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Supporting data for "Regulation of osteoblast function with osteoinductive aptamers"

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posted on 2022-09-26, 08:21 authored by Wai Ling Winnie TongWai Ling Winnie Tong

  

Bone mass is maintained constant by the coupled action of osteoclasts and osteoblasts through bone remodeling cycles. Patients with low bone mass disorders such as osteoporosis and osteogenesis imperfecta are prone to bone fractures and in severe cases anti-resorptive therapy is ineffective. With limited choices of existing anabolic agents, it is beneficial to develop alternative improved therapeutic approaches. Targeting functional proteins regulating differentiation and activities of osteoblasts with therapeutic antibodies showed promising outcomes in promoting bone growth. Oligonucleotide aptamers are “chemical antibodies” with advantages over conventional antibodies such as specificity and ease of production.

Aptamers inhibiting negative regulators of bone formation such as sclerostin (SOST) and WW-containing protein 1 (WWP1) have been generated and functionally tested in vitro. To extend this for in vivo and translational studies, we aim to assess the functions of these aptamers with stabilization modifications and to evaluate their pharmacokinetics and bioavailability. 

Serum stability of aptamers for SOST and WWP1 were enhanced with inverted thymidine modification at the 3’ end, namely SOST-dT and WWP1-dT, and were tested to verify that the modification did not affect aptamers’ functionality. Metatarsal and femur bones were cultured ex vivo in osteogenic medium supplemented with aptamer SOST-dT. Bone morphometric analysis revealed no significant difference in cortical bone mineral density with and without aptamer SOST-dT treatment, immunohistochemistry examination suggested reduced sclerostin protein expression in an ex vivo setting. Osteoblastic cells incubated with aptamer WWP1-dT in culture medium seemed to have increased RUNX2 protein expression yet one-way ANOVA analysis suggested that it was not statistically significant. Before we can conclude the effect of these aptamers, we elect to evaluate the efficacy of aptamer SOST-dT and WWPT-dT directly in mouse model. 

Aptamer extraction and quantification assay with SYBR Green qPCR were developed and optimized for monitoring bioavailability and pharmacokinetics in tissues. Aptamer retrieval from serum, bone marrow, liver and kidney have been successfully optimized with various incubation conditions, tissue volume, tissue homogenization methods and effect of DNA carrier tested out. To investigate for PCR interference by inhibiting factors presence in tissues, correction factors and detection range were deduced. These assessments provided key information on optimal injection frequency and dosage for evaluating the efficacy of aptamers in promoting bone formation in mouse models, demonstrating their therapeutic potentials in treating low bone mass disorders.

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