #1 (antibody to the carboxy terminus) are 
subtracted from values obtained with kit #2 (antibody to the amino 
terminus) to obtain an accurate and sensitive measurements. The level of 
recombinant peptide is thus correlated with the degree of new bone 
formation. 
3. BMP Transgene 
Preferably, BMP proteins, such as the mouse BMP-4 transgene peptide 
product, are detected immunohistochemically using a specific antibody that 
recognizes the HA epitope (Majmudar et al., 1991), such as the monoclonal 
antibody available from Boehringer-Mannheim. Antibodies to BMP proteins 
themselves may also be used. Such antibodies, along with various 
immunoassay methods, are described in U.S. Pat. No. 4,857,456, 
incorporated herein by reference. 
Osteotomy tissue specimens are fixed in Bouins fixative, demineralized, and 
then split in half along the longitudinal plane. One-half of each specimen 
is embedded in paraffin for subsequent immunohistochemical identification 
of the recombinant mouse BMP-4 molecule. 
EXAMPLE VIII 
Direct Gene Transfer into Regenerating Bone In Vivo 
To assess the feasibility of direct gene transfer into regenerating bone in 
vivo, marker gene transfer into cells in the rat osteotomy model was 
employed. These studies involved two marker genes: bacterial 
.beta.-galactosidase and insect luciferase. 
Aliquots of a fibrous collagen implant material were soaked in solutions of 
pure marker gene DNA. The implant materials were then placed in the 
osteotomy site, and their expression determined as described above. 
It was found that bot marker genes were successfully transferred and 
expressed, without any failures, as demonstrated by substrate utilization 
assays (FIG. 5 and FIG. 6). Since mammalian cells do not normally 
synthesize either marker gene product, this provides direct evidence that 
osteotomy repair cells were transfected in vivo and then expressed the 
.beta.-galactosidase and luciferase transgenes as a functional enzymes. 
EXAMPLE IX 
Adenoviral Gene Transfer into Regenerating Bone in vivo 
One of the alternative methods to achieve in vivo gene transfer into 
regenerating bone is to utilize an adenovirus-mediated transfer event. 
Successful adenoviral gene transfer of a marker gene construct into bone 
repair cells in the rat osteotomy model has been achieved. 
The inventors employed the adenoviral vector pAd. CMVlacZ, which is an 
example of a replication-defective adenoviral vector which can replicate 
in permissive cells (Stratford-Perricaudet et al., 1992). In pAd.CMVlacZ, 
the early enhancer/promoter of the cytomegalovirus (CMV) is used to drive 
transcription of lacZ with an SV40 polyadenylation sequence cloned 
downstream from this reporter (Davidson et al., 1993). 
The vector pAd.RSV4 is also utilized by the inventors. This vector 
essentially has the same backbone as pAdCMVlacZ, however the CMV promoter 
and the single BglII cloning site have been replaced in a cassette-like 
fashion with BglII fragment that consists of an RSV promoter, a multiple 
cloning site, and a poly(A.sup.+) site. The greater flexibility of this 
vector is contemplated to be useful in subcloning osteotropic genes, such 
as the hPTH1-34 cDNA fragment, for use in further studies. 
To generate recombinant PTH adenovirus, a 100 mm dish of 293 cells is 
transfected using calcium phosphate with 20 mg of a plasmid construct, 
e.g., the plasmid containing the hPTH1-34 insert linearized with NheI, 
plus 2 mg of wild type adenovirus DNA digested with XbaI and ClaI. The 
adenovirus DNA is derived from adenovirus type 5, which contains only a 
single XbaI and ClaI sites and has a partial deletion of the E3 region. 
Approximately 7 days post-transfection, cells and media are harvested and 
a lysate prepared by repeated freeze-thaw cycles. This lysate is diluted 
and used to infect 60 mm dishes of confluent 293 cells for 1 hour. The 
cells are then overlaid with 0.8% agar/1.times. MEM/2% calf serum/12.5 mM 
MgCl.sub.2. Ten days post-infection, individual plaques are to be picked 
and used to infect 60 mm dishes of 293 cells to expand the amount of 
virus. Positive plaques are selected for further purification and the 
generation of adenoviral stocks. 
To purify recombinant adenovirus, 150 mm dishes of 75-90% confluent 293 
cells are infected with 2-5 PFU/cell, a titer that avoids the potential 
cytotoxic effects of adenovirus. Thirty hours post-infection, the cells 
are rinsed, removed from the dishes, pelleted, and resuspended in 10 mM 
Tris-HCl, pH 8.1. A viral lysate is generated by three freeze-thaw cycles, 
cell debris is removed by centrifugation for 10 min. at 2,000 rpm, and the 
adenovirus is purified by density gradient centrifugation. The adenovirus 
band is stored at -20.degree. C. in sterile glycerol/BSA until needed. 
The solution of virus particles was sterilized and incubated with the 
implant material (from 6 mins to overnight), and the virus-impregnated 
material was implanted into the osteotomy gap, where viral infection of 
cells clearly occurred. The results obtained clearly demonstrated the 
exquisite specificity of the anti-.beta.-gal antibody (Sambrook et al., 
1989), and conclusively demonstrated expression of the marker gene product 
in chondrocyte-like cells of the osteotomy gap (FIG. 10A and FIG. 10C). 
The nuclear-targeted signal has also been observed in pre-osteoblasts. 
EXAMPLE X 
Transfer of an Osteotropic Gene Stimulates Bone Regeneration/Repair In Vivo 
In order for a parathyroid hormone (PTH) transgene to function as an 
osteotropic agent, it is likely that there is a requirement for the 
PTH/PTHrP receptor to be expressed in the bone repair tissue itself. 
Therefore, the inventors investigated PTH/PTHrP receptor expression in the 
rat osteotomy model. 
A Northern analysis of poly-A(.sup.+) RNA was conducted which demonstrated 
that the PTH/PHTrP receptor was expression in osteotomy repair tissue 
(FIG. 7). 
The inventors next investigated whether gene transfer could be employed to 
create transfected cells that constitutively express recombinant hPTH1-34 
in vivo, and whether this transgene can stimulate bone formation. The rate 
of new bone formation is analyzed as follows. At necropsy the osteotomy 
site is carefully dissected for histomorphometric analysis. The A-P and 
M-L dimensions of the callus tissue are measured using calipers. Specimens 
are then immersion fixed in Bouins fixative, washed in ethanol, and 
demineralized in buffered formic acid. Plastic embedding of decalcified 
materials is used because of the superior dimensional stability of 
methacrylate during sample preparation and sectioning. 
Tissue blocks are dehydrated in increasing alcohol concentrations and 
embedded. 5 mm thick sections are cut in the coronal plane using a 
Reichert Polycut microtome. Sections are prepared from midway through the 
width of the marrow cavity to guard against a sampling bias. Sections for 
light microscopy are stained using a modified Goldner's trichrome stain, 
to differentiate bone, osteoid, cartilage, and fibrous tissue. Sections 
are cover-slipped using Eukitt's mounting medium (Calibrated Instruments, 
Ardsley, N.Y.). Histomorphometric analyses are performed under brightfield 
using a Nikon Optiphot Research microscope. Standard point count 
stereology techniques using a 10 mm.times.10 mm eyepiece grid reticular 
are used. 
Total callus area is measured at 125.times. magnification as an index of 
the overall intensity of the healing reaction. Area fractions of bone, 
cartilage, and fibrous tissue are measured at 250.times. magnification to 
examine the relative contribution of each tissue to callus formation. 
Since the dimensions of the oste6tomy gap reflect the baseline (time 0), a 
measurement of bone area at subsequent time intervals is used to indicate 
the rate of bone infill. Statistical significance is assessed using 
analysis of variance, with post-hoc comparisons between groups conducted 
using Tukey's studentized range t test. 
In the 5 mm rat osteotomy model described above, it was found that PTH 
transgene expression can stimulate bone regeneration/repair in live 
animals (FIG. 8A and FIG. 8B). This is a particularly important finding as 
it is known that hPTH1-34 is a more powerful anabolic agent when given 
intermittently as opposed to continuously, and it is the continuous-type 
delivery that results from the gene transfer methods used here. 
Although the present inventors have already demonstrated success of direct 
gene transfer into regenerating bone in vivo, the use of ex vivo treatment 
protocols is also contemplated. In such embodiments, bone progenitor cells 
would be isolated from a particular animal or human subject and maintained 
in an in vitro environment. Suitable areas of the body from which to 
obtain bone progenitor cells are areas such as the bone tissue and fluid 
surrounding a fracture or other skeletal defect (whether or not this is an 
artificially created site) and from the bone marrow. Isolated cells would 
then be contacted with the DNA (or recombinant viral) composition, with, 
or preferably without, a matrix, when the cells would take up the DNA (or 
be infected by the recombinant virus). The stimulated cells would then be 
returned to the site in the animal or patient where bone repair is to be 
stimulated. 
EXAMPLE XI 
Transfer of Genes to Achilles' Tendon and to Cruciate Ligament In Vivo 
The studies on regenerating bone described above complement others by the 
inventors in which gene transfer was successfully employed to introduce 
genes into Achilles' tendon and cruciate ligament. 
The Achilles' tendon consist of cells and extracellular matrix organized in 
a characteristic tissue architecture. Tissue wounding can disrupt this 
architecture and stimulate a wound healing response. The wounded tendon 
will regenerate, as opposed to scar, if its connective tissue elements 
remain approximately intact. Regeneration is advantageous because scar 
tissue is not optimally designed to support normal mechanical function. 
Segmental defects in tendon due to traumatic injury may be treated with 
biological or synthetic implants that encourage neo-tendon formation. This 
strategy is limited, however, by the availability of effective 
(autologous) biological grafts, the long term stability and compatibility 
of synthetic prostheses, and the slow rate of incorporation often observed 
with both types of implants. 
The inventors hypothesized that the effectiveness of biological grafts may 
be enhanced by the over-expression of molecules that regulate the tissue 
regeneration response. Toward this end, they developed a model system in 
which segmental defects in Achilles' tendon are created and a novel 
biomaterial, small intestinal submucosa or SIS, is used as a tendon 
implant/molecular delivery agent. In the present example, the ability to 
deliver and express marker gene constructs into regenerating tendon tissue 
using the SIS graft is demonstrated. 
Plasmid (pSV.beta.gal, Promega) stock solutions were prepared according to 
standard protocols (Sambrook et al., 1989). SIS graft material was 
prepared from a segment of jejunum of adult pigs (Badylak et al., 1989). 
At harvest, mesenteric tissues were removed, the segment was inverted, and 
the mucosa and superficial submucosa were removed by a mechanical abrasion 
technique. After returning the segment to its original orientation, the 
serosa and muscle layers were rinsed, sterilized by treatment with dilute 
peracetic acid, and stored at 4.degree. C. until use. 
Mongrel dogs (all studies) were anesthetized, intubated, placed in 
right-lateral recumbency upon a heating pad, and maintained with inhalant 
anesthesia. A lateral incision from the musculotendinous junction to the 
plantar fascia was used to expose the Achilles' tendon. A double thickness 
sheet of SIS was wrapped around a central portion of the tendon, both ends 
were sutured, a 1.5 cm segment of the tendon was removed through a lateral 
opening in the graft material, and the graft and surgical site were 
closed. The leg was immobilized for 6 weeks and then used freely for 6 
weeks. Graft tissues were harvested at time points indicated below, fixed 
in Bouins solution, and embedded in paraffin. Tissue sections (8 .mu.m) 
were cut and used for immunohistochemistry. 
In an initial study, SIS material alone (SIS-alone graft) engrafted and 
promoted the regeneration of Achilles' tendon following the creation of a 
segmental defect in mongrel dogs as long as 6 months post surgery. The 
remodeling process involved the rapid formation of granulation tissue and 
eventual degradation of the graft. Scar tissue did not form, and evidence 
of immune-mediated rejection was not observed. 
In a second study, SIS was soaked in a plasmid DNA solution (SIS+plasmid 
graft) and subsequently implanted as an Achilles' tendon graft (n=2 dogs) 
or a cruciate ligament graft (n=2 dogs) in normal mongrel dogs. A 
pSV.beta.gal plasmid that employs simian virus 40 regulatory sequences to 
drive .beta.-galactosidase (.beta.-gal) activity was detectable by 
immunohistochemistry using a specific antibody in 4/4 animals. As a 
negative control, .beta.-gal activity was not detected in the unoperated 
Achilles' tendon and cruciate ligament of these animals. It appeared, 
therefore, that SIS facilitated the uptake and subsequent expression of 
plasmid DNA by wound healing cells in both tendon and ligament.l 
A third study was designed to evaluate the time course of .beta.-gal 
transgene expression. SIS+plasmid grafts were implanted for 3, 6, 9, and 
12 weeks (n=2 dogs pr time point) and transgene expression was assayed by 
immunohistochemistry (FIG. 11A and FIG. 11B) and by in situ hybridization. 
Cross-sections (8 .mu.m) of Bouins fixed, paraffin embedded tissue were 
cut and mounted on ProbeOn Plus slides (Fisher). Immunohistochemistry was 
performed according to the protocol provided with the Histostain-SP kit 
(Zymed). In brief, slides were incubated with a well characterized 
anti-.beta.-galactosidase antibody (1:200 dilution, 5 Prime.fwdarw.3 
Prime), washed in PBS, incubated with a biotinylated second antibody, 
washed, stained with the enzyme conjugate plus a substrate-chromogen 
mixture, and then counterstained with hematoxylin and eosin. 
Bacterial .beta.-gal activity was detected in tendons that received the 
SIS+plasmid graft (8/8 animals). Although not rigorously quantitative, 
transgene expression appeared to peak at 9-12 weeks. Bacterial .beta.-gal 
gene expression was not detected in animals that received SIS-alone grafts 
(N=2, 3 weeks and 12 weeks). Again, scar tissue did not form and evidence 
of immune-mediated rejection was not observed. 
This study has demonstrated that (i) a novel biomaterial, SIS, can 
effectively function as an autologous graft which promotes the 
regeneration of tissues such as Achilles' tendon and anterior cruciate 
ligament, and (ii) SIS can be used to deliver a marker gene construct to 
regenerating tissue. 
EXAMPLE XIII 
Mechanical Properties of New Bone Formation 
The mechanical properties of new bone formed during gene transfer may be 
measured using, e.g., whole bone torsion tests which create a stress state 
in which the maximum tensile stresses will occur on planes that lie 
obliquely to the bone's longitudinal axis. Such tests may provide 
important inferences about the mechanical anisotropy of callus tissue and 
the degree of osseous integration of new bone tissue. These tests are 
particularly advantageous in the evaluation of fracture specimens, e.g., 
the irregular shape of callus tissue typically precludes the use of whole 
bone 4-point bending tests because it is impossible to reproducibly align 
the points from specimen to specimen. 
Femurs are tested on an MTS Servohydraulic Testing Machine while moist and 
at room temperature. A torque sensor and rotary variable displacement 
transduces provides data for torque-angular displacement curves. Specially 
designed fixtures support each bone near the metaphyseal-diaphyseal 
junctions, and apply a 2-point load to the diaphysis. Tests are conducted 
at a constant rate of displacement equal to 20 degrees/sec. A 250 
inch-ounce load cell measures the total applied force. All bones are 
tested while moist and room temperature. Torque and angular displacement 
data are acquired using an analog-to-digital converter and a Macintosh 
computer and software. From this data, the following variables are 
calculated: a) maximum torque, b) torsional stiffness, the slope of the 
pre-yield portion of the curve determined from a linear regression of the 
data, c) energy to failure, the area under the torque-angular displacement 
curve to the point of failure, and d) the angular displacement ratio, the 
ratio of displacement at failure to displacement at yield. Statistical 
significance is determined Analysis of Variance followed by multiple 
comparisons with appropriate corrections (e.g., Bonferroni). 
This invention also provides a means of using osteotropic gene transfer in 
connection with reconstructive surgery and various bone remodelling 
procedures. The techniques described herein may thus be employed in 
connection with the technology described by Yasko et al., (1992), Chen et 
al., (1991) and Beck et al. (1991), each incorporated herein by reference. 
EXAMPLE XIV 
Identification of Further Osteotropic Genes: Isolation of a Novel Latent 
TGF-.beta. Binding Protein-Like Gene 
The extracellular matrix contains a heterogenous population of 3-20 nm 
filaments termed microfibrils. The inventors recently isolated and 
characterized the mouse and human genes for several microfibril components 
and characterized their expression pattern during mouse development. This 
example concerns the isolation and characterization of a new member of the 
fibrillin gene family. 
A. Methods 
1. cDNA Cloning 
Aliquots (typically 40-50,000 PFU) of phage particles from a CDNA library 
in the .lambda.ZAP II vector made from 3T3 cell mRNA (Stratagene) and 
fresh overnight XL1-blue cells (grown in Luria broth supplemented with 
0.4% maltose in 10 mM MgSO.sub.4) were mixed, incubated for 15 min. at 
37.degree. C., mixed again with 9 ml of liquid (50.degree. C.) top layer 
agarose (NZY broth plus 0.75% agarose), and then spread evenly onto a 
freshly poured 150 mm NZY-agar plate. Standard methods were used for the 
preparation of plaque-lifts and filter hybridization (42.degree. C., in 
buffer containing 50% formamide, 5.times. SSPE, 1.times. Denhardt's, 0.1% 
SDS, 100 mg/ml salmon sperm DNA, 100 mg/ml heparin). cDNA probes were 
radiolabeled by the nick translation method (Sambrook et al., 1989). 
Purified phage clones were converted to pBluescript plasmid clones, which 
were sequenced using Sequenase.TM. (version 2.0) as described (Chen et 
al., 1993). 
2. Polymerase Chain Reaction 
Poly(A.sup.+) RNA was prepared from mouse embryo and rat tissues as 
indicated using commercially available reagents (Fast Track.TM., 
Invitrogen). cDNA synthesis was performed as described (cDNA Synthesis 
System Plus kit protocol, Amersham), except that the reverse transcriptase 
enzyme was purchased separately (Seikagaku). Aliquots of cDNA (.about.10%) 
were PCR.TM. amplified using commercially available reagents (Perkin 
Elmer-Cetus). Amplification proceeded through 30 cycles of denaturation, 
annealing, and elongation. The annealing temperature of the reaction was 
determined by the equation 4(G+C)+2(A+T)-6=T.sub.A (Chen et al., 1993). 
3. Northern Analysis 
Poly(A.sup.+) RNA (2-10 .mu.g aliquots) was electrophoresed on a 1.25% 
agarose/2.2M formaldehyde gel and then transferred to a nylon membrane 
(Hybond-N, Amersham). The RNA was cross-linked to the membrane by exposure 
to a UV light source (1.2.times.10.sup.6 mJ/cm.sup.2, UV Stratalinker 
2400, Stratagene) and then prehybridized for &gt;15 min. at 65.degree. C. in 
Rapid-Hyb buffer (Amersham, Inc.). Specific CDNA probes were .sup.32 
P-labeled by random priming and used for hybridization (2 h at 65.degree. 
C.). Blots were washed progressively to high stringency (0.1.times. 
SSC/0.1% SDS, 65.degree. C.), and then placed against x-ray film with 
intensifying screens (X-OMAT XAR, Eastman Kodak, Inc.) at -86.degree. C. 
4. Isolation and Sequencing of a Mouse Genomic Clone 
A genomic library in the Lambda Fix II vector (made from mouse strain SV129 
liver DNA, Stratagene, Inc.) was plated at .about.60,000 plaques/plate, 
and nitrocellulose replicas were screened at high stringency (42.degree. 
C., in buffer containing 50% formamide, 5.times. SSPE, 1.times. 
Denhardt's, 0.1% SDS, 100 mg/ml salmon sperm DNA, 100 mg/ml heparin) using 
a 2770 bp cDNA fragment as the probe (derived from clone "#18",). Filters 
were washed in 0.1.times. SSPE+0.1% SDS at 42.degree. C. and 
autoradiographed. Duplicate positive plaques from one independent clone 
(WY-G-1-1) were re-screened until purified, and DNA was prepared as 
described in the Magic Lambda Prep kit protocol (Promega, Inc.). The 
insert consisted of &gt;10 kb of genomic DNA. BamHI digestion of the phage 
clone yielded several DNA fragments that could be resolved by agarose gel 
electrophoresis, and all of these were subcloned into BamHI-digested 
pGEM3Z (Promega, Inc.). Selected regions of the subcloned insert were 
sequenced as described above to verify the nature of the clone. 
5. Isolation and Sequencing of a Human Genomic Clone 
A human genomic library (Lambda EMBL3 EcoRI) was plated at .about.40,000 
plaques/plate, and nitrocellulose replicas were screened at moderate 
stringency (as described above) using a 3.8 kb mouse cDNA fragment as the 
probe (clone "#18",). Filters were washed in 1.times. SSC+0.1% SDS at 
48.degree. C. and autoradiographed. Duplicate positive plaques from one 
independent clone (ES-C) were re-screened until purified, and DNA was 
prepared as described in the Magic Lambda Prep kit protocol (Promega). 
BamHI digestion of the purified phage clone yielded a single 4.0 kb DNA 
fragment that could be resolved by Southern analysis. This fragment was 
then cut with SmaI, a 872 bp BamHI-SmaI fragment was subcloned into pGEM7Z 
(Promega), and selected regions of the subcloned insert were sequenced as 
described above to verify the nature of the clone. 
6. Tissue In Situ Hybridization 
To prepare sense and antisense probes, a 382 bp fragment from the 3' 
untranslated region (+3753 to +4134, counting the "A" of the initiator Met 
codon as +1; see FIG. 14, clone "ish") was subcloned into the pBluescript 
KS+ plasmid (Stratagene, Inc.). Template DNA was linearized with either 
EcoRI or BamHI, extracted, and precipitated with ethanol. Sense and 
antisense transcripts were generated from 1 .mu.g template with T3 and T7 
polymerases in the presence of .sup.35 S!UTP at &gt;6 mCi/ml (Amersham, 
&gt;1200 Ci/mmol) and 1.6 U/ml RNasin (Promega), with the remaining in vitro 
transcription reagents provided in a kit (SureSite, Novagen, Inc.). After 
transcription at 37.degree. C. for 1 h, DNA templates were removed by a 15 
min. digest at 37.degree. C. with 0.5 U/ml RNase-free DNase I, extracted, 
and precipitated with ethanol. Riboprobes were hydrolyzed to an average 
final length of 150 bp by incubating in 40 mM NaHCO.sub.3, 60 mM Na.sub.2 
CO.sub.3, 80 mM DTT for 40 min. at 60.degree. C. Hydrolysis was terminated 
by addition of sodium acetate, pH 6.0, and glacial acetic acid to 0.09M 
and 0.56% (vol./vol.), respectively, and the probes were then ethanol 
precipitated, dissolved in 0.1M DTT, counted, and stored at 20.degree. C. 
until use. Day 8.5-9.0, day 13.5, and day 16.5 mouse embryo tissue 
sections (Novagen) and the in situ hybridization protocol were employed as 
described (Chen et al., 1993). 
B. Results 
Microfibrils 10 nm in diameter assist in elastic fiber assembly, serve an 
anchoring function in non-elastic tissues, and play a role in tissue 
remodeling. Consistent with a possible role in wound healing, it was found 
that the new fibrillin gene is expressed as alternatively spliced 
transcripts in fracture tissue. 
In this study, the inventors isolated and characterized a novel mouse 
fibrillin-like cDNA. It provides a unique mRNA of 4,314 nucleotides, with 
an open reading frame of 3,756 nucleotides (SEQ ID NO:2). The deduced 
molecule is a unique polypeptide of 1,252 amino acids (SEQ ID NO:3). 
Excluding the signal peptide (est. 18 amino acids), the novel 
fibrillin-like molecule consists of five structurally distinct regions 
(A-E), a schematic representation of the domain structure of the new 
sequence is shown in FIG. 12A. The largest region (region D) extends for 
635 amino acids and comprises an uninterrupted series of 12 cysteine-rich 
repeats. Based on structural homologies, this sequence includes ten 
epidermal growth factor-calcium binding repeats and two transforming 
growth factor-.beta.1-binding protein repeats. 
A second cysteine-rich region (region B), more near the amino terminus, 
spans 392 amino acids. Between the two cysteine-rich regions is a 154 
amino acid segment (region C) that has a high proline content (21%). The 
last two predicted regions of the novel fibrillin are a 22 amino acid 
carboxy-terminus (region E) and a 31 amino acid stretch at the 
amino-terminus (region A). Northern blot analysis of mouse embryo RNA 
agrees with the deduced size of the transcript, showing a single band of 
4.5-5.0 kb. The corresponding schematic of the human LTBP is shown in FIG. 
12B. 
The first indication of alternative splicing came from molecular cloning 
studies in the mouse, in which independent cDNA clones were isolated with 
a deletion of 51 bp from the coding sequence. PCR/Southern blot analysis 
provided additional evidence that the homologous 51 bp sequence was 
alternatively spliced in normal mouse embryo tissues. 
Northern blot analysis demonstrated that the novel fibrillin gene was also 
expressed in rat callus three weeks after osteotomy, after mineralization 
has begun. Gene expression in normal adult rat bone tissue was 
insignificant, which suggests that microfibrils are an important part of 
the bone fracture healing response. The novel fibrillin-like gene was 
expressed in callus as a pair of alternatively spliced transcripts. This 
result has been independently reproduced on three occasions. Molecular 
cloning of the novel fibrillin gene in both mouse and rat has identified 
potential splice junction sites for the alternative splicing event. 
This new fibrillin-like gene is present in both the mouse and rat, and is 
expressed in callus tissue as a pair of alternatively spliced transcripts. 
This is the first evidence that fibrillin-associated microfibrils are 
present in the extracellular matrix of callus. 
This new fibrillin gene is expressed during mouse development. The 
transcript is widely expressed in connective tissue and mesenchyme (FIG. 
13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E and FIG. 13F.), it is also 
expressed in liver, heart and CNS tissues. 
As part of a study designed to clone the mouse fibrillin-1 gene, cDNA from 
a 3T3 cell cDNA library was amplified using human fibrillin-1 PCR.TM. 
primers under low stringency conditions (i.e. annealing at 37.degree. C. 
initially for 10 cycles, followed by annealing at 60.degree. C. for 30 
cycles). The initial results were surprising in that a mouse DNA fragment 
of unexpectedly low homology (.about.50%) to human fibrillin-1 was 
obtained. Molecular cloning of the authentic mouse fibrillin-1 transcript 
was eventually completed, and this study confirmed that the human and 
mouse fibrillin-1 coding sequences share &gt;95% sequence identity (Yin et 
al., submitted for publication). The mouse fibrillin-1 and PCR.TM. 
sequences were different, which suggested that the PCR.TM. product may 
have been derived from a related, fibrillin-like cDNA. The 3T3 cell CDNA 
library was screened at high stringency using the mouse PCR.TM. product as 
the probe in order to test this hypothesis. A cDNA walking strategy 
eventually yielded seven overlapping cDNA clones (clone no. 3 extending 
from -156 to +1,055; clone no. 19 extending from +200 to +1643; clone no. 
18-5 extending from +200 to +3195; clone no. B extending from +1759 to 
+3236; clone no. A extending from +3231 to +4035; clone ish extending from 
+3973 to +4314; clone no. 18 extending from +498 to +4289; the numbering 
system assumes that the "A" of the initiator Met codon is nucleotide No. 
1.) which coded a unique transcript of 4.6 kb (SEQ ID NO:2) and a 
polypeptide of 1,252 amino acids (SEQ ID NO:3). 
Sequence analysis of these clones revealed an open reading frame of 3,753 
nt. A methionine codon in a favorable context for translation initiation 
was provisionally designated +1. The initiator methionine was followed by 
a characteristic signal sequence of 21 amino acids (von Heijne, 1986). 
Beyond this, the conceptual amino acid sequence appeared to be organized 
into five structurally distinct domains (#-5). Domain #1 is a 28 amino 
acid segment with a net basic charge (est. pI, 12.36) that may allow for 
binding acidic molecules in the extracellular matrix (e.g., acidic 
proteoglycans). Sequences rich in basic amino acids may also function as 
endoproteolytic processing signals (Barr, 1991; Steiner et al., 1992), 
which suggests that the NH.sub.2 -terminus may be proteolytically 
processed. Domain #2 extends for of 390 amino acids, consisting of an 
EGF-like repeat, a 135 amino acid segment that was proline-rich (20.7%) 
and glycine-rich (11.8%) but not cysteine-rich, a Fibmotif (Pereira et 
al., 1993), an EGF-CB repeat, and a TGF-bp repeat. Domain #3 is a 113 
amino acid segment characterized by its high proline content (21%). Domain 
#4 extends for 678 amino acids and consists of 14 consecutive 
cysteine-rich repeats. Based on structural homologies, 12/14 repeats were 
epidermal growth factor-calcium binding (EGF-CB) motifs (Handford et al., 
1991), whereas 2/14 were transforming growth factor-.beta.-binding protein 
(TGF-bp) motifs (Kanzaki et al., 1990). Finally, domain #5 is a 22 amino 
acid segment at the carboxy-terminus. 
The conceptual amino acid sequence encoded by the open reading frame 
consisted of 1,252 amino acids (FIG. 12A) with an estimated pI of 5.92, a 
predicted molecular mass of 134,710 Da, and five potential N-linked 
glycosylation sites. No RGD sequence was present. Northern blot analysis 
of mouse embryo RNA using a 3' untranslated region probe identified a 
transcript band of .about.4.6 kb. In this regard, 4,310 nt have been 
isolated by cDNA cloning, including a 3' untranslated region of 401 nt and 
a 5' upstream sequence of 156 not. The apparent discrepancy between the 
Northern analysis result and the cDNA sequence analysis suggested that the 
t' upstream sequence may include .about.300 nt of additional upstream 
sequence. This estimate was consistent with preliminary primer extension 
mapping studies indicating that the 5' upstream sequence is 400-500 nt in 
length. 
A total of 19 cysteine-rich repeats were found in domains #2 and #4 of the 
mouse LTBP-like polypeptide. Thirteen were EGF-like and 11/13 contained 
the calcium binding consensus sequence. This consensus was derived from an 
analysis of 154 EGF-CB repeats in 23 different proteins and from 
structural analyses of the EGF-CB repeat, both bound and unbound to 
calcium ion (Selander-Sunnerhagen et al., 1992). Variations on the 
consensus have been noted previously and one of these, D-L-N/D-E-C.sub.1, 
was identified in the third EGF-like repeat of domain #4. In addition, a 
potential calcium binding sequence which has not previously been reported 
(E-T-N/D-E-C.sub.1) was identified in the first EGF-like repeat of domain 
#4. Ten of thirteen EGF-CB repeats also contained a second consensus 
sequence which represents a recognition sequence for an Asp/Asn 
hydroxylase that co-and posttranslationally modifies D/N residues (Stenflo 
et al., 1987; Gronke et al., 1989). 
Although about one-half the size, the deduced polypeptide was organized 
like fibrillin-1 in that it consisted of a signal peptide followed by 5 
structurally distinct domains, i.e. two domains with numerous EGF-like, 
EGF-CB and Fib repeats and a third with a proline-rich sequence (Pereira 
et al., 1993). However, comparison of each of these domains using the GAP 
and BESTFIT programs (Genetics Computer Group) has revealed a low level of 
amino acid homology of only 27% over the five structural domains shared by 
the deduced mouse polypeptide and human fibrillin-2. These values are low 
for a putative fibrillin family member because fibrillin-1 and fibrillin-2 
share .about.50% identity (zhang et al., 1994). 
A search of available databases revealed that the deduced mouse polypeptide 
was most similar to the human and rat latent TGF-.beta. binding proteins 
(Kanzaki et al., 1990; Tsuji et al., 1990). In this regard LTBP was found 
to be similar to fibrillin in that it could also be divided into five 
structurally distinct domains (FIG. 12A and FIG. 12B). These include a 
relatively short domain downstream of the signal peptide with a net basic 
charge (amino acids 21-33, est. pI, 11.14); a domain consisting of 
EGF-like, EGF-CB, TGF-bp, and Fib motifs plus a proline-rich and 
glycine-rich sequence (amino acids 34-407); a proline-rich domain (amino 
acids 408-545); a large, domain consisting of EGF-CB, TGF-bp, and 
TGF-bp-like repeat motifs (amino acids 546-1379); and a relatively short 
domain at the carboxy terminus (amino acids 1380-1394). Amino acid 
sequence comparison of the deduced mouse and human polypeptides shows 60% 
identity for domain #1, 52% identity for domain #2, 30% identity for 
domain #3, 43% identity for domain #4, and 7% identity for domain #5. The 
average identity over the five domains shared by the mouse polypeptide and 
human LTBP was 38.4%. Significantly, cysteine residues in both polypeptide 
sequences were highly conserved. 
The fibrillins are exclusively expressed by connective cells in developing 
tissues (zhang et al., 1994), whereas LTBP should be expressed along with 
TGF-.beta. by both epithelial and connective cells (Tsuji et al., 1990). 
The structural homology data therefore predict that the mouse gene shown 
in FIG. 12A should be expressed by both epithelial and connective tissue 
cells. Tissue in situ hybridization was used to test this hypothesis. 
An overview of the expression pattern as determined by tissue in situ 
hybridization is presented in FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 
13E and FIG. 13F. Approximate mid-sagittal sections of normal mouse 
embryos at days 8.5-9.0, 13.5 and 16.5 p.c. of development were hybridized 
with a .sup.35 S-labeled single stranded normal sense riboprobe from the 
same cDNA construct was used. At day 8.5-9.0 of development, intense gene 
expression was observed in the mesometrial and antimesometrial uterine 
tissues, ectoplacental cone, placenta, placental membranes. The transcript 
appeared to be widely expressed in mouse embryo mesenchymal/connective 
tissue compartments, including the facial mesenchyme, at days 8.5-9.0, 
13.5 and 16.5 of development. Particularly intense expression of the 
transcript was noted in the liver. 
Microscopy of day 8.5-9.0 embryos confirmed the widespread expression of 
the mouse gene by mesenchymal cells. Significant expression of the 
transcript by cells of the developing central nervous system, somites and 
cardiovascular tissue (myocardium plus endocardium) was also observed. 
Microscopy of day 13.5 and day 16.5 embryos demonstrated expression of the 
mouse gene by skeletal muscle cells and by cells involved in 
intramembranous and endochondral bone formation. The transcript was 
expressed by osteoblasts and by periosteal cells of the calvarium, 
mandible and maxilla. The transcript was also identified in both cartilage 
and bone of the lower extremity. A positive signal was detected in 
perichondrial cells and chondrocytes (proliferating&gt;mature&gt;hypertrophic) 
of articular cartilage, the presumptive growth plate, and the cartilage 
model within the central canal. The positive signal was also expressed by 
blood vessel endothelial cells within the mid-diaphysis. 
Respiratory epithelial cells lining developing small airways and connective 
tissue cells in the pulmonary interstitium expressed the mouse transcript, 
as did myocardial cells (atria and ventricles) and endocardial cushion 
tissue. Cells within the walls of large arteries also expressed the 
transcript. Expression of the mouse gene was identified in several organs 
of the alimentary system, including the tongue, esophagus, stomach, small 
and large intestine, pancreas and liver. Mucosal epithelial cells lining 
the upper and lower digestive tract plus the smooth muscle and connective 
tissue cells found in the submucosa expressed the transcript, as did 
acinar cells of the exocrine pancreas. Despite the high level of 
transcript expression in the liver, these results suggest both cell 
populations express the LTBP-like transcript. 
In the kidney, expression above the basal level was observed in cells of 
developing nephrons, the ureteric bud, kidney blastema and the kidney 
interstitium. In the skin, epidermal and adnexal keratinocytes, dermal 
connective tissue cells, and brown fat cells within the dorsal subcutis 
expressed the mouse transcript. In the central and peripheral nervous 
systems, ganglion cells within the cerebrum, brainstem, spinal cord, and 
peripheral nerves expressed the mouse transcript. The transcript was also 
intensely expressed by cells of the developing mouse retina. 
Thus, the mouse gene is widely expressed by both epithelial and connective 
tissue cells, a pattern that would be expected for a latent TGF-.beta. 
binding protein. Three final observations argue that the LTBP-like 
sequence presented in FIG. 12A is not simply the mouse homologue of human 
LTBP. First, domain #4 of the mouse LTBP-like sequence has a smaller 
number of EGF-like repeat motifs than human and rat LTBP (8 versus 11). 
Second, portions of the human and rat LTBP-like coding sequence were 
characterized and found to share .about.90% identity with human and rat 
LTBP but only 65% identity with the mouse LTBP-like gene. The first and 
second observations came from studies using oligonucleotide primers from 
rat LTBP to amplify mouse embryo cDNA to generate a single 311 bp PCR.TM. 
product. Primer sequences were as follows: 
(upstream, outer), ATGCCTAAACTCTACCAGCACG (SEQ ID NO: 7); 
(upstream, inner) GAGTCACGTCATCCATTCCACA (SEQ ID NO: 8); 
(downstream), CGTCCAAGTTGTGTCTTAGCAG (SEQ ID NO: 9); and the fragment of 
mouse LTBP was generated by PCRTM as described previously. Third, the 
human LTBP and LTBP-like genes are localized to separate chromosomes. 
Human LTBP was assigned to human chromosome 2 based on the analysis of 
human x rodent somatic cell hybrid lines (Stenman et al., 1994). To our 
knowledge, no LTBP gene has been mapped in the mouse. The human LTBP-like 
genes was recently localized to chromosome 11 band q12, while the mouse 
gene was mapped to mouse chromosome 19, band B (a region of conserved 
synteny), using several independent approaches. 
C. Discussion 
This study reports the molecular cloning of a novel LTBP-like gene that 
contains numerous EGF-like repeats. Northern analysis indicates that the 
gene encodes a single transcript of .about.4.6 kb in mouse embryo tissues. 
The deduced amino acid sequence of the mouse gene product appears to be a 
secreted polypeptide of 1,251 amino acids. Although it is similar to 
fibrillin, the overall structural organization and expression pattern of 
this gene product most resembles LTBP, a latent TGF-.beta. binding protein 
that was originally isolated and characterized by Heldin and co-workers 
(see Kanzaki et al., 1990). Several observations strongly suggest that 
LTBP and the mouse LTBP-like gene product are therefore derived from 
related but distinct genetic loci. First, LTBP and the LTBP-like coding 
sequence share .about.40% identity and differences exist in the number of 
EGF-CB repeats in the deduced polypeptide sequence of the two molecules. 
Second, a portion of the mouse LTBP gene has been cloned and shown to 
share .about.90% identity with human and rat LTBP. Conversely, portions of 
the human and rat LTBP-like genes have been cloned and shown to share 
.about.90% identity with the mouse LTBP-like gene. Third, LTBP and the 
LTBP-like gene reside on different human chromosomes (Stenman et al., 
1994). Taken together, these data suggest that a family of at least two 
LTBP genes exists. 
Similarities in the structural organization of LTBP-1 and the fibrillin-1 
and fibrillin-2 polypeptides have been noted previously (Pereira et al., 
1993; Zhang et al., 1994; Taipale et al., 1994). For example, LTBP-1 and 
the fibrillins are all secreted extracellular matrix constituents. 
Moreover, each polypeptide can be organized into five domains, two of 
which consists predominantly of EGF-CB and TGF-bp repeat motifs. LTBP-1 
and fibrillin-1 also share a domain that is proline-rich, and LTBP 
possesses an 8-cysteine repeat previously referred to as the "Fib motif" 
because it was assumed to be unique to fibrillin (Pereira et al., 1993). 
These similarities likely explain the initial isolation and cloning of the 
LTBP-2 PCR.TM. product, especially since the human oligonucleotide primers 
used to initially amplify mouse cDNA were designed to direct the synthesis 
of an EGF-CB repeat in domain #4. 
Another point of distinction between LTBP-2 and fibrillin concerns the 
spacing of conserved cysteines C4 and C5 in EGF-like repeats. Fibrillin-1 
and fibrillin-2 each contain &gt;50 such repeats, and in every one the 
spacing is C.sub.4 -X-C.sub.5. While this pattern is repeated in a 
majority of the EGF-like repeats in LTBP-1 and LTBP-2, both genes also 
contain repeats with the spacing C.sub.4 -X-X-C.sub.5. Although the 
significance of this observation is unclear, variation in the number of 
amino acids between C.sub.4 and C.sub.5 would not be expected to alter the 
function of the EGF-like repeat. Mature EGF is a 48 amino acid secreted 
polypeptide consisting of two subdomains that have few interdomain 
contacts (Engel, 1989; Davis, 1990). The larger NH.sub.2 -terminal 
subdomain consists of residues 1-32 and is stabilized by a pair of 
disulfide bonds (C.sub.1 -C.sub.3 and C.sub.2 -C.sub.4), whereas the 
smaller COOH-terminal subdomain (amino acids 33-48) is stabilized by a 
single disulfide bond (C.sub.5 -C.sub.6). The COOH-terminal subdomain has 
a highly conserved conformation that only is possible if certain residues 
and the distances between them are well conserved, while 
conformation-sequence requirements for the NH2-terminal subdomain are 
relatively relaxed. Variation in C.sub.4 -C.sub.5 spacing would not be 
expected to alter conformation because these residues do not normally form 
a disulfide bond and the spacing variation occurs at the interface of 
subdomains that would not be predicted to interact. The cloning of 
additional genes will decide whether variation in C.sub.4 -C.sub.5 spacing 
is a reliable discriminator between members of the LTBP and fibrillin gene 
families. 
The LTBP-2 gene is expressed more widely during development than 
fibrillin-1 or fibrillin-2. Studies in developing mouse tissues have shown 
that the Fbn-1 gene is expressed by mesenchymal cells of developing 
connective tissue, whereas the mouse LTBP-like gene is intensely expressed 
by epithelial, parenchymal and stromal cells. Earlier reports have 
suggested that TGF-.beta. plays a role in differentiation and 
morphogenesis during mouse development (Lyons and Moses, 1990), when 
TGF-.beta. is produced by epithelial, parenchymal and stromal cells. Tsuji 
et al. (1990) and others have suggested that the expression of TGF-.beta. 
binding proteins should mirror that of TGF-.beta.0 itself; the expression 
pattern of the LTBP-2 gene over the course of murine development is 
consistent with this expectation. However, the LTBP-2 gene may not be 
completely co-regulated with TGF-.beta.. TGF-.beta. gene and protein 
expression during mouse development has been surveyed extensively (Heine 
et al., 1987; Lehnert and Akhurst, 1988; Pelton et al., 1989; Pelton et 
al., 1990a and b; Millan et al., 1991); these studies have not identified 
expression by skeletal muscle cells, chondrocytes, hepatocytes, ganglion 
cells, mucosal cells lining the gut, and epithelial cells of developing 
nephrons. It is conceivable that the LTBP-2 molecule has an additional 
function in certain connective tissues besides targeting TGF-.beta.. 
The binding properties of the LTBP-2 gene product are under investigation. 
Formally, the LTBP-2 polypeptide may bind a specific TGF-.beta. isoform, 
another member of the TGF-.beta. superfamily (e.g., a bone morphogenetic 
protein, inhibin, activin, or Mullerian inhibiting factor), or a growth 
factor unrelated to TGF-.beta.. Anti-peptide antibodies to the mouse 
LTBP-2 polypeptide have been generated and osteoblast cell lines that 
express the molecule at relatively high levels have been identified. 
Studies with these reagents suggest that LTBP-2 assembles intracellularly 
into large latent complexes with a growth factor that is being 
characterized by immunological methods. 
The presence of dibasic amino acids in the LTBP-2 sequence suggests that it 
may undergo cell- and tissue-specific proteolysis. TGF-.beta. regulates 
extracellular matrix production by suppressing matrix degradation (through 
a decrease in the expression of proteases such as collagenase, plasminogen 
activator, and stromelysin plus an increase in the expression of 
proteinase inhibitors such as plasminogen activator inhibitor-1 and tissue 
inhibitor of metalloproteinase-1) and by stimulating matrix macromolecule 
synthesis (for recent review, see Lyons and Moses, 1990; Massague, 1990; 
Laiho and Keski-Oja, 1992; and Miyazono et al., 1993). Conversely, 
production of extracellular matrix has been shown to down regulate 
TGF-.beta. gene expression (Streuli et al., 1993). TGF-.beta. may 
therefore regulate extracellular matrix production through a sophisticated 
feedback loop that influences the expression of a relatively large number 
of genes. LTBP-1 and LTBP-2 may contribute to this regulation by 
facilitating the assembly and secretion of large latent growth factor 
complexes and then targeting the complex to specific connective tissues 
(Taipale et al., 1994). 
EXAMPLE XV 
Type II Collagen Promotes New Bone Growth 
Certain matrix materials are capable of stimulating at least some new 
growth in their own right, i.e., are "osteoconductive materials". 
Potential examples of such materials are well known in the field of 
orthopedic research and include preparations of hydroxyapatite; 
preparations of crushed bone and mineralized collagen; polymers of 
polylactic acid and polyannhydride. The ability of these materials to 
stimulate new bone formation distinguishes them from inert implant 
materials such as methylcellulose, which have in the past been used to 
deliver BMPs to sites of fracture repair. 
This Example relates to a study using the rat osteotomy model with implants 
made of collagen type I (Sigma), collagen type II (Sigma), SIS (small 
intestinal submucosa), and UltraFiber.TM. (Norian Corp.). These materials 
have been placed in situ without DNA of any type. Five animals received an 
osteotomy with 10 mg of a type II collagen implant alone (10 mg refers to 
the original quantity of lyophilized collagen). Five of five control 
animals received an osteotomy with 10 mg of a type I collagen implant 
alone. Animals were housed for three weeks after surgery and then 
sacrificed. 
The results of these studies were that SIS appeared to retard new bone 
formation; type I collagen incited a moderately intense inflammatory 
response; and UltraFiber.TM. acted as an osteoconductive agent. The type 
II collagen implant studies yielded surprising results in that 10 mg of 
this collagen was found to promote new bone formation in the 5 mm 
osteotomy model. New bone--bridging the osteotomy gap--was identified 
three weeks after surgery in 5/5 animals that received a type II collagen 
implant alone (i.e. minus DNA of any type). In contrast, fibrous 
granulation tissue, but no evidence of new bone formation, was obtained in 
5/5 animals receiving a type I collagen implant alone. 
Radiographic analysis demonstrated conclusively that all animals receiving 
an osteotomy with a type II collagen implant without exception showed 
radio-dense material in the osteotomy gap. In sharp contrast, radiographic 
analysis of all animals receiving a type I collagen implant revealed no 
radio-dense material forming in the osteotomy gap. New bone growth is 
formed in the osteotomy gap of type II collagen implanted-animals. No such 
new bone growth was observed in the animals receiving type I collagen 
implants (all animals were examined three weeks after surgery). 
The results of the ostetomy with a type II collagen implant resulted in 
areas of new bone formed in the osteotomy gap using histological analysis. 
In contrast, only fibrous granulation tissue was identified in the type I 
collagen gap. 
Previous studies have suggested that type II collagen plays only a 
structural role in the extracellular matrix. The results of the type II 
collagen implant studies are interesting because they demonstrate a novel 
and osteoconductive role for type II collagen during endochondral bone 
repair. To further optimize the osteoconductive potential of type II 
collagen, a yeast expression vector that encodes for type II collagen 
(full length .alpha.1 (II) collagen) will be employed to produce 
recombinant .alpha.1 (II) collagen protein. 
EXAMPLE XIV 
Expression of Recombinant Type II Collagen 
The Pichia Expression Kit (Invitrogen, Inc.) may be used to prepare 
recombinant type II collagen. This kit, based on the methylotrophic yeast, 
Pichia pastoris, allows high-level expression of recombinant protein in an 
easy-to-use relatively inexpensive system. In the absence of the preferred 
carbon source, glucose, P. pastoris utilizes methanol as a carbon source. 
The AOX1 promoter controls the gene that codes for the expression of the 
enzyme alcohol oxidase, which catalyzes the first step in the metabolism 
of methanol. This promoter, which is induced by methanol, has been 
characterized and incorporated into a series of Pichia expression vectors. 
This feature of Pichia has been exploited to express high levels of 
recombinant proteins often in the range of grams per liter. Because it is 
eukaryotic, P. pastoris utilizes posttranslational modification pathways 
that are similar to those used by mammalian cells. This implies that the 
recombinant type II collagen will be glycosylated and will contain 
disulfide bonds. 
The inventors contemplate the following particular elements to be useful in 
the expression of recombinant type II collagen: the DNA sequence of human 
type II collagen (SEQ ID NO:11) (Lee et al., 1989); rat type II collagen 
(SEQ ID NO:13) (Michaelson, et al., 1994); and/or mouse type II collagen 
(SEQ ID NO:15) (Ortman, et al., 1994). As other sources of DNA sequences 
encoding type II collagen are available, these three are examples of many 
sequence elements that may be useful in the present invention. 
For preparation of a recombinant type II collagen, the native type II 
collagen cDNA is modified by the addition of a commercially available 
epitope tag (the HA epitope, Pharmacia, LKB Biotechnology, Inc.). Such 
fragments may be readily prepared by, for example, directly synthesizing 
the fragment by chemical means, by application of nucleic acid 
reproduction technology, such as the PCR technology of U.S. Pat. No. 
4,603,102 (herein incorporated by reference) or by introducing selected 
sequences into recombinant vectors for recombinant production. (PCR.TM. is 
a registered trademark of Hoffmann-LaRoche, Inc.). This is followed by 
cloning into the Pichia expression vector. The resulting plasmid is 
characterized by DNA sequence analysis, linearized by digestion with NotI, 
and spheroplasts will be prepared and transformed with the linearized 
construct according to the manufacturer's recommendations. 
Transformation facilitates a recombination event in vivo between the 5' and 
3' AOX1 sequences in the Pichia vector and those in the Pichia genome. The 
result is the replacement of AOX1 with the gene of interest. 
Transformants are then plated on histidine-deficient media, which will 
select for successfully transformed cells. Transformants are further 
selected against slow growth on growth media containing methanol. Positive 
transformants are grown for 2 days in liquid culture and then for 2-6 days 
in broth that uses methanol as the sole carbon source. Protein expression 
is evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis 
(SDS-PAGE) and Western hybridization using a commercially available 
polyclonal antisera to the HA epitope (Pharmacia). 
Recombinant type II collagen protein can be purified according to the 
manufacturer's recommendations, dialyzed against double distilled, 
deionized water and lyophilized in 10 mg aliquots. The aliquots are 
sterilized and used as implant material for the osteoconductive matrices. 
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. 
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__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 15 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 417 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- Met Ile Pro Gly Asn Arg Met Leu Met Val Va - #l Leu Leu Cys Gln Val 
# 15 
- Leu Leu Gly Gly Ala Thr Asp Ala Ser Leu Me - #t Pro Glu Thr Gly Lys 
# 30 
- Lys Lys Val Ala Glu Ile Gln Gly His Ala Gl - #y Gly Arg Arg Ser Gly 
# 45 
- Gln Ser His Glu Leu Leu Arg Asp Phe Glu Al - #a Thr Leu Leu Gln Met 
# 60 
- Phe Gly Leu Arg Arg Arg Pro Gln Pro Ser Ly - #s Ser Ala Val Ile Pro 
#80 
- Asp Tyr Met Ser Asp Leu Tyr Arg Leu Gln Se - #r Gly Glu Glu Glu Glu 
# 95 
- Glu Glu Gln Ser Gln Gly Thr Gly Leu Glu Ty - #r Pro Glu Arg Pro Ala 
# 110 
- Ser Ser Ala Asn Thr Val Ser Ser Phe His Hi - #s Glu Glu His Leu Glu 
# 125 
- Asn Ile Pro Gly Thr Ser Glu Ser Ser Ala Ph - #e Arg Phe Phe Phe Asn 
# 140 
- Leu Ser Ser Ile Pro Glu Asn Glu Val Ile Se - #r Ser Ala Glu Leu Arg 
145 1 - #50 1 - #55 1 - 
#60 
- Leu Phe Arg Glu Gln Val Asp Gln Gly Pro As - #p Trp Glu Gln Gly Phe 
# 175 
- His Arg Met Asn Ile Tyr Glu Val Met Lys Pr - #o Pro Ala Glu Met Val 
# 190 
- Pro Gly His Leu Ile Thr Arg Leu Leu Asp Th - #r Ser Leu Val Arg His 
# 205 
- Asn Val Thr Arg Trp Glu Thr Phe Asp Val Se - #r Pro Ala Val Leu Arg 
# 220 
- Trp Thr Arg Glu Lys Gln Pro Asn Tyr Gly Le - #u Ala Ile Glu Val Thr 
225 2 - #30 2 - #35 2 - 
#40 
- His Leu His Gln Thr Arg Thr His Gln Gly Gl - #n His Val Ser Ile Ser 
# 255 
- Arg Ser Leu Pro Gln Gly Ser Gly Asn Trp Al - #a Gln Leu Arg Pro Leu 
# 270 
- Leu Val Thr Phe Gly His Asp Gly Arg Gly Hi - #s Thr Leu Thr Arg Arg 
# 285 
- Ser Ala Lys Arg Ser Pro Lys His His Pro Gl - #n Arg Ser Ser Lys Lys 
# 300 
- Asn Lys Asn Cys Arg Arg His Ser Leu Tyr Va - #l Asp Phe Ser Asp Val 
305 3 - #10 3 - #15 3 - 
#20 
- Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gl - #y Tyr Gln Ala Phe Tyr 
# 335 
- Cys His Gly Asp Cys Pro Phe Pro Leu Ala As - #p His Leu Asn Ser Thr 
# 350 
- Asn His Ala Ile Val Gln Thr Leu Val Asn Se - #r Val Asn Ser Ser Ile 
# 365 
- Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Se - #r Ala Ile Ser Met Leu 
# 380 
- Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Ly - #s Asn Tyr Gln Glu Met 
385 3 - #90 3 - #95 4 - 
#00 
- Val Val Glu Gly Cys Gly Cys Arg Tyr Pro Ty - #r Asp Val Pro Asp Tyr 
# 415 
- Ala 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 4314 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 157..3912 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- CCTCCTGCTG TCCCCTCCCT ACCCTTGGCT TCTCGCCCCG CTCTGCCCTC TG - #CTACCAAC 
60 
- ACTCGATCCC CTGCTCGGGC TCGACCTCCA ATCTCCGAGG GTCGTGCGGC CC - #CGGATGCC 
120 
#GCC GGC GGA 174CA CGGCCTGGCC CCTGCG ATG CGC CAG 
# Met Arg Gln Ala Gly Gly 
# 5 1 
- TTG GGG CTG CTG GCA CTA CTC CTG CTG GCG CT - #G CTG GGC CCC GGC GGC 
222 
Leu Gly Leu Leu Ala Leu Leu Leu Leu Ala Le - #u Leu Gly Pro Gly Gly 
# 20 
- CGA GGG GTG GGC CGG CCG GGC AGC GGG GCA CA - #G GCG GGG GCG GGG CGC 
270 
Arg Gly Val Gly Arg Pro Gly Ser Gly Ala Gl - #n Ala Gly Ala Gly Arg 
# 35 
- TGG GCC CAA CGC TTC AAG GTG GTC TTT GCG CC - #T GTG ATC TGC AAG CGG 
318 
Trp Ala Gln Arg Phe Lys Val Val Phe Ala Pr - #o Val Ile Cys Lys Arg 
# 50 
- ACC TGT CTG AAG GGC CAG TGT CGG GAC AGC TG - #T CAG CAG GGC TCC AAC 
366 
Thr Cys Leu Lys Gly Gln Cys Arg Asp Ser Cy - #s Gln Gln Gly Ser Asn 
# 70 
- ATG ACG CTC ATC GGA GAG AAC GGC CAC AGC AC - #C GAC ACG CTC ACC GGT 
414 
Met Thr Leu Ile Gly Glu Asn Gly His Ser Th - #r Asp Thr Leu Thr Gly 
# 85 
- TCT GCC TTC CGC GTG GTG GTG TGC CCT CTA CC - #C TGC ATG AAC GGT GGC 
462 
Ser Ala Phe Arg Val Val Val Cys Pro Leu Pr - #o Cys Met Asn Gly Gly 
# 100 
- CAG TGC TCT TCC CGA AAC CAG TGC CTG TGT CC - #C CCG GAT TTC ACG GGG 
510 
Gln Cys Ser Ser Arg Asn Gln Cys Leu Cys Pr - #o Pro Asp Phe Thr Gly 
# 115 
- CGC TTC TGC CAG GTG CCT GCT GCA GGA ACC GG - #A GCT GGC ACC GGG AGT 
558 
Arg Phe Cys Gln Val Pro Ala Ala Gly Thr Gl - #y Ala Gly Thr Gly Ser 
# 130 
- TCA GGC CCC GGC TGG CCC GAC CGG GCC ATG TC - #C ACA GGC CCG CTG CCG 
606 
Ser Gly Pro Gly Trp Pro Asp Arg Ala Met Se - #r Thr Gly Pro Leu Pro 
135 1 - #40 1 - #45 1 - 
#50 
- CCC CTT GCC CCA GAA GGA GAG TCT GTG GCT AG - #C AAA CAC GCC ATT TAC 
654 
Pro Leu Ala Pro Glu Gly Glu Ser Val Ala Se - #r Lys His Ala Ile Tyr 
# 165 
- GCG GTG CAG GTG ATC GCA GAT CCT CCC GGG CC - #G GGG GAG GGT CCT CCT 
702 
Ala Val Gln Val Ile Ala Asp Pro Pro Gly Pr - #o Gly Glu Gly Pro Pro 
# 180 
- GCA CAA CAT GCA GCC TTC TTG GTG CCC CTG GG - #G CCA GGA CAA ATC TCG 
750 
Ala Gln His Ala Ala Phe Leu Val Pro Leu Gl - #y Pro Gly Gln Ile Ser 
# 195 
- GCA GAA GTG CAG GCT CCG CCC CCC GTG GTG AA - #C GTG CGT GTC CAT CAC 
798 
Ala Glu Val Gln Ala Pro Pro Pro Val Val As - #n Val Arg Val His His 
# 210 
- CCT CCT GAA GCT TCC GTT CAG GTG CAC CGC AT - #C GAG GGG CCG AAC GCT 
846 
Pro Pro Glu Ala Ser Val Gln Val His Arg Il - #e Glu Gly Pro Asn Ala 
215 2 - #20 2 - #25 2 - 
#30 
- GAA GGC CCA GCC TCT TCC CAG CAC TTG CTG CC - #G CAT CCC AAG CCC CCG 
894 
Glu Gly Pro Ala Ser Ser Gln His Leu Leu Pr - #o His Pro Lys Pro Pro 
# 245 
- CAC CCG AGG CCA CCC ACT CAA AAG CCA CTG GG - #C CGC TGC TTC CAG GAC 
942 
His Pro Arg Pro Pro Thr Gln Lys Pro Leu Gl - #y Arg Cys Phe Gln Asp 
# 260 
- ACA TTG CCC AAG CAG CCT TGT GGC AGC AAC CC - #T TTG CCT GGC CTT ACC 
990 
Thr Leu Pro Lys Gln Pro Cys Gly Ser Asn Pr - #o Leu Pro Gly Leu Thr 
# 275 
- AAG CAG GAA GAT TGC TGC GGT AGC ATC GGT AC - #T GCC TGG GGA CAA AGC 
1038 
Lys Gln Glu Asp Cys Cys Gly Ser Ile Gly Th - #r Ala Trp Gly Gln Ser 
# 290 
- AAG TGT CAC AAG TGC CCA CAG CTT CAG TAT AC - #A GGG GTG CAG AAG CCT 
1086 
Lys Cys His Lys Cys Pro Gln Leu Gln Tyr Th - #r Gly Val Gln Lys Pro 
295 3 - #00 3 - #05 3 - 
#10 
- GTA CCT GTA CGT GGG GAG GTG GGT GCT GAC TG - #C CCC CAG GGC TAC AAG 
1134 
Val Pro Val Arg Gly Glu Val Gly Ala Asp Cy - #s Pro Gln Gly Tyr Lys 
# 325 
- AGG CTC AAC AGC ACC CAC TGC CAG GAT ATC AA - #C GAA TGT GCG ATG CCC 
1182 
Arg Leu Asn Ser Thr His Cys Gln Asp Ile As - #n Glu Cys Ala Met Pro 
# 340 
- GGG AAT GTG TGC CAT GGT GAC TGC CTC AAC AA - #C CCT GGC TCT TAT CGC 
1230 
Gly Asn Val Cys His Gly Asp Cys Leu Asn As - #n Pro Gly Ser Tyr Arg 
# 355 
- TGT GTC TGC CCG CCC GGT CAT AGC TTG GGT CC - #C CTC GCA GCA CAG TGC 
1278 
Cys Val Cys Pro Pro Gly His Ser Leu Gly Pr - #o Leu Ala Ala Gln Cys 
# 370 
- ATT GCC GAC AAA CCA GAG GAG AAG AGC CTG TG - #T TTC CGC CTT GTG AGC 
1326 
Ile Ala Asp Lys Pro Glu Glu Lys Ser Leu Cy - #s Phe Arg Leu Val Ser 
375 3 - #80 3 - #85 3 - 
#90 
- ACC GAA CAC CAG TGC CAG CAC CCT CTG ACC AC - #A CGC CTA ACC CGC CAG 
1374 
Thr Glu His Gln Cys Gln His Pro Leu Thr Th - #r Arg Leu Thr Arg Gln 
# 405 
- CTC TGC TGC TGT AGT GTG GGT AAA GCC TGG GG - #T GCC CGG TGC CAG CGC 
1422 
Leu Cys Cys Cys Ser Val Gly Lys Ala Trp Gl - #y Ala Arg Cys Gln Arg 
# 420 
- TGC CCG GCA GAT GGT ACA GCA GCC TTC AAG GA - #G ATC TGC CCC GGC TGG 
1470 
Cys Pro Ala Asp Gly Thr Ala Ala Phe Lys Gl - #u Ile Cys Pro Gly Trp 
# 435 
- GAA AGG GTA CCA TAT CCT CAC CTC CCA CCA GA - #C GCT CAC CAT CCA GGG 
1518 
Glu Arg Val Pro Tyr Pro His Leu Pro Pro As - #p Ala His His Pro Gly 
# 450 
- GGA AAG CGA CTT CTC CCT CTT CCT GCA CCC GA - #C GGG CCA CCC AAA CCC 
1566 
Gly Lys Arg Leu Leu Pro Leu Pro Ala Pro As - #p Gly Pro Pro Lys Pro 
455 4 - #60 4 - #65 4 - 
#70 
- CAG CAG CTT CCT GAA AGC CCC AGC CGA GCA CC - #A CCC CTC GAG GAC ACA 
1614 
Gln Gln Leu Pro Glu Ser Pro Ser Arg Ala Pr - #o Pro Leu Glu Asp Thr 
# 485 
- GAG GAA GAG AGA GGA GTG ACC ATG GAT CCA CC - #A GTG AGT GAG GAG CGA 
1662 
Glu Glu Glu Arg Gly Val Thr Met Asp Pro Pr - #o Val Ser Glu Glu Arg 
# 500 
- TCG GTG CAG CAG AGC CAC CCC ACT ACC ACC AC - #C TCA CCC CCC CGG CCT 
1710 
Ser Val Gln Gln Ser His Pro Thr Thr Thr Th - #r Ser Pro Pro Arg Pro 
# 515 
- TAC CCA GAG CTC ATC TCT CGC CCC TCC CCA CC - #T ACC TTC CAC CGG TTC 
1758 
Tyr Pro Glu Leu Ile Ser Arg Pro Ser Pro Pr - #o Thr Phe His Arg Phe 
# 530 
- CTG CCA GAC TTG CCC CCA TCC CGA AGT GCA GT - #G GAG ATC GCC CCC ACT 
1806 
Leu Pro Asp Leu Pro Pro Ser Arg Ser Ala Va - #l Glu Ile Ala Pro Thr 
535 5 - #40 5 - #45 5 - 
#50 
- CAG GTC ACA GAG ACC GAT GAG TGC CGA TTG AA - #C CAG AAT ATC TGT GGC 
1854 
Gln Val Thr Glu Thr Asp Glu Cys Arg Leu As - #n Gln Asn Ile Cys Gly 
# 565 
- CAT GGA CAG TGT GTG CCT GGC CCC TCG GAT TA - #C TCC TGC CAC TGC AAC 
1902 
His Gly Gln Cys Val Pro Gly Pro Ser Asp Ty - #r Ser Cys His Cys Asn 
# 580 
- GCT GGC TAC CGG TCA CAC CCG CAG CAC CGC TA - #C TGT GTT GAT GTG AAC 
1950 
Ala Gly Tyr Arg Ser His Pro Gln His Arg Ty - #r Cys Val Asp Val Asn 
# 595 
- GAG TGC GAG GCA GAG CCC TGC GGC CCC GGG AA - #A GGC ATC TGT ATG AAC 
1998 
Glu Cys Glu Ala Glu Pro Cys Gly Pro Gly Ly - #s Gly Ile Cys Met Asn 
# 610 
- ACT GGT GGC TCC TAC AAT TGT CAC TGC AAC CG - #A GGC TAC CGC CTC CAC 
2046 
Thr Gly Gly Ser Tyr Asn Cys His Cys Asn Ar - #g Gly Tyr Arg Leu His 
615 6 - #20 6 - #25 6 - 
#30 
- GTG GGT GCA GGG GGC CGC TCG TGC GTG GAC CT - #G AAC GAG TGC GCC AAG 
2094 
Val Gly Ala Gly Gly Arg Ser Cys Val Asp Le - #u Asn Glu Cys Ala Lys 
# 645 
- CCT CAC CTG TGT GGG GAC GGT GGC TTC TGC AT - #C AAC TTC CCT GGT CAC 
2142 
Pro His Leu Cys Gly Asp Gly Gly Phe Cys Il - #e Asn Phe Pro Gly His 
# 660 
- TAC AAA TGC AAC TGC TAT CCT GGC TAC CGG CT - #C AAG GCC TCC CGA CCG 
2190 
Tyr Lys Cys Asn Cys Tyr Pro Gly Tyr Arg Le - #u Lys Ala Ser Arg Pro 
# 675 
- CCC ATT TGC GAA GAC ATC GAC GAG TGT CGC GA - #C CCT AGC ACC TGC CCT 
2238 
Pro Ile Cys Glu Asp Ile Asp Glu Cys Arg As - #p Pro Ser Thr Cys Pro 
# 690 
- GAT GGC AAA TGT GAA AAC AAA CCT GGC AGC TT - #C AAG TGC ATC GCC TGC 
2286 
Asp Gly Lys Cys Glu Asn Lys Pro Gly Ser Ph - #e Lys Cys Ile Ala Cys 
695 7 - #00 7 - #05 7 - 
#10 
- CAG CCT GGC TAC CGT AGC CAG GGG GGC GGG GC - #C TGT CGT GAT GTC AAC 
2334 
Gln Pro Gly Tyr Arg Ser Gln Gly Gly Gly Al - #a Cys Arg Asp Val Asn 
# 725 
- GAA TGC TCC GAA GGT ACC CCC TGC TCT CCT GG - #A TGG TGT GAG AAA CTT 
2382 
Glu Cys Ser Glu Gly Thr Pro Cys Ser Pro Gl - #y Trp Cys Glu Lys Leu 
# 740 
- CCG GGT TCT TAC CGT TGC ACG TGT GCC CAG GG - #G ATA CGA ACC CGC ACA 
2430 
Pro Gly Ser Tyr Arg Cys Thr Cys Ala Gln Gl - #y Ile Arg Thr Arg Thr 
# 755 
- GGA CGC CTC AGT TGC ATA GAC GTG GAT GAC TG - #T GAG GCT GGG AAA GTG 
2478 
Gly Arg Leu Ser Cys Ile Asp Val Asp Asp Cy - #s Glu Ala Gly Lys Val 
# 770 
- TGC CAA GAT GGC ATC TGC ACG AAC ACA CCA GG - #C TCT TTC CAG TGT CAG 
2526 
Cys Gln Asp Gly Ile Cys Thr Asn Thr Pro Gl - #y Ser Phe Gln Cys Gln 
775 7 - #80 7 - #85 7 - 
#90 
- TGC CTC TCC GGC TAT CAT CTG TCA AGG GAT CG - #G AGC CGC TGT GAG GAC 
2574 
Cys Leu Ser Gly Tyr His Leu Ser Arg Asp Ar - #g Ser Arg Cys Glu Asp 
# 805 
- ATT GAT GAA TGT GAC TTC CCT GCG GCC TGC AT - #C GGG GGT GAC TGC ATC 
2622 
Ile Asp Glu Cys Asp Phe Pro Ala Ala Cys Il - #e Gly Gly Asp Cys Ile 
# 820 
- AAT ACC AAT GGT TCC TAC AGA TGT CTC TGT CC - #C CTG GGT CAT CGG TTG 
2670 
Asn Thr Asn Gly Ser Tyr Arg Cys Leu Cys Pr - #o Leu Gly His Arg Leu 
# 835 
- GTG GGC GGC AGG AAG TGC AAG AAA GAT ATA GA - #T GAG TGC AGC CAG GAC 
2718 
Val Gly Gly Arg Lys Cys Lys Lys Asp Ile As - #p Glu Cys Ser Gln Asp 
# 850 
- CCA GGC CTG TGC CTG CCC CAT GCC TGC GAG AA - #C CTC CAG GGC TCC TAT 
2766 
Pro Gly Leu Cys Leu Pro His Ala Cys Glu As - #n Leu Gln Gly Ser Tyr 
855 8 - #60 8 - #65 8 - 
#70 
- GTC TGT GTC TGT GAT GAG GGT TTC ACA CTC AC - #C CAG GAC CAG CAT GGG 
2814 
Val Cys Val Cys Asp Glu Gly Phe Thr Leu Th - #r Gln Asp Gln His Gly 
# 885 
- TGT GAG GAG GTG GAG CAG CCC CAC CAC AAG AA - #G GAG TGC TAC CTT AAC 
2862 
Cys Glu Glu Val Glu Gln Pro His His Lys Ly - #s Glu Cys Tyr Leu Asn 
# 900 
- TTC GAT GAC ACA GTG TTC TGT GAC AGC GTA TT - #G GCT ACC AAT GTC ACT 
2910 
Phe Asp Asp Thr Val Phe Cys Asp Ser Val Le - #u Ala Thr Asn Val Thr 
# 915 
- CAG CAG GAA TGC TGT TGC TCT CTG GGA GCT GG - #C TGG GGA GAC CAC TGC 
2958 
Gln Gln Glu Cys Cys Cys Ser Leu Gly Ala Gl - #y Trp Gly Asp His Cys 
# 930 
- GAA ATC TAT CCC TGT CCA GTC TAC AGC TCA GC - #C GAA TTT CAC AGC CTG 
3006 
Glu Ile Tyr Pro Cys Pro Val Tyr Ser Ser Al - #a Glu Phe His Ser Leu 
935 9 - #40 9 - #45 9 - 
#50 
- GTG CCT GAT GGG AAA AGG CTA CAC TCA GGA CA - #A CAA CAT TGT GAA CTA 
3054 
Val Pro Asp Gly Lys Arg Leu His Ser Gly Gl - #n Gln His Cys Glu Leu 
# 965 
- TGC ATT CCT GCC CAC CGT GAC ATC GAC GAA TG - #C ATA TTG TTT GGG GCA 
3102 
Cys Ile Pro Ala His Arg Asp Ile Asp Glu Cy - #s Ile Leu Phe Gly Ala 
# 980 
- GAG ATC TGC AAG GAG GGC AAG TGT GTG AAC TC - #G CAG CCC GGC TAC GAG 
3150 
Glu Ile Cys Lys Glu Gly Lys Cys Val Asn Se - #r Gln Pro Gly Tyr Glu 
# 995 
- TGC TAC TGC AAG CAG GGC TTC TAC TAC GAT GG - #C AAC CTG CTG GAG TGC 
3198 
Cys Tyr Cys Lys Gln Gly Phe Tyr Tyr Asp Gl - #y Asn Leu Leu Glu Cys 
# 10105 
- GTG GAC GTG GAC GAG TGC TTG GAT GAG TCT AA - #C TGC AGG AAC GGA GTG 
3246 
Val Asp Val Asp Glu Cys Leu Asp Glu Ser As - #n Cys Arg Asn Gly Val 
# 10301020 - # 1025 
- TGT GAG AAC ACG TGG CGG CTA CCG TGT GCC TG - #C ACT CCG CCG GCA GAG 
3294 
Cys Glu Asn Thr Trp Arg Leu Pro Cys Ala Cy - #s Thr Pro Pro Ala Glu 
# 10450 
- TAC AGT CCC GCA CAG GCC CAG TGT CTG ATC CC - #G GAG AGA TGG AGC ACG 
3342 
Tyr Ser Pro Ala Gln Ala Gln Cys Leu Ile Pr - #o Glu Arg Trp Ser Thr 
# 10605 
- CCC CAG AGA GAC GTG AAG TGT GCT GGG GCC AG - #C GAG GAG AGG ACG GCA 
3390 
Pro Gln Arg Asp Val Lys Cys Ala Gly Ala Se - #r Glu Glu Arg Thr Ala 
# 10750 
- TGT GTA TGG GGC CCC TGG GCG GGA CCT GCC CT - #C ACT TTT GAT GAC TGC 
3438 
Cys Val Trp Gly Pro Trp Ala Gly Pro Ala Le - #u Thr Phe Asp Asp Cys 
# 10905 
- TGC TGC CGC CAG CCG CGG CTG GGT ACC CAG TG - #C AGA CCG TGC CCG CCA 
3486 
Cys Cys Arg Gln Pro Arg Leu Gly Thr Gln Cy - #s Arg Pro Cys Pro Pro 
# 11101100 - # 1105 
- CGT GGC ACC GGG TCC CAG TGC CCG ACT TCA CA - #G AGT GAG AGC AAT TCT 
3534 
Arg Gly Thr Gly Ser Gln Cys Pro Thr Ser Gl - #n Ser Glu Ser Asn Ser 
# 11250 
- TTC TGG GAC ACA AGC CCC CTG CTA CTG GGG AA - #G TCT CCG CGA GAC GAA 
3582 
Phe Trp Asp Thr Ser Pro Leu Leu Leu Gly Ly - #s Ser Pro Arg Asp Glu 
# 11405 
- GAC AGC TCA GAG GAG GAT TCA GAT GAG TGC CG - #T TGT GTG AGC GGA CCG 
3630 
Asp Ser Ser Glu Glu Asp Ser Asp Glu Cys Ar - #g Cys Val Ser Gly Pro 
# 11550 
- TGT GTG CCA CGG CCA GGC GGG GCG GTA TGC GA - #G TGT CCT GGA GGC TTT 
3678 
Cys Val Pro Arg Pro Gly Gly Ala Val Cys Gl - #u Cys Pro Gly Gly Phe 
# 11705 
- CAG CTG GAC GCC TCC CGT GCC CGC TGC GTG GA - #C ATT GAT GAG TGC CGA 
3726 
Gln Leu Asp Ala Ser Arg Ala Arg Cys Val As - #p Ile Asp Glu Cys Arg 
# 11901180 - # 1185 
- GAA CTG AAC CAG CGG GGA CTG CTG TGT AAG AG - #C GAG CGG TGC GTG AAC 
3774 
Glu Leu Asn Gln Arg Gly Leu Leu Cys Lys Se - #r Glu Arg Cys Val Asn 
# 12050 
- ACC AGT GGA TCC TTC CGC TGT GTC TGC AAA GC - #T GGC TTC ACG CGC AGC 
3822 
Thr Ser Gly Ser Phe Arg Cys Val Cys Lys Al - #a Gly Phe Thr Arg Ser 
# 12205 
- CGC CCT CAC GGG CCT GCG TGC CTC AGC GCC GC - #C GCT GAT GAT GCA GCC 
3870 
Arg Pro His Gly Pro Ala Cys Leu Ser Ala Al - #a Ala Asp Asp Ala Ala 
# 12350 
- ATA GCC CAC ACC TCA GTG ATC GAT CAT CGA GG - #G TAT TTT CAC 
#3912 
Ile Ala His Thr Ser Val Ile Asp His Arg Gl - #y Tyr Phe His 
# 12505 
- TGAAAGTGGA GACAGACAAG TACATCCTTT GCTCCTGACC AAACGAGAGC AT - #GGACCCAA 
3972 
- GGATCCTTCA GGGCCCACAA ATCTCCTTCC CACACCCCAA ACCCAAGGTG CT - #CCTGTCTG 
4032 
- CAGAGTGCTG TCTGCTTTCT CCCAAGGGTG ATTCCTAGAA ACTTCGACAT CA - #GATCTGCC 
4092 
- CCTTTAATTT ACTCTTGGCT TTCAAGGCAA ATTGATATTC ACATCCAAAG CG - #GGCAGCAT 
4152 
- CAACTGCTTG GCGGGTTGGA CTGAGCTGGG ACCCAGGATG TGAAATAGAA TT - #TATTGTGG 
4212 
- CTCTGATTAT GTACACTAGA TGTGCCTGAC CTGCTGACCA GGCTCACATG GT - #TTGTACAA 
4272 
#4314 GAAA AAAAAAAAAA AAAAAAAAAA AA 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 1252 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- Met Arg Gln Ala Gly Gly Leu Gly Leu Leu Al - #a Leu Leu Leu Leu Ala 
# 15 
- Leu Leu Gly Pro Gly Gly Arg Gly Val Gly Ar - #g Pro Gly Ser Gly Ala 
# 30 
- Gln Ala Gly Ala Gly Arg Trp Ala Gln Arg Ph - #e Lys Val Val Phe Ala 
# 45 
- Pro Val Ile Cys Lys Arg Thr Cys Leu Lys Gl - #y Gln Cys Arg Asp Ser 
# 60 
- Cys Gln Gln Gly Ser Asn Met Thr Leu Ile Gl - #y Glu Asn Gly His Ser 
# 80 
- Thr Asp Thr Leu Thr Gly Ser Ala Phe Arg Va - #l Val Val Cys Pro Leu 
# 95 
- Pro Cys Met Asn Gly Gly Gln Cys Ser Ser Ar - #g Asn Gln Cys Leu Cys 
# 110 
- Pro Pro Asp Phe Thr Gly Arg Phe Cys Gln Va - #l Pro Ala Ala Gly Thr 
# 125 
- Gly Ala Gly Thr Gly Ser Ser Gly Pro Gly Tr - #p Pro Asp Arg Ala Met 
# 140 
- Ser Thr Gly Pro Leu Pro Pro Leu Ala Pro Gl - #u Gly Glu Ser Val Ala 
145 1 - #50 1 - #55 1 - 
#60 
- Ser Lys His Ala Ile Tyr Ala Val Gln Val Il - #e Ala Asp Pro Pro Gly 
# 175 
- Pro Gly Glu Gly Pro Pro Ala Gln His Ala Al - #a Phe Leu Val Pro Leu 
# 190 
- Gly Pro Gly Gln Ile Ser Ala Glu Val Gln Al - #a Pro Pro Pro Val Val 
# 205 
- Asn Val Arg Val His His Pro Pro Glu Ala Se - #r Val Gln Val His Arg 
# 220 
- Ile Glu Gly Pro Asn Ala Glu Gly Pro Ala Se - #r Ser Gln His Leu Leu 
225 2 - #30 2 - #35 2 - 
#40 
- Pro His Pro Lys Pro Pro His Pro Arg Pro Pr - #o Thr Gln Lys Pro Leu 
# 255 
- Gly Arg Cys Phe Gln Asp Thr Leu Pro Lys Gl - #n Pro Cys Gly Ser Asn 
# 270 
- Pro Leu Pro Gly Leu Thr Lys Gln Glu Asp Cy - #s Cys Gly Ser Ile Gly 
# 285 
- Thr Ala Trp Gly Gln Ser Lys Cys His Lys Cy - #s Pro Gln Leu Gln Tyr 
# 300 
- Thr Gly Val Gln Lys Pro Val Pro Val Arg Gl - #y Glu Val Gly Ala Asp 
305 3 - #10 3 - #15 3 - 
#20 
- Cys Pro Gln Gly Tyr Lys Arg Leu Asn Ser Th - #r His Cys Gln Asp Ile 
# 335 
- Asn Glu Cys Ala Met Pro Gly Asn Val Cys Hi - #s Gly Asp Cys Leu Asn 
# 350 
- Asn Pro Gly Ser Tyr Arg Cys Val Cys Pro Pr - #o Gly His Ser Leu Gly 
# 365 
- Pro Leu Ala Ala Gln Cys Ile Ala Asp Lys Pr - #o Glu Glu Lys Ser Leu 
# 380 
- Cys Phe Arg Leu Val Ser Thr Glu His Gln Cy - #s Gln His Pro Leu Thr 
385 3 - #90 3 - #95 4 - 
#00 
- Thr Arg Leu Thr Arg Gln Leu Cys Cys Cys Se - #r Val Gly Lys Ala Trp 
# 415 
- Gly Ala Arg Cys Gln Arg Cys Pro Ala Asp Gl - #y Thr Ala Ala Phe Lys 
# 430 
- Glu Ile Cys Pro Gly Trp Glu Arg Val Pro Ty - #r Pro His Leu Pro Pro 
# 445 
- Asp Ala His His Pro Gly Gly Lys Arg Leu Le - #u Pro Leu Pro Ala Pro 
# 460 
- Asp Gly Pro Pro Lys Pro Gln Gln Leu Pro Gl - #u Ser Pro Ser Arg Ala 
465 4 - #70 4 - #75 4 - 
#80 
- Pro Pro Leu Glu Asp Thr Glu Glu Glu Arg Gl - #y Val Thr Met Asp Pro 
# 495 
- Pro Val Ser Glu Glu Arg Ser Val Gln Gln Se - #r His Pro Thr Thr Thr 
# 510 
- Thr Ser Pro Pro Arg Pro Tyr Pro Glu Leu Il - #e Ser Arg Pro Ser Pro 
# 525 
- Pro Thr Phe His Arg Phe Leu Pro Asp Leu Pr - #o Pro Ser Arg Ser Ala 
# 540 
- Val Glu Ile Ala Pro Thr Gln Val Thr Glu Th - #r Asp Glu Cys Arg Leu 
545 5 - #50 5 - #55 5 - 
#60 
- Asn Gln Asn Ile Cys Gly His Gly Gln Cys Va - #l Pro Gly Pro Ser Asp 
# 575 
- Tyr Ser Cys His Cys Asn Ala Gly Tyr Arg Se - #r His Pro Gln His Arg 
# 590 
- Tyr Cys Val Asp Val Asn Glu Cys Glu Ala Gl - #u Pro Cys Gly Pro Gly 
# 605 
- Lys Gly Ile Cys Met Asn Thr Gly Gly Ser Ty - #r Asn Cys His Cys Asn 
# 620 
- Arg Gly Tyr Arg Leu His Val Gly Ala Gly Gl - #y Arg Ser Cys Val Asp 
625 6 - #30 6 - #35 6 - 
#40 
- Leu Asn Glu Cys Ala Lys Pro His Leu Cys Gl - #y Asp Gly Gly Phe Cys 
# 655 
- Ile Asn Phe Pro Gly His Tyr Lys Cys Asn Cy - #s Tyr Pro Gly Tyr Arg 
# 670 
- Leu Lys Ala Ser Arg Pro Pro Ile Cys Glu As - #p Ile Asp Glu Cys Arg 
# 685 
- Asp Pro Ser Thr Cys Pro Asp Gly Lys Cys Gl - #u Asn Lys Pro Gly Ser 
# 700 
- Phe Lys Cys Ile Ala Cys Gln Pro Gly Tyr Ar - #g Ser Gln Gly Gly Gly 
705 7 - #10 7 - #15 7 - 
#20 
- Ala Cys Arg Asp Val Asn Glu Cys Ser Glu Gl - #y Thr Pro Cys Ser Pro 
# 735 
- Gly Trp Cys Glu Lys Leu Pro Gly Ser Tyr Ar - #g Cys Thr Cys Ala Gln 
# 750 
- Gly Ile Arg Thr Arg Thr Gly Arg Leu Ser Cy - #s Ile Asp Val Asp Asp 
# 765 
- Cys Glu Ala Gly Lys Val Cys Gln Asp Gly Il - #e Cys Thr Asn Thr Pro 
# 780 
- Gly Ser Phe Gln Cys Gln Cys Leu Ser Gly Ty - #r His Leu Ser Arg Asp 
785 7 - #90 7 - #95 8 - 
#00 
- Arg Ser Arg Cys Glu Asp Ile Asp Glu Cys As - #p Phe Pro Ala Ala Cys 
# 815 
- Ile Gly Gly Asp Cys Ile Asn Thr Asn Gly Se - #r Tyr Arg Cys Leu Cys 
# 830 
- Pro Leu Gly His Arg Leu Val Gly Gly Arg Ly - #s Cys Lys Lys Asp Ile 
# 845 
- Asp Glu Cys Ser Gln Asp Pro Gly Leu Cys Le - #u Pro His Ala Cys Glu 
# 860 
- Asn Leu Gln Gly Ser Tyr Val Cys Val Cys As - #p Glu Gly Phe Thr Leu 
865 8 - #70 8 - #75 8 - 
#80 
- Thr Gln Asp Gln His Gly Cys Glu Glu Val Gl - #u Gln Pro His His Lys 
# 895 
- Lys Glu Cys Tyr Leu Asn Phe Asp Asp Thr Va - #l Phe Cys Asp Ser Val 
# 910 
- Leu Ala Thr Asn Val Thr Gln Gln Glu Cys Cy - #s Cys Ser Leu Gly Ala 
# 925 
- Gly Trp Gly Asp His Cys Glu Ile Tyr Pro Cy - #s Pro Val Tyr Ser Ser 
# 940 
- Ala Glu Phe His Ser Leu Val Pro Asp Gly Ly - #s Arg Leu His Ser Gly 
945 9 - #50 9 - #55 9 - 
#60 
- Gln Gln His Cys Glu Leu Cys Ile Pro Ala Hi - #s Arg Asp Ile Asp Glu 
# 975 
- Cys Ile Leu Phe Gly Ala Glu Ile Cys Lys Gl - #u Gly Lys Cys Val Asn 
# 990 
- Ser Gln Pro Gly Tyr Glu Cys Tyr Cys Lys Gl - #n Gly Phe Tyr Tyr Asp 
# 10050 
- Gly Asn Leu Leu Glu Cys Val Asp Val Asp Gl - #u Cys Leu Asp Glu Ser 
# 10205 
- Asn Cys Arg Asn Gly Val Cys Glu Asn Thr Tr - #p Arg Leu Pro Cys Ala 
# 10401030 - # 1035 
- Cys Thr Pro Pro Ala Glu Tyr Ser Pro Ala Gl - #n Ala Gln Cys Leu Ile 
# 10550 
- Pro Glu Arg Trp Ser Thr Pro Gln Arg Asp Va - #l Lys Cys Ala Gly Ala 
# 10705 
- Ser Glu Glu Arg Thr Ala Cys Val Trp Gly Pr - #o Trp Ala Gly Pro Ala 
# 10850 
- Leu Thr Phe Asp Asp Cys Cys Cys Arg Gln Pr - #o Arg Leu Gly Thr Gln 
# 11005 
- Cys Arg Pro Cys Pro Pro Arg Gly Thr Gly Se - #r Gln Cys Pro Thr Ser 
# 11201110 - # 1115 
- Gln Ser Glu Ser Asn Ser Phe Trp Asp Thr Se - #r Pro Leu Leu Leu Gly 
# 11350 
- Lys Ser Pro Arg Asp Glu Asp Ser Ser Glu Gl - #u Asp Ser Asp Glu Cys 
# 11505 
- Arg Cys Val Ser Gly Pro Cys Val Pro Arg Pr - #o Gly Gly Ala Val Cys 
# 11650 
- Glu Cys Pro Gly Gly Phe Gln Leu Asp Ala Se - #r Arg Ala Arg Cys Val 
# 11805 
- Asp Ile Asp Glu Cys Arg Glu Leu Asn Gln Ar - #g Gly Leu Leu Cys Lys 
# 12001190 - # 1195 
- Ser Glu Arg Cys Val Asn Thr Ser Gly Ser Ph - #e Arg Cys Val Cys Lys 
# 12150 
- Ala Gly Phe Thr Arg Ser Arg Pro His Gly Pr - #o Ala Cys Leu Ser Ala 
# 12305 
- Ala Ala Asp Asp Ala Ala Ile Ala His Thr Se - #r Val Ile Asp His Arg 
# 12450 
- Gly Tyr Phe His 
1250 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 24 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 24GAGA GAAC 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 18 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 18 TC 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 24 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 24GCAA TCTT 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 22 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
# 22GCA CG 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 22 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
# 22CCA CA 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 22 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
# 22AGC AG 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 53 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- Gly Pro Pro Gly Pro Gln Gly Ala Thr Gly Pr - #o Leu Gly Pro Lys Gly 
# 15 
- Gln Thr Gly Glu Pro Gly Ile Ala Gly Phe Ly - #s Gly Glu Gln Gly Pro 
# 30 
- Lys Gly Glu Thr Gly Pro Ala Gly Pro Gln Gl - #y Ala Pro Gly Pro Ala 
# 45 
- Gly Glu Glu Gly Lys 
50 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 159 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- GGCCCTCCCG GTCCTCAAGG TGCAACTGGT CCTCTGGGCC CCAAAGGTCA GA - #CGGGTGAG 
60 
- CCCGGCATCG CTGGCTTCAA AGGTGAACAA GGCCCCAAGG GAGAGACTGG AC - #CTGCTGGG 
120 
# 159 GCCC TGCTGGTGAA GAAGGAAAA 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 1442 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- Met Ile Arg Leu Gly Ala Pro Gln Ser Leu Va - #l Leu Leu Thr Leu Leu 
# 15 
- Ile Ala Ala Val Leu Arg Cys Gln Gly Gln As - #p Ala Gln Glu Ala Gly 
# 30 
- Ser Cys Leu Gln Asn Gly Gln Arg Tyr Lys As - #p Lys Asp Val Trp Lys 
# 45 
- Pro Ser Ser Cys Arg Ile Cys Val Cys Asp Th - #r Gly Asn Val Leu Cys 
# 60 
- Asp Asp Ile Ile Cys Glu Asp Pro Asp Cys Le - #u Asn Pro Glu Ile Pro 
#80 
- Phe Gly Glu Cys Cys Pro Ile Cys Pro Ala As - #p Leu Ala Thr Ala Ser 
# 95 
- Gly Arg Lys Leu Gly Pro Lys Gly Gln Lys Gl - #y Glu Pro Gly Asp Ile 
# 110 
- Arg Asp Gly Pro Ala Gly Glu Gln Gly Pro Ar - #g Gly Asp Arg Gly Asp 
# 125 
- Lys Gly Glu Lys Asn Phe Ala Ala Gln Met Al - #a Gly Gly Tyr Asp Glu 
# 140 
- Lys Ala Gly Gly Ala Gln Met Gly Val Met Gl - #n Gly Pro Met Gly Pro 
145 1 - #50 1 - #55 1 - 
#60 
- Met Gly Pro Arg Gly Pro Pro Gly Pro Ala Gl - #y Ala Pro Gly Pro Gln 
# 175 
- Gly Phe Gln Gly Asn Pro Gly Glu Pro Gly Gl - #u Pro Gly Val Ser Gly 
# 190 
- Pro Met Gly Pro Arg Gly Pro Pro Gly Pro Al - #a Gly Lys Pro Gly Asp 
# 205 
- Asp Gly Glu Ala Gly Lys Pro Gly Lys Ser Gl - #y Glu Arg Gly Leu Pro 
# 220 
- Gly Pro Met Gly Ala Arg Gly Phe Pro Gly Th - #r Pro Gly Leu Pro Gly 
225 2 - #30 2 - #35 2 - 
#40 
- Val Lys Gly His Arg Gly Tyr Pro Gly Leu As - #p Gly Ala Lys Gly Glu 
# 255 
- Ala Gly Ala Pro Gly Val Lys Gly Glu Ser Gl - #y Ser Pro Gly Glu Asn 
# 270 
- Gly Ser Pro Gly Pro Met Gly Pro Arg Gly Le - #u Pro Gly Glu Arg Gly 
# 285 
- Arg Thr Gly Pro Ala Gly Ala Ala Gly Ala Ar - #g Gly Asn Asp Gly Gln 
# 300 
- Pro Gly Pro Ala Gly Pro Pro Gly Pro Val Gl - #y Pro Ala Gly Gly Pro 
305 3 - #10 3 - #15 3 - 
#20 
- Gly Phe Pro Gly Ala Pro Gly Ala Lys Gly Gl - #u Ala Gly Pro Thr Gly 
# 335 
- Ala Arg Gly Pro Glu Gly Ala Gln Gly Ser Ar - #g Gly Glu Pro Gly Asn 
# 350 
- Pro Gly Ser Pro Gly Pro Ala Gly Ala Ser Gl - #y Asn Pro Gly Thr Asp 
# 365 
- Gly Ile Pro Gly Ala Lys Gly Ser Ala Gly Al - #a Pro Gly Ile Ala Gly 
# 380 
- Ala Pro Gly Phe Pro Gly Pro Arg Gly Pro Pr - #o Gly Pro Gln Gly Ala 
385 3 - #90 3 - #95 4 - 
#00 
- Thr Gly Pro Leu Gly Pro Lys Gly Gln Ala Gl - #y Glu Pro Gly Ile Ala 
# 415 
- Gly Phe Lys Gly Asp Gln Gly Pro Lys Gly Gl - #u Thr Gly Pro Ala Gly 
# 430 
- Pro Gln Gly Ala Pro Gly Pro Ala Gly Glu Gl - #u Gly Lys Arg Gly Ala 
# 445 
- Arg Gly Glu Pro Gly Gly Ala Gly Pro Ile Gl - #y Pro Pro Gly Glu Arg 
# 460 
- Gly Ala Pro Gly Asn Arg Gly Phe Pro Gly Gl - #n Asp Gly Leu Ala Gly 
465 4 - #70 4 - #75 4 - 
#80 
- Pro Lys Gly Ala Pro Gly Glu Arg Gly Pro Se - #r Gly Leu Ala Gly Pro 
# 495 
- Lys Gly Ala Asn Gly Asp Pro Gly Arg Pro Gl - #y Glu Pro Gly Leu Pro 
# 510 
- Gly Ala Arg Gly Leu Thr Gly Arg Pro Gly As - #p Ala Gly Pro Gln Gly 
# 525 
- Lys Val Gly Pro Ser Gly Ala Pro Gly Glu As - #p Gly Arg Pro Gly Pro 
# 540 
- Pro Gly Pro Gln Gly Ala Arg Gly Gln Pro Gl - #y Val Met Gly Phe Pro 
545 5 - #50 5 - #55 5 - 
#60 
- Gly Pro Lys Gly Ala Asn Gly Glu Pro Gly Ly - #s Ala Gly Glu Lys Gly 
# 575 
- Leu Ala Gly Ala Pro Gly Leu Arg Gly Leu Pr - #o Gly Lys Asp Gly Glu 
# 590 
- Thr Gly Ala Ala Gly Pro Pro Gly Pro Ser Gl - #y Pro Ala Gly Glu Arg 
# 605 
- Gly Glu Gln Gly Ala Pro Gly Pro Ser Gly Ph - #e Gln Gly Leu Pro Gly 
# 620 
- Pro Pro Gly Pro Pro Gly Glu Gly Gly Lys Gl - #n Gly Asp Gln Gly Ile 
625 6 - #30 6 - #35 6 - 
#40 
- Pro Gly Glu Ala Gly Ala Pro Gly Leu Val Gl - #y Pro Arg Gly Glu Arg 
# 655 
- Gly Phe Pro Gly Glu Arg Gly Ser Pro Gly Al - #a Gln Gly Leu Gln Gly 
# 670 
- Pro Arg Gly Leu Pro Gly Thr Pro Gly Thr As - #p Gly Pro Lys Gly Ala 
# 685 
- Ala Gly Pro Asp Gly Pro Pro Gly Ala Gln Gl - #y Pro Pro Gly Leu Gln 
# 700 
- Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Il - #e Ala Gly Pro Lys Gly 
705 7 - #10 7 - #15 7 - 
#20 
- Asp Arg Gly Asp Val Gly Glu Lys Gly Pro Gl - #u Gly Ala Pro Gly Lys 
# 735 
- Asp Gly Gly Arg Gly Leu Thr Gly Pro Ile Gl - #y Pro Pro Gly Pro Ala 
# 750 
- Gly Ala Asn Gly Glu Lys Gly Glu Ala Gly Pr - #o Pro Gly Pro Ser Gly 
# 765 
- Ser Thr Gly Ala Arg Gly Ala Pro Gly Glu Pr - #o Gly Glu Thr Gly Pro 
# 780 
- Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro Gl - #y Ala Asp Gly Gln Pro 
785 7 - #90 7 - #95 8 - 
#00 
- Gly Ala Lys Gly Asp Gln Gly Glu Ala Gly Gl - #n Lys Gly Asp Ala Gly 
# 815 
- Ala Pro Gly Pro Gln Gly Pro Ser Gly Ala Pr - #o Gly Pro Gln Gly Pro 
# 830 
- Thr Gly Val Thr Gly Pro Lys Gly Ala Arg Gl - #y Ala Gln Gly Pro Pro 
# 845 
- Gly Ala Thr Gly Phe Pro Gly Ala Ala Gly Ar - #g Val Gly Pro Pro Gly 
# 860 
- Ala Asn Gly Asn Pro Gly Pro Ala Gly Pro Pr - #o Gly Pro Ala Gly Lys 
865 8 - #70 8 - #75 8 - 
#80 
- Asp Gly Pro Lys Gly Val Arg Gly Asp Ser Gl - #y Pro Pro Gly Arg Ala 
# 895 
- Gly Asp Pro Gly Leu Glu Gly Pro Ala Gly Al - #a Pro Gly Glu Lys Gly 
# 910 
- Glu Pro Gly Asp Asp Gly Pro Ser Gly Leu As - #p Gly Pro Pro Gly Pro 
# 925 
- Gln Gly Leu Ala Gly Gln Arg Gly Ile Val Gl - #y Leu Pro Gly Gln Arg 
# 940 
- Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly Pr - #o Ser Gly Glu Pro Gly 
945 9 - #50 9 - #55 9 - 
#60 
- Lys Gln Gly Ala Pro Gly Ala Ser Gly Asp Ar - #g Gly Pro Pro Gly Pro 
# 975 
- Val Gly Pro Pro Gly Leu Thr Gly Pro Ala Gl - #y Glu Pro Gly Arg Glu 
# 990 
- Gly Ser Pro Gly Ala Asp Gly Pro Pro Gly Ar - #g Asp Gly Ala Ala Gly 
# 10050 
- Val Lys Gly Asp Arg Gly Glu Thr Gly Ala Le - #u Gly Ala Pro Gly Ala 
# 10205 
- Pro Gly Pro Pro Gly Ser Pro Gly Pro Ala Gl - #y Pro Thr Gly Lys Gln 
# 10401030 - # 1035 
- Gly Asp Arg Gly Glu Ala Gly Ala Gln Gly Pr - #o Met Gly Pro Ser Gly 
# 10550 
- Pro Ala Gly Ala Arg Gly Ile Ala Gly Pro Gl - #n Gly Pro Arg Gly Asp 
# 10705 
- Lys Gly Glu Ser Gly Glu Gln Gly Glu Arg Gl - #y Leu Lys Gly His Arg 
# 10850 
- Gly Phe Thr Gly Leu Gln Gly Leu Pro Gly Pr - #o Pro Gly Pro Ser Gly 
# 11005 
- Asp Gln Gly Ala Ser Gly Pro Ala Gly Pro Se - #r Gly Pro Arg Gly Pro 
# 11201110 - # 1115 
- Pro Gly Pro Val Gly Pro Ser Gly Lys Asp Gl - #y Ser Asn Gly Ile Pro 
# 11350 
- Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Ar - #g Ser Gly Glu Thr Gly 
# 11505 
- Pro Val Gly Pro Pro Gly Ser Pro Gly Pro Pr - #o Gly Pro Pro Gly Pro 
# 11650 
- Pro Gly Pro Gly Ile Asp Met Ser Ala Phe Al - #a Gly Leu Gly Gln Arg 
# 11805 
- Glu Lys Gly Pro Asp Pro Met Gln Tyr Met Ar - #g Ala Asp Glu Ala Asp 
# 12001190 - # 1195 
- Ser Thr Leu Arg Gln His Asp Val Glu Val As - #p Ala Thr Leu Lys Ser 
# 12150 
- Leu Asn Asn Gln Ile Glu Ser Ile Arg Ser Pr - #o Asp Gly Ser Arg Lys 
# 12305 
- Asn Pro Ala Arg Thr Cys Gln Asp Leu Lys Le - #u Cys His Pro Glu Trp 
# 12450 
- Lys Ser Gly Asp Tyr Trp Ile Asp Pro Asn Gl - #n Gly Cys Thr Leu Asp 
# 12605 
- Ala Met Lys Val Phe Cys Asn Met Glu Thr Gl - #y Glu Thr Cys Val Tyr 
# 12801270 - # 1275 
- Pro Asn Pro Ala Thr Val Pro Arg Lys Asn Tr - #p Trp Ser Ser Lys Ser 
# 12950 
- Lys Glu Lys Lys His Ile Trp Phe Gly Glu Th - #r Met Asn Gly Gly Phe 
# 13105 
- His Phe Ser Tyr Gly Asp Gly Asn Leu Ala Pr - #o Asn Thr Ala Asn Val 
# 13250 
- Gln Met Thr Phe Leu Arg Leu Leu Ser Thr Gl - #u Gly Ser Gln Asn Ile 
# 13405 
- Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr Le - #u Asp Glu Ala Ala Gly 
# 13601350 - # 1355 
- Asn Leu Lys Lys Ala Leu Leu Ile Gln Gly Se - #r Asn Asp Val Glu Met 
# 13750 
- Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Th - #r Ala Leu Lys Asp Gly 
# 13905 
- Cys Thr Lys His Thr Gly Lys Trp Gly Lys Th - #r Val Ile Glu Tyr Arg 
# 14050 
- Ser Gln Lys Thr Ser Arg Leu Pro Ile Ile As - #p Ile Ala Pro Met Asp 
# 14205 
- Ile Gly Gly Ala Glu Gln Glu Phe Gly Val As - #p Ile Gly Pro Val Cys 
# 14401430 - # 1435 
- Phe Leu 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 267 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- ATAGGCCCTT TGGAGACGGC TGTTTTCCAG ACTCCAAACT ATCGTGTCAC AC - #GTGTGGGA 
60 
- AATGAAGTGT CTTTCAATTG TGAGCAAACC CTGGACCACA ATACTATGTA CT - #GGTACAAG 
120 
- CAAGACTCTA AGAAATTGCT GAAGATTATG TTTAGCTACA ATAATAAGCA AC - #TCATTGTA 
180 
- AACGAAACAG TTCCAAGGCG CTTCTCACCT CAGTCTTCAG ATAAAGCTCA TT - #TGAATCTT 
240 
# 267 AGCT GGAGGAC 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 54 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- Gly Pro Ser Gly Asp Gln Gly Ala Ser Gly Pr - #o Ala Gly Pro Ser Gly 
# 15 
- Pro Arg Gly Pro Pro Gly Pro Val Gly Pro Se - #r Gly Lys Asp Gly Ala 
# 30 
- Asn Gly Ile Pro Gly Pro Ile Gly Pro Pro Gl - #y Pro Arg Gly Arg Ser 
# 45 
- Gly Glu Thr Gly Pro Ala 
50 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 731 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- AGAATATAGA TAGATATGTC TGTGCTGACC GTGGCCTTTT GCCTCTTCCT TC - #TACACAGG 
60 
- GTCCTTCTGG AGACCAAGGT GCTTCTGGTC CTGCTGGTCC TTCTGGCCCT AG - #AGTAAGTG 
120 
- ACATGGAGTT GGAAGATGGA GGGGGCCCTT CAGAGAGTGT GGGCCTGTGT TC - #CCATGGGG 
180 
- AGGGAAATGC TGCTGCTTCT GGGGAAGCTG TGGGCTCAGG GGTCCTCACT CA - #GTAATGGG 
240 
- GGCAGGACTG GCTCATGTGC CTATGGCCAG AAAAGCGCCT GAGGCCACAA TG - #GCTGTAAG 
300 
- ACAAACATGA ATCAGCCTCT CGCTGTCAGA CAGAACAGCA TTTTACAAAG AG - #GAGCTTAG 
360 
- GAGGGTAGGC AAGCCATGGA GCTATCCTGC TGGTTCTTGG CCAAATAGAG AC - #CAACTTAG 
420 
- GGTTCCATGA CTGAGCATGT GAAGAACTGG GGGCGGAGTG GCTGGTGCTA TC - #AGGACAGC 
480 
- CACCTACCCA GCCCCAGCGA CTCCCCAGCC TTCCCTGTGG TGACCACTCT TT - #CCTCACGA 
540 
- CCTCTCTCTC TTGCAGGGTC CTCCTGGCCC CGTCGGTCCC TCTGGCAAAG AT - #GGTGCTAA 
600 
- TGGAATCCCT GGCCCCATTG GGCCTCCTGG TCCCCGTGGA CGATCAGGCG AA - #ACCGGCCC 
660 
- TGCTGTAAGT GTCCTGACTC CTTCCCTGCT GTCGAGGTGT CCCTACCATC CG - #GGAGGCTT 
720 
# 731 
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