Lipid nanoparticle compositions incorporating an immunosuppressant for the delivery of self-amplifying RNA

Abstract

A lipid nanoparticle composition for use in the delivery of a self-amplifying RNA (saRNA) construct in one or more target cells is disclosed. The lipid nanoparticle composition comprises a lipid mixture comprising at least one (ionizable) cationic lipid, at least one helper lipid, a sterol, a corticosteroid such as Dexamethasone, and a least one lipid-polyethylene glycol conjugate. The saRNA construct comprises a first open reading frame which encodes one or more non-structural proteins, and a second open reading frame operatively linked to the first open reading frame. The second open reading frame comprises a coding region which encodes one or more target proteins. In some embodiments, the target proteins comprise one or more therapeutic proteins. In some embodiments, the target proteins comprise one or more one or more Cas proteins and / or variants thereof for use in CRISPR-based gene editing.

Description

LIPID NANOPARTICLE COMPOSITIONS INCORPORATING AN IMMUNOSUPPRESSANT FOR THE DELIVERY OF SELF-AMPLIFYING RNAThis invention was made with Government support under W81XWH-20-2-0041 awarded by the Medical Research and Development Command. The government has certain rights in the invention.Cross-Reference to Related Applications

[0001] This application claims priority from US application No. 63 / 562,900 filed 8 March 2024 and entitled INCREASING CIRCULATING LEVELS OF COAGULATION FACTOR FVII and US application No. 63 / 564,748 filed 13 March 2024 and entitled PHARMACOLOGICAL APPROACH TO INCREASE CIRCULATING PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) LEVELS, 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 / 562,900 filed 8 March 2024 and entitled INCREASING CIRCULATING LEVELS OF COAGULATION FACTOR FVII and US application No. 63 / 564,748 filed 13 March 2024 and entitled PHARMACOLOGICAL APPROACH TO INCREASE CIRCULATING PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) LEVELS which are hereby incorporated herein by reference for all purposes.Field

[0002] The present invention pertains to the delivery of nucleic acids such as saRNA and mRNA to target cells using lipid nanoparticles.Background

[0003] Self-amplifying RNA (saRNA) contains a viral replicase and conserved sequence elements derived from alphaviruses. Alphavirus genome consists of ~10-12 kb single-stranded, positive sense RNA segmented into two cistrons. The first cistron encodes for the non-structural proteins 1 through 4 (nsP1-4) that make up the replication machinery. The second cistron, the subgenomic RNA (sgRNA), encodes for the structural. In saRNA vectors derived from alphaviruses, the structural proteinsare entirely removed and replaced by a gene of interest. Upon entry into the cell, nsP1-4 polyproteins are translated initially. The nsPs form the replicative complex or replicase and generate more saRNA copies through a negative sense intermediate. The replicase utilizes the subgenomic promoter to create the positive sense subgenomic RNA at a higher rate than the genomic strand. The subgenomic RNA is translated to produce the therapeutic GOI.

[0004] The inventors have recognized a general need for improved delivery of saRNA using lipid nanoparticles (LNPs) into target cells so the saRNA can achieve extended functionality in the cells with low reactogenicity being triggered.Summary

[0005] One aspect of the invention pertains to a lipid nanoparticle composition for use in the delivery of a self-amplifying RNA (saRNA) construct in one or more target cells. In some embodiments, the target cells comprise liver cells. The lipid nanoparticle composition comprises a lipid mixture comprising at least one (ionizable) cationic lipid, at least one helper lipid, a sterol, a corticosteroid, and a least one lipidpolyethylene glycol conjugate. In some embodiments, the molar concentration of the corticosteroid in the lipid mixture is in the range of about 0.1 to 50 mol %, and in some embodiments, about 0.1 to 20 mol %, and in some embodiments, 0.1 to 10 mol %. In some embodiments, the corticosteroid comprises Dexamethasone. The saRNA construct comprises a first open reading frame which encodes one or more non- structural proteins, and a second open reading frame operatively linked to the first open reading frame. The second open reading frame comprises a coding region which encodes one or more target proteins.

[0006] The lipid nanoparticle may be used to deliver a plurality of mRNA constructs. In some embodiments, the plurality of mRNA constructs encode the same protein. In some embodiments, the plurality of mRNA constructs encode one or more different proteins.

[0007] In some embodiments, the target proteins comprise one or more therapeutic proteins. The one or more therapeutic proteins may comprise one or more of coagulation factor VII, plasminogen activator inhibitor-1 (PAI-1), lipoprotein lipase (LPL), alpha-1 antitrypsin (A1AT), protein S (PROS1), and tissue plasminogenactivator (TPA), plasminogen (PLG), fibrinogen (FGA), anti-GLP-1 , and insulin.

[0008] In some embodiments, the target proteins comprise one or more Cas proteins and / or variants thereof for use in CRISPR-based gene editing.

[0009] In some embodiments, the target proteins comprise one or more of SadCas9, AsdCas12a, SpCas9, NlaCas11 , Cas9 nickase, Prime editor max (PEmax), and human fibroblast growth factor receptor 3 (FGFR3) (transmembrane deletion).

[0010] One aspect of the invention pertains to use of a pharmaceutical composition comprising the lipid nanoparticle for the treatment of one or more disorders and diseases.

[0011] One aspect of the invention pertains to an mRNA construct comprising a coding region which encodes an open reading frame of coagulation factor VII (FVII). Some aspects of the invention pertain to use of a pharmaceutical composition comprising the mRNA construct which encodes FVII and a lipid nanoparticle for the treatment of bleeding disorder and / or excessive bleeding in a subject.

[0012] One aspect of the invention pertains to an mRNA construct comprising a coding region which encodes an open reading frame of plasminogen activator inhibitor (PAI-1). Some aspects pertain to use of a pharmaceutical composition comprising the mRNA construct which encodes PAI-1 and a lipid nanoparticle for the treatment of one or more of excessive bleeding, a bleeding disorder, cardiac fibrosis, and PAI-1 deficiency in a subject.

[0013] Further aspects and example embodiments are illustrated in the accompanying drawings and / or described in the following description.

[0014] 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

[0015] The accompanying drawings illustrate non-limiting example embodiments of the invention.

[0016] FIG. 1 illustrate results from treating wildtype mice with standard LNP containing saRNA encoding Firefly Luciferase.

[0017] FIG. 2 is a plot of bioluminescence emitted by the luciferase as a function of the number of days post-injection in mice that were treated with LNP-Dex formulationcontaining saRNA or standard LNP formulation containing saRNA.

[0018] FIG. 3 illustrate results from testing various ionizable lipids in the LNP-Dex formulation to evaluate compatibility of the various ionizable lipids with 3.85% of Dexamethasone in vitro (a,b) and in vivo (c).

[0019] FIG. 4 are in vivo imaging data showing wildtype mice that were treated subcutaneously with 12 or 17 pg of saRNA-Fluc encapsulated in LNP-Dex formulation 4- and 24-hours post-injection.

[0020] FIG. 5 are in vivo imaging data showing wildtype mice were treated with 12 or 3 pg of two different saRNA-Fluc constructs encapsulated in LNP-Dex formulation 4- and 24-hours post-injection.

[0021] FIG. 6A is a schematic diagram illustrating an example saRNA construct for bicistronic expression of a therapeutic protein and Flue. Fusion to Flue allows for inferring expression of the protein by using a luciferase assay. FIG. 6B a plot showing the results of a luciferase assay performed 16 hours after HEK cells were transfected with 100 nanograms of each of the saRNA constructs which encode the proteins of interest (SEAP, A1AT, LPL, tPA, and PROS1).

[0022] FIG. 7 is a plot comparing the expression levels of Flue encoded by saRNA constructs that are encapsulated using LNP-Dex formulations each containing different ratios of Dexamethasone.

[0023] FIG. 8A is a plot comparing the amount of blood loss in rats which have Von Willebrand disease (VWD) Type 3 that have been treated with either mRNA-FVIl LNP or mRNA NanoLuciferase (nLuc) as a control, after being induced with a tail transection bleeding

[0024] FIG. 8B is a plot illustrating results from a Rotational thromboelastometry (ROTEM) analysis of plasma collected healthy (normal), FVI I- / -, FVII I- / -, FIX- / -, and Von Willebrand Disease (VWD) Type 1-3, and which recombinant human FVII was added at final concentrations of 0 (left bar), 0.5 (middle bar) or 1 (right bar) ug / mL

[0025] FIG. 9A is a plot of plasma FVII (ng / mL) as a function of days post-injection of 30 pg of saRNA encoding mouse FVII ORF encapsulated in LNP-Dex.

[0026] FIG. 9B is a plot of plasma FVII (ng / mL) as a function of days post-injection of 20 pg of saRNA encoding mouse FVII ORF encapsulated in LNP-Dex.

[0027] FIG. 10 are results showing that hepatic delivery of FVII mRNA using LNPsincreases circulating levels of FVI I. Wildtype mice were treated with LNP containing mRNA encoding mouse FVII ORF and were euthanized at 6-, 24-, and 48-hours postinjection to quantify hepatic mRNA by qPCR (a) and plasma FVII levels by western blot (b). Wildtype swine were treated with LNP containing mRNA encoding human FVII ORF, and blood was collected 5 hours and 24 hours post-injection to quantify plasma FVII levels by western blot (c).

[0028] FIG. 11 are results showing that increasing circulating levels of FVII decreases blood loss after injury in a mouse model of hemophilia A. Wildtype (WT) or hemophilia A (FVI 11- / -) mice were treated with LNP containing mRNA encoding mouse FVII ORF 24 hours prior to inducing bleeding by transecting the tails 4 mm from the distal tip. Blood loss during the 20-minute monitoring period following tail transection, normalized by body weight are plotted in (a). Hepatic FVII mRNA levels were quantified by qPCR as shown in (b).

[0029] FIG. 12 are results from expressing enhanced variants of FVII in rodent models using mRNA-LNP. Wild-type mice were treated with LNP containing mRNA encoding wild-type human FVII (‘FVII’) or a human FVII variant designed for enhanced activity, expression, and / or half-life (‘FVII-Var.’). Mice were sacrificed at 7 hours post-treatment, and plasma levels of heterologously expressed FVII was assessed by immunoblotting for a C-terminal epitope tag as shown in (a). Control mice were sham injected with PBS (Untreated; ‘UT’). Rats deficient in Von Willebrand Factor were treated with LNP containing mRNA encoding human FVII variant designed for enhanced activity, expression, and / or half-life. Rats were sacrificed at the times indicated following treatment, plasma was collected for protein detection by immunoblotting using antibody recognizing C-terminal epitope tag as shown in (b).

[0030] FIG. 13 are results showing that excessive fibrinolysis in human PAI-1 deficient plasma can be corrected with recombinant PAI-1 protein. The percentage of clot remaining (also known as lysis index, LI) at 60 min (LI60) in normal human plasma or in PAI-1 deficient human plasma are shown in plot (a), (b) is a representative ROTEM curve of PAI-1 deficient plasma (blue) and PAI-1 deficient plasma with recombinant PAI-1 (400 ng / mL). c) is a plot showing LI60 of PAI-1 deficient plasma with the addition of recombinant PAI-1 protein.

[0031] FIG. 14 are results showing increased expression of circulating PAI-1 in micefollowing intravenous injection of mRNA PAI-1 LNP. a) WT mouse plasma PAI-1 concentrations following administration of mRNA PAI-1 LNP (mPAI-1) at 0.5 mg / kg or 0 mg / kg (PBS). Healthy normal mouse PAI-1 levels indicated by the shaded region. b) plot showing percentage of clot remaining in the presence of tPA in blood collected from mice administered PBS or mPAI-1 at 0.5 mg / kg. c) WT mouse plasma PAI-1 concentrations following administration of PBS or mPAI-1 at 0.1 mg / kg, 0.5 mg / kg, or 1 mg / kg at 6, 24, and 48 hours post-injection.

[0032] FIG. 15 are results showing that mRNA PAI-1 LNP results in circulating PAI-1 protein in PAI-1- / - mice, a) PAI-1- / - mouse plasma PAI-1 concentrations following administration of mRNA PAI-1 LNP (mPAI-1) at 1 mg / kg or saline at 6- and 24-hours post-injection. Healthy normal mouse PAI-1 levels indicated in the shaded region, b) PAI-1 + / + mouse plasma PAI-1 concentrations following administration of mPAI-1 (1 mg / kg) or saline at 6- and 24-hours post-injection. Accurate limit of detection of assay indicated by dashed line.

[0033] FIG. 16 is a plot showing inferred replication of saRNA containing the coding sequence of PAI-1 , AsdCAs12a, SadCas9 and FLuc as a control.Detailed Description

[0034] 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

[0035] “Untranslated region” or “UTR” refers to RNA sequences located on the 5' or 3' of a saRNA or 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 saRNA or mRNA coding sequence (a “5' UTR”) or 3' to (or downstream of) the saRNA or mRNA coding sequence (a “3' UTR”).

[0036] “Downstream” means a location in a nucleic acid sequence located 3' to a given reference point in that nucleic acid sequence.

[0037] “Upstream” means a location in a nucleic acid sequence located 5' to a given reference point in that nucleic acid sequence.

[0038] “Open reading frame” or “ORF” means a stretch of nucleic acid which comprises a series of codons capable of being translated into a polypeptide by the appropriate cellular transcription / translation machinery.

[0039] “Sequence identity” refers to the degree of identify between nucleotides in two or more aligned sequences, when aligned using a sequence alignment program.Example embodiments of saRNA constructs encapsulated in immunosuppressantincorporated LNP formulations such as LNP-Dex formulations

[0040] Some aspects of the present invention pertain to a particularly efficient and robust system for delivering self-amplifying RNA (saRNA) into target cells using lipid nanoparticles (LNP) for the expression of one or more target proteins that are encoded by the saRNA.

[0041] Experimental results produced by the inventors have shown that the use of lipid nanoparticles (LNPs) to deliver saRNA triggers the innate response and results in acute inflammation (FIG. 1). The inflammation caused by the delivery platform is believed to be exacerbated by the encapsulated synthetic saRNA that triggers an antiviral response. Due to replication through double-stranded intermediates and production of viral non-structural proteins, more pro-inflammatory innate sensors are believed to be triggered.

[0042] Proof of concept experiments have demonstrated that extended saRNA expression (e.g., saRNA expression of more than 30 days) and low immunogenicity (e.g., low levels of inflammatory response triggered by the LNP delivery platform) in target cells can be achieved by using an immunosuppressant-incorporated LNP such as a Dexamethasone-incorporated LNP (referred to herein as a “LNP-Dex formulation”) as the saRNA delivery system. In some embodiments, the target cells comprise liver cells.

[0043] An immunosuppressant-incorporated LNP formulation such as the LNP-Dex formulation comprises a lipid mixture which comprises at least one (ionizable) cationiclipid, at least one helper lipid, a sterol, a corticosteroid such as Dexamethasone, and a least one lipid-polyethylene glycol conjugate.

[0044] In some embodiments, the immunosuppressant-incorporated LNP formulation comprises a molar concentration of corticosteroid in the range of from about 0.1 mol % to about 50 mol %, and in some embodiments, in the range of from about 0.1 mol % to about 25 mol %, and in some embodiments, in the range of from about 0.1 mol% to about 10 mol %, and in some embodiments, in the range of from about 0.1 mol% to about 5 mol %.

[0045] In some embodiments, the lipid mixture comprises about 30-70 mol % of the at least one (ionizable) cationic lipid, about 0.1-50 mol% of the sterol, about 5-20 mol% of the at least one helper lipid, about 0.1-50 mol% of corticosteroid, and about 0.5-4 mol% of the at least one lipid-polyethylene glycol conjugate.

[0046] In some embodiments, the lipid mixture comprises about 30-55 mol % of the at least one (ionizable) cationic lipid, about 25-50 mol % of sterol, about 5-20 mol % of the at least one helper lipid, about 0.1 to 50 mol % of corticosteroid and about 0.5-3 mol % of at least one lipid-polyethylene glycol conjugate.

[0047] In some example embodiments, the molar ratio of the at least one (ionizable) cationic lipid, the sterol, the at least one helper lipid, the corticosteroid, and the at least one lipid-polyethylene glycol conjugate is 50: 34.65: 10: 3.85: 1.5 %.

[0048] In some embodiments, the corticosteroid comprises Dexamethasone. In some embodiments, the molar ratio of sterol to corticosteroid such as Dexamethasone is adjusted to optimize one or more of duration of saRNA expression, level of saRNA expression, level of immunogenicity and / or toxicity of the immunosuppressantincorporated LNP formulation in the delivery of saRNA in target cells. In some embodiments, the combined molar ratio of sterol and corticosteroid such as Dexamethasone in the immunosuppressant-incorporated LNP formulation is in the range of from about 10 to about 50 mol %, including any values and subranges therebetween. In some embodiments, the molar ratio of the corticosteroid such as Dexamethasone and the sterol in the immunosuppressant-incorporated LNP formulation is in the range of from about 1 :7 to about 1 :12, and in some embodiments, in the range of from about 1 :8 to about 1 :11 , and in some example embodiments, about 1 :9.

[0049] Non-limiting examples of at least one ionizable cationic lipid in the lipid mixture include one or more of DLin-MC3-DMA, C12-200, SM-102, A18-2C18, DOPE-based ionizable lipids, C16-18, DOTAP derivatives, DODAP, ALC-0315, DODMA, CKK-E12 and its analogs, CL4H6, CL1 H6, CL15H6, CL1 D6, ALC-0159, Lipid A9, CL1 , and 7C1.

[0050] In some embodiments, the sterol in the lipid mixture is cholesterol and / or a cholesterol derivative.

[0051] In some embodiments, 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).

[0052] Non-limiting examples of at least one corticosteroid in the lipid mixture include one or more of Dexamethasone, 9a-Fluoro-16a-methyl-11 p, 17a, 21 -trihydroxy-1 , 4- pregnadiene-3, 20-dione (CAS Number: 50-02-2), betamethasone (CAS Number: 378- 44-9), Flumethasone (CAS Number:2135-17-3), Prednisolone (CAS Number: 50-24- 8), isoflupredone (CAS Number: 338-98-7) and / or analogs and derivatives thereof that retain at least about 50% (or in some embodiments, between 50% to 99% including any subrange or value therebetween) of the activity of 9a-Fluoro-16a- methyl-1 i p, 17a, 21 -trihydroxy-1 , 4-pregnadiene-3, 20-dione.

[0053] 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).

[0054] In some embodiments, a saRNA construct may be encapsulated in the immunosuppressant-incorporated LNP formulation for delivery into target cells. The saRNA construct comprises a first open reading frame which encodes one or more non-structural proteins, and a second open reading frame or the subgenomic RNA (sgRNA) which encodes the one or more target proteins.

[0055] The second open reading frame may be operatively linked downstream of the first open reading frame.

[0056] In some embodiments, the second open reading frame is operatively linked to a subgenomic promoter (SGP). In some embodiments, the subgenomic promoter is or is derived from the genome of an alphavirus. The alphaviruses are small, spherical, enveloped viruses with single-stranded, positive-sense, RNA genomes.

[0057] Non-limiting examples of “alphaviruses” include the Barmah Forest virus complex (e.g., Barmah Forest virus), Eastern equine encephalitis complex (e.g., seven antigenic types of Eastern equine encephalitis virus, Middelburg virus complex (e.g., Middelburg virus), Ndumu virus complex (e.g., Ndumu virus), Semliki Forest virus complex (e.g., Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong Nyong virus, and its subtype Igbo-Ora virus, Ross River virus and its subtypes Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Me Tri virus), Venezuelan equine encephalitis complex (e.g., Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Rio Negro virus, Trocara virus and its subtype Bijou Bridge virus, Venezuelan equine encephalitis virus); Western equine encephalitis complex (e.g., Aura virus, Babanki virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Whataroa virus, Buggy Creek virus, Fort Morgan virus, Highlands J virus, Western equine encephalitis virus), unclassified viruses such as Salmon pancreatic disease virus, Sleeping Disease virus and Southern elephant seal virus, Tonate virus.

[0058] In some embodiments, the one or more non-structural proteins that are encoded by the first open reading frame form a replicase complex or replicase. The replicase complex comprises a plurality of polyproteins which function to generate additional copies of the saRNA by initially synthesizing a negative-sensed RNA as an intermediate. The replicase complex utilizes the subgenomic promoter to create a positive sense subgenomic RNA. The subgenomic RNA is then translated to produce the one or more target proteins.

[0059] In some embodiments, the one or more non-structural proteins comprise an alphavirus replicase. The alphavirus replicase comprises one or more nsP1 , nsP2, nsP3 and nsP4. The alphavirus replicase is or is derived from a replicase encoded by the genome of an alphavirus.

[0060] In some example embodiments, the first open reading frame encodes a replicase that is or is derived from the genome of Venezuelan equine encephalitisvirus (VEEV). In such embodiments, the first open reading frame comprises a sequence set forth in SEQ ID NO. 1 , or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange there between) to SEQ ID NO. 1.

[0061] In some embodiments, the saRNA construct additionally comprise a 5’ UTR. In some embodiments, the 5’ UTR comprises conserved sequence elements (CSEs). The 5’ UTR may be operatively linked upstream of the first open reading frame.

[0062] In some embodiments, the saRNA construct additionally comprise a 3’ UTR. In some embodiments, the 3’ UTR comprises conserved sequence elements. The 3’ UTR may be operatively linked downstream of the second open reading frame.

[0063] In some embodiments, the conserved sequence elements are or are derived from the conserved sequence elements encoded by the genome of an alphavirus. In some embodiments, the conserved sequence elements are selected from CSE-1 , CSE-2, CSE-3 and / or CSE-4, or is derived from one or more of CSE-1 , CSE-2, CSE- 3 and / or CSE-4. Such conserved sequence elements are understood to be required for viral replication.

[0064] In some example embodiments, the 5’ UTR comprises conserved sequence elements that are derived from the genome of Venezuelan equine encephalitis virus (VEEV). In some embodiments, the 5’ UTR comprises a sequence set forth in SEQ ID NO. 2 or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange there between) to SEQ ID NO. 2.

[0065] In some example embodiments, the subgenomic promoter is or is derived from the genome of Venezuelan equine encephalitis virus (VEEV). In some embodiments, the subgenomic promoter comprises a sequence set forth in SEQ ID NO. 3 or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of fromabout 50% to about 99% sequence identity (including any value and subrange there between) to SEQ ID NO. 3.

[0066] In some example embodiments, the 3’UTR comprises conserved sequence elements that are derived from the genome of Venezuelan equine encephalitis virus (VEEV). In some embodiments, the 3’ UTR comprises a sequence set forth in SEQ ID NO. 4 or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange there between) to SEQ ID NO. 4.

[0067] It will be understood that the expression “derived from” means that the nucleic acid sequence which is derived from (another) nucleic acid sequence, shares at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, at least about 99% with the nucleic acid sequence from which it is derived.

[0068] The saRNA construct additionally comprises one or more additional RNA elements operatively linked upstream of the 5’ UTR and / or downstream of the 3’ UTR. The one or more additional RNA elements may comprise one or more sequences which, in combination with the first and second open reading frames and the 5’ UTR and 3’ UTR, form a functional saRNA. A functional saRNA allows for proper translation of the saRNA into the encoded target protein.

[0069] In some embodiments, the additional one or more RNA elements comprise a 5' cap structure operatively linked upstream of the 5’ UTR. In some embodiments, the 5' cap structure is positioned at the 5' end of the 5’ UTR. 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.

[0070] In some embodiments, the additional one or more RNA elements comprise apoly (A) tail operatively linked downstream of 3’ UTR. In some embodiments, the poly (A) tail is positioned at the 3' end of the 3’ UTR. 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. In some example embodiments, the poly (A) tail comprises a sequence set forth in SEQ ID NO. 5 or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 5.

[0071] The second open reading frame of the saRNA construct comprises a coding region which encodes for one or more target proteins. The one or more target proteins may comprise one or more therapeutic proteins. The one or more therapeutic proteins may be expressed in the one or more target cells for the treatment of one or more disorders and / or diseases.

[0072] In some embodiments, the coding region encodes an open reading frame of coagulation factor VII (FVII). In some embodiments, the FVII comprises wildtype human coagulation factor VII (hFVIl). In some example embodiments, the nucleic acid sequence which encodes the wildtype hFVIl is set forth in SEQ ID NO: 6, or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 70% to about 99% sequence identity (including any value and subrange there between) to SEQ ID NO. 6.

[0073] In some embodiments, the FVII comprises a variant of hFVIl. A “variant” covers a polypeptide which differs in one or more amino acid residues from the wildtype hFVIl by one or more modifications. As used herein “modification” encompasses insertions, deletions, substitutions including but are not limited to nucleoside base modifications, and combinations thereof. In some example embodiments, the nucleic acid sequence of the variant of hFVIl comprises one or more modified nucleosides. The one or more modified nucleosides may be selectedfrom pseudouridine and / or 1-methyl-pseudouridine. In some embodiments, one or more of the uridine bases in the nucleic acid sequence of the wildtype hFVI I are replaced with one or more pseudouridine and 1-methyl-pseudouridine. In some example embodiments, the nucleic acid sequence of the wildtype hFVH is depleted of all or substantially all uridine (U) bases. In some example embodiments, the nucleic acid sequence which encodes the variant hFVH is set forth in SEQ ID NO: 7, or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 7

[0074] In some embodiments, the FVII protein comprises murine coagulation factor VII (mFVIl). In some example embodiments, the nucleic acid sequence which encodes the mFVIl is set forth in SEQ ID NO: 8, or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 8.

[0075] In some embodiments, the saRNA construct which encodes an open reading frame of the murine FVII protein comprises a nucleic acid sequence set forth in SEQ ID NO: 9, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 9.

[0076] In some embodiments, the coding region encodes an open reading frame of plasminogen activator inhibitor-1 (PAI-1). In some embodiments, the PAI-1 protein comprises wildtype murine PAI-1 (mPAI-1). In some example embodiments, the nucleic acid sequence which encodes the wildtype mPAI-1 is set forth in SEQ ID NO: 10, or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in therange of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 10.

[0077] In some embodiments, the PAI-1 protein comprises wildtype human PAI-1 (hPAI-1). In some example embodiments, the nucleic acid sequence which encodes the wildtype hPAI-1 is set forth in SEQ ID NO: 11 , or a sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 11.

[0078] In some embodiments, the saRNA construct which encodes an open reading frame of the murine PA1-1 protein comprises a nucleic acid sequence set forth in SEQ ID NO: 12, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 12.

[0079] In some embodiments, the saRNA construct which encodes an open reading frame of the human PA1-1 protein comprises a nucleic acid sequence set forth in SEQ ID NO: 13, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 13.

[0080] In some embodiments, the saRNA construct encodes an open reading frame of one or more coagulation proteins, metabolic proteins, age-associated proteins.

[0081] In some embodiments, the saRNA construct encodes an open reading frame of one or more therapeutic proteins for the treatment of a excessive bleeding, bleeding disorder, a metabolic disorder, obesity and / or diabetes.

[0082] In some example embodiments, the saRNA construct encodes an open reading frame of one or more of lipoprotein lipase (LPL), alpha-1 antitrypsin (A1AT), protein S (PROS1), and tissue plasminogen activator (TPA), plasminogen (PLG),fibrinogen (FGA), anti-GLP-1 , and insulin.

[0083] In some embodiments, the saRNA construct encodes an open reading frame of LPL. In some example embodiments, the saRNA construct encodes an open reading frame of human LPL-S447X variant which comprises a nucleic acid sequence set forth in SEQ ID NO. 15, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 15.

[0084] In some embodiments, the saRNA construct which encodes an open reading frame of human A1AT comprises a nucleic acid sequence set forth in SEQ ID NO. 16, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 16.

[0085] In some embodiments, the saRNA construct which encodes an open reading frame of human PROS1 comprises a nucleic acid sequence set forth in SEQ ID NO. 17, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 17.

[0086] In some embodiments, the saRNA construct which encodes an open reading frame of human TPA comprises a nucleic acid sequence set forth in SEQ ID NO. 18, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 18.

[0087] In some embodiments, the saRNA construct which encodes an open reading frame of human PLG comprises a nucleic acid sequence set forth in SEQ ID NO. 21 ,or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 21.

[0088] In some embodiments, the saRNA construct which encodes an open reading frame of human FGA comprises a nucleic acid sequence set forth in SEQ ID NO. 24, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 24.

[0089] In some embodiments, the saRNA construct which encodes an open reading frame of two linked copies of human GLP-1 agonist comprises a nucleic acid sequence set forth in SEQ ID NO.30, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 30.

[0090] In some embodiments, the saRNA construct which encodes an open reading frame of human insulin comprises a nucleic acid sequence set forth in SEQ ID NO. 25, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 25.

[0091] It will be understood that the described saRNA construct may be adapted to express different therapeutic proteins which may be used to treat various diseases and disorders. The described saRNA construct may for example be adapted to express different therapeutic proteins which may be used to treat various bleeding diseases, aging diseases, cardiovascular diseases, metabolic diseases, and / orgenetic disorders. The described saRNA construct may be adapted to express different therapeutic proteins which may be used for enzyme replacement therapies such as but are not limited to Gaucher Disease, Pompe disease and Mucopolyssaccharidosis, protein replacement therapies, cancer treatment and / or other immunotherapies.

[0092] Some aspects of the invention pertain to the use of a pharmaceutical composition comprising the described immunosuppressant-incorporated LNP formulations such as LNP-Dex formulations for the delivery of the described saRNA constructs into one or more target cells for the treatment of one or more disorders and / or diseases. In some embodiments, the one or more target cells comprise liver cells.

[0093] In some embodiments, the pharmaceutical composition is formulated for administration to a subject in need thereof by one or more of intravenous, subcutaneous, intraperitoneal, intratumoral and intramuscular administration.

[0094] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of FVII for the delivery of the saRNA construct into one or more target cells for the treatment of excessive bleeding such as from a bleeding disorder and / or bleeding resulting from other etiology (e.g., trauma). FVII which are encoded by the saRNA are expressed in the one or more target cells. The expressed FVII increases circulating levels of FVII in the target cells, thereby decreases bleeding in a mammalian subject being treated with such immunosuppressant-incorporated LNP encapsulated saRNA.

[0095] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of PAI-1 for the delivery of the saRNA construct into one or more target cells for the treatment of excessive bleeding and / or cardiac fibrosis and / or mammalian subjects which has one or more mutations in the SERPINE-1 gene which may lead to PAI-1 deficiency. PAI-1 which are encoded by the saRNA are expressed in the one or more target cells. The expressed PAI-1 increases circulating levels of PAI-1 in the target cells, thereby inhibiting fibrinolysis in a subject treated with such immunosuppressant-incorporated LNP encapsulated saRNA.

[0096] In some embodiments, the immunosuppressant-incorporated LNPencapsulated saRNA encodes an open reading frame of LPL for the delivery of the saRNA construct into one or more target cells for the treatment of lipoprotein lipase deficiency (LPL-D). LPL which are encoded by the saRNA are expressed in the one or more target cells.

[0097] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of A1 AT for the delivery of the saRNA construct into one or more target cells for the treatment of Alpha-1 antitrypsin (A1AT) deficiency. A1AT which are encoded by the saRNA are expressed in the one or more target cells.

[0098] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of PROS1 for the delivery of the saRNA construct into one or more target cells for the treatment of thrombophilia. PROS1 which are encoded by the saRNA are expressed in the one or more target cells.

[0099] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of TPA for the delivery of the saRNA construct into one or more target cells for the treatment of ischemic stroke. TPA which are encoded by the saRNA are expressed in the one or more target cells.

[0100] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of PLG for the delivery of the saRNA construct into one or more target cells for the treatment of plasminogen deficiency. Plasminogen which are encoded by the saRNA are expressed in the one or more target cells.

[0101] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of FGA for the delivery of the saRNA construct into one or more target cells for the treatment of fibrinogen deficiency. Fibrinogen which are encoded by the saRNA are expressed in the one or more target cells.

[0102] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of anti-GLP1 or GLP-agonist for the delivery of the saRNA construct into one or more target cells for the treatment of one or more of metabolic disorders, obesity and diabetes. Anti-GLP1 which areencoded by the saRNA are expressed in the one or more target cells.

[0103] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of insulin for the delivery of the saRNA construct into one or more target cells for the treatment of diabetes. Insulin which are encoded by the saRNA are expressed in the one or more target cells.

[0104] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of insulin for the delivery of the saRNA construct into one or more target cells for the treatment of Fabry’s disease. Ceramide trihexosidase (a-galactosidase-A) which are encoded by the saRNA may be expressed in the one or more target cells.

[0105] In some embodiments, the immunosuppressant-incorporated LNP encapsulated saRNA encodes an open reading frame of a coagulation protein (such as prothrombin, tissue factor, thrombomodulin, Protein C, anti-thrombin and Factors V, IX, VIII, XI, XII and XIII) for the delivery of the saRNA construct into one or more target cells.

[0106] Some aspects of the invention pertain to saRNA constructs that are adapted for use in gene editing, in particular, allowing the targeted and site-directed manipulation of a genome of interest. In some embodiments, the saRNA construct encodes an open reading frame of one or more nucleases. Some nucleases may be used to break a nucleic acid of interest at a defined position to induce either a doublestrand break (DSB) or one or more single-strand breaks. Alternatively, some nucleases can be chimeric or mutated variants, which no longer comprises a nuclease function, but rather operates as recognition molecules in combination with another enzyme. In some embodiments, the one or more nucleases comprise one or more endonucleases. Non-limiting examples of suitable endonucleases include meganucleases, zinc finger nucleases, TALE nucleases and CRISPR nucleases as part of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.

[0107] In some embodiments, the saRNA construct encodes an open reading frame of one or more CRISPR nucleases. Non-limiting examples of CRISPR nucleases include a Cas protein and or variants thereof, such as SpCas9, HiFi Cas9, spRY Cas9, AsCas12a, AsdCas12a LbCas12a, SaCas9, SadCas9, DpbCas12e,PlmCas12e, Cas12j3, Cas11 , and Cas12j2, etc.

[0108] In some embodiments, the saRNA construct which encodes an open reading frame of SpCas9 comprises a nucleic acid sequence set forth in SEQ ID NO. 26, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 26.

[0109] In some embodiments, the saRNA construct which encodes an open reading frame of NlaCas11 comprises a nucleic acid sequence set forth in SEQ ID NO. 27, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 27.

[0110] In some embodiments, the saRNA construct which encodes an open reading frame of Cas9 nickase comprises a nucleic acid sequence set forth in SEQ ID NO. 28, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 28.

[0111] In some embodiments, the saRNA construct which encodes an open reading frame of Prime editor max (PEmax) comprises a nucleic acid sequence set forth in SEQ ID NO. 29, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 29.

[0112] In some embodiments, the saRNA construct which encodes an open reading frame of human fibroblast growth factor receptor 3 (FGFR3) (transmembrane deletion) comprises a nucleic acid sequence set forth in SEQ ID NO. 31 , or a nucleicacid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 31.

[0113] In some example embodiments, the saRNA construct encodes an open reading frame of SadCas9 comprising a nucleic acid sequence set forth in SEQ ID NO. 19, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 19.

[0114] In some example embodiments, the saRNA construct encodes an open reading frame of AsdCas12a comprising a nucleic acid sequence set forth in SEQ ID NO. 20, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value and subrange therebetween) to SEQ ID NO. 20.

[0115] Some aspects of the invention pertain to encapsulating the described saRNA construct which encodes the open reading frame of the one or more nucleases in the described immunosuppressant-incorporated LNP formulation for use in gene editing such as by a CRISPR-based gene editing system. In some embodiments, the saRNA construct which encodes the open reading frame of the one or more nucleases is coencapsulated into the immunosuppressant-incorporated LNP formulation with one or more guide RNAs (gRNA). In some embodiments, the gRNA is encapsulated into the immunosuppressant-incorporated LNP formulation separately from the saRNA. The gRNA comprises a RNA sequence which recognizes a target DNA region of interest. The RNA sequence of the gRNA may be complementary to the sequence of the target DNA region of interest. The gRNA functions to direct the one or more nucleases to such target DNA region of interest for editingExample embodiments of mRNA constructs for increasing circulating levels of coagulation factor FVII

[0116] Some aspects of the invention pertain to mRNA constructs for increasing circulating levels of coagulation factor VII (FVII). In some embodiments, the mRNA construct is encapsulated in a LNP for delivery into one or more target cells. In some embodiments, the one or more target cells comprise liver cells.

[0117] Bleeding remains a concern for most people with bleeding disorders. This is particularly important in hemophilia with neutralizing antibodies and in other rare bleeding disorders (RBDs), such as FVII deficiency, due to limited therapeutic options. The bleeding risk in hemophilia patients is primarily managed by factor replacement therapy, but neutralizing antibodies can develop against the replaced factor and render this therapy ineffective. Current solutions include administrating “bypassing agents”, including recombinant activated coagulation factor VII (rFVIla), which activate the coagulation cascade downstream of the affected coagulation factor. rFVIla and other bypassing agents, however, are less suitable for prophylactic use and carry risk of thrombosis. An alternative approach to using rFVIla, an activated enzyme, is to increase circulating levels of zymogen FVII, which we expect will be longer-acting and safer for patients with bleeding disorders.

[0118] Proof of concept experiments have demonstrated that lipid nanoparticle (LNP)- mediated hepatic delivery of mRNA encoding FVII increases circulating FVII zymogen and thus have broad utility to decrease bleeding in patients with bleeding disorders.

[0119] The mRNA construct comprises a coding sequence which encodes an open reading frame of FVII.

[0120] In some embodiments, the FVII comprises a variant of FVII. A “variant” covers a polypeptide which differs in one or more amino acid residues from the wildtype FVII by one or more modifications. In some example embodiments, the nucleic acid sequence of the variant of FVII comprises one or more modified nucleosides. The one or more modified nucleosides may be selected from pseudouridine and / or 1-methyl- pseudouridine. In some embodiments, one or more of the uridine bases in the nucleic acid sequence of the wildtype FVII are replaced with one or more pseudouridine and 1-methyl-pseudouridine. In some example embodiments, the nucleic acid sequence of the wildtype FVII is depleted of all or substantially all uridine (U) bases.

[0121] In some embodiments, the FVII comprises wildtype human coagulation factor VII (hFVIl) or a variant of hFVI I .

[0122] In some embodiments, the mRNA construct additionally comprises a 5’ UTR operatively linked upstream of the coding sequence.

[0123] In some embodiments, the mRNA construct additionally comprises a 3’ UTR operatively linked downstream of the coding sequence.

[0124] The 5’ UTR and 3’ UTR may be any suitable naturally-occurring sequences (i.e. , sequences which may be found or derived from a natural source such as eukaryotic cells and / or viruses) or synthetic sequences (i.e., sequences which are not found or derived from natural sources) that facilitate the stabilization and translation of the mRNA. In some embodiments, the 5’ UTR is or is derived from a human alpha gene and / or beta globin gene. In some embodiments, the 3’ UTR comprises one or more of the human mitochondrial 12S ribosomal RNA gene, the AES / TLE5 gene and segments thereof.

[0125] The mRNA construct additionally comprises one or more additional RNA elements operatively linked upstream of the 5’ UTR and / or downstream of the 3’ UTR. The one or more additional RNA elements may comprise one or more sequences which, in combination with the 5’ UTR and 3’ UTR, form a functional mRNA. A functional mRNA allows for proper translation of the mRNA into the encoded FVII protein.

[0126] In some embodiments, the additional one or more RNA elements comprise a 5' cap structure operatively linked upstream of the 5’ UTR. The 5’ cap structure has been previously described herein so such description will not be repeated for brevity.

[0127] In some embodiments, the additional one or more RNA elements comprise a poly (A) tail operatively linked downstream of 3’ UTR. The poly(A) tail has been previously described herein so such description will not be repeated for brevity.

[0128] In some embodiments, the mRNA construct which encodes an open reading frame of the FVII protein comprises a nucleic acid sequence set forth in SEQ ID NO: 22, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, about 70% sequence identity, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity(including any value or subrange therebetween) to SEQ ID NO. 22.

[0129] Some aspects of the invention pertain to the use of the described immunosuppressant-incorporated LNP formulations such as LNP-Dex formulations or standard LNP formulations for the delivery of the described mRNA which encodes FVII into one or more target cells for the treatment of excessive bleeding such as from a bleeding disorder and / or bleeding resulting from other etiology (e.g., trauma). FVII which are encoded by the mRNA are expressed in the one or more target cells. The expressed FVII increases circulating levels of FVII in the target cells, thereby decreases bleeding in a mammalian subject being treated with such immunosuppressant-incorporated LNP encapsulated mRNA or LNP encapsulated mRNA. In some embodiments, the one or more target cells comprise liver cells.

[0130] As used herein, a standard LNP formulation is free of a corticosteroid such as a Dexamethasone. In some embodiments, the standard LNP formulation comprises a lipid mixture which comprises at least one (ionizable) cationic lipid, at least one helper lipid, a sterol, and a least one lipid-polyethylene glycol conjugate.

[0131] In some embodiments, the lipid mixture comprises about 30-70 mol % of the at least one (ionizable) cationic lipid, about 0.1-50 mol % of sterol, about 5-20 mol % of the at least one helper lipid, and about 0.5-4 mol % of at least one lipid-polyethylene glycol conjugate.Example embodiments of mRNA constructs for reversing PAI-1 deficiency

[0132]

[0133] Some aspects of the invention pertain to mRNA constructs for increasing circulating levels of plasminogen activator inhibitor (PAI-1). In some embodiments, the mRNA construct is encapsulated in a LNP for delivery into one or more target cells. In some embodiments, the one or more target cells comprise liver cells.

[0134] Plasminogen activator inhibitor-1 (PAI-1) deficiency is a rare disorder that causes moderate to severe bleeding and life-threatening cardiac fibrosis, caused by mutation in the SERPINE-1 gene and no detectable circulating PAI-1 protein. There are currently no therapies that can effectively replace PAI-1 because the protein has a short half-life of under 45 minutes. An alternative approach to using recombinant protein is to endogenously increase circulating levels of PAI-1 using mRNA therapy,which expresses protein for days in vivo.

[0135] Proof of concept experiments demonstrate that delivering mRNA encoding PAI-1 to the liver, a major site of PAI-1 synthesis, using lipid nanoparticles expresses and increases circulating PAI-1 protein. In some embodiments, methods of treating a subject that is deficient in PAI-1 comprise repeated dosing of the described mRNA which encodes PAI-1 and / or the described mRNA which encodes PAI-1 contained in LNPs. Such repeated dosing may be a preventative treatment of bleeding and cardiac fibrosis. “Repeated dosing” means the subject will be dosed with the described mRNA over a period of time, which may in some embodiments, extend over several days, weeks, months or years.

[0136] In some embodiments, the mRNA construct additionally comprises a 5’ UTR operatively linked upstream of the coding sequence.

[0137] In some embodiments, the mRNA construct additionally comprises a 3’ UTR operatively linked downstream of the coding sequence.

[0138] The 5’ UTR and 3’ UTR may be any suitable naturally-occurring sequences (i.e. , sequences which may be found or derived from a natural source such as eukaryotic cells and / or viruses) or synthetic sequences (i.e., sequences which are not found or derived from natural sources) that facilitate the stabilization and translation of the mRNA. In some embodiments, the 5’ UTR is or is derived from a human alpha gene and / or beta globin gene. In some embodiments, the 3’ UTR comprises one or more of the human mitochondrial 12S ribosomal RNA gene, the AES / TLE5 gene and segments thereof.

[0139] The mRNA construct additionally comprises one or more additional RNA elements operatively linked upstream of the 5’ UTR and / or downstream of the 3’ UTR. The one or more additional RNA elements may comprise one or more sequences which, in combination with the 5’ UTR and 3’ UTR, form a functional mRNA. A functional mRNA allows for proper translation of the mRNA into the encoded FVII protein.

[0140] In some embodiments, the additional one or more RNA elements comprise a 5' cap structure operatively linked upstream of the 5’ UTR. The 5’ cap structure has been previously described herein so such description will not be repeated for brevity.

[0141] In some embodiments, the additional one or more RNA elements comprise apoly (A) tail operatively linked downstream of 3’ UTR. The poly(A) tail has been previously described herein so such description will not be repeated for brevity.

[0142] In some embodiments, the mRNA construct which encodes an open reading frame of the human PAI-1 protein comprises a nucleic acid sequence set forth in SEQ ID NO: 14, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value or subrange therebetween) to SEQ ID NO. 14.

[0143] In some embodiments, the mRNA construct which encodes an open reading frame of the murine PAI-1 protein comprises a nucleic acid sequence set forth in SEQ ID NO: 23, or a nucleic acid sequence having at least about 50% sequence identity, and in some embodiments, at least about 70%, and in some embodiments, at least about 80%, and in some embodiments, at least about 90%, and in some embodiments, in the range of from about 50% to about 99% sequence identity (including any value or subrange therebetween) to SEQ ID NO. 23.

[0144] Some aspects of the invention pertain to the use of the described immunosuppressant-incorporated LNP formulations such as LNP-Dex formulations or standard LNP formulations for the delivery of the described mRNA which encodes PAI-1 into one or more target cells for the treatment of excessive bleeding and / or cardiac fibrosis and / or mammalian subjects which has one or more mutations in the SERPINE-1 gene which may lead to PAI-1 deficiency. PAI-1 which are encoded by the saRNA are expressed in the one or more target cells. The expressed PAI-1 increases circulating levels of PAI-1 in the target cells, thereby inhibiting fibrinolysis in a subject treated with such immunosuppressant-incorporated LNP encapsulated mRNA or standard LNP encapsulated mRNA. In some embodiments, the one or more target cells comprise liver cells.

[0145] As used herein, a standard LNP formulation is free of a corticosteroid such as a Dexamethasone. In some embodiments, the standard LNP formulation comprises a lipid mixture which comprises at least one (ionizable) cationic lipid, at least one helper lipid, a sterol, and a least one lipid-polyethylene glycol conjugate.

[0146] In some embodiments, the lipid mixture comprises about 30-55 mol % of the atleast one (ionizable) cationic lipid, about 25-50 mol % of sterol, about 5-20 mol % of the at least one helper lipid, and about 0.5-3 mol % of at least one lipid-polyethylene glycol conjugate.

[0147] 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.ExamplesExample 1 - saRNA constructs encapsulated in LNP-Dex formulations

[0148] Self-amplifying RNA (saRNA) encoding firefly luciferase (Flue) were encapsulated in standard LNP formulations. Standard LNP formulations do not comprise Dexamethasone. The standard LNP formulation used in the Examples comprise ALC-0315, PEG-DMG, DSPC, and Cholesterol at molar ratio of 50: 1.5: 10: 38.5 %.

[0149] Wildtype mice were treated with 15 pg of the standard LNP containing saRNA encoding Firefly Luciferase. Referring to FIG. 1 , the mice were imaged 24 hours (a) and (b) 48 hours post injection.

[0150] FIG. 1 show that systemic administration of standard LNPs containing saRNA encoding firefly luciferase (Flue) induces acute inflammation resulting in fast clearance of the saRNA in the liver. saRNA expression in the liver (boxed region) significantly decreases after 1 day post-injection and is undetectable by in vivo imaging (c).

[0151] saRNA encoding Flue were encapsulated in a LNP-Dex formulation comprising Dexamethasone (Dex). Such LNP-Dex formulation may be referred to as “immune-suppressive LNP”. The LNP-Dex formulation used in the Examples comprise ALC-0315, PEG-DMG, DSPC, Dexamethasone and Cholesterol at molar ratio of 50: 1.5: 10: 3.85: 34.65.

[0152] Wildtype mice were then treated with saRNA encoding Firefly Luciferase encapsulated in standard LNPs or immune-suppressive LNPs containing Dexamethasone (Dex). The mice were imaged 4 hrs, 5-, 8-, 15- and 20-days postinjection. FIG. 2 is a plot of bioluminescence emitted by the luciferase as a function of the number of days post-injection in mice that were treated with LNP-Dex formulationcontaining saRNA or standard LNP formulation containing saRNA. The FIG. 2 plot shows that modifying the ionizable lipid in the LNP-Dex formulation results in differential expression. The results show that the addition of Dexamethasone in the LNP formulation resulted in extended saRNA expression in the liver for the duration of the study.

[0153] The FIG. 3 results show that various ionizable lipids are compatible with 3.85% of Dexamethasone. Human embryonic kidney cells and mouse myoblasts were transfected with 40 nanograms of LNP-Dex encapsulating saRNA-Fluc. Bioluminescence assay was performed 20 hours after transfection (FIG. 3a, b). Wildtype mice were treated with saRNA-Fluc encapsulated in LNP-Dex formulation with ALC-0135, D-Lin-MC3-DMA or SM-102 as ionizable lipids. 4 hours post-injection in vivo imaging indicated different levels of saRNA expression in the liver (FIG. 3c).

[0154] FIG. 4 are in vivo imaging data which show that dexamethasone incorporated in the LNP can deliver saRNA subcutaneously. Wildtype mice were treated with 12 or 17 pg of saRNA-Fluc encapsulated in LNP-Dex formulation. 4- and 24-hours postinjection in vivo imaging indicated increasing levels of saRNA expression in the liver. saRNA Construct 1 is derived from Trinidad and saRNA Construct 2 is derived from TC83. Both constructs are from the same VEEV alphavirus family. The FIG. 4 images support that saRNA delivered in LNP-Dex formulation allows for robust expression of encoding protein after subcutaneous injection.

[0155] FIG. 5 are in vivo imaging data which show that Dexamethasone incorporated in the LNP can systemically deliver different saRNA constructs encoding for the same target protein. Wildtype mice were treated with 12 or 3 pg of saRNA-Fluc encapsulated in LNP-Dex formulation. 4- and 24-hours post-injection in vivo imaging indicated increasing levels of saRNA expression in the liver.

[0156] FIG. 6A is a schematic diagram illustrating a design of saRNA construct for bicistronic expression of a therapeutic protein and Flue. Fusion to Flue allows for infering expression of the protein by using a luciferase assay. To assess whether the described saRNA construct may be adapted to express different proteins, HEK cells were transfected with 100 nanograms of the saRNA construct encoding each of SEAP, A1AT, LPL, TPA, PROS1 shown in FIG. 6A. A luciferase assay was performed after 16 hours and the results are shown in FIG. 6B. The results in FIGS.6A and 6B show that the saRNA construct can be adapted to express different proteins and this can be used for a wide range of diseases.

[0157] FIG. 7 is a plot comparing the expression levels of Flue encoded by saRNA constructs that are encapsulated using LNP-Dex formulations each containing different ratios of Dexamethasone, specifically 2 molar % Dex, 3.85 molar % Dex, 3.85 molar% Pro-Dex, and 5 molar% Dex. The results show that inclusion of different Dexamethasone ratio and dexamethasone forms enables a range of systemic expression.

[0158] FIG. 16 is a plot showing inferred replication of saRNA containing the coding sequence of PAI-1 , AsdCAs12a, SadCas9 and FLuc as a control. NSP3 transcript were quantified and no statistical significance was for all samples except AsdCas12a. Similar levels of NSP3 transcripts as compared to a functional saRNA construct infers similar levels of replication of saRNA transcripts 24 hours after transfection.Example 2 - Overexpression of Factor VII (FVI I)

[0159] Synthetic messenger RNA (mRNA) encoding mouse FVII (FVI I) were encapsulated in standard LNP formulations. The sequence of SEQ ID NO. 8 is used as the mouse FVII sequence. The LNP formulation used in this example comprises ALC-0315, Cholesterol, PEG-DMG and DSPC at molar ratio of 50: 38.5: 1.5: 10 %.

[0160] Von Willebrand disease (VWD) Type 3 rats were treated with mRNA-FVIl LNP or mRNA NanoLuciferase (nLuc), as a control. A tail transection bleeding was induced 16 hours post-injection. Blood loss was recorded over 20 minutes and recorded based on weight as shown in FIG. 8A. The results demonstrate that the treatment of mRNA-FVIl encapsulated in LNP in a bleeding disorder rat model decreases blood loss in the model.

[0161] Plasma from healthy (normal), FVII- / -, FVI 11- / -, FIX- / -, and Von Willebrand Disease (VWD) Type 1-3 were then analyzed via ROTEM. Recombinant human FVII was added at final concentrations of 0 (left bar), 0.5 (middle bar) or 1 (right bar) ug / mL (FIG. 8B). The FIG. 8B results show that increasing FVII concentrations in plasma from patients with various bleeding disorders improves clotting time.

[0162] Wildtype mice were treated with LNP containing saRNA encoding mouse FVI 11 ORF and euthanized 1 , 3, 6, 9, 15, 21 , 27, 33 days post-injection. Plasma FVII levelswere measured by ELISA and the results are shown in FIGS. 9A and 9B. Mice were injected with 30 ug of saRNA, with a LNP-Dex formulation (FIG. 9A). Mice were injected with 20 ug of saRNA, with a LNP-Dex formulation (FIG. 9B). The LNP-Dex formulation was composed of ALC-0315, Cholesterol, DSPC, Dexamethasone and PEG at molar ratio of 50: 34.65: 10: 3.85: 1.5 %. FIGS. 9A and 9B show that “optimized” hepatic delivery of mouse FVII saRNA using LNP-Dex increases circulating levels of FVII.

[0163] FIG. 10 are results showing that hepatic delivery of FVII mRNA using LNPs increases circulating levels of FVII. Wildtype mice were treated with LNP containing mRNA encoding mouse FVII ORF and euthanized at 6-, 24-, and 48-hours postinjection to quantify hepatic mRNA by qPCR (a) and plasma FVII levels by western blot (b) The levels were compared to untreated (UT) mice. The LNP used in these experiments composed of MC3, Cholesterol, DSPC, and PEG at molar ratio of: 50: 38.5: 10: 1.5 %. Wildtype swine were treated with LNP containing mRNA encoding human FVII ORF, and blood was collected 5 hours and 24 hours post-injection to quantify plasma FVII levels by western blot (c). Levels of FVII in the mRNA-FVIl- treated pigs (Pig 1 and 2) were compared to PBS treated pigs (Ctrl Pig). The LNP used in these experiments composed of ALC-0315, Cholesterol, DSPC, and PEG at molar ratio of: 50: 38.5: 10: 1.5 %.

[0009] FIG. 11 are results demonstrating increasing circulating levels of FVII decreases blood loss after injury in a mouse model of hemophilia A. Wildtype (WT) or hemophilia A (FVII I- / -) mice were treated with LNP containing mRNA encoding mouse FVII ORF 24 hours prior to inducing bleeding by transecting the tails 4 mm from the distal tip. Blood loss during the 20-minute monitoring period following tail transection, normalized by body weight is plotted in (a). Hepatic FVII mRNA levels were quantified by qPCR. Levels were compared to untreated (UT) mice as shown in (b). The LNP formulation used in these experiments composed of MC3, Cholesterol, DSPC, and PEG at molar ratio of: 50: 38.5: 10: 1.5 %.

[0164] FIG. 12 are results demonstrating expressing enhanced variants of FVII in rodent models using mRNA-LNP. Wild-type mice were treated with LNP containing mRNA encoding wild-type human FVII (‘FVII’) or a human FVII variant designed for enhanced activity, expression, and / or half-life (‘FVII-Var.’). Mice were sacrificed at 7hours post-treatment, and plasma levels of heterologously expressed FVII was assessed by immunoblotting for a C-terminal epitope tag as shown in (a). Control mice were sham injected with PBS (Untreated; ‘UT’). Rats deficient in Von Willebrand Factor were treated with LNP containing mRNA encoding human FVII variant designed for enhanced activity, expression, and / or half-life. Rats were sacrificed at the times indicated following treatment, plasma was collected for protein detection by immunoblotting using antibody recognizing C-terminal epitope tag as shown in (b).Example 3 - Overexpression of Plasminogen Activator Inhibitor 1 (PAI-1)

[0165] To determine whether protein replacement with mRNA is a viable therapeutic approach for PAI-1 deficient patients, the inventors first tested if the excessive fibrinolytic characteristics of PAI-1 deficient plasma can be overcome with recombinant PAI-1 protein. Fibrinolysis was examined using rotational thromboelastometry (ROTEM), which measures viscoelastic properties in blood to assess clot formation and lysis. Normal and PAI-1 depleted human plasma were analyzed ex vivo with ROTEM with the addition of tPA. In the absence of tPA no fibrinolysis was expected, and PAI-1 depleted plasma and normal human plasma exhibited no fibrinolytic differences within 60 min following the initiation of the clot, as measured by the lysis index at 60 min after initiation of clotting (LI60). In the presence of tPA, PAI-1 depleted plasma had significantly higher fibrinolysis (19 ± 9 % LI60) compared to normal plasma (73 ± 15 % LI60, P<0.05) (FIG. 13a). PAI-1 depleted plasma spiked with recombinant PAI-1 protein (400 ng / mL) had significantly less fibrinolysis (LI60 of 72 ± 17 %, P<0.05) compared to PAI-1 depleted plasma with no recombinant protein (22 ± 10 %) (FIG. 13b,c).

[0166] The inventors then examined whether mRNA encoding PAI-1 encapsulated in LNP can overexpress PAI-1 in the liver and increase plasma PAI-1 concentrations. The PAI-1 coding sequence was uridine depleted, and mRNA was encapsulated in LNP. Wild-type (WT) mice were intravenously injected with either mRNA PAI-1 LNP (mPAI-1) at 0.5 mg of mRNA per kg of mouse body weight (mg / kg) or PBS as a vehicle control. Plasma was analyzed for total protein PAI-1 levels 24 hours postinjection (FIG. 14a). Mice treated with mPAI-1 had significantly higher PAI-1 levels compared to mice treated with PBS (7.14 ± 0.9 ng / mL mPAI-1 versus 1.5 ± 0.1ng / mL, P<0.05).

[0167] To test if increasing circulating PAI-1 concentrations in mice alters fibrinolysis, whole blood from WT mice treated with mPAI-1 was assessed by ROTEM. Blood from mice treated with mPAI-1 had significantly less fibrinolysis (LI60 of 81 ± 7 %) compared to mice treated with PBS (33 ± 8 %, P<0.05) (FIG. 14b).

[0168] To assess the longevity and dosing regimen of mPAI-1 , mice were injected with mPAI-1 at 0, 0.1 , 0.5, or 1 mg / kg dose and plasma PAI-1 protein concentrations were assessed 6-, 24-, and 48-hours post-injection (FIG. 14c). Mice treated with mPAI-1 at 0.5 and 1 mg / kg had significantly higher PAI-1 levels at 6 hours (28 ± 4, 24 ± 2 ng / mL, P<0.05) and 24 hours (13 ± 2 ng / mL, 29 ± 7 ng / mL P<0.05) post-injection compared to mice treated with PBS (1.7 ± 0.1 , 3 ± 0.6 ng / mL, respectively). Mice treated with mPAI-1 at 0.1 mg / kg had significantly higher PAI-1 levels 6 hours postinjection (15 ± 1 ng / mL, P<0.05) compared to PBS, but similar levels at 24 hours post-injection. All treated mPAI-1 mice had similar PAI-1 levels at 48 hours postinjection compared to mice treated with PBS.

[0169] The inventors then examined whether mPAI-1 can express PAI-1 in complete PAI-1 knockout (PAI-1- / -) mice. PAI-1- / - mice were intravenously injected with mPAI-1 at a dose of 1 mg / kg or 200 pL of saline as a vehicle control and blood was collected at 6- and 24-hours post-injection (FIG. 15a). PAI-1- / - mice treated with mPAI-1 had significantly higher PAI-1 levels at both timepoints (>25, 5 ± 1 ng / mL versus <0.3 ng / mL, P<0.05). In parallel to this study, PAI-1 + / + mice were administered saline or mPAI-1 at a dose of 1 mg / kg and plasma PAI-1 protein concentration was analyzed 6- and 24-hours post-injection (FIG. 15b). PAI-1 + / + mice treated with mPAI-1 had significantly higher PAI-1 levels at both timepoints (>30, 13 ± 1 ng / mL versus 3 ± 0.3, 1 ± 0.2 ng / mL). Some of the values in these analyses were outside of the dynamic range of the assay; therefore, they are presented as larger or smaller than a specific value.

[0170] In summary, a novel agent, mRNA encoding PAI-1 encapsulated in LNP (mPAI-1), which offers a potential solution to the short-lived effects of recombinant PAI-1 protein by sustaining PAI-1 protein levels for more than 24 hours was developed. The increased circulating PAI-1 protein is functional and ameliorates the disease phenotype, as blood from mice 24 hours post-treatment exhibitedsignificantly reduced fibrinolysis, demonstrating the therapeutic potential of mPAI-1 to stabilize clots. mPAI-1 offers a distinct advantage over current treatment options by enabling endogenous production of PAI-1 protein which provides extended therapeutic effects and reduces the need for continuous administration, without the potential for inhibitory antibody production commonly developed in protein replacement therapies. Furthermore, mPAI-1 treatment restored PAI-1 levels to physiological and supra-physiological concentrations in PAI-1 knockout mice, underscoring its utility in managing PAI-1 deficiency.

[0171] mPAI-1 can be as a preventative treatment for patients undergoing high-risk procedures or anticipated trauma, commonly seen in athletes or certain occupations, as well as for chronic conditions like heavy menstrual bleeding in PAI-1 deficient women. Furthermore, due to the short LNP circulation time and rapid onset of mRNA expression, exogenous protein expression can be observed as early as 30 minutes post-administration. Thus, mPAI-1 could also be suitable during acute bleeding events such as those occurring during surgical procedures or trauma. The implications of mPAI-1 as a potential therapeutic extends beyond bleeding management. PAI-1 deficiency is implicated in the pathogenesis of cardiac fibrosis, suggesting that mPAI-1 could provide a novel research tool for studying the role of circulating PAI-1 in fibrotic processes and potentially offer a therapeutic benefit for the prevention of cardiac fibrosis.

[0172] These experiments establish the foundation for an mRNA-based PAI-1 replacement therapy as a versatile therapeutic modality for PAI-1 deficient patients. mPAI-1 addresses significant limitations of conventional protein replacement therapies, offering sustained effects and broader applicability.Methods used for Example 3 Experiments mRNA Synthesis

[0173] Messenger RNA (mRNA) for encapsulation was synthesized in bulk by in vitro transcription. Briefly, plasmid DNA template encoding a CleanCap AG bacteriophage T7 promoter site and uridine-depleted, codon-optimized mouse PAI-1 coding sequence was linearized with Sapl enzyme. RNA was produced by in vitro transcription reactions containing CleanCap AG reagent and N1 methylpseudouridine-5' -triphosphate (TriLink BioTechnologies™, San Diego, CA). DNA template was digested by DNAsel and purified using an RNeasy Kit (Qiagen™, Toronto, ON) prior to enzymatic tailing using an A-Plus Poly(A) Polymerase Tailing Kit (CellScript™, Madison, Wl). mRNA was purified a final time and integrity monitored by bioanalyzer (Agilent Technologies™, Santa Clara, CA) before encapsulation into LNP. mRNA-LNP formulation

[0174] N1 methylpseudouridine mRNA encoding PAI-1 was encapsulated in LNP as previously described23. mRNA was dissolved in sodium acetate buffer (pH 4) and lipids were dissolved in pure ethanol. The lipid solution consisted of ALC-0315, DSPC, cholesterol and PEG-DMG, (Avanti Lipids) at a 50:10:38.5:1.5 % molar ratio. The mRNA and lipid solutions were combined at an amine-to-phosphate (N / P) ratio of 6. The LNP were dialyzed overnight against Dulbecco’s phosphate buffered saline (PBS) in 500-fold volume excess. To determine mRNA concentration and encapsulation efficiency, RiboGreen assay (Quant-IT Ribogreen™ RNA Assay Kit, ThermoFisher™) was performed. The Malvern Zeta Particle Sizer™ was used to determine the size and polydispersity index (PDI) of the particles. The LNP were diluted to a final concentration of 0.1 mg mRNA per mL in 10 % sucrose and 10 mM L-Histidine buffer and frozen at -80 oC prior to intravenous (IV) injection.Mice

[0175] All murine studies were conducted in accordance with institutional animal care guidelines, approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee (IACUC) (Protocols # AUA00007758) and the University of Ottawa Heart Institute (Protocol #2909). Male WTC57BL / 6J mice (Jackson Labs™, Bar Harbor, ME stock # 000664), between ages 6-11 weeks were used in wildtype mouse studies. PAI-1- / - littermates B6.129S2-Serpine1tm1 Mlg / J (Jackson Labs, Bar Harbor, ME, Strain #002507), between ages 6-10 weeks were used.Mice injections and plasma extraction

[0176] Mice were administered LNP intravenously by retroorbital or tail vein injection at doses of 1 , 0.5 or 0.1 mg mRNA per kg body weight (mg / kg). Non-terminal blooddraws were collected either retro-orbitally under isoflurane anaesthesia or by tail-nick. Blood samples taken at endpoint were collected via cardiac puncture. Blood was collected into a pipette tip or syringe containing sodium citrate (0.32 % final) for retro- orbital and cardiac puncture blood collection or in heparin coated tubes for tail-nick blood collection. Plasma was separated from whole blood by spinning at 1500 x g for 10 minutes at room temperature.Analysis of PAI-1 levels in plasma

[0177] The plasma PAI-1 concentration was analyzed with a mouse total PAI-1 ELISA kit (IMSPAI1 KTT™, Innovative Research, Novi, Ml) following the manufacturer’s guidelines. Alterations to the manufacturer’s protocol included diluting plasma 1 :1 for each sample and increasing primary antibody incubation time to 1.5 hours.Fibrinolysis analysis ex vivo

[0178] Rotational thromboelastometry (ROTEM) (Rotem Delta™, Werfen S.A., Spain) was performed according to the manufacturer’s instructions. All reagents were first warmed to 37 °C. Each test was allowed to proceed for 1 .5 hours. For mouse experiments, whole blood was mixed with 20 pL of 0.2 M CaCI2, 20 pL EXTEM reagent containing tissue factor (Werfen S.A., Spain), and recombinant mouse tPA (AB92715, Abeam) at a final concentration of 350 ng / mL. For human sample experiments, plasma was mixed with 20 pL of 0.2 M CaCI2, 20 pL EXTEM reagent containing tissue factor (Werfen S.A., Spain), recombinant human tPA (AB92637, Abeam) at 180 ng / mL and in the absence or presence of purified recombinant PAI-1 protein (1786-PI-010, R&D Systems) at 400 ng / mL.Statistical Analysis

[0179] The statistical analysis was completed using GraphPad Prism™ (Version 10.0.3). The F-test was performed to confirm the standard deviation (SD) between groups was not statistically significant. Comparisons between the mean of two groups were performed with a one-tailed unpaired parametric t-test and two-way analysis of variance (ANOVA) was used to compare 2 data sets over time. Welch’s t-test or Welch’s two-way ANOVA were used respectively if the SD between groups wassignificantly different. Significance was designated at P values < 0.05Interpretation of Terms

[0180] 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”;• “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 more unless 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 beunderstood as indicating or implying relative importance or indicating the number of indicated technical features.

[0181] 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.

[0182] 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 also included in the invention.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] Any aspects described above in reference to apparatus may also apply to methods and vice versa.

[0187] 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.

[0188] Various features are described herein as being present in “someembodiments”. 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.

[0189] 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 lipid nanoparticle composition for use in the delivery of a self-amplifying RNA (saRNA) construct in one or more target cells, the lipid nanoparticle composition comprising a lipid mixture comprising at least one (ionizable) cationic lipid, at least one helper lipid, a sterol, a corticosteroid, and a least one lipid-polyethylene glycol conjugate, wherein the molar concentration of the corticosteroid in the lipid mixture is in the range of from about 0.1 to 50 mol %, and wherein the saRNA construct comprises a first open reading frame which encodes one or more non-structural proteins, and a second open reading frame operatively linked to the first open reading frame, the second open reading frame comprising a coding region which encodes one or more target proteins.

2. The lipid nanoparticle composition as defined in claim 1 , wherein the molar concentration of the corticosteroid in the lipid mixture is in the range of from about 0.1 to 20 mol %.

3. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the corticosteroid comprises one or more of dexamethasone, betamethasone, prednisone, and prednisolone.

4. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the one or more target cells comprise liver cells.

5. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the lipid mixture comprises: about 30-70 mol % of the at least one (ionizable) cationic lipid; about 0.1-50 mol% of the sterol; about 5-20 mol% of the at least one helper lipid; about 0.1-50 mol% of the corticosteroid; and about 0.5-4 mol% of the at least one lipid-polyethylene glycol conjugate.

6. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the molar ratio of the at least one (ionizable) cationic lipid, the sterol, the at least one helper lipid, the corticosteroid, and the at least one lipidpolyethylene glycol conjugate is 50: 34.65: 10: 3.85: 1.5 %.

7. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the lipid nanoparticle is used to deliver a plurality of the saRNA constructs, wherein the saRNA constructs encode the same target protein or one or more different target proteins.

8. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the one or more non-structural proteins encoded by the first open reading frame form a replicase complex.

9. The lipid nanoparticle composition as defined in claim 8, wherein the replicase complex is or is derived from a replicase encoded by the genome of an alphavirus.

10. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the first open reading frame encodes a replicase that is or is derived from the genome of Venezuelan equine encephalitis virus (VEEV) which comprises a sequence set forth in SEQ ID NO. 1 , or a sequence having at least about 50% sequence identity to SEQ ID NO. 1.11 . The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the second open reading frame of the saRNA construct is operatively linked to a subgenomic promoter (SGP).

12. The lipid nanoparticle composition as defined in claim 11 , wherein the subgenomic promoter is or is derived from the genome of an alphavirus which comprises a sequence set forth in SEQ ID NO. 3, or a sequence having at least about 50% sequence identity to SEQ ID NO.3.

13. The lipid nanoparticle composition as defined in any one of the precedingclaims, wherein the saRNA construct additionally comprises a 5’ UTR operatively linked upstream of the first open reading frame which encodes the one or more non-structural proteins.

14. The lipid nanoparticle composition as defined in claim 13, wherein the 5’ UTR comprises conserved sequence elements (CSEs) that are or are derived from the genome of an alphavirus.

15. The lipid nanoparticle composition as defined in claim 13 or 14, wherein the 5’ UTR comprises conserved sequence elements that are or are derived from the genome of Venezuelan equine encephalitis virus (VEEV) which comprises a sequence set forth in SEQ ID NO. 2 or a sequence having at least about 50% sequence identity to SEQ ID NO. 2.

16. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the saRNA construct additionally comprises a 3’ UTR operatively linked downstream of the second open reading frame which encodes the one or more target proteins.

17. The lipid nanoparticle composition as defined in claim 16, wherein the 3’ UTR comprises conserved sequence elements that are or are derived from the genome of an alphavirus.

18. The lipid nanoparticle composition as defined in claim 16 or 17, wherein the 3’ UTR comprises conserved sequence elements (CSEs) that are or are derived from the genome of Venezuelan equine encephalitis virus (VEEV) which comprises a sequence set forth in SEQ ID NO. 4 or a sequence having at least about 50% sequence identity to SEQ ID NO. 4.

19. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the saRNA construct additionally comprises a 5’ cap structure operatively linked upstream of the 5’ UTR.

20. The lipid nanoparticle composition as defined in any one of the precedingclaims, wherein the saRNA construct additionally comprises a poly(A) tail operatively linked downstream of the 3’ UTR.21 . The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the coding region of the saRNA construct encodes an open reading frame of a coagulation factor involved in hemostasis.

22. The lipid nanoparticle composition as defined in any one of the preceding claims, wherein the coding region of the saRNA construct encodes an open reading frame of coagulation factor VII (FVI I).

23. The lipid nanoparticle composition as defined in claim 21 or 22, wherein the coagulation factor comprises a wildtype human coagulation factor VII (hFVIl).

24. The lipid nanoparticle composition as defined in claim 23, wherein the coding region of the saRNA construct which encodes an open reading frame of FVI I comprises a sequence set forth in SEQ ID NO: 6, or a sequence having at least about 50% sequence identity to SEQ ID NO: 6.

25. The lipid nanoparticle composition as defined in claim 22, wherein the FVII comprises a mutant variant of human coagulation factor VII (hFVIl).

26. The lipid nanoparticle composition as defined in claim 25, wherein the coding region of the saRNA construct which encodes an open reading frame of FVII comprises a sequence set forth in SEQ ID NO: 7, or a sequence having at least about 50% sequence identity to SEQ ID NO: 7.

27. The lipid nanoparticle composition as defined in claim 22, wherein the FVII comprises a murine coagulation factor VII (mFVIl).

28. The lipid nanoparticle composition as defined in claim 27, wherein the coding region of the saRNA construct which encodes an open reading frame of FVII comprises a sequence set forth in SEQ ID NO: 8, or a sequence having at least about 50% sequence identity to SEQ ID NO: 8.

29. The lipid nanoparticle composition as defined in claim 22, wherein the saRNA construct which encodes an open reading frame of FVII comprises a nucleic acid sequence set forth in SEQ ID NO: 9, or a sequence having at least about 50% sequence identity to SEQ ID NO: 9.

30. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of plasminogen activator inhibitor-1 (PAI-1).31 . The lipid nanoparticle composition as defined in claim 30, wherein the coding region of the saRNA construct which encodes an open reading frame of PAI-1 comprises a sequence set forth in SEQ ID NO: 10, or a sequence having at least about 50% sequence identity to SEQ ID NO: 10.

32. The lipid nanoparticle composition as defined in claim 30, wherein the coding region of the saRNA construct which encodes an open reading frame of PAI-1 comprises a sequence set forth in SEQ ID NO: 11 , or a sequence having at least about 50% sequence identity to SEQ ID NO: 11 .

33. The lipid nanoparticle composition as defined in claim 30, wherein the saRNA construct which encodes an open reading frame of PAI-1 comprises a nucleic acid sequence set forth in SEQ ID NO: 12, or a sequence having at least about 50% sequence identity to SEQ ID NO: 12.

34. The lipid nanoparticle composition as defined in claim 30, wherein the saRNA construct which encodes an open reading frame of PAI-1 comprises a nucleic acid sequence set forth in SEQ ID NO: 13, or a sequence having at least about 50% sequence identity to SEQ ID NO: 13.

35. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more of a coagulation protein, metabolic protein and age- associated protein.

36. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more coagulation proteins.

37. The lipid nanoparticle composition as defined in claim 36, wherein the one or more coagulation proteins comprise one or more of prothrombin, tissue factor, thrombomodulin, Protein C, anti-thrombin and Factors V, IX, VIII, XI, XII and XIII.

38. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more metabolic proteins.

39. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more of an age-associated protein.

40. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more therapeutic proteins for the treatment of excessive bleeding.41 . The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more therapeutic proteins for the treatment of a bleeding disorder.

42. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more therapeutic proteins for the treatment of a metabolic disorder.

43. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open readingframe of one or more therapeutic proteins for the treatment of a genetic disorder.

44. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more therapeutic proteins for the treatment of obesity.

45. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more therapeutic proteins for the treatment of diabetes.

46. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more therapeutic proteins for the treatment of Fabry’s Disease.

47. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more therapeutic proteins for immuntherapies.

48. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of one or more therapeutic proteins for the treatment of cancer.

49. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes a lipoprotein lipase (LPL).

50. The lipid nanoparticle composition as defined in claim 49, wherein the saRNA construct which encodes an open reading frame of LPL comprises a nucleic acid sequence set forth in SEQ ID NO: 15, or a sequence having at least about 50% sequence identity to SEQ ID NO: 15.51 . The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of alpha-1 antitrypsin (A1AT),52. The lipid nanoparticle composition as defined in claim 51 , wherein the saRNA construct which encodes an open reading frame of A1AT comprises a nucleic acid sequence set forth in SEQ ID NO: 16, or a sequence having at least about 50% sequence identity to SEQ ID NO: 16.

53. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of protein S (PROS1),54. The lipid nanoparticle composition as defined in claim 53, wherein the saRNA construct which encodes an open reading frame of PROS1 comprises a nucleic acid sequence set forth in SEQ ID NO: 17, or a sequence having at least about 50% sequence identity to SEQ ID NO: 17.

55. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of tissue plasminogen activator (TPA).

56. The lipid nanoparticle composition as defined in claim 55, wherein the saRNA construct which encodes an open reading frame of TPA comprises a nucleic acid sequence set forth in SEQ ID NO: 18, or a sequence having at least about 50% sequence identity to SEQ ID NO: 18.

57. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of plasminogen (PLG).

58. The lipid nanoparticle composition as defined in claim 57, wherein the saRNA construct which encodes an open reading frame of PLG comprises a nucleic acid sequence set forth in SEQ ID NO: 21 , or a sequence having at least about 50% sequence identity to SEQ ID NO: 21 .

59. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open readingframe of GLP-1 (glucagon-like peptide-1) agonist.

60. The lipid nanoparticle composition as defined in claim 59, wherein the saRNA construct which encodes an open reading frame of GLP-1 agonist comprises a nucleic acid sequence set forth in SEQ ID NO: 30, or a sequence having at least about 50% sequence identity to SEQ ID NO: 30.61 . The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of insulin.

62. The lipid nanoparticle composition as defined in claim 61 , wherein the saRNA construct which encodes an open reading frame of insulin comprises a nucleic acid sequence set forth in SEQ ID NO: 25, or a sequence having at least about 50% sequence identity to SEQ ID NO: 25.

63. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of fibrinogen alpha-chain (FGA).

64. The lipid nanoparticle composition as defined in claim 63, wherein the saRNA construct which encodes an open reading frame of FGA comprises a nucleic acid sequence set forth in SEQ ID NO: 24, or a sequence having at least about 50% sequence identity to SEQ ID NO: 24.

65. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of a Cas protein and or variants thereof.

66. The lipid nanoparticle composition as defined in claim 65, wherein the Cas protein or variant thereof comprises SadCas9.

67. The lipid nanoparticle composition as defined in claim 66, wherein the saRNA construct which encodes an open reading frame of SadCas9 comprises a nucleic acid sequence set forth in SEQ ID NO: 19, or a sequence having atleast about 50% sequence identity to SEQ ID NO: 19.

68. The lipid nanoparticle composition as defined in claim 65, wherein the Cas protein or variant thereof comprises AsdCas12a.

69. The lipid nanoparticle composition as defined in claim 68, wherein the saRNA construct which encodes an open reading frame of AsdCas12a comprises a nucleic acid sequence set forth in SEQ ID NO: 20, or a sequence having at least about 50% sequence identity to SEQ ID NO: 20.

70. The lipid nanoparticle composition as defined in claim 65, wherein the Cas protein or variant thereof comprises SpCas9.71 . The lipid nanoparticle composition as defined in claim 70, wherein the saRNA construct which encodes an open reading frame of SpCas9 comprises a nucleic acid sequence set forth in SEQ ID NO: 26, or a sequence having at least about 50% sequence identity to SEQ ID NO: 26.

72. The lipid nanoparticle composition as defined in claim 65, wherein the Cas protein or variant thereof comprises NlaCas11 .

73. The lipid nanoparticle composition as defined in claim 71 , wherein the saRNA construct which encodes an open reading frame of NlaCas11 comprises a nucleic acid sequence set forth in SEQ ID NO: 27, or a sequence having at least about 50% sequence identity to SEQ ID NO: 27.

74. The lipid nanoparticle composition as defined in claim 65, wherein the Cas protein or variant thereof comprises Cas9 nickase.

75. The lipid nanoparticle composition as defined in claim 74, wherein the saRNA construct which encodes an open reading frame of Cas9 nickase comprises a nucleic acid sequence set forth in SEQ ID NO: 28, or a sequence having at least about 50% sequence identity to SEQ ID NO: 28.

76. The lipid nanoparticle composition as defined in any one of claims 1 to 20,wherein the coding region of the saRNA construct encodes an open reading frame of Prime Editor max (PEmax).

77. The lipid nanoparticle composition as defined in claim 76, wherein the saRNA construct which encodes an open reading frame of PEmax comprises a nucleic acid sequence set forth in SEQ ID NO: 29, or a sequence having at least about 50% sequence identity to SEQ ID NO: 29.

78. The lipid nanoparticle composition as defined in any one of claims 1 to 20, wherein the coding region of the saRNA construct encodes an open reading frame of fibroblast growth factor 3 (FGFR3) (transmembrane deletion).

79. The lipid nanoparticle composition as defined in claim 78, wherein the saRNA construct which encodes an open reading frame of FGFR3 (transmembrane deletion) comprises a nucleic acid sequence set forth in SEQ ID NO: 31 , or a sequence having at least about 50% sequence identity to SEQ ID NO: 31.

80. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of a bleeding disorder in a subject.

81. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of excessive bleeding in a subject.

82. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for prophylactic treatment of bleeding in a subject.

83. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of cardiac fibrosis in a subject.

84. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of a subject with PAI-1deficiency.

85. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of excessive bleeding in a subject.

86. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of a subject with lipoprotein lipase deficiency (LPL-D).

87. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of a subject with Alpha-1 antitrypsin (A1AT) deficiency.

88. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of thrombophilia in a subject.

89. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of ischemic stroke in a subject.

90. Use of a composition comprising the lipid nanoparticle as defined in any one of any one of claims 1 to 20 for gene editing using a CRISPR-based system.

91. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of any one of claims 1 to 20 for the treatment of a subject with a genetic disorder or disease.

92. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of a subject with plasminogen deficiency.

93. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of a subject withdiabetes.

94. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of a subject with obesity.

95. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for the treatment of a subject with a metabolic disorder.

96. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined any one of claims 1 to 20 for the treatment of a subject with fibrinogen deficiency.

97. Use of a pharmaceutical composition comprising the lipid nanoparticle as defined any one of claims 1 to 20 for the treatment of a subject with FV deficiency.

98. The use as defined in any one of claims 80 to 97, wherein the pharmaceutical composition is formulated for administration to a subject in need thereof by one or more of intravenous, subcutaneous, intraperitoneal, intratumoral and intramuscular administration.

99. Use of a composition comprising the lipid nanoparticle as defined in any one of claims 1 to 20 for prime editing.

100. The use as defined in claim 99, wherein the composition is formulated for administration to a subject by one or more of intravenous, subcutaneous, intraperitoneal, intratumoral and intramuscular administration.

101. An mRNA construct comprising a coding region which encodes an open reading frame of coagulation factor VII, the open reading frame comprising a sequence set forth in SEQ ID NO: 6, or a sequence having at least about 50% sequence identity to SEQ ID NO: 6.

102. An mRNA construct comprising a coding region which encodes anopen reading frame of coagulation factor VII, the open reading frame comprising a sequence set forth in SEQ ID NO: 7, or a sequence having at least about 50% sequence identity to SEQ ID NO: 7.

103. An mRNA construct comprising a coding region which encodes an open reading frame of coagulation factor VII, the open reading frame comprising a sequence set forth in SEQ ID NO: 8, or a sequence having at least about 50% sequence identity to SEQ ID NO: 8.

104. An mRNA construct which encodes an open reading frame of coagulation factor VII comprises a sequence set forth in SEQ ID NO: 22, or a sequence having at least about 50% sequence identity to SEQ ID NO: 22.

105. Use of a pharmaceutical composition comprising the mRNA construct as defined in any one of claims 101 to 104 and a lipid nanoparticle for the treatment of bleeding disorder in a subject.

106. Use of a pharmaceutical composition comprising the mRNA construct as defined in any one of claims 101 to 104 and a lipid nanoparticle for the treatment of excessive bleeding in a subject.

107. The use as defined in claim 105 or 106, wherein the lipid nanoparticle comprises least one (ionizable) cationic lipid, at least one helper lipid, a sterol, and a least one lipid-polyethylene glycol conjugate.

108. The use as defined in any one of claims 105 to 107, wherein the pharmaceutical composition is formulated for administration to a subject in need thereof by one or more of intravenous, subcutaneous, intraperitoneal, intratumoral and intramuscular administration.

109. An mRNA construct comprising a coding region which encodes an open reading frame of plasminogen activator inhibitor (PAI-1), the open reading frame comprising a sequence set forth in SEQ ID NO: 10, or a sequence having at least about 50% sequence identity to SEQ ID NO: 10.

110. An mRNA construct comprising a coding region which encodes an open reading frame of plasminogen activator inhibitor (PAI-1), the open reading frame comprising a sequence set forth in SEQ ID NO: 11 , or a sequence having at least about 50% sequence identity to SEQ ID NO: 11 .

111. An mRNA construct which encodes an open reading frame of PAI-1 comprises a sequence set forth in SEQ ID NO: 14, or a sequence having at least about 50% sequence identity to SEQ ID NO: 14.

112. An mRNA construct which encodes an open reading frame of PAI-1 comprises a sequence set forth in SEQ ID NO: 23, or a sequence having at least about 50% sequence identity to SEQ ID NO: 23.

113. Use of a pharmaceutical composition comprising the mRNA construct as defined in any one of claims 109 to 112 and a lipid nanoparticle for the treatment of bleeding disorder in a subject.

114. Use of a pharmaceutical composition comprising the mRNA construct as defined in any one of claims 109 to 112 and a lipid nanoparticle for the treatment of excessive bleeding in a subject.

115. Use of a pharmaceutical composition comprising the mRNA construct as defined in any one of claims 109 to 112 and a lipid nanoparticle for the treatment of cardiac fibrosis in a subject.

116. Use of a pharmaceutical composition comprising the mRNA construct as defined in any one of claims 109 to 112 and a lipid nanoparticle for the treatment of a subject with PAI-1 deficiency.

117. The use as defined in any one of claims 113 to 116, wherein the lipid nanoparticle comprises least one (ionizable) cationic lipid, at least one helper lipid, a sterol, and a least one lipid-polyethylene glycol conjugate.

118. The use as defined in any one of claims 113 to 117, wherein the pharmaceutical composition is formulated for administration to a subject inneed thereof by one or more of intravenous, subcutaneous, intraperitoneal, intratumoral and intramuscular administration.

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