RNA sequence elements for boosting expression of target proteins in platelets and platelet-interacting cells

Abstract

mRNA-containing constructs for use in the expression of target proteins in platelet cells and / or platelet-interacting cells are disclosed. The mRNA-containing constructs comprise a 5ʹ RNA element operatively linked to an mRNA coding sequence being an mRNA sequence which encodes the amino acids of a target protein. In some embodiments, the 5ʹ RNA element comprises one or more copies of a 5ʹ-untranslated region (5ʹ UTR) of an endogenous mRNA sequence and / or a 5ʹ-untranslated region (5ʹ UTR) of an exogenous mRNA sequence. Methods of transfecting the mRNA- containing construct into platelet cells and transfected platelet cells and platelet- interacting cells containing the mRNA-containing construct are also disclosed.

Description

RNA SEQUENCE ELEMENTS FOR BOOSTING EXPRESSION OF TARGET PROTEINS IN PLATELETS AND PLATELET-INTERACTING CELLSCross-Reference to Related Applications

[0001] This application claims priority from US application No. 63 / 560,238 filed 1 March 2024 and entitled RNA SEQUENCE ELEMENTS FOR BOOSTING EXPRESSION OF EXOGENOUS PROTEINS IN PLATELETS and 63 / 560,382 filed 1 March 2024 and entitled MODULATION OF PLATELET FUNCTION BYEXPRESSION OF EXOGENOUS PROTEINS, both of which are hereby incorporated herein by reference for all purposes. For purposes of the United States of America, this application claims the benefit under 35 U.S.C. §119 of US application No. 63 / 560,238 filed 1 March 2024 and entitled RNA SEQUENCE ELEMENTS FOR BOOSTING EXPRESSION OF EXOGENOUS PROTEINS IN PLATELETS and 63 / 560,382 filed 1 March 2024 and entitled MODULATION OF PLATELET FUNCTION BY EXPRESSION OF EXOGENOUS PROTEINS which are hereby incorporated herein by reference for all purposes.Field

[0002] The present invention pertain to target protein expression in platelet and platelet-interacting cells, in particular, to mRNA-containing construct used for increasing or modulating the expression of target proteins in such cells.Background

[0003] Platelets are small cells that build the foundation for blood clot formation.Following vascular injury, platelets are recruited to damage sites where they release pro-coagulant agents that initiate blood clotting. Due to this key role in hemostasis, platelets can be used for transfusions to prevent bleeding. Platelets are frequently transfused to control hemorrhage during major trauma, prevent excessive blood loss prior to major surgery, or restore normal platelet concentrations in cancer and blood disorder patients. Current platelet transfusion practices are limited by short unit lifespan, variable donor-based hemostatic efficacy, and decreased unit efficacy duringsevere hemorrhage. These limitations could be overcome by directly manipulating different characteristics of donor platelets through gene therapy.

[0004] Despite being anucleate, platelets possess the necessary machinery for mRNA translation and actively synthesize proteins from mRNA. This pathway presents an attractive target for genetic engineering, to tune platelet function through protein expression or to modulate activities of cells which interact with platelets and take up their RNA cargo. However, protein expression from delivered exogenous mRNA in platelets is limited.

[0005] The inventors have recognized a general need for improved RNA-containing constructs which can increase mRNA stability and / or target protein expression levels particularly in platelets and / or platelet-interacting cells.Summary

[0006] One aspect of the invention pertains to an mRNA-containing construct for use in the expression of a target protein in a platelet cell and / or platelet-interacting cell. The mRNA-containing construct comprises a 5' RNA element operatively linked to an mRNA coding sequence. The mRNA coding sequence comprises an mRNA sequence which encodes the amino acids of a target protein. In some embodiments, the 5' RNA element comprises one or more copies of a 5'-untranslated region (5' UTR) of an endogenous mRNA sequence and / or a 5'-untranslated region (5'UTR) of an exogenous mRNA sequence.

[0007] In some embodiments, the 5' UTR of an endogenous mRNA sequence comprises the 5' UTR of the mRNA sequence which encodes eukaryotic translation initiation factor 4E (elF4E) comprising SEQ ID NO: 1 , hemoglobin beta (HBB) comprising SEQ ID NO: 2, cyclooxygenase 1 (COX-1) comprising SEQ ID NO: 3, thymosin beta-4 (TMSB4X) comprising SEQ ID NO: 4, glycoprotein 1 B (GP1 BA) comprising SEQ ID NO: 5, glycoprotein II lb (CD36) comprising SEQ ID NO: 6, and plasminogen activator inhibitor-1 (PAI-1) comprising SEQ ID NO: 7, and combinations thereof.

[0008] In some embodiments, the 5' UTR of an exogenous mRNA sequence comprises the 5' UTR of the RNA genome of Dengue fever virus (DV), comprising SEQ ID NO: 8 and / or the 5' UTR of the RNA genome of Tobacco mosaic virus (TMV),comprising SEQ ID NO: 9. In some embodiments, the 5' UTR of an exogenous mRNA sequence comprises one or more internal ribosome entry sites (IRES). The IRES may be an IRES from a viral RNA genome. In some embodiments, the internal IRES comprises one or more of the IRES from the RNA genome of the encephalomyocarditis virus (EMCV), comprising SEQ ID NO: 10, the IRES from the RNA genome of the Hepatitis C virus (HCV), comprising SEQ ID NO: 11 , the IRES from the RNA genome of the Cricket paralysis virus (CrPV), comprising SEQ ID NO: 12, and combinations thereof.

[0009] Some aspects of the invention pertain to methods of transfecting the described mRNA-containing construct into a platelet cell to produce a transfected platelet cell which contains the mRNA-containing construct, wherein the method comprises encapsulating the mRNA-containing construct in a lipid nanoparticle (LNP).

[0010] Some aspects of the invention pertain to transfected platelet cells and / or transfected platelet-interacting cells which contain the described mRNA-containing construct.

[0011] Further aspects and example embodiments are illustrated in the accompanying drawings and / or described in the following description.

[0012] It is emphasized that the invention relates to all combinations of the above features, even if these are recited in different claims.Brief Description of the Drawings

[0013] The accompanying drawings illustrate non-limiting example embodiments of the invention.

[0014] FIG. 1 is a schematic diagram illustrating the basic components of an mRNA- containing construct according to an example embodiment of the invention.

[0015] FIG. 2 is a schematic diagram illustrating a messenger RNA (mRNA) which comprises an mRNA-containing construct according to an example embodiment of the invention.

[0016] FIG. 3 is a schematic diagram illustrating a self-amplifying mRNA (samRNA) which comprises an mRNA-containing construct according to an example embodiment of the invention.

[0017] FIG. 4 is a schematic diagram illustrating a circular mRNA (circmRNA) whichcomprises an mRNA-containing construct according to an example embodiment of the invention.

[0018] FIG. 5A is a graph showing exogenous reporter protein expression in hepatocytes, megakaryoblasts, and human platelets transfected with mRNA-lipid nanoparticles, encoding reporter protein Nanoluciferase with varied endogenous platelet 5' untranslated regions (UTRs).

[0019] FIG. 5B is a graph showing exogenous reporter protein expression in human donor platelets transfected with mRNA-lipid nanoparticles, encoding reporter protein Nanoluciferase with varied endogenous 5' untranslated regions (UTRs).

[0020] FIG. 5C is a graph showing exogenous reporter protein expression in human donor platelets transfected with mRNA-lipid nanoparticles, encoding reporter protein Nanoluciferase with varied endogenous 5' untranslated regions (UTRs), at various time points.

[0021] FIG. 6A is a graph showing exogenous reporter protein expression from mRNA encoding Nanoluciferase using varied exogenous Internal Ribosome Entry Sites (IRES) as the 5' RNA element, in a cell-free rabbit reticulocyte lysate expression system.

[0022] FIG. 6B is a graph showing exogenous reporter protein expression in HEK293 cells transfected with mRNA encoding Nanoluciferase using varied exogenous Internal Ribosome Entry Sites (IRES) as the 5' RNA element (X-axis), using mRNA - lipid nanoparticles.

[0023] FIG. 6C is a graph showing exogenous reporter protein expression in human donor platelets transfected with mRNA encoding Nanoluciferase varied exogenous Internal Ribosome Entry Sites (IRES) as the 5' RNA element (X-axis), using mRNA - lipid nanoparticles.

[0024] FIG. 7 is a graph showing exogenous reporter protein expression in human donor platelets transfected with self-amplifying RNA (saRNA) encoding Nanoluciferase.

[0025] FIG. 8A, FIG. 8B and FIG. 8C are representative ROTEM curves, with clotting initiated by EXTEM reagent (tissue factor) and clot lysis accelerated through the addition of tissue plasminogen activator (tPA). In each plot, one of the lines represents the ROTEM tracing of aprotinin LNP engineered platelets (LEPs) and theother one of the lines represent Nanoluciferase LNP engineered platelets. The plots demonstrate that platelets engineered to express aprotinin are resistant to clot lysis in vitro.

[0026] FIG. 8D is a graph correlating NLuc expression in platelets at 4 h to clot stability induced by aprotinin expression.

[0027] FIG. 8E is a graph showing correlation from N = 9 biological replicates. The graph demonstrates that reporter protein expression correlates to lysis differences between NanoLuc control platelets and aprotinin engineered platelets.

[0028] FIG. 9 is a graph comparing the expression of Nanoluciferase in platelets engineered with LNPs containing an RNA encoding both Myglobin and Nanoluciferase and a Nanoluciferase only control (i.e. , the LNP contains an RNA which encodes only Nanoluciferase). Expression of Nanoluciferase is dependent on the successful translation of the Myoglobin protein. P-values were determined by oneway ANOVA. Data reported as mean ± SD. *P < 0.05.Detailed Description

[0029] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive sense.Definitions

[0030] “Platelets” are small blood cells that build the foundation for blood clot formation. Following vascular injury, platelets are recruited to damage sites such as damaged blood vessels where they release coagulant agents and other cargo that initiates blood clotting and hemostasis.

[0031] “Platelet-interacting cells” are cells that can naturally interact with platelet cells, or be induced to interact with platelet cells. The interaction may be directly such as by contact-dependent mechanisms, and indirectly such as by secreted immune mediator-driven mechanisms. Non-limiting examples of platelet-interacting cellsinclude inflammatory cells, e.g., white blood cells, vascular endothelial cells, etc.

[0032] “Endogenous”, as used herein, refers to a messenger RNA (mRNA) or protein sequence that is inherent to or naturally produced by unmodified, non-transfected platelet cells or platelet precursor cells.

[0033] “Exogenous” means a messenger RNA (mRNA) or protein sequence that is not inherent to, or is not naturally produced, by unmodified, non-transfected platelet cells or platelet precursor cells.

[0034] “Internal Ribosome Entry Site” or “IRES” means structural RNA elements that are located upstream of (or 5' to) an mRNA coding sequence which directly promotes ribosome binding and mRNA translation, independent of a cap structure, and recruits few or no initiation factors. IRESs are non-coding mRNA sequences that may be associated with viruses, exogenous to the human transcriptome. Viruses use these IRESs to hijack cellular ribosomes from endogenous RNA translation and promote expression of its own viral genome.

[0035] “Lipid nanoparticle(s)” or “LNP(s)” are spherical vesicles made of one or more ionizable cationic lipid, one or more helper lipid, cholesterol, and one more polyethylene glycol (PEG)-lipid conjugate, plus the nucleic acid (DNA or RNA) cargo within the particle.

[0036] “mRNA coding sequence” means a RNA polynucleotide which directly specifies the amino acid sequence of a polypeptide.

[0037] “5' RNA element” refers to an RNA sequence located upstream of (or 5' to) an mRNA coding sequence.

[0038] “Untranslated region” or “UTR” refers to RNA sequences located on the 5' or 3' of mRNA, outside the coding region, that are important for RNA stability, and translational regulation. A UTR may be located 5' to (or upstream of) the mRNA coding sequence (a “5' UTR”) or 3' to (or downstream of) the mRNA coding sequence (a “3' UTR”). A UTR may be endogenous (naturally found in human cells, including platelet cells) or exogenous (not found naturally in human platelet cells).

[0039] “Downstream” means a location in a nucleic acid sequence located 3' to a given reference point in that nucleic acid sequence.

[0040] “Upstream” means a location in a nucleic acid sequence located 5' to a given reference point in that nucleic acid sequence.Example embodiments

[0041] Aspects of the invention relate to mRNA-containing constructs that are particularly effective in increasing, modulating and / or enhancing expression of target proteins in transfected platelet and platelet-interacting cells. In some embodiments, the target protein is an exogenous protein, i.e. , a protein that is not found naturally in human platelet cells. In some embodiments, the target protein is an endogenous protein, i.e., a protein that is found naturally in human platelet cells. Referring to FIG. 1 , the mRNA-containing construct comprises a 5' RNA element operatively linked upstream of an mRNA coding sequence. The mRNA coding sequence is an mRNA sequence which encodes an amino acid sequence of a desired target protein.

[0042] In some embodiments, the 5' RNA element comprises one or more copies of an 5' untranslated region (5' UTR) of an endogenous mRNA sequence. In some embodiments, the 5' UTR of an endogenous mRNA sequence comprises the 5' UTR of the mRNA sequence which encodes eukaryotic translation initiation factor 4E (elF4E) comprising SEQ ID NO: 1 , hemoglobin beta (HBB) comprising SEQ ID NO: 2, cyclooxygenase 1 (COX-1) comprising SEQ ID NO: 3, thymosin beta-4 (TMSB4X) comprising SEQ ID NO: 4, glycoprotein 1 B (GP1 BA) comprising SEQ ID NO: 5, glycoprotein I lib (CD36) comprising SEQ ID NO: 6, and plasminogen activator inhibitor-1 (PAI-1) comprising SEQ ID NO: 7, and combinations thereof.

[0043] In some embodiments, the 5' UTR of an endogenous mRNA sequence comprises a full length sequence and / or partial length sequence of one or more of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7. In some embodiments, the 5' UTR of an endogenous mRNA sequence comprises one or more segments of one or more of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7.

[0044] In some embodiments, the 5' UTR of an endogenous mRNA sequence comprises a sequence that has at least about 80% sequence identity to one or more of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, including any value and range therebetween, including for example, 85%, 90%, 95%, 99% sequence identity, etc., and between 80 and100%, 85 and 100%, 90 and 100%, 95 and 100% sequence identity, etc.

[0045] In some embodiments, the RNA element comprises one or more copies of a 5' untranslated region (5' UTR) of an exogenous mRNA sequence. In some embodiments, the 5' UTR of an exogenous mRNA sequence comprises the 5' UTR of the RNA genome of Dengue fever virus (DV), comprising SEQ ID NO: 8 and / or the 5' UTR of the RNA genome of Tobacco mosaic virus (TMV), comprising SEQ ID NO: 9.

[0046] In some embodiments, the 5' UTR of an exogenous mRNA sequence comprises one or more internal ribosome entry sites (IRES). In some embodiments, the IRES is an IRES from a viral RNA genome. In some embodiments, IRES comprises one or more of IRESs from the RNA genome of the encephalomyocarditis virus (EMCV), comprising SEQ ID NO: 10, the internal ribosome entry site (IRES) from the RNA genome of the Hepatitus C virus (HCV), comprising SEQ ID NO: 11 , the internal ribosome entry site (IRES) from the RNA genome of the Cricket paralysis virus (CrPV), comprising SEQ ID NO: 12, and combinations thereof.

[0047] In some embodiments, the 5' UTR of an exogenous mRNA sequence comprises a full length sequence and / or partial length sequence of one or more of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 , and SEQ ID NO. 12. In some embodiments, the 5' UTR of an exogenous mRNA sequence comprises segments of one or more of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 , and SEQ ID NO. 12.

[0048] In some embodiments, the 5' UTR of an exogenous mRNA sequence comprises a sequence that has at least about 80% sequence identity to one or more of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 , and SEQ ID NO. 12, including any value and range therebetween, including for example, 85%, 90%, 95%, 99% sequence identity, etc., and between 80 and 100%, 85 and 100%, 90 and 100%, 95 and 100% sequence identity, etc.

[0049] The 5' RNA element comprises (and in some embodiments, consists essentially of) a RNA sequence that forms a secondary structure which is favorable for recruitment of ribosomes to internal mRNA sites to initiate translation.

[0050] In some embodiments, the 5' RNA element is a synthetic RNA sequence. The synthetic RNA sequence may form highly folded secondary structures. In some example embodiments, the synthetic RNA sequence comprise folded structures withone or more pseudoknots. An example of a synthetic RNA sequence that is suitable for use as the 5' RNA element is the sequence that is set forth in SEQ ID NO: 13. The 5' RNA element may comprise a full length sequence or partial length sequence of SEQ ID NO: 13, or segments of SEQ ID NO: 13. In some embodiments, the 5' RNA element comprises a sequence that has at least about 80% sequence identity to SEQ ID NO: 13, including any value and range therebetween, including for example, 85%, 90%, 95%, 99% sequence identity, etc., and between 80 and 100%, 85 and 100%, 90 and 100%, 95 and 100% sequence, identity, etc.

[0051] In some embodiments, the 5' RNA element comprises an initiator sequence. The initiator sequence may be provided to enable the linking of one or more additional RNA elements to the 5' RNA element. Such one or more additional RNA elements may for example comprise a 5' cap as described in detail below. In some example embodiments, the initiator sequence comprises the sequence “AGG”. An initiator sequence may be operatively linked to one or more of the sequences defined in SEQ ID NOs: 1-13. For clarity, SEQ ID NOs: 1-13 as defined do not include the initiator sequence.

[0052] The 5' RNA element may comprise a combination of 5' untranslated region (5' UTR) of an endogenous mRNA sequence(s) and 5' untranslated region (5' UTR) of an exogenous mRNA sequence(s). The 5' RNA element may comprise a combination of one or more of 5' untranslated region (5' UTR) of an endogenous mRNA sequence(s), 5' untranslated region (5' UTR) of an exogenous mRNA sequence(s), and synthetic RNA sequence(s).

[0053] The mRNA-containing construct comprises one or more additional RNA elements operatively linked to the 5' RNA element and the mRNA coding sequence. The one or more additional RNA elements may comprise one or more sequences which, in combination with the 5' RNA element and the mRNA coding sequence, form a functional mRNA. A functional mRNA allows for proper translation of the mRNA into the encoded protein.

[0054] In some embodiments, the additional one or more RNA elements comprise one or more copies of a 3' UTR operatively linked downstream of the mRNA coding sequence. The 3' UTR sequence may for example be a full length sequence and / or partial length sequence and / or segments of one or more 3' UTRs of the mRNAsequence which encode a target protein, such as but are not limited to eukaryotic translation initiation factor 4E (elF4E), hemoglobin beta (HBB), hemoglobin alpha, human growth hormone (hGH), albumin, cyclooxygenase 1 (COX-1), thymosin beta-4 (TMSB4X), glycoprotein 1 B (GP1 BA), glycoprotein lllb (CD36), and / or plasminogen activator inhibitor-1 (PAI-1), etc., and / or one or more 3' UTRs from the RNA genome of a virus, such as but is not limited to Dengue fever virus (DV), Tobacco mosaic virus (TMV), encephalomyocarditis virus (EMCV), Hepatitus C virus (HCV), Cricket paralysis virus (CrPV), etc.

[0055] In some embodiments, the additional one or more RNA elements comprise a 5' cap structure operatively linked upstream of the 5' RNA element. In some embodiments, the 5' cap structure is positioned at the 5' end of the mRNA-containing construct. The 5' cap structure comprises any suitable 5' cap structure that is present in eukaryotic cellular mRNAs, and / or a 5' cap analog. Examples of a 5' cap include m7G(5')ppp(5')N which comprises a 7-methylguanosine moiety attached via a 5-5 triphosphate bridge to the 5'-terminal nucleoside of the RNA strand, and "N" is any nucleotide, and / or 5' cap analogs, such as m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7GpppAmpG, m7GpppAmpG ammonium, etc.

[0056] In some embodiments, the additional one or more RNA elements comprise a poly (A) tail operatively linked downstream of the mRNA coding sequence. In some embodiments, the poly (A) tail is positioned at the 3' end of the mRNA-containing construct. The poly (A) tail comprises a long chain of adenine nucleotides. The length of the poly (A) tail is variable, and may range from about 20 nucleotides to about 250 nucleotides, and in some embodiments, between about 150 to 250 nucleotides.

[0057] In some embodiments, the mRNA-containing construct is in the form of a linear structure. Such an mRNA-containing construct may be referred to as a “linear mRNA”.

[0058] Referring to FIG. 2, in some embodiments, the mRNA comprises the 5' RNA element operatively linked upstream of the mRNA encoding sequence. The mRNA comprises additional RNA elements including a 3' UTR is operative linked downstream of the mRNA encoding sequence, a poly (A) tail operatively linked downstream of the 3' UTR, and a 5' cap structure operatively linked upstream of the 5' RNA element.

[0059] Referring to FIG. 3, in some embodiments, the mRNA-containing construct is in the form of a self-amplifying RNA (saRNA). In some embodiments, the saRNA comprises the 5' RNA element operatively linked upstream of the mRNA encoding sequence. The saRNA additionally comprises a saRNA specific sequence element. The saRNA specific sequence element comprises one or more sequences which encode one or more self-replicating proteins. The saRNA specific sequence element may be operatively linked upstream of the mRNA encoding sequence. In some embodiments, the saRNA specific sequence element is operatively linked downstream of the 5' RNA element, and placed between the 5' RNA element and the mRNA coding sequence. In some embodiments, self-replicating proteins are non- structural proteins which function to generate additional strands of saRNA, and / or to synthesize additional RNA which comprises the mRNA encoding sequence, thereby increasing the production of the desired target protein.

[0060] In some example embodiments, the one or more self-replicating proteins comprise viral replicase. Suitable one or more promoters may be operatively linked to between the mRNA encoding sequence and the one or more sequences which encode the one or more self-replicating proteins. The one or more promoters may be arranged for binding to the replicase so as to control the synthesis of the mRNA coding sequence. It will be understood that other self-replicating proteins are within the scope of the invention.

[0061] The saRNA may comprise additional RNA elements including a 3' UTR operative linked downstream of the mRNA encoding sequence, a poly (A) tail operatively linked downstream of the 3' UTR, and a 5' cap operatively linked upstream of the 5' RNA element.

[0062] In some embodiments, the mRNA-containing construct is in the form of a circular structure. Such an mRNA-containing construct may be referred to as a “circular mRNA” or “circmRNA”.

[0063] Referring to FIG. 4, in some embodiments, circmRNA comprises the 5' RNA element operatively linked to the mRNA encoding sequence, and the 3' UTR operatively linked between the 5' RNA element and the mRNA encoding sequence. The 3' terminus at the 3' UTR and the 5' terminus at the 5' RNA element may be ligated to form a covalently closed circle.

[0064] In some embodiments, the mRNA-containing construct further comprises a translation initiation sequence. A translation initiation sequence is a sequence which serves as an initiation site where protein translation begins in an mRNA that is produced from transcription. The translation initiation sequence may be operatively linked to and placed between the 5' RNA element and the mRNA coding sequence. In some example embodiments, the translation initiation sequence comprises a Kozak sequence. As used herein, a “Kozak sequence” may be a consensus Kozak sequence which comprises the sequence GCC(A / G)CC positioned immediately before the AUG start codon for translation or a variant of the consensus Kozak sequence.

[0065] In some embodiments, the target protein that is encoded by the mRNA coding sequence comprises a coagulation factor, an antifibrinolytic, an anticoagulant, a fibrinolytic, an antimicrobial, an immunomodulator, an anti-cancer agent, an aptamer, a genetic editor, and / or a reporter protein. Non-limiting examples of target proteins that may be encoded by the mRNA coding sequence in the mRNA-containing construct are listed in Table 1 below.Table 1. Examples of target proteins that may be encoded by the mRNA coding sequence in the mRNA-containing construct.

[0066] Some aspects of the invention pertain to the use of the described mRNA- containing constructs to transfect eukaryotic cells to produce a population of transfected cells which may express the target protein encoded by the mRNA coding sequence. Non-limiting examples of cells which may be transfected with the described mRNA-containing sequence include platelet cells, HEK293 cells, hepatocytes, or megakaryoblasts, etc. Proof of concept experiments demonstrate that the described mRNA-containing constructs can be a promising approach for use in modulating the expression of a desired target protein in transfected human donor platelets.

[0067] Any suitable mRNA delivery mechanisms may be used to transfect the mRNA- containing construct into the desired cells. In some example embodiments, the mRNA-containing constructs are transfected into eukaryotic cells with lipid nanoparticles (LNPs) using LNP compositions and methods described in International Publication No. WO 2023 / 220815 A1 , the contents of which are incorporated by reference herein.

[0068] For example, in some embodiments of the invention, the mRNA-containing construct is encapsulated in a LNP. The LNP may comprise a composition comprising a lipid mixture which comprises at least one (ionizable) cationic lipid; at least onehelper lipid; a sterol; and a least one lipid-polyethylene glycol conjugate. In some embodiments, the lipid mixture comprises about 30-55 mol % of the at least one (ionizable) cationic lipid; about 5-20 mol % of the at least one helper lipid; about 25-50 mol % of sterol; and about 0.5 - 3 mol % of the at least one lipid-polyethylene glycol conjugate. In some embodiments, the at least one ionizable cationic lipid in the lipid mixture is one or more of CL4H6, SM-102, ALC-0315, CL1 H6, CL15H6, CL1 D6, and ALC-0159. In some embodiments, the at least one helper lipid in the lipid mixture is one or more of phosphatidylcholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), and egg sphingomyelin (ESM). In some embodiments, the sterol in the lipid mixture is cholesterol and / or a cholesterol derivative. In some embodiments, the at least one lipidpolyethylene glycol conjugate is one or more of l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE-PEG2000), and PEG-l,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG).

[0069] However, it will however be understood that other suitable methods and / or LNP compositions may be used to transfect the described mRNA-containing construct into the desired cells.

[0070] In some embodiments, transfected platelet cells (i.e. , platelets that are transfected with the described mRNA-containing construct) may be caused to transfer or deliver the mRNA-containing construct to platelet-interacting cells.

[0071] Some aspects of the invention pertain to transfected platelet cells and / or transfected platelet-interacting cells which contain the mRNA-containing construct. The population of transfected platelet cells are caused to express the desired target protein which is encoded by the mRNA coding sequence of the mRNA-containing construct.

[0072] Some aspects of the invention pertain to platelet cells and platelet-interacting cells being transfected with the described mRNA-containing constructs.

[0073] The invention is further described with reference to the following specific examples, which are not meant to limit the invention, but rather to further illustrate it.Examples

[0074] mRNA-containing constructs such as the one schematically illustrated in FIG. 1 were prepared.Example 1 : Platelet-Specific preferences of endogenous 5' UTRs for target protein expression.

[0075] mRNA-containing constructs each comprising a different endogenous 5' UTR as the 5' RNA element were transfected into human donor platelet cells and the megakaryoblast and hepatocyte cell lines MEG-01 and HUH7. In these examples, the mRNA-containing constructs were transfected into the cells using lipid nanoparticles (or LNPs). Enzymatic assay of reporter protein nanoluciferase (NanoLuc) activity was used to measure target protein levels. RNA encoding nanoluciferase with endogenous 5' UTR as the 5' RNA element was delivered encapsulated in lipid nanoparticles. The endogenous 5' UTR that were tested are listed in Table 2 below.Table 2. Endogenous 5' UTR as the 5' RNA element tested in the Examples

[0076] After a 24 h incubation, significant differences in the best performing sequences were evident between the cell lines, with variations depending on the cell type (FIG. 5A). For both cell lines tested, all sequences had similar or lower levels of expression compared to the standard HBB (FIG. 5A). For both hepatocyte and megakaryoblast cell lines, elF4E had the highest expression of the UTRs tested after HBB. In platelets, elF4E, COX-1 , GP1 BA and are the top expressing UTRs after a 24 h incubation (FIG. 5A). However, TMSB4X was one of the lowest expressing UTRs.The difference in expression, relative to HBB, of the GP1 BA UTR for megakaryoblasts was 0.4-fold, while in platelets it was a 2.4-fold increase. Similarly, the difference of expression for elF4E, relative to HBB, for megakaryoblasts and platelets were 0.9-fold, and 1.7-fold, respectively. In hepatocytes, the differences between elF4E and GP1 BA UTRs versus HBB were less pronounced, with 0.8-fold and 0.7-fold differences respectively. These results indicate that GP1 BA, elF4E and COX-1 UTRs show promise over other 5' UTRs to improve protein expression in platelets, a cell-type specific observation. UTRs elF4E, COX1 and GPIBA were confirmed to be superior to HBB which was sustained over 24 h in platelets (Figure 5B-C). The described endogenous UTRs demonstrate as proof-of concept that target protein translation in platelets can be altered through UTR elements which is cell type specific.

[0077] In summary, as can be seen in FIG. 5A, endogenous RNA elements at the 5' end of transfected mRNA can preferentially and specifically drive mRNA translation and protein synthesis in platelet cells. Performance of the endogenous UTRs from hemoglobin beta (HBB), eukaryotic translation initiation factor 4E (elF4E), cyclooxygenase 1 (COX1), thymosin beta-4 (TMSB4X), glycoprotein Ib-alpha (GP1 BA), platelet glycoprotein 4 (CD36), and plasminogen activator inhibitor-1 (PAI- 1) differ between platelets and nucleated cell types, including platelet precursor cells megakaryoblasts.

[0078] As seen in FIG. 5B, endogenous platelet RNA elements (i.e. , the endogenous 5' UTR) can significantly modulate the translation of exogenous mRNA, and the resulting protein expression in transfected human donor platelets. This example demonstrates that endogenous platelet 5' UTRs can drive translation of exogenous mRNA and target protein expression in platelets.Example 2: Platelets transfected with mRNA-LNP express target proteins

[0079] mRNA-containing constructs each comprising a different exogenous 5' UTR as the 5' RNA element were delivered into uncomplexed (cell-free rabbit reticulocyte lysate) or encapsulated in lipid nanoparticles (HEK293 cells and human donor platelets). Enzymatic assay of reporter protein nanoluciferase (NanoLuc) activity was used to measure target protein levels. The tested exogenous RNA elements are listedin Table 2 below. The tested exogenous RNA elements include the listed exogenous 5' UTRs and a synthetic UTR which comprises a non-natural 39 base sequence that forms a short secondary structure hairpin with high activity in platelets specifically.Table 3. Exogenous 5' UTR as the 5' RNA element tested in the Examples

[0080] Viral IRES sequences from three groups with differing initiation factor requirements were screened in rabbit reticulocyte lysate, cultured human kidney cells (HEK293), and human platelets (FIGS. 6A-6C). In contrast to reticulocyte lysate and HEK293 cell expression systems, some of the highest expression in human platelets was achieved with inclusion of a representative group III IRES (FIG 6C). High expression was also achieved with use of the 5' UTR from the Dengue virus and Tobacco Mosaic virus. The most notable contrast in synthesis between platelets and the other systems tested was with the synthetic UTR sequence.

[0081] In summary, as can be seen in FIGS. 6A-C, operatively linking an exogenous RNA elements at the 5' end (or exogenous 5' UTRs) to an mRNA coding sequence can preferentially and specifically drive mRNA translation and protein synthesis in platelet cells. A short, synthetic RNA element (“synthetic UTR”) shows high translational activity in platelets, relative to its performance in other cell lines. Representative IRES sequences from Group I (Cricket Paralysis Virus; CrPV), Group II (Hepatitis C; HCV) and Group III (Encephalomyocarditis virus; EMCV) show differences in platelet-specific translational patterns. Additional viral RNA elements TMV (Tobacco Mosaic Virus) and the Dengue Virus untranslated region display translational activity in transfected platelets.

[0082] As seen in FIG. 6C, the influence of 5' exogenous RNA elements on plateletexpression of target protein is time-dependent, with varied profiles at 4 and 24 h following transfection.

[0083] This example demonstrates that exogenous 5' UTRs can drive mRNA translation and target protein expression in platelets.Example 3: Platelets transfected with mRNA-LNP express target protein

[0084] Self-amplifying RNA constructs such as the one illustrated in FIG. 3 was prepared. FIG. 7 demonstrates that self-amplifying RNA (saRNA) constructs encoding Nanoluciferase were also able to support target protein expression in transfected platelets. As seen with other saRNA models, protein expression occurs early during transfection, drops off, and then returns at later time points, supporting the selfamplifying feature of these mRNAs.Materials and MethodsPreparing Platelets for Transfection

[0085] Washed platelets (WP) were prepared for transfection from pooled platelet concentrate (PC) units. PC was sampled through a coupling port (Fresenius Kabi, Bad Homburg, Germany) with a sterile Leur-Lok™ syringe (BD, Franklin Lakes, NJ). PC was centrifuged at 250 x g for 20 min at room temperature. The supernatant was removed, and the remaining platelet pellet was washed once in anticoagulant citrate dextrose solution, USP, Formula A (ACD-A) (Haemonetics, Boston, MA) and once in Tyrode’s-HEPES buffer pH 6.5 (134 mM NaCI, 2.9 mM KCI, 0.34 mM NaH2PO4, 10 mM HEPES, 5 mM D-glucose) at 250 x g for 10 min at 22 °C. WP were suspended in Tyrode’s-HEPES buffer pH 6.5 and counted using a Sysmex XN-550 hematology analyzer (Sysmex Corporation, Kobe, Japan). For all experiments, three PC units were washed and transfected within 24 h. Single-donor apheresis platelet units were prepared in the same manner following collection.Synthesizing mRNA in vitro

[0086] Messenger RNA (or mRNA-containing constructs) was synthesized in bulk by in vitro transcription. Briefly, plasmid DNA templates, some encoding a CleanCap® AG bacteriophage T7 promoter site, with various 5' UTRs and NanoLuc luciferase(NanoLuc) coding sequence were linearized with Sapl or BamHI. RNA was produced by in vitro transcription reactions and purified using an RNeasy Kit (Qiagen, Toronto, ON) or with lithium chloride precipitation. RNA was capped co-transcriptionally using CleanCap® AG reagent when necessary (TriLink BioTechnologies, San Diego, CA), or post-transcriptionally capped using an enzymatic-based capping kit (CELLSCRIPT, Madison, Wl) following purification. RNA was tailed using a post-transcriptional tailing kit (CELLSCRIPT, Madison, Wl), prior to an additional purification. RNA purity, integrity, and tailing efficiency were monitored by agarose gel electrophoresis.Preparing Lipid Nanoparticles Containing mRNA

[0087] An ethanolic lipid mixture containing an ionizable lipid, helper lipid, cholesterol, and 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (50: 10:38.5: 1 .5 mol%) was mixed via a T-junction mixer at a 1 :3 ratio with an aqueous solution of 25 mM sodium acetate pH 4 containing the desired mRNA- containing construct at an amine-to-phosphate (N / P) ratio of 6. The resulting mixture was dialyzed (Spectra / Por 2 Dialysis Tubing 12-14 kDa MWCO, Spectrum Labs, San Francisco, CA) 500-fold against 1X phosphate-buffered saline (PBS) and then sterile- filtered through an Acrodisc® 0.2 pm syringe filter (Pall Corporation, Mississauga, ON). The formulations were concentrated in Amicon® 10,000 kDa MWCO ultracentrifugation units (EMD Millipore Corporation, Billerica, MA) and the RNA content and encapsulation determined using a Quant-it™ Ribogreen RNA Assay Kit (Thermo Fisher Scientific, Eugene, OR). Total lipid content was determined with the Cholesterol E kit (Fujifilm Wako Diagnostics, Mountain View, CA). Particle size and polydispersity index were measured via dynamic light scattering on the Malvern Zetasizer Nano (Malvern Panalytical, Worcestershire, England).Treating Platelets or Cell Lines with mRNA-LNP

[0088] Washed platelets were treated with RNA-LNP at an RNA dose of 7 pg / mL (endogenous UTR experiments) per 30 x 106platelets mL-1, or 12 pg / mL per 40 x 106platelets mL-1(exogenous UTR experiments) in Tyrode’s-HEPES buffer pH 6.5 and incubated at 37 °C for 0, 4, 6, 16, or 24 h before further downstream processing. Cell lines HUH7 and MEG01 were seeded at 10 000 cells / well 24 h prior to treatment andwere treated at a dose of 1 pg / mL. HEK293 cells were plated at 70% confluency, 4 h prior to transfection at 1 pg RNA per mL media. When treating with the commercial reagents, Lipofectamine™ MessengerMAX™ (Thermo Fisher Scientific, Carlsbad, CA) and RiboJuice™ (EMD Millipore Corporation, Burlington, MA), the platelets were treated with equivalent amounts of mRNA-containing construct and according to the manufacturer’s recommended protocols. Following transfection, target protein expression was assessed by enzymatic assay of NanoLuc activity.Luciferase Assay

[0089] Washed and treated platelets were pelleted by centrifugation at 300 x g for 10 min at room temperature before lysing in 100 pL of Gio Lysis buffer (Promega Corporation, Madison, Wl). Lysates were transferred to a 96-Flat Well white microplate and mixed with prepared NanoLuc substrate (Promega Corporation, Madison, Wl) at a 1 :1 ratio. Luminescence was recorded on the Tecan Spark® Multimode Microplate Reader (Paramit Corporation, Morgan Hill, CA) with an integration time of 1000 ms and zero attenuation. The total protein content in each lysate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s protocol, with absorbance measured at 562 nm on the Tecan Spark Multimode Microplate Reader.Example 4: Lipid nanoparticles (LNPs) containing RNA that encodes protein targets enables the deliberate modulation of platelet function and the introduction of new functions.

[0090] This example demonstrates methods to modulate platelet function through the delivery of RNA using LNPs. To provide an example of how platelet function can be deliberately enhanced, mRNA encoding the small peptide, aprotinin, was delivered to donor platelets via the LNP engineering platform described herein and in International Publication No. WO 2023 / 220815. The LNP composition used in these examples comprise ALC-0315, DOPC, PEG2000 and cholesterol. Aprotinin is a small antifibrinolytic peptide used clinically to prevent excessive clot breakdown (fibrinolysis). Aprotinin is a serine protease inhibitor with activity against plasmin, an enzyme that drives clot degradation. The inventors delivered mRNA encodingaprotinin into platelets with the goal of enhancing resistance to clot breakdown, thereby increasing clot stability, something that may be clinically valuable in the future. To test this, platelets were treated with platelet-specific LNPs containing mRNA encoding for aprotinin or the reporter protein NanoLuc as a control. Platelets were then incubated for 4 hrs to enable target protein expression. To determine if aprotinin engineered platelets were more resistant to clot degradation, the inventors used rotational thromboelastometry (ROTEM), a common in vitro clinical tool used to measure clot formation kinetics and stability. Using an adapted ROTEM test, the inventors evaluated the sensitivity of aprotinin engineered platelets versus control platelets to clot degradation based on the firmness of the clot over time. Referring to FIGS. 8A and 8B, aprotinin engineered platelets showed an increased resistance to clot degradation compared to control platelets. This difference was observed in one other biological replicate experiment for a total of two successful trials. In the third replicate, platelet bag 3, no difference was observed between the control and treated platelets (FIG. 8C). This discrepancy from earlier results may be due to an overall decreased level of protein expression in bag 3, possibly due to donor variability. The inventors have noticed that platelet reporter protein expression can vary with different donors, indicating that some platelets are more amenable to transfection. To gauge the protein expression capabilities per bag, the NanoLuc expression in the control samples were measured. Both platelet bags 1 and 2 showed higher expression compared to bag 3 (FIG. 8D). Additionally, the NanoLuc expression correlated with the degree of clot stability in aprotinin engineered platelets (FIG. 8E). Collectively, these findings indicate that aprotinin expression is the driving force behind the increased resistance to clot degradation observed in the treated platelets.

[0091] To provide an example of how new functions could be introduced into platelets the inventors expressed myoglobin, an iron- and oxygen-binding protein found in the cardiac and skeletal muscle tissue. Within platelets, delivery and transport of oxygen is not a reported function; however adding this feature could potentially increase oxygen capacity in circulation. Platelets were transfected with a specialized mRNA sequence that encoded for both myoglobin and the reporter NanoLuc using the LNP engineering platform described herein and in International Publication No. WO 2023 / 220815. Myoglobin and NanoLuc were expressed as individual proteins from asingle mRNA transcript. Based on the transcript design, NanoLuc expression is entirely dependent on the ability of the cell machinery to successfully process and express myoglobin. After treatment with LNPs containing this specialized mRNA, NanoLuc was expressed (FIG. 9). Although the detection of NanoLuc is an indirect indicator that Myoglobin is being expressed, these results suggest that platelets have been endowed with a protein that may enable a new function: to bind and carry oxygen. This new function may be beneficial for enhancing oxygen carrying capacity in the blood after transfusion of the myoglobin engineered platelets as a future clinical application.

[0092] In summary, these experimental results demonstrate a lipid nanoparticle engineering platform that can be used to deliver RNA agents enabling downstream expression of target proteins in platelets which can specifically modulate existing platelet function or introduce new functions.Interpretation of Terms

[0093] Unless the context clearly requires otherwise, throughout the description and the claims:• “comprise”, “comprising”, and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”;• “connected”, “coupled”, or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements can be physical, logical, or a combination thereof;• “herein”, “above”, “below”, and words of similar import, when used to describe this specification, shall refer to this specification as a whole, and not to any particular portions of this specification;• “or”, in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list;• the singular forms “a”, “an”, and “the” also include the meaning of any appropriate plural forms. These terms (“a”, “an”, and “the”) mean one or moreunless stated otherwise;• “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes both (A and B) and (A or B);• “approximately” when applied to a numerical value means the numerical value ± 10%;• where a feature is described as being “optional” or “optionally” present or described as being present “in some embodiments” it is intended that the present disclosure encompasses embodiments where that feature is present and other embodiments where that feature is not necessarily present and other embodiments where that feature is excluded. Further, where any combination of features is described in this application this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only" and the like in relation to the combination of features as well as the use of "negative" limitation(s)” to exclude the presence of other features; and• “first” and “second” are used for descriptive purposes and cannot be understood as indicating or implying relative importance or indicating the number of indicated technical features.

[0094] Words that indicate directions such as “vertical”, “transverse”, “horizontal”, “upward”, “downward”, “forward”, “backward”, “inward”, “outward”, “left”, “right”, “front”, “back”, “top”, “bottom”, “below”, “above”, “under”, and the like, used in this description and any accompanying claims (where present), depend on the specific orientation of the apparatus described and illustrated. The subject matter described herein may assume various alternative orientations. Accordingly, these directional terms are not strictly defined and should not be interpreted narrowly.

[0095] Where a range for a value is stated, the stated range includes all sub-ranges of the range. It is intended that the statement of a range supports the value being at an endpoint of the range as well as at any intervening value to the tenth of the unit of the lower limit of the range, as well as any subrange or sets of sub ranges of the range unless the context clearly dictates otherwise or any portion(s) of the stated range is specifically excluded. Where the stated range includes one or both endpoints of the range, ranges excluding either or both of those included endpoints are alsoincluded in the invention.

[0096] Certain numerical values described herein are preceded by "about". In this context, "about" provides literal support for the exact numerical value that it precedes, the exact numerical value ±5%, as well as all other numerical values that are near to or approximately equal to that numerical value. Unless otherwise indicated a particular numerical value is included in “about” a specifically recited numerical value where the particular numerical value provides the substantial equivalent of the specifically recited numerical value in the context in which the specifically recited numerical value is presented. For example, a statement that something has the numerical value of “about 10” is to be interpreted as: the set of statements:• in some embodiments the numerical value is 10;• in some embodiments the numerical value is in the range of 9.5 to 10.5; and if from the context the person of ordinary skill in the art would understand that values within a certain range are substantially equivalent to 10 because the values with the range would be understood to provide substantially the same result as the value 10 then “about 10” also includes:• in some embodiments the numerical value is in the range of C to D where C and D are respectively lower and upper endpoints of the range that encompasses all of those values that provide a substantial equivalent to the value 10.

[0097] Specific examples of systems, methods and apparatus have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to systems other than the example systems described above. Many alterations, modifications, additions, omissions, and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and / or acts with equivalent features, elements and / or acts; mixing and matching of features, elements and / or acts from different embodiments; combining features, elements and / or acts from embodiments as described herein with features, elements and / or acts of other technology; and / or omitting combining features, elements and / or acts from described embodiments.

[0098] As will be apparent to those of skill in the art upon reading this disclosure,each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any other described embodiment(s) without departing from the scope of the present invention.

[0099] Any aspects described above in reference to apparatus may also apply to methods and vice versa.

[0100] Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, while processes or blocks are presented in a given order, alternative examples may perform routines having steps, or employ systems having blocks, in a different order, and some processes or blocks may be deleted, moved, added, subdivided, combined, and / or modified to provide alternative or subcombinations. Each of these processes or blocks may be implemented in a variety of different ways. Also, while processes or blocks are at times shown as being performed in series, these processes or blocks may instead be performed in parallel, simultaneously or at different times.

[0101] Various features are described herein as being present in “some embodiments”. Such features are not mandatory and may not be present in all embodiments. Embodiments of the invention may include zero, any one or any combination of two or more of such features. All possible combinations of such features are contemplated by this disclosure even where such features are shown in different drawings and / or described in different sections or paragraphs. This is limited only to the extent that certain ones of such features are incompatible with other ones of such features in the sense that it would be impossible for a person of ordinary skill in the art to construct a practical embodiment that combines such incompatible features. Consequently, the description that “some embodiments” possess feature A and “some embodiments” possess feature B should be interpreted as an express indication that the inventors also contemplate embodiments which combine features A and B (unless the description states otherwise or features A and B are fundamentally incompatible). This is the case even if features A and B are illustrated in different drawings and / or mentioned in different paragraphs, sections or sentences.

[0102] It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations,additions, omissions, and sub-combinations as may reasonably be inferred. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS1 . A mRNA-containing construct for use in the expression of a target protein in a platelet cell and / or platelet-interacting cell, wherein the mRNA-containing construct comprises a 5' RNA element operatively linked to a mRNA coding sequence being a mRNA sequence which encodes the amino acids of the target protein, wherein the 5' RNA element comprises one or more copies of a 5'-untranslated region (5' UTR) of an endogenous mRNA sequence and / or a 5'-untranslated region (5'UTR) of an exogenous mRNA sequence.

2. The mRNA-containing construct as defined in claim 1 , wherein the 5' UTR of an endogenous mRNA sequence comprises the 5' UTR of the mRNA sequence which encodes eukaryotic initiation factor 4E (elF4E) comprising SEQ ID NO: 1 , hemoglobin beta (HBB) comprising SEQ ID NO: 2, cyclooxygenase 1 (COX-1) comprising SEQ ID NO: 3, thymosin beta-4 (TMSB4X) comprising SEQ ID NO: 4, glycoprotein 1 B (GP1 BA) comprising SEQ ID NO: 5, glycoprotein I lib (CD36) comprising SEQ ID NO: 6, and plasminogen activator inhibitor-1 (PAI-1) comprising SEQ ID NO: 7, and combinations thereof.

3. The mRNA-containing construct as defined in claim 1 or 2, wherein the 5' UTR of the endogenous mRNA sequence comprises a full length sequence and / or a partial length sequence and / or segments of one or more of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7.

4. The mRNA-containing construct as defined in any one of claims 1 to 3, wherein the 5' UTR of the endogenous mRNA sequence comprises a sequence that has at least about 80% sequence identity to one or more of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7.

5. The mRNA-containing construct as defined in any one of claims 1 to 4, wherein the 5' UTR of an exogenous mRNA sequence comprises the 5' UTR of the RNA genome of Dengue fever virus (DV), comprising SEQ ID NO: 8 and / or the 5' UTR of the RNA genome of Tobacco mosaic virus (TMV),comprising SEQ ID NO: 9.

6. The mRNA-containing construct as defined in any one of claims 1 to 5, wherein the 5' UTR of an exogenous mRNA sequence comprises one or more internal ribosome entry sites (IRES).

7. The mRNA-containing construct as defined in claim 6, wherein the IRES is an IRES from a viral RNA genome.

8. The mRNA-containing construct as defined in claim 6 or 7, wherein the internal ribosome entry site (IRES) comprises one or more of the internal ribosome entry site (IRES) from the RNA genome of the encephalomyocarditis virus (EMCV), comprising SEQ ID NO: 10, the internal ribosome entry site (IRES) from the RNA genome of the Hepatitis C virus (HCV), comprising SEQ ID NO: 11 , the internal ribosome entry site (IRES) from the RNA genome of the Cricket paralysis virus (CrPV), comprising SEQ ID NO: 12, and combinations thereof.

9. The mRNA-containing construct as defined in any one of claims 1 to 8, wherein the 5' UTR of an exogenous mRNA sequence comprises a full length sequence and / or partial length sequence and / or segments of one or more of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 , and SEQ ID NO. 12.

10. The mRNA-containing construct as defined in any one of claims 1 to 9, wherein the 5' UTR of an exogenous mRNA sequence comprises a sequence that has at least about 80% sequence identity to one or more of SEQ ID NO.8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 , and SEQ ID NO. 12.11 . The mRNA-containing construct as defined in any one of claims 1 to 10, wherein the 5' UTR of the exogenous mRNA sequence comprises a synthetic RNA sequence which forms a highly folded secondary structure.

12. The mRNA-containing construct as defined in claim 11 , wherein the synthetic RNA sequence comprises a full length sequence and / or partial length sequence of SEQ ID NO: 13.

13. The mRNA-containing construct as defined in any one of claims 1 to 12, wherein the mRNA-containing construct additionally comprises a translation initiation sequence operatively linked to and placed between the 5' RNAelement and the mRNA coding sequence.

14. The mRNA-containing construct as defined in claim 13, wherein the translation initiation sequence comprises a Kozak consensus sequence or a variant thereof.

15. The mRNA-containing construct as defined in any one of claims 1 to 14, wherein the mRNA-containing construct comprises one or more additional RNA elements operatively linked to the 5' RNA element and / or the mRNA coding region.

16. The mRNA-containing construct as defined in claim 15, wherein the one or more additional RNA elements comprise one or more of a 5' cap operatively linked upstream of the 5' RNA element, a 3' UTR operatively linked downstream of the mRNA coding sequence, and a Poly(A) tail operatively linked downstream of the 3' UTR.

17. The mRNA-containing construct as defined in claim 16, wherein the 3' UTR comprises a full length sequence and / or partial length sequence and / or fragments of a 3' UTR of an mRNA sequence which encode an endogenous protein.

18. The mRNA-containing construct as defined in claim 16 or 17, wherein the 3' UTR comprises a full length sequence and / or partial length sequence and / or fragments of a 3' UTR of an mRNA sequence which encode one or more of eukaryotic translation initiation factor 4E (elF4E), hemoglobin beta (HBB), cyclooxygenase 1 (COX-1), thymosin beta-4 (TMSB4X), glycoprotein 1 B (GP1 BA), glycoprotein I lib (CD36), and plasminogen activator inhibitor-1 (PAI- 1).

19. The mRNA-containing construct as defined in any one of claims 16 to 18, wherein the 3' UTR comprises a full length sequence and / or partial length sequence and / or fragments of a 3' UTR of an mRNA sequence which is found in a RNA genome of one or more viruses.

20. The mRNA-containing construct as defined in any one of claims 16 to 19, wherein the 3' UTR comprises a full length sequence and / or partial length sequence and / or fragments of a 3' UTR of an mRNA sequence which is found in a RNA genome of one or more of Dengue fever virus (DV), Tobacco mosaicvirus (TMV), encephalomyocarditis virus (EMCV), Hepatitus C virus (HCV), and Cricket paralysis virus (CrPV),21. The mRNA-containing construct as defined in any one of claims 1 to 20, wherein the mRNA-containing construct is in the form of a linear messenger RNA (mRNA).

22. The mRNA-containing construct as defined any one of claims 1 to 20, wherein the one or more additional RNA elements comprise one or more selfamplifying specific sequence elements, the one or more self-amplifying specific sequence elements encode one or more of self-replicating proteins.

23. The mRNA-containing construct as defined in claim 22, wherein the one or more self-amplifying specific sequence elements are operatively linked and placed between the 5' RNA element and the mRNA coding sequence.

24. The mRNA-containing construct as defined in claim 22 or 23, wherein the one or more self-replicating proteins comprise viral replicase.

25. The mRNA-containing construct as defined in any one of claims 22 to 24, wherein the mRNA-containing construct is in the form of a self-amplifying RNA (saRNA).

26. The mRNA-containing construct as defined in any one of claims 1 to 20, wherein the mRNA-containing construct is in the form of a circular mRNA (circmRNA).

27. The mRNA-containing construct as defined in claim 26, wherein the 3' UTR and the 5' RNA element of the mRNA-containing construct are ligated to produce a covalently closed circle.

28. The mRNA-containing construct as defined in claim 26 or 27, wherein the circmRNA consists essentially of the 3' UTR, the 5' RNA element operatively linked to the 3' UTR, and the mRNA coding sequence operatively linked to both the 3' UTR and the 5' RNA element and placed therebetween.

29. The mRNA-containing construct as defined any one of claims 1 to 28, wherein the target protein encoded by the mRNA coding sequence comprises one or more of a coagulation factor, an antifibrinolytic, an anticoagulant, a fibrinolytic, an antimicrobial, an immunomodulator, an anti-cancer agent, an aptamer, a genetic editor, and / or a reporter protein.

30. A method of transfecting the mRNA-containing construct as defined in any one of claims 1 to 29 into a platelet cell to produce a transfected platelet cell which contains the mRNA-containing construct, wherein the method comprises encapsulating the mRNA-containing construct in a lipid nanoparticle (LNP).

31. The method as defined in claim 30, wherein the lipid nanoparticle comprises a composition which comprises a lipid mixture, the lipid mixture comprises: a. at least one (ionizable) cationic lipid; b. at least one helper lipid; c. a sterol; and d. at least one lipid-polyethylene glycol conjugate.

32. The method as defined in claim 30 or 31 , wherein the lipid mixture comprises: a. 30-55 mol % of the at least one (ionizable) cationic lipid; b. 5-20 mol % of the at least one helper lipid; c. 25-50 mol % of sterol; and d. 0.5 - 3 mol % of the at least one lipid-polyethylene glycol conjugate.

33. The method as defined in claim 31 or 32, wherein the at least one ionizable cationic lipid in the lipid mixture is one or more of CL4H6, SM-102, ALC-0315, CL1 H6, CL15H6, CL1 D6, or ALC-0159.

34. The method as defined in any one of claims 31 to 33, wherein the at least one helper lipid in the lipid mixture is one or more of phosphatidylcholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), egg sphingomyelin (ESM).

35. The method as defined in any one of claims 31 to 34, wherein the sterol in the lipid mixture is one or more of cholesterol and a cholesterol derivative.

36. The method as defined in any one of claims 31 to 35, wherein the at least one lipid polyethylene glycol conjugate is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000), l,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE- PEG2000), or PEG-l,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG).

37. A population of transfected platelet cells and / or platelet-interacting cellscontaining the mRNA-containing construct as defined in any one of claims 1- 28.