TIn a unique integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which primary collagen defects cause skeletal fragility and other significant connective tissue symptoms and then to apply the knowledge gained from our studies to the treatment of children with these conditions. An understanding of the interactions of mutant collagen molecules with the normal collagenous and non-collagenous components of extracellular matrix will also enhance our understanding of normal bone function and may yield insights applicable to the more common forms of osteoporosis. We recently focused on the development of a non-lethal animal model for OI with a classical collagen mutation. This non-lethal knock-in mouse, the Brtl mouse (Brtl), with a glycine substitution mutation in the a1(I) chain, is an excellent model for pharmacological treatment trials, for approaches to gene therapy suitable for dominant disorders, and for investigations of the skeletal matrix of OI. Our clinical studies involve children with types III and IV OI who are enrolled in age-appropriate clinical protocols for treatment and form a longitudinal study group.

The Brtl Mouse Model for OI
Marini, Uveges, Moriarty, Zhao; in collabora-tion with Raggio, Forlino, Goldstein
We have generated a knock-in murine model for OI that carries a classical OI mutation in type I collagen under the control of the endogenous promoter; we named the model the Brittle (Brtl) mouse. We introduced a point mutation into one murine col1a1 allele causing a gly349cys substitution that was modeled on the collagen defect present in one of our type IV OI patients. We selected it for duplication in the mouse because it was non-lethal in humans and was typical of the glycine substitutions in type I collagen that cause about 85 percent of OI cases.

We have investigated the skeletal adaptation of the Brtl mouse during puberty by using a combination of bone density measurements (BDM), biomechanics, and histomorphometry. It is well known that fractures in type IV OI decrease after puberty, but the mechanism of the change remains unknown. BDM of the Brtl femur and spine is about 70 percent of wild type before puberty. After puberty, the BMD of the wild-type mice does not change significantly while that of the post-pubertal Brtl mouse is significantly greater than the pre-pubertal Brtl, attaining 90 percent of wild-type BMD. The change in the amount, composition, or organization of the mineral phase of the skeleton during puberty in the Brtl mouse is distinct from pubertal development in the wild-type mouse. The increased strength of the Brtl bone in the context of weak geometry points to changes in the composition of the Brtl bone material itself as the key to post-pubertal adaptation. Understanding the physiological processes that cause improved bone strength and the mechanisms that control them may provide novel approaches to the therapy of OI.

In collaboration with investigators at the Hospital for Special Surgery, we are comparing the effects of bisphosphonate alendronate on the Brtl mouse with those on a recessive mouse model for OI, the oim mouse. The treatment of Brtl mice with alendronate is complementary to our patient treatment trial with bisphosphonate. The murine study will provide skeletal material for biomechanics, histology, and matrix composition investigations. For example, the inhibition of bone remodeling by the bisphosphonate drugs might cause increased brittleness of OI bone, a potential side effect that cannot be studied in patients. Our preliminary data indicate that bisphosphonate treatment does not alter the growth patterns of either Brtl or wild-type mice, with the treated Brtl remaining about 60 percent of the wild-type size through puberty.

Ribozyme Approach to OI Gene Therapy
Uveges, Marini
We have taken a mutation suppression approach to gene therapy of the dominant negative connective tissue disorders. Suppression of the level of mutant collagen transcripts would, in principle, transform a structural collagen mutation with severe clinical consequences into a quantitative mutation with mild to undetectable clinical symptoms. We are using hammerhead ribozymes as the mutation suppression agent. Previously, we demonstrated the specificity and effectiveness of hammerhead ribozymes in vitro and in cultured OI cells. We are currently engaged in generating transgenic mice with the ribozyme under control to achieve high levels of ribozyme expression. The mice will be bred to the Brtl mice for investigation of the effect of ribozyme on collagen mutations and the OI phenotype in vivo.

Parental Mosaicism as a Model for OI Cell Therapy
Cabral, Marini
Parents who are mosaics for the collagen mutations that cause clinically significant OI in their children are models for the cell therapy approach to OI. Although these individuals carry the collagen mutation in a fraction of their cells, they are clinically normal or minimally affected. In cell therapy, a portion of the osteocytes in an affected individual would be replaced with normal cells, resulting in a mosaic individual who might have improved skeletal function. One major deficit in the scientific rationale for cell therapy has been a lack of information about the proportion of heterozygous bone cells present in genetic mosaic individuals. We have examined skeletal cells from two asymptomatic mosaic carriers with COL1A1 mutations. Each mosaic carrier has a high proportion of dermal fibroblasts that are heterozygous for the collagen defect that causes OI in their children. Carriers for types IV OI and III OI were studied by using both labeled and single-cell PCR. Despite a 45 to 75 percent burden of mutant cells, both women had normal bone growth, density, and histology and minimal clinical signs. Thus, our data provide the first demonstration that a significant burden of mutant osteoblasts is compatible with normal skeletal functioning, allowing us to set the goal for cell therapy of OI at tentatively 50 to 60 percent normal cells.

Study of Collagen Mutations Causing OI
Cabral, Masella, Moriarty, Marini; in collaboration with Leikin
We have been investigating the consequences of a rare type of collagen mutation on collagen assembly, stability, and incorporation into fibrils and matrix. We delineated a triplet duplication in COL1A1 exon 44; the normal allele encodes three identical Gly-Ala-Hyp triplets while the mutant allele encodes four. This mutation shifts the register of the collagen chains with respect to each other but does not interrupt the triplet sequence, and yet it causes a lethal phenotype. The realignment of X and Y positions caused by the register shift delays helix formation, causing overmodification. Differential scanning calorimetry yields 2°C decreased stability of helices with one mutant chain and 6°C decreased stability of helices with two mutant chains. The register shift persists throughout the entire helix and decreases the rate of N-proteinase processing. The shift also disrupts incorporation of mutant collagen into fibrils and matrix. Collagen helices with two mutant chains and a significant portion of helices with one mutant a1(I) chain do not participate in fibril formation. This exclusion of mutant chains would be expected to cause dramatically decreased matrix production in vivo. In matrix deposited by proband fibroblasts in culture, mutant chains were well incorporated into the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. The profound biochemical effects of shifting the collagen register correlate with the severe clinical consequences. Given that double mutant molecules were most severely impaired in stability and fibrillogenesis, alignment of the a1(I) chains with the a2(I) X and Y positions appears to be critical.

Other investigations involve collagen mutations at the amino end of the helical region of a1(I) collagen. These mutations do not result in collagen overmodification but do cause destabilization of the helix as detected by differential scanning calorimetry. In addition, at the opposite end of pro-a1(I), an NIH OI patient with type IV OI has a notable collagen mutation located in the carboxy-terminal propeptide. Since the mutation itself would not be present in the mature collagen that is incorporated into helix, it is interesting that the patient has significant bone disease. We are examining the biochemical and matrix formation consequences of this mutation. Finally, we are investigating possible modifying factors for OI. In particular, we are exploring several polymorphisms that are known to be associated with post-menopausal osteoporosis.

Treatment of Children with OI with Bisphosphonate and/or Growth Hormone
Letocha, Marini
We have been conducting a four-arm treatment trial of the bisphosphonate pamidronate and recombinant growth hormone (rGH). Children with types III and IV OI are randomized among four groups: pamidronate alone, rGH alone, both drugs, or no drugs. Given that growth hormone stimulates osteoblasts to produce bone matrix and that the bisphosphonate inhibits resorption by osteoclasts, the two drugs could act synergistically to increase quantities of bone matrix.
In the wake of uncontrolled pilot studies, the OI community has voiced strong demand for bisphosphonate administration. Our study, however, is the only controlled trial of pamidronate in OI children. Major endpoints include lumbar spine bone density and vertebral compressions. Because it is possible that increased quantities of bone matrix containing abnormal type I collagen might lead to increased brittleness of bone, we will devote special effort to determining whether the quality of the bone matrix is improved. Other protocols investigate the natural history of basilar invagination in OI and the incidence and progression of pulmonary and cardiac complications