Research we propose here is a continuation of our current study on the effects of mutations in fibrillar collagens on the structure of extracellular matrices and behavior of cells with special emphasis on mutations in collagen II and their effects on cartilage. The broad, long-term objective of the proposed study is to employ three-dimensional cartilage-like constructs to determine effects of the presence of mutant collagen molecules on the structure of ECM, cell behavior, and to define target parameters for cell and gene therapies that should be reached to change abnormal phenotypes developed in the presence of collagen mutants. Our hypotheses are that mutations in fibrillar collagens alter not only the structure of extracellular matrices, but also impact the behavior of cells, and that requirements for therapy approaches to be successful vary for different collagen mutants. To meet our long-term objectives and test stated hypotheses we formulated the following Specific Aims: (1) To create a biologically relevant experimental model to study effects of collagen mutants on degeneration and repair of affected tissues, (2) To determine effects of suppression of expression of collagen mutants on remodeling of abnormal connective tissue, (3) To define conditions for cell therapies needed to override pathological changes caused by collagen mutants. A fundamental barrier to move forward development of therapies for dominant negative effects of mutations in collagen genes is a lack of information about minimal conditions needed to be achieved to drive affected tissues toward their remodeling into normal structures in response to cell or gene therapies. In our studies we will address this problem by creating a biologically relevant model, which will resemble the complexity of cartilage. This model will consist of engineered cells that, in addition to endogenous wild type procollagen II will conditionally express cDNA constructs encoding procollagen II mutants found in patients with various forms of chondrodysplasias. These cells will be employed to create cartilage-like constructs in cell culture conditions and in athymic nude mice. Subsequently, changes in morphological, biological, and biomechanical characteristics of the cartilage-like constructs formed in the presence of mutant collagen II variants will be studied after switching off mutant cDNAs or after experiments simulating """"""""delivery"""""""" of cells expressing wild type procollagen II. By employing models for gene and cell therapies, studies we propose will determine intrinsic capacity of cartilaginous tissues to repair and regenerate. Moreover, these studies will provide important information for designing therapeutic approaches to counterbalance not only dominant negative effects of mutations in collagen II but also, most likely, effects of mutations in other collagenous and noncollagenous extracellular matrix macromolecules that are associated with heritable diseases of skeletal tissues. Thus, the relevance of the proposed study to public health is high.
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