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Modulating clot stability by regulating lysis with lipid nanoparticle-delivered siRNA Strilchuk, Amy Wong
Abstract
Despite the importance of the thrombolytic and fibrinolytic pathways in regulating thrombosis, hemostasis, and inflammation, there exist distinct gaps in our ability to regulate these processes in the lab and the clinic. This work utilizes RNA interference machinery, delivered via lipid nanoparticle (LNP), to suppress the expression of ADAMTS13, plasminogen, and FXIII, proteins that control the degradation of VWF and fibrin multimers. We used LNPs composed of recently-developed lipid compositions, including ionizable cationic lipids DLin-MC3-DMA (MC3) and ALC-0315, to reach targets in hepatocytes and hepatic stellate cells. This thesis shows that ALC-0315 enables the knockdown of novel targets in hepatic stellate cells, such as ADAMTS13, of which the antigen and activity levels in plasma were depleted by over 50%, while MC3 is equally efficient at infiltrating hepatocytes, and induces less hepatotoxicity at high doses. Using MC3 LNPs, siRNA was used to knock-down the non-enzymatic B-subunit of FXIII (FXIII-B), causing a depletion of both FXIII-B and FXIII-A subunits from blood plasma, without impacting FXIII-A levels in platelets. Subsequently formed clots were more susceptible to lysis, including in a mouse model of arterial thrombosis. In both mice and rabbits, knockdown of plasma FXIII did not exacerbate blood loss after injury, though mice exhibited more re-bleeding events due to clot failure. siRNA-LNPs were also used to knock-down plasminogen, the precursor to the fibrinolytic protease, plasmin. Accordingly, depletion of plasminogen protein from plasma, and consequent suppression of plasmin activity showed clot stabilizing effects in mouse and dog models of FVIII deficiency. This suggests that fibrinolysis may contribute to impaired early stages of hemostasis in hemophilia A. It was also observed that sustained plasminogen levels < 25% were not associated with symptoms of plasminogen deficiency, providing further insight into the minimal plasminogen necessary for normal control of fibrin deposition. This work develops a method for controlling three proteins involved in clot lysis, and provides insight into their roles in thrombosis and hemostasis. This method may be extended to countless other targets in hepatocytes and hepatic stellate cells.
Item Metadata
| Title |
Modulating clot stability by regulating lysis with lipid nanoparticle-delivered siRNA
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| Creator | |
| Supervisor | |
| Publisher |
University of British Columbia
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| Date Issued |
2022
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| Description |
Despite the importance of the thrombolytic and fibrinolytic pathways in regulating thrombosis, hemostasis, and inflammation, there exist distinct gaps in our ability to regulate these processes in the lab and the clinic. This work utilizes RNA interference machinery, delivered via lipid nanoparticle (LNP), to suppress the expression of ADAMTS13, plasminogen, and FXIII, proteins that control the degradation of VWF and fibrin multimers.
We used LNPs composed of recently-developed lipid compositions, including ionizable cationic lipids DLin-MC3-DMA (MC3) and ALC-0315, to reach targets in hepatocytes and hepatic stellate cells. This thesis shows that ALC-0315 enables the knockdown of novel targets in hepatic stellate cells, such as ADAMTS13, of which the antigen and activity levels in plasma were depleted by over 50%, while MC3 is equally efficient at infiltrating hepatocytes, and induces less hepatotoxicity at high doses.
Using MC3 LNPs, siRNA was used to knock-down the non-enzymatic B-subunit of FXIII (FXIII-B), causing a depletion of both FXIII-B and FXIII-A subunits from blood plasma, without impacting FXIII-A levels in platelets. Subsequently formed clots were more susceptible to lysis, including in a mouse model of arterial thrombosis. In both mice and rabbits, knockdown of plasma FXIII did not exacerbate blood loss after injury, though mice exhibited more re-bleeding events due to clot failure.
siRNA-LNPs were also used to knock-down plasminogen, the precursor to the fibrinolytic protease, plasmin. Accordingly, depletion of plasminogen protein from plasma, and consequent suppression of plasmin activity showed clot stabilizing effects in mouse and dog models of FVIII deficiency. This suggests that fibrinolysis may contribute to impaired early stages of hemostasis in hemophilia A. It was also observed that sustained plasminogen levels < 25% were not associated with symptoms of plasminogen deficiency, providing further insight into the minimal plasminogen necessary for normal control of fibrin deposition.
This work develops a method for controlling three proteins involved in clot lysis, and provides insight into their roles in thrombosis and hemostasis. This method may be extended to countless other targets in hepatocytes and hepatic stellate cells.
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| Genre | |
| Type | |
| Language |
eng
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| Date Available |
2024-04-30
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| Provider |
Vancouver : University of British Columbia Library
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| Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
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| DOI |
10.14288/1.0421305
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| URI | |
| Degree | |
| Program | |
| Affiliation | |
| Degree Grantor |
University of British Columbia
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| Graduation Date |
2022-11
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| Campus | |
| Scholarly Level |
Graduate
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| Rights URI | |
| Aggregated Source Repository |
DSpace
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Attribution-NonCommercial-NoDerivatives 4.0 International