ABSTRACT
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.
ABSTRACT
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: 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.