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postgraduate thesis: Development of inhibitory nucleic acid aptamers against aggrecanase for therapeutic applications

TitleDevelopment of inhibitory nucleic acid aptamers against aggrecanase for therapeutic applications
Authors
Issue Date2015
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Yu, Y. [于媛媛]. (2015). Development of inhibitory nucleic acid aptamers against aggrecanase for therapeutic applications. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5570782
AbstractIntervertebral degenerative disc disease (DDD) is a disease that occurs not only in the elderly as part of aging, but also in younger people due to overuse or misuse of discs. DDD has become a major health problem affecting every population and is the second leading cause of seeking medical help. DDD seriously affects quality of life and may cause psychological distress especially for younger people. The major proteoglycan in discs is aggrecan. Aggrecan degradation is the major pathological mechanism for DDD. ADAMTS-5 and ADAMTS-4 are two aggrecanases which can cleave aggrecan with high efficiency and cause cartilage degradation in degenerative disc disease as well as osteoarthritis. Both single ADAMTS-5 knockout and double ADAMTS-5/ADAMTS-4 knockout mice are protected from cartilage breakdown and have less severe osteoarthritis. Therefore, aggrecanases have been proposed as ideal therapeutic targets for degenerative disc disease and osteoarthritis. Small molecule inhibitors have previously been discovered that inhibit aggrecanases but have shown high toxicity in previous clinical trials due to their low specificities against related targets. Aptamers are short single-stranded nucleic acids which have often been described as being nucleic acid versions of antibodies due to their high affinity and specificity. Due to their smaller size compared to antibodies, they are less likely to stimulate an immune response, and potentially have minimal side effects due to tailored specificity. Therefore, the aim of this project was to develop novel nucleic acid aptamers that specifically inhibit aggrecanase ADAMTS-5 for therapeutics of degenerative disc disease and osteoarthritis. Previous studies on aggrecanases have been limited by low expression yields and low solubilities of the catalytic domain. In the first part of this research, aggrecanase expression and purification were optimized. ADAMTS-5CDT and ADAMTS-4CDT were both expressed as inclusion bodies in E. coli expression system. Both of them were successfully refolded and purified with high purity and high yield. Successful purification of aggrecanase catalytic domain fused with disintegrin domain provided the foundation for aptamer selection. Aptamers against aggrecanase ADAMTS-5CDT were then selected through 20 rounds of Systematic Evolution of Ligands by EXponential enrichment (SELEX). To increase the specificity and to remove aptamers which bind to same structure of ADAMTS-4CDT, 2 rounds of counter SELEX against ADAMTS-4CDT were performed. Selected aptamers were found to be rich in guanine and formed particular parallel G-quadruplex structures as determined by circular dichroism. Aptamers apt21, 25, 30 and aptc17 bound to ADAMTS-5CDT with dissociation constants (KD) at 1.54±0.16 nM, 1.79±0.08 nM, 1.45±0.29 nM and 0.79±0.08 nM respectively as determined by an enzyme linked oligonucleotide binding assay (ELONA). Aptamers did not bind to ADAMTS-4CDT, indicating high binding affinity and high specificity. Apt21 and Apts25 inhibited ADAMTS-5CDT activity with IC50 at 13.5 μM and 14.2 μM respectively using an in vitro enzymatic Fluorescence Resonance Energy Transfer (FRET) assay. In future study, inhibition efficacy of aptamers will be evaluated in human cartilage explant models as well as mouse models. Development of aptamers against aggrecanase is a promising potential approach for therapy of degenerative diseases with minimal toxicities and minimal side effects.
DegreeDoctor of Philosophy
SubjectOligonucleotides - Therapeutic use
Intervertebral disk - Diseases - Treatment
Dept/ProgramBiochemistry
Persistent Identifierhttp://hdl.handle.net/10722/226673

 

DC FieldValueLanguage
dc.contributor.authorYu, Yuanyuan-
dc.contributor.author于媛媛-
dc.date.accessioned2016-06-24T23:16:33Z-
dc.date.available2016-06-24T23:16:33Z-
dc.date.issued2015-
dc.identifier.citationYu, Y. [于媛媛]. (2015). Development of inhibitory nucleic acid aptamers against aggrecanase for therapeutic applications. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5570782-
dc.identifier.urihttp://hdl.handle.net/10722/226673-
dc.description.abstractIntervertebral degenerative disc disease (DDD) is a disease that occurs not only in the elderly as part of aging, but also in younger people due to overuse or misuse of discs. DDD has become a major health problem affecting every population and is the second leading cause of seeking medical help. DDD seriously affects quality of life and may cause psychological distress especially for younger people. The major proteoglycan in discs is aggrecan. Aggrecan degradation is the major pathological mechanism for DDD. ADAMTS-5 and ADAMTS-4 are two aggrecanases which can cleave aggrecan with high efficiency and cause cartilage degradation in degenerative disc disease as well as osteoarthritis. Both single ADAMTS-5 knockout and double ADAMTS-5/ADAMTS-4 knockout mice are protected from cartilage breakdown and have less severe osteoarthritis. Therefore, aggrecanases have been proposed as ideal therapeutic targets for degenerative disc disease and osteoarthritis. Small molecule inhibitors have previously been discovered that inhibit aggrecanases but have shown high toxicity in previous clinical trials due to their low specificities against related targets. Aptamers are short single-stranded nucleic acids which have often been described as being nucleic acid versions of antibodies due to their high affinity and specificity. Due to their smaller size compared to antibodies, they are less likely to stimulate an immune response, and potentially have minimal side effects due to tailored specificity. Therefore, the aim of this project was to develop novel nucleic acid aptamers that specifically inhibit aggrecanase ADAMTS-5 for therapeutics of degenerative disc disease and osteoarthritis. Previous studies on aggrecanases have been limited by low expression yields and low solubilities of the catalytic domain. In the first part of this research, aggrecanase expression and purification were optimized. ADAMTS-5CDT and ADAMTS-4CDT were both expressed as inclusion bodies in E. coli expression system. Both of them were successfully refolded and purified with high purity and high yield. Successful purification of aggrecanase catalytic domain fused with disintegrin domain provided the foundation for aptamer selection. Aptamers against aggrecanase ADAMTS-5CDT were then selected through 20 rounds of Systematic Evolution of Ligands by EXponential enrichment (SELEX). To increase the specificity and to remove aptamers which bind to same structure of ADAMTS-4CDT, 2 rounds of counter SELEX against ADAMTS-4CDT were performed. Selected aptamers were found to be rich in guanine and formed particular parallel G-quadruplex structures as determined by circular dichroism. Aptamers apt21, 25, 30 and aptc17 bound to ADAMTS-5CDT with dissociation constants (KD) at 1.54±0.16 nM, 1.79±0.08 nM, 1.45±0.29 nM and 0.79±0.08 nM respectively as determined by an enzyme linked oligonucleotide binding assay (ELONA). Aptamers did not bind to ADAMTS-4CDT, indicating high binding affinity and high specificity. Apt21 and Apts25 inhibited ADAMTS-5CDT activity with IC50 at 13.5 μM and 14.2 μM respectively using an in vitro enzymatic Fluorescence Resonance Energy Transfer (FRET) assay. In future study, inhibition efficacy of aptamers will be evaluated in human cartilage explant models as well as mouse models. Development of aptamers against aggrecanase is a promising potential approach for therapy of degenerative diseases with minimal toxicities and minimal side effects.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.lcshOligonucleotides - Therapeutic use-
dc.subject.lcshIntervertebral disk - Diseases - Treatment-
dc.titleDevelopment of inhibitory nucleic acid aptamers against aggrecanase for therapeutic applications-
dc.typePG_Thesis-
dc.identifier.hkulb5570782-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiochemistry-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5570782-

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