For more information on the protocols used in the experiments, please refer to here. Knowledge of abbreviations stated in the experimental protocols will be assumed in this page. Other abbreviations will be defined.
We have proposedfor our Ostamer to be used in patients who require bone anabolic treatment. Hence, it is necessary for us to perform experiments to determine the efficacy of the ostamer in increasing bone anabolism.
We compared the binding affinities of the precursor of the pre-chemically modified ostamer (O) with full-length sclerostin (FL-SOST) in controlled environments and in human serum collected from OI patients and healthy controls to determine if the aptamer is desirable for use in humans.
As a control, we have also designed a mutant version of O (Om) which will exhibit effects directly inverse of O to act as a control to ensure any interactions between and FL-SOST variants is due to the unique binding properties of O towards FL-SOST and its variants.
It has been shownthat O has been successfully bound to the loop 3 sequence in FL-SOST. Further testing had confirmed these results in the presence of human serum, ensuring that the effects exhibited by O can also be expressed when the unmodified aptamer is present in the human body.
Hence, it is determined that O can bind to recombinant FL-SOST via targeting loop 3 in sclerostin, and this is also true in the serum of OI patients and healthy controls via the same process.
As normal aptamers have a very short half-life when present in human serum[1], we have performed additional chemical modifications to O to increase serum stability. Please note that O is marked as "aptscl56" and the chemically modified ostamer (O') is marked as "Apc001PE" in the following diagrams.
Details about the chemical modifications can be found here.
Fig PoC.1: Electrophoresis of O and O' samples in 10% and 100% human serum. [unpublished data]
From the diagram, it can be ascertained that O' has a higher serum stability than O, as the O' samples had travelled further along the solvent, suggesting that it was much more resilient to human serum than O.
Fig PoC.2: Concentration of O and O' across time when immersed in OI mice. [unpublished data]
From the graph, it can be seen that O only persisted in OI mice for roughly 10 hours, with wildly fluctuating levels, but O' had remained at roughly the same concentration for 100 hours, more than 10-fold of that of O. Not only that, but the half-life of O' in OI mice was 72 times longer than that of O.
This shows that
Fig PoC.3: Light absorbance and luciferase (bioluminescent oxidative enzymes) activity for O and O'. [unpublished data]
In conclusion, we have determined that the chemical modifications made not only prolonged the half-life of the ostamer in the human body, its affinity with FL-SOST has increased by nearly a factor of three.
Once we have determined that O' can bind to FL-SOST with high affinity, we can begin to determine its efficacy in mice.
Fig PoC.4: Graphs and pictures from micro-CT analysis of trabecular bones in mice. [unpublished data]
Using micro-CT analysis, we are able to determine and compare the bone density of the trabecular bone of the mice, displayed above in R.1. From the images and the graphs in R.1, it is clear that the bone density due to the effects induced by the addition of O' is significantly higher than that of the age-matched baseline. The femur Tb.Sp, Tb.BV/TV and Tb.vBMD of the bone exhibiting effects of O' were comparable to the age-matched baseline. Femur Tb.BV/TV and Tb.vBMD for those without the introduction of O' and those with the introduction of a mutant variant of loop 3 of scleorstin (loop3m, does not inhibit osteoblast activity, but binds to O', thereby inhibiting O') remained at significantly lower levels, while their Tb.Sp values were higher compared to that of O', which suggests that the trabecular bones in mice after the introduction of O' were comparable in density to normal mice.
This shows that the introduction of O' has increased bone anabolism in trabecular bones in OI-ApoE-/- mice.
Fig PoC.5: Graphs and pictures from bone histomorphometry of trabecular bones in mice. [unpublished data]
Bone histomorphometry also confirms these results, as shown in R.2.
The femur Tb.BFR/BS and Tb.Mar of the bones under the influence of O' were both comparable to the age-matched baseline, while those of without the introduction of O' and with loop3m were also significantly lower. Not only that, but the images above (green = initial level, red = final, change = marked by white arrows) also agree with the data in the graphs.
This also shows that the introduction of O' has increased bone anabolism in trabecular bones in OI-ApoE-/- mice.
Fig PoC.6: Graphs demonstrating the mechanical strengths of femoral mid-shafts. [unpublished data]
These conclusions are further supported by additional mechanical tests, as shown in R.3.
The femur failure force and stiffness of the O' group were higher than that of the age-matched baseline, while the fracture energy of the O' group were comparable to that of the baseline. This was not the case with the loop3m group and the OI group. Not only that, but the force-position curves of these groups show that the forces required to break the bones in the O' group (marked in green) were comparable to that of the age-matched baseline (marked in navy blue), while those of the loop3m group (marked in bright blue) and the OI group (marked in black) required less force to fracture.
Once again, this also shows that the introduction of O' has increased bone anabolism in trabecular bones in OI-ApoE-/- mice.
Combining the results from above, it can be concluded that the induced bone anabolic effects by the addition of the ostamer vastly increased bone density in OI-ApoE-/- mice, up to healthy levels. This shows that our ostamer is effective.
[1] Thiviyanathan, V., & Gorenstein, D. G. (2012). Aptamers and the next generation of diagnostic reagents. Proteomics. Clinical applications, 6(11-12), 563–573.
