Targeting gene-based therapeutics for canine osteoarthritis

Campbell, Sarah Elizabeth (2002) Targeting gene-based therapeutics for canine osteoarthritis. PhD thesis, University of Glasgow.

Abstract

Osteoarthritis (OA) is a chronic, painful condition and is of major concern to both human and veterinary medicine. This degenerative joint disorder is characterised by pathological changes in articular cartilage, the underlying subchondral bone and synovial membrane. To date, treatment modalities are generally limited to 'symptom-modifying' therapies that only address joint pain. The primary aim of this project was to develop a targeted, 'structure-modifying' gene-based therapy for the future treatment of OA in the dog, with the prospect of developing an animal model for human disease. To modify disease progression at the molecular level therapeutic genes can be introduced into arthritic joints to regulate those enzymes responsible for extra-cellular matrix (ECM) turnover. Since the matrix metalloproteinases (MMPs) play a central role in the development of OA, gene-based therapeutics should include mediators for controlling the synthesis and activity of these enzymes at the transcriptional and/or post-translational levels. Candidate proteins that may be of therapeutic benefit in treating OA include interleukin-1 receptor antagonist (IL-lRa), soluble tumour necrosis factor receptors (sTNFR) and various tissue inhibitors of MMPs (TIMPs). The cDNA encoding the canine homologue of IL-lRa was isolated using reverse transcription polymerase chain reaction (RT-PCR) and RNA harvested from canine peripheral blood mononuclear cells (PBMCs) as a template. Sequence analysis of the canine IL-lRa demonstrated an open reading frame of 531 base pairs (bp) encoding a protein of 177 amino acids showing considerable sequence similarity with the homologous sequences published for other species. The canine-specific therapeutic proteins including IL-lRa (19.5 kDa), TIMP-1 (22.8 kDa) and TIMP-2 9(24.3 kDa) were expressed using the in vitro transcription and translation techniques, however, sTNFRI (23.2 kDa) was unable to be expressed using this system. Levels of therapeutic gene expression are strictly controlled at the transcriptional level by promoter regions of DNA. Candidate promoters that have potential for driving disease-specific expression in arthritic joints include those that are upregulated by pro-inflammatory cytokines and growth factors associated with the pathogenesis of OA. The canine homologues of MMP-9 and -13 promoters were cloned for use in a homologous species-specific targeted gene transfer study for canine disease. The 5' untranslated regions (UTRs) were obtained by genome walking upstream of the canine MMP-9 and -13 translation start sites using genomic DNA (gDNA) as template. DNA fragments of 1894 and 1494 bp were isolated and on analysis demonstrated regions of sequence homology with the equivalent promoter sequences already determined for other species. In general, conserved regions correlated with numerous putative DNA binding motifs including Activator Protein-1 (AP-1) sites and a Nuclear Factor (NF)-KB binding domain. A consensus TATA-(like) box was identified in each case and shown to direct transcription initiation from specific positions upstream of the translation start site. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: David Bennett
• Keywords: Veterinary science, Animal diseases
• Date of Award: 2002
• Depositing User: Enlighten Team
• Unique ID: glathesis:2002-72758
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jun 2019 11:06
• URI: https://theses.gla.ac.uk/id/eprint/72758