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Background: Platelets are transfused therapeutically for hemostasis, and are an integral part of hemorrhage management. However, transfusions can be ineffective in the most severe cases of hemorrhage. Platelets are also a potential cell therapy in other applications, but development has been hindered by inadequate methods to control which proteins are expressed by platelets. Currently, there are no methods to express exogenous proteins in transfusable platelets, which would expand their use to help treat the diseases they modulate. A method is therefore needed to modify transfusable platelets, and thus enhance their protein composition for specific applications. Aim(s): To produce engineered, transfusable platelets to enhance their natural coagulability and functional repertoire by directly transfecting donor-derived platelets with mRNA via lipid nanoparticle (LNP)-mediated delivery. The recent advances through the COVID-19 mRNA vaccines demonstrates the clinical safety and efficacy of LNP-mediated gene therapy, and thus offers a promising strategy to effectively engineer modified platelets. Method(s): Donor-derived platelets were washed and subsequently incubated with a systematic array of LNPs encapsulating Cy5-labeled mRNA encoding for nanoluciferase in comparison to commercial transfection reagents. LNP uptake and platelet activation via CD62p levels was assessed following 4 hours by flow cytometry, while luciferase expression was assessed by normalizing the luminescence intensity to the total protein content. Result(s): Platelets took up the mRNA through all conditions tested;nanoluciferase was only expressed, however, in platelets treated with LNPs and not commercial reagents. Systematically optimizing LNPs increased nanoluciferase expression nine-fold relative to pre-optimized LNPs. Exogenous protein expression did not appear to correlate with mRNA uptake nor platelet activation. Conclusion(s): Platelets transfected with LNPs can express exogenous protein. Further optimization can eventually lead to the creation of a platform technology that in the long-term will allow platelets to deliver therapeutic proteins and yield more effective platelet products.
ABSTRACT
Background/Case Studies: Platelet transfusions are an essential treatment for attenuating bleeding but are often ineffective in cases of intractable hemorrhage. Although anucleate, mature platelets synthesize protein de novo, making them amenable to mRNA gene therapy;however, there remains to be an effective transfection technique. Advancements in lipid nanoparticle (LNP) technology has enabled leading COVID vaccines and is an efficient method to deliver nucleic acids into target cells. Recently, a LNP approach to successfully express exogenous protein in donor platelets [unpublished data] has been developed, a first step towards demonstrating that donor platelet coagulability can be engineered. However, the effects of LNP treatment on platelet function has yet to be investigated. Study Design/Methods: Donated, pooled platelets were obtained from a regional blood for research centre. The hemostatic profiles of LNP-treated and clinical donor platelets were assessed using an adapted rotational thromboelastometry model of dilutional coagulopathy. Coagulability of whole blood (WB) and hemodiluted WB were assessed. Untreated and LNP-treated platelets were then supplemented into hemodiluted WB and activated using INTEM (ellagic acid) to generate a hemostatic profile. LNP-treated platelets were also stimulated with platelet agonists thrombin (0.1U/mL) and ADP (10 mM) and CD62p levels were evaluated to test if the activation response was similar to clinical platelets using flow cytometry. Statistical analysis was conducted by one-way ANOVA and significance defined by P < 0.05. Results/Findings: LNP-treated platelets have a comparable hemostatic profile to clinically transfused platelets and significantly contributed to clot strength when spiked into hemodiluted WB. After INTEM activation, the maximum clot firmness (MCF) of LNP-treated (45.67 +/-1.15) and untreated platelets (49.77 +/- 0.58) was significantly increased (P < 0.05) compared to diluted WB alone (35.00 +/- 1.00). No significant difference between untreated and LNP-treated donor platelets was observed although MCF trended down. No statistical difference in thrombin or ADP responsiveness, indicated by median fluorescent intensity of CD62p surface presentation, was observed between LNP treated and untreated platelets (P > 0.05). Conclusion(s): LNPs are an effective way to deliver exogenous nucleic acids into platelets;they do not significantly impair the platelet contribution to clot strength or responsiveness to agonists stimulation. The minimal effect of LNP exposure on in vitro platelet characteristics demonstrate that LNP engineering is a promising new approach to load platelets with nucleic acids encoding therapeutic protein to enhance their function.
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Background/Case Studies: Platelets are transfused therapeutically for hemostasis and are an integral part of hemorrhage management. Transfusions, however, can be ineffective in the most severe cases of hemorrhage. Platelets are also a potential cell therapy in other applications, but development has been hindered by inadequate methods to control which proteins are expressed by platelets. Currently, there are no methods to express exogenous proteins in transfusable platelets, which would expand their use to help treat the diseases they modulate. A method is therefore needed to modify transfusable platelets, and thus enhance their protein composition for specific applications. Lipid nanoparticles (LNPs) represent the most clinically advanced system for non-viral gene delivery, and can potentially be used to transfect donor-derived platelets with mRNA to produce transfusable platelets with an enhanced functional repertoire. The recent advances through the COVID-19 mRNA vaccines demonstrates the clinical safety and efficacy of LNP-mediated gene therapy, and thus offers a promising strategy to effectively engineer modified platelets. Study Design/Methods: Donor-derived platelets were washed and subsequently incubated with a systematic array of LNPs encapsulating Cy5-labeled mRNA encoding for nanoluciferase in comparison to the commercial transfection reagents, lipofectamine and Ribojuice. LNP uptake and platelet activation via CD62p levels was assessed following 4 h by flow cytometry, while nanoluciferase expression was assessed by normalizing the luminescence intensity (RLU) to the total protein content. Data was analyzed via a one-way unpaired Student or Welch's t-test or one-way analysis of variance (ANOVA) as appropriate. A p-value < 0.05 was considered significant. Results/Findings: Platelets internalized the mRNA through all conditions tested, with Ribojuice yielding the highest significant increase in Cy5 median fluorescence intensity relative to the LNP (59815 +/- 6466 A.U. vs. 1253 +/- 44 A.U., respectively, p = 0.002). Nanoluciferase was only expressed, however, in platelets treated with LNPs, yielding a normalized luminescence signal of 62 +/- 17 RLU/mug protein, and not with either of the commercial reagents. Systematically optimizing LNPs increased nanoluciferase expression nine-fold to 589 +/- 241 RLU/mug protein relative to pre-optimized LNPs (p = 0.031). A Pearson correlation analysis revealed that the expression of exogenous protein expression did not appear to correlate with the mRNA uptake (Pearson correlation coefficient, r = -0.35) nor platelet activation (r = -0.07). Conclusion(s): Transfecting platelets with LNPs containing mRNA enable the expression of exogenous protein. Further optimization can eventually lead to the creation of a platform technology that in the long-term will allow platelets to deliver therapeutic proteins and yield more effective platelet products.
ABSTRACT
Background: Platelet transfusions are an essential treatment for attenuating bleeding but are often ineffective in cases of intractable haemorrhage. Although anucleate, mature platelets synthesize protein de novo, making them amenable to mRNA gene therapy;however, there remains to be an effective transfection technique. Advancements in lipid nanoparticle technology has enabled leading COVID vaccines and is an efficient method to deliver nucleic acids into target cells. Recently, we developed a LNP approach to successfully express exogenous protein in platelets [unpublished data], a first step towards demonstrating that donor platelet coagulability can be engineered. However, the effects of LNP treatment on platelet function has yet to be investigated. Aims: To determine whether LNP-treated donor platelets are functionally similar, or better, in vitro, than platelets currently transfused clinically as a next step to establish LNP engineered platelets as a new cell therapy. Methods: The hemostatic profiles of LNP-treated and clinical donor platelets were assessed using an adapted rotational thromboelastometry model of dilutional coagulopathy. LNP-treated platelets were also stimulated with conventional platelet agonists to test if responsiveness is similar, or better than clinical platelets using flow cytometry. Results: LNP-treated platelets have a comparable hemostatic profile to clinically transfused platelets and significantly improved clotting dynamics when spiked into hemodiluted whole blood in an in vitro transfusion simulation. LNP-treated platelets also respond comparably, and in some cases more potently to agonist simulation compared to untreated platelets as indicated by similar p-selectin surface presentation. Summary/Conclusions: LNPs are an effective way to deliver exogenous nucleic acids into platelets;they do not significantly change platelet coagulability or responsiveness to agonists. LNP platelet engineering is a promising new approach to load platelets with procoagulant protein to enhance their function.
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A game-changing intervention in the COVID-19 pandemic has been the rapid implementation of highly effective vaccines against SARS-CoV-2. The 2022 Canada Gairdner International Award recognizes Pieter Cullis, Katalin Kariko, and Drew Weissman "for their pioneering work developing nucleoside-modified mRNA and lipid nanoparticle (LNP) drug delivery: the foundational technologies for the highly effective COVID-19 mRNA vaccines.'' Cell editor Cheri Sirois caught up with Pieter to discuss how a long interest in basic and applied questions in lipid biology led to this fortuitous collaboration. Excerpts of the conversation are presented below.
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INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19. METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging. RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control. CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.
Subject(s)
COVID-19 , Nanoparticles , Animals , Green Fluorescent Proteins/genetics , Humans , Lipids , Liposomes , Male , Mice , RNA, Messenger/genetics , RNA, Small Interfering , RNA, Viral , Respiratory System/metabolism , SARS-CoV-2ABSTRACT
Background: Fibrinogen is an acute phase protein dramatically elevated in several diseases and during severe inflammation, such as COVID-19, cancer and sepsis. While fibrinogen is essential for maintaining hemostasis following vascular injury, overexpression of fibrinogen contributes to thrombosis. Decreasing elevated fibrinogen back to near normal levels may be a way to attenuate both inflammation and thrombosis, but has not been tested, presumably due to lack of a suitable agent that can deplete fibrinogen for long durations. Aims: To develop a potent siRNA approach that safely knocks down fibrinogen for long durations in vivo and to determine if decreasing the concentration of fibrinogen in blood plasma effectively modulates fibrinogen-mediated inflammation. Methods : Three siRNA sequences targeting mouse fibrinogen (siFbg), each encapsulated in lipid nanoparticles, were administered to separate cohorts of mice, from which hepatic mRNA and plasma protein levels were analyzed one-week post-injection. The most potent siFbg was used in subsequent studies in mice, including a monthlong dose titration study, a saphenous vein bleeding model, a lipopolysaccharide (LPS)-induced inflammatory model, and a sterile peritonitis model. Results: siFbg significantly knocked down fibrinogen in blood plasma in mice by one-week post-injection and lasted for at least three weeks, compared to mice treated with siRNA against luciferase as negative control. Dose-response studies with the most effective siFbg are ongoing as to offer unique control of the degree of fibrinogen knockdown. Mice treated with siFbg formed clots with decreased clot strength ex vivo , but hemostasis was not impaired in vivo in a saphenous vein puncture model. Treatment with siFbg protected wild-type C57BL/6J mice from increased fibrinogen 24-hours after challenging them with LPS, and restored macrophage migration in plasminogen deficient mice after thioglycollate-induced peritonitis. Conclusions: siRNA is an effective strategy for decreasing the concentration of fibrinogen in blood plasma long-term without impairing hemostasis, and can effectively modulate fibrinogen-mediated inflammation in mouse models.