mRNA expression method and composition thereof

Abstract

Provided is a lipid nanoparticle comprising capped mRNA bearing one or more ribose modifications, and 20 to 70 mol % of a neutral lipid, an ionizable lipid; and a sterol and optionally a hydrophilic polymer-lipid conjugate, the lipid nanoparticles exhibiting at least a 10% increase in extrahepatic protein expression of the mRNA in vivo, as measured in one or more extrahepatic organs or tissues. In some examples, the mRNA comprises a 5' cap having an N7-methylated guanosine at a position 0, and a nucleoside at a position 1 linked to the N7-methylated guanosine by a 5' to 5' bridge, wherein the N7-methylated guanosine has a modification at a 3' carbon of its ribose and / or the nucleoside at the position 1 has a modification at a 2' carbon of its ribose.

Description

MRNA EXPRESSION METHOD AND COMPOSITION THEREOFTechnical Field

[0001] The present disclosure relates to methods of extrahepatic expression of mRNA using lipid nanoparticles comprising modifications in the mRNA cap and compositions thereof for delivery to extrahepatic tissue.Background

[0002] Lipid nanoparticle (LNP) formulations represent a revolution in the field of nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro™. Onpattro™ is a lipid nanoparticle-based short interfering RNA (siRNA) drug formulation for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis. The success of this LNP delivery system paved the way for the clinical development of the leading LNP -based COVID-19 mRNA vaccines.

[0003] The Onpattro™ LNP formulation consists of four main lipid components, namely: ionizable amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol conjugated lipids (PEG-lipids) at respective molar amounts of 50 / 10 / 38.5 / 1.5. Onpattro™ is still considered the gold standard for comparison in studies of LNP-mediated efficacy and current approaches to LNP design make few deviations from the four-component system.

[0004] Of these four components, the ionizable lipid makes up the bulk of the Onpattro™ formulation (50 mol%) and is considered important for the in vitro and in vivo activity of the LNP. Accordingly, most work in the field has focused primarily on improving this lipid component. The ionizable lipid, which is typically an amino lipid, has been carefully designed so that it is charged at low pH and near-neutral at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid during initial formulation. Since the ionizable lipid is near neutral at physiological pH, toxicity and renal clearance is reduced. After cellular uptake by endocytosis, the acidic environment of the endosome leads to an increase in the net positive charge of the ionizable amino lipids, which promotes fusion with the anionic lipids of the endosomal membrane and subsequent membrane destabilization and release of the nucleic acidbased therapeutics into the cytoplasm to exert their effects.

[0005] With respect to the remaining three lipid components, the PEG-lipid is well known for improving circulation longevity of the LNP and cholesterol functions to stabilize the particle. Generally, however, comparatively less attention has been devoted to studying DSPC beyond its role as a structural lipid.

[0006] While strides have been made in research relating to LNP -mediated nucleic acid delivery, it is widely known that the Onpattro™ formulation largely accumulates in liver (hepatic) tissues. The ability of LNPs to accumulate in organs and tissues beyond the liver would greatly expand the clinical utility of these delivery systems. In order to improve the delivery of nucleic acid cargo to extrahepatic tissues, such as the bone marrow, the particles should exhibit enhanced circulation lifetimes. Traditional approaches to achieve this rely on optimizing the levels of PEG-lipid in the LNP, but the inclusion of PEG-lipids in LNPs often results in transfection potencies that are low or unfavorable immune responses.

[0007] Studies have been conducted in vivo to investigate the ability of four-component, Onpattro™ LNP -type formulations to deliver siRNA beyond the liver. In particular, siRNA gene silencing beyond the liver was investigated with Onpattro™-type LNPs (MC3 / Cholesterol / DSPC / PEG-DMG) incorporating DSPC at 10 and 40 mol% (Ordobadi, Lipid Nanoparticles for Delivery of Bioactive Molecules, 2019, A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy, The University of British Columbia). It was shown that the 10 mol% DSPC Onpattro™-like formulations had similar liver accumulation and blood circulation lifetimes as 40 mol% DSPC formulations (MC3 / Cholesterol / DSPC / PEG-DMG: 18.5 / 40 / 40 / 1.5 mol%). Further, the 40 mol% DSPC siRNA-LNP only performed comparably to 10 mol% DSPC formulations in bone marrow gene silencing. These previous studies thus showed no clear benefit resulting from adjusting the levels of neutral lipids such as DSPC to improve extrahepatic delivery of nucleic acid cargo.

[0008] The above studies investigating extrahepatic delivery were confined to LNPs having siRNA cargo. However, messenger RNA (mRNA) therapy is increasingly becoming an important tool to treat disease and the delivery of mRNA to extrahepatic tissues would expand the clinical utility of mRNA therapeutics beyond the liver. Similar to siRNA, the mRNA molecule rapidly degrades in the body, and so LNPs are used to reduce such degradation. Nonetheless, there areinherent chemical and structural differences between mRNA and siRNA in terms of length, stability and charge density of the nucleic acid (Kauffman et al., 2015, NanoLetters, 15(1 l):7300- 7306). Consequently, siRNA-LNP studies may not be informative for the design of LNPs for mRNA delivery. Further, present work on LNP mRNA systems for intravenous administration focusses primarily on developing improved ionizable cationic lipids (Semple et al., Nat Biotechnol 2010, 28: 172). In addition, these systems use the Onpattro™ lipid composition (see above) and have short circulation lifetimes with most of the cargo accumulating in the liver within 30 min (Akinc et al., 2019, Nat Nanotechnol., 14: 1084).

[0009] Recent years have witnessed major advances in our understanding of how LNP formulations can be redesigned for optimal extrahepatic delivery of nucleic acid payloads. It is becoming increasingly apparent that chemical modification of mRNA will similarly impact protein expression in different tissues. Much of the work carried out on mRNA expression has focused on chemical modification of internal nucleobases, namely uridine modifications (Curr Opin Drug Discov Devel., 2007, Sep;10(5):523-32). By comparison, considerably less is known about how regulatory elements of the mRNA contribute to protein expression in vivo, particularly in the context of LNP-mediated delivery. Today, it is widely accepted that the mRNA cap structure, present in all nuclear-encoded eukaryotic mRNAs, plays a fundamental role in protein expression both in vitro and in vivo. However, the relationship between cap modifications and high neutral lipid containing LNPs has not been explored.Summary

[0010] The present disclosure addresses one or more of the foregoing problems in the prior art and / or provides useful alternatives to known compositions for the delivery of mRNA.

[0011] The present disclosure is based on the finding that an ionizable LNP formulation (herein “IcLNP”) that includes elevated levels of neutral lipid and bearing one or more mRNA cap modifications described herein can significantly improve the in vivo extrahepatic delivery of mRNA. In some embodiments, the present disclosure provides an mRNA-LNP formulation that has improved in vivo expression of mRNA in extrahepatic tissues over a baseline LNP (Onpattro™-type formulation), as measured in a subset of bone marrow and spleen tissue at predetermined times after administration, such as at 4 hours and 24 hours post-administration. In some embodiments, the present disclosure provides an mRNA-LNP formulation with a modifiedcap at its 5’ end that has an improved in vivo expression of mRNA in extrahepatic tissues over an mRNA-LNP formulation that has a cap lacking the one or more modifications.

[0012] In one aspect, the present disclosure provides a lipid nanoparticle for extrahepatic delivery of mRNA, the lipid nanoparticle comprising: (i) a neutral lipid content of from 30 mol% to 70 mol%; (ii) an ionizable cationic lipid content of from 5 mol% to 50 mol%; wherein the mRNA is encapsulated in the lipid nanoparticle, the mRNA comprising a 5’ cap comprising a guanosine at position 0, and a nucleoside at position 1 linked to the guanosine by a 5’ to 5’ bridge, wherein the guanosine has a modification at a 3’ carbon of its ribose and / or the nucleoside at position 1 has a modification at a 2’ carbon of its ribose; and wherein each mol% is measured relative to a total lipid content of the lipid nanoparticle. In some embodiments, the guanosine at position 0 is methylated at nitrogen 7 of its purine ring. In some embodiments, the 3’ ribose modification of the guanosine at position 0 enhances mRNA translation. In some embodiments, the 3’ ribose modification offers increased mRNA stability. In another embodiment, 3’ ribose modification offers increased mRNA translation.

[0013] In one embodiment, the modification at any one of the ribose blocks chain initiation. In one embodiment, the modification of the guanosine at position 0 blocks chain initiation.

[0014] In one embodiment, the guanosine at position 0 further comprises a modification at the 2’ carbon of its ribose.

[0015] In one embodiment, the ribose modifications are added / incorporated or substituted precapping of the mRNA. In another embodiment, the ribose modifications on the mRNA cap are added / incorporated or substituted post-capping of the mRNA.

[0016] In one embodiment, the 5’ cap is a mononucleotide. In another embodiment, the 5’ cap is a dinucleotide cap. In yet another embodiment, the 5’ cap is a trinucleotide cap. In one embodiment, the 5’ cap is a tetranucleotide cap. In another embodiment, the 5’ cap is an oligonucleotide. In one embodiment, the cap is a trinucleotide bearing a further modification at 2’ ribose of its nucleotide at a position 2. In another embodiment, the 5’ cap is a tetranucleotide bearing a 2’ ribose modification at a nucleotide at position 2 and a nucleotide at position 3.

[0017] In one embodiment, the guanosine at position 0 is modified. In a further embodiment, the modification is an alkylation. In another embodiment, the modification is an arylation. In a furtherembodiment, the arylation is a cyclic modification. In one embodiment, the 3’ carbon is modified with an H group. In another embodiment, the 3’ carbon is modified with an OCH3 group. In one embodiment, the 3 ’ carbon is modified with an OC2H5 group. In another embodiment, the 3 ’ carbon is modified with an OC3H7 group.

[0018] In one embodiment, the nucleoside at position 1 is a guanosine. In another embodiment, the nucleoside at position 1 is an adenosine. In one embodiment, the ribose of the nucleoside at position 1 has an O-alkylation at its 2’ carbon. In a further embodiment, the ribose of the nucleoside at position 1 has an O-methylation. In another embodiment, the ribose has an O-ethylation. In one embodiment, the ribose of nucleoside at position 1 has a halogenation. In yet a further embodiment, the halogenation is done using a F, Cl, Br or I atom.

[0019] In another embodiment, the 5’ to 5’ bridge has a structure as defined by the following moiety:X is O, S, CH2, or N-R5, wherein R5is optionally H or an optionally substituted C1-C3 alkyl,A1is OH, SH, SeH, BH3, or a salt thereof, wherein if A1is the salt, a counterion is present optionally selected from Na+, NH4+, Et3NH+; n is 3 to 5, and wherein the X and A1of the moiety X-P(=O)-A1are, independently, as defined above;Y is O, S, CH2, or N-R6, wherein R6can be H or an optionally substituted C1-C3 alkyl.

[0020] In one embodiment, the bridge is a triphosphate moiety. In another embodiment, the bridge is a tetraphosphate moiety. In one embodiment, the bridge is modified with a BH3 group. In another embodiment, the bridge is modified with an S group. In one embodiment, Se, CH3, or an NH2 group is used for bridge modification.

[0021] In some embodiments, the 5’ cap has a structure of Formula A:Formula A or a pharmaceutically acceptable salt thereof, wherein the R1moiety is a lone pair of electrons or the R1of the -R1moiety is an optionally substituted C1-C10 alkyl, cycloalkyl or aryl, optionally a methyl;A2and A3are, independently, absent or present, and if A2is absent, then R2is H directly bonded to the 2’ carbon of the ribose, and if A2is present, then A2is O and R2is H or an optionally substituted alkyl, which alkyl is optionally C1-C3 alkyl; if A3is absent, then R3is H directly bonded to the 3’ carbon of the ribose, and if A3is present, then A3is O and R3is H or an optionally substituted alkyl, which alkyl is optionally C1-C3 alkyl; if A2and A3are both present, then both A2and A3are O and R2and R3are either not bonded to each other or bonded to each other, as indicated by a dashed bond, wherein if R2and R3are not bonded to each other, then R2and R3are, independently, H or optionally substituted C1-C3 alkyl, and if R2and R3are bonded to each other, then R2and R3together form a moiety ofFormula 1:wherein L is an optionally substituted C1-C3 alkylidene, optionally methylene (CH2), 1,1 -ethylene (CH-CH3), 1,2-ethylene (CH2-CH2), 1,1 -propylene (CH-CH2- CH3), 1,2-propylene (CH2-CH-CH3), 1,3-propylene (CH2-CH2-CH2), 2,2- propylene (CH3-C-CH3), and substituted derivatives thereof;X is O, S, CH2, or N-R5, wherein R5is optionally H or an optionally substituted C1-C3 alkyl,A1is OH, SH, SeH, BH3, or a salt thereof, wherein if A1is the salt, a counterion is present, optionally selected from Na+, NH4+and Et3NH+; and n is 3 to 5, andY is O, S, CH2, or N-R6, wherein R6is H or an optionally substituted C1-C3 alkyl;B1is a purine or a pyrimidine, optionally a 9-adeninyl, a N6-methyl 9-adeninyl, a 9-guaninyl, a 1-cytosinyl, a 1 -uracilyl, or variants of the purine or pyrimidine thereof;R4is H, OH or O-R6, wherein R6is an optionally substituted alkyl, which alkyl is optionally Ci- C3 alkyl; wherein if R2and R3are not bonded to one another, then at least one of A3-R3and R4is an O- alkyl;G1is H or a nucleotide having a structure of Formula 2:Formula 2 wherein theof Formula 2 represents a bond to the O atom to which G1is bonded;A4is as defined above for A1;Z is as defined above for Y;B2is as defined above for B1;R7is as defined above for R4; andG2is as defined above for G1, wherein the compound of Formula A comprises from 0 to 3 moi eties of Formula 2, and wherein if Formula 2 is present, a terminal moiety of Formula 2 is such that G2of the terminal moiety is H.

[0022] In some embodiments, the 5’ cap has a structure of Formula BFormula B or a pharmaceutically acceptable salt thereof, wherein the -R1moiety is a lone pair of electrons or R1an optionally substituted Ci-Cio alkyl, cycloalkyl or aryl, optionally a methyl;A2and A3are, independently, absent or present, and if A2is absent, then R2is H directly bonded to 2’ carbon of the ribose, and if A2is present, then A2is O and R2is H or an alkyl, which alkyl is optionally substituted C1-C3 alkyl; if A3is absent, then R3is H directly bonded to 3’ carbon of the ribose, and if A3is present, then A3is O and R3is H or an alkyl, which alkyl is optionally substituted C1-C3 alkyl; if A2and A3are both present, then both A2and A3are O and R2and R3are either not bonded to each other or bonded to each other, as indicated by a dashed bond, whereinif R2and R3are not bonded to each other, then R2and R3are, independently, H or optionally substituted C1-C3 alkyl, and if R2and R3are bonded to each other, then R2and R3together form a moiety of Formula 3:Formula 3 wherein L is an optionally substituted C1-C3 alkylidene, optionally methylene (CH2), 1,1 -ethylene (CH-CH3), 1,2-ethylene (CH2-CH2), 1,1 -propylene (CH-CH2-CH3), 1,2-propylene (CH2-CH-CH3), 1,3-propylene (CH2-CH2-CH2), 2,2- propylene (CH3-C-CH3), and derivatives thereof;B1is a purine or a pyrimidine moiety, optionally 9-adeninyl, N6-methyl 9-adeninyl, 9-guaninyl, 1-cytosinyl, 1 -uracilyl, or variants of the purine or pyrimidine thereof;R4is H, OH or O-R6, wherein R6is an alkyl, which alkyl is an optionally substituted C1-C3 alkyl; wherein if R2and R3are not bonded to one another, then at least one of A3-R3and R4 is an O- alkyl,G1is H or a nucleotide having a structure of Formula 4:Formula 4 wherein theof Formula 4 represents a bond to the O atom to which the G1is bonded;A4is OH, SH, SeH, BH3, or a salt thereof, wherein if A4is the salt, a counterion is present optionally selected from Na+, NH4+or Et3NH+;Z is O, S, CH2, or N-R6, wherein R6is H or an optionally substituted C1-C3 alkyl;B2is as defined above for B1;R7is as defined above for R4; andG2is as defined above for G1; wherein Z2and Z3are independently present or absent, wherein Z1is: a moiety of Formula bla:Formula bla wherein the wavy line connected to the CH2 group of Formula bla represents a bond between the CH2 group and the ribose of the nucleotide at position 0, the wavy line connecting to the O atom represents a bond to Z2, to Z3if Z2is absent or to Z4if both Z2and Z3are absent; p is 0-5, or a moiety of Formula bibO11—L-P-O— 00Formula bib wherein L is CH=CH of either E or Z configuration or L is C=C, the wavy line connected to L represents a bond to the ribose of the nucleotide at position 0, and the wavy line connected to the O atom represents a bond to Z2, or to Z3if Z2is absent, or to Z4if both Z2and Z3are absent, or a triazole moiety of Formula i or Formula ii:Formula iFormula ii wherein q, r and t are, independently, 1-5, s is 0-5, the wavy lines connecting to (CH2)qand to (CH2)Srepresent bonds to the ribose of the nucleotide at position 0, and the wavy lines connected to the O atoms of Formula i or Formula ii represent bonds between the O atoms and Z2, or to Z3if Z2is absent, or to Z4if both Z2and Z3are absent;Z2and Z3if present are, independently: a moiety of structurewherein u is 1-5, or a triazole moiety of Formula iii or Formula iv:Formula iv wherein the wavy lines connected to (CH2)qand to (CH2)Srepresent bonds to Z1when Z2is the triazole moiety or to Z2when Z3is the triazole moiety, the wavy lines connected to the respective O atoms of (CH2)r-0 and to (CH2)t-0 represent bonds between the respective O atoms and Z3when Z2is the triazole moiety or Z4when Z3is the triazole moiety;Z4is:a moiety of structurewherein the wavy line connected to the P atom represents a chemical bond to Z3if present or to Z2if present or to Z1and the wavy line connected to the CH2 group represents a chemical bond between the CH2 group and the ribose of the nucleotide at position 1; and wherein v is 0-5, or a moiety of structurewherein the wavy line connected to the P atom represents a chemical bond to Z3if present or to Z2if present or to Z1and the wavy line connected to the CH2 group represents a bond between the CH2 group and the ribose of the nucleotide at position 1; and wherein v is 0-5, or a moiety of structureO« / W P 11-M « / w© 6 wherein M is CH=CH, of either E or Z configuration or M is C=C, the wavy line connected to M represents a chemical bond to the ribose of the nucleotide at position 1, and the wavy line connected to the P atom represents a chemical bond to Z1or to Z2if present or to Z3if present, or a triazole moiety of Formula v or Formula vi:Formula vFormula vi wherein the wavy lines connected to (CH2)Wand to (CH2)yrepresent respective bonds to Z 1 or to Z2 if present or to Z3if present, the wavy lines connected to (CH2)Xand to (CH2)Zrepresent chemical bonds to the ribose of nucleotide at position 1; w is 2-5; x and y are, independently, 0-5, and z is 1-5; wherein none or at most one of Z1, Z2, Z3, Z4is the triazole moiety of the Formula i-vi as defined above; and wherein the compound of Formula B comprises from 0 to 3 moi eties of Formula 4, and wherein if Formula 4 is present, a terminal moiety of Formula 4 is such that G2of the terminal moiety is H.

[0023] In one embodiment, the bridge includes further modifications.

[0024] According to some non-limiting examples of the embodiment, the cap may have one of the following structures:

[0025] In one embodiment, the 5’ cap has the following structure:m7G3’OMepppm6A2’OMepG, wherein the adenosine at position 1 is modified at nitrogen 6 with a methyl group.

[0026] In one embodiment, the 5’ cap has a further nucleotide at position 2 that bears a modification. In a further embodiment, the modification in the nucleotide at position 2 is in its ribose ring. In yet a further embodiment, the modification is at the 2’ carbon of the ribose ring. In another embodiment, the modification in the nucleotide at position 2 is in its nucleobase. In a further embodiment, the 5’ cap has a modified nucleotide at position 3. In yet a further embodiment, the 5’ cap has a modified nucleotide at position 4.

[0027] In one embodiment, the 5 ’cap has a further nucleotide at position 2 that is unmodified. In a further embodiment, the 5’ cap has an unmodified nucleotide at position 3. In yet a further embodiment, the 5’ cap has an unmodified nucleotide at position 4.

[0028] In one embodiment, the lipid nanoparticle further comprises a guide RNA. The lipid nanoparticle comprising the mRNA modification is delivered to extrahepatic tissue for gene editing applications along with the guide RNA.

[0029] In one embodiment, the neutral lipid is a phosphatidylcholine lipid selected from di stearoylphosphatidylcholine (DSPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC).

[0030] In another embodiment, the neutral lipid is a mixture of two phosphatidylcholine lipids. In a further embodiment, the mixture comprises distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC).

[0031] In one embodiment, the phosphatidylcholine lipid content is between 40 mol % and 60 mol %. In another embodiment, the phosphatidylcholine lipid content is between 43 mol % and 57 mol %. In yet a further embodiment, the phosphatidylcholine lipid content is between 44 mol % and 56 mol %.

[0032] In one embodiment, the cationic lipid is an amino lipid. In another embodiment, the ionizable cationic lipid is present at less than 40 mol% based on the total lipid present in the lipid nanoparticle. In a further embodiment, the ionizable cationic lipid is present at less than 30 mol%.

[0033] In one embodiment, the lipid nanoparticle further comprises a sterol. In one embodiment, the sterol is a cholesterol or a derivative thereof.

[0034] In one embodiment, the sterol is present between 17.5 mol% to 42.5 mol% based on the total lipid present in the lipid nanoparticle. In a further embodiment, the sterol is present between 18.5 mol% to 39.5 mol% based on the total lipid present in the lipid nanoparticle.

[0035] In one embodiment, the lipid nanoparticle further comprises a hydrophilic polymer-lipid conjugate.

[0036] In one embodiment, the hydrophilic polymer-lipid conjugate is a polyethylene glycollipid conjugate.

[0037] In another aspect, the present disclosure provides a lipid nanoparticle for extrahepatic expression comprising an encapsulated mRNA with a 5’ cap comprising a guanosine at position 0, and a nucleoside at position 1 linked to the guanosine by a 5’ to 5’ bridge, wherein the guanosine has a modification at a 3 ’ carbon of its ribose and / or the nucleoside at position 1 of the mRNA has a modification at a 2’ carbon of its ribose, 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid, the lipid nanoparticle having at least 1.2-fold increase in expression in extrahepatic tissue at 24 hours relative to the same lipid nanoparticle having an mRNA cap lacking the modifications, wherein the expression is measured by firefly luciferase protein levels.

[0038] In another aspect, there is provided a method for extrahepatic expression of a polypeptide in a tissue or organ of a subject, the method comprising administering to the subject ffia lipid nanoparticle encapsulating a 5’ cap comprising a guanosine at position 0, and a nucleoside at position 1 linked to the guanosine by a 5’ to 5’ bridge, wherein the guanosine has a modificationat a 3’ carbon of its ribose and / or the nucleoside at position 1 of the mRNA has a modification at a 2’ carbon of its ribose, the lipid nanoparticle having 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid, the lipid nanoparticle having at least 1.2-fold increase in expression in extrahepatic tissue at 24 hours relative to the same lipid nanoparticle with an mRNA cap lacking the modifications, wherein the expression is measured by firefly luciferase protein levels.

[0039] In one embodiment, the capping of mRNA is performed co-transcriptionally prior to the encapsulation. In another embodiment, the capping of mRNA is performed post-transcriptionally prior to the encapsulation. In yet a further embodiment, the capping is done enzymatically.

[0040] In one embodiment, the capping is done using an enzyme selected from vaccinia virus or faustovirus.

[0041] In another aspect, there is provided a lipid nanoparticle for extrahepatic expression of mRNA in a subject comprising a 5’ cap comprising a guanosine at position 0, and a nucleoside at position 1 linked to the guanosine by a 5’ to 5’ bridge, wherein the guanosine has a modification at a 3’ carbon of its ribose and / or the nucleoside at position 1 of the mRNA has a modification at a 2’ carbon of its ribose, the lipid nanoparticle having 30 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle, and an ionizable cationic lipid, the lipid nanoparticle having at least 1.2-fold increase in expression in extrahepatic tissue at 24 hours post injection relative to a lipid nanoparticle formulation comprising less than 30 mol% of neutral lipid encapsulating the same capped mRNA, but otherwise measured under identical conditions, wherein the expression is measured by firefly luciferase protein levels. The lipid nanoparticle formulation comprising less than 30 mol% of neutral lipid is an Onpattro™-type formulation having a composition of ionizable lipid (norMC3):DSPC:Cholesterol:PEG2ooo-DMG (50: 10:38.5: 1.5) as described in Table 2 herein.

[0042] In one embodiment, the extrahepatic tissue or organ for delivery is spleen, bone marrow, lungs, kidney, heart, abdominal skin, back skin and / or ear.

[0043] In one embodiment, the extrahepatic organ is bone marrow or tissue thereof.

[0044] In one embodiment, the lipid nanoparticle provides a prophylactic, ameliorative or a therapeutic benefit. In a further embodiment, the lipid nanoparticle is used to treat a disease or disorder that is an autoimmune disorder.

[0045] In one embodiment, the lipid nanoparticle is used to treat a disease or disorder that is an infectious disease.

[0046] In another embodiment, the lipid nanoparticle is used to treat a disease or disorder that is cancer.

[0047] In one aspect, the present disclosure suggests the use of a lipid nanoparticle for in vivo or in vitro delivery and expression of mRNA to mammalian cells.

[0048] In one embodiment, the use of the lipid nanoparticle for the manufacture of a medicament for in vivo or in vitro delivery of the mRNA to mammalian cells is disclosed.

[0049] In one embodiment, the lipid nanoparticle is used to target an autoimmune disorder in vivo.

[0050] In another embodiment, the lipid nanoparticle is used to target an infectious disease in vivo.

[0051] In one embodiment, the lipid nanoparticle is used to target cancer in vivo.

[0052] In another embodiment, the lipid nanoparticle is used for the manufacture of a vaccine composition.

[0053] In another aspect, the present disclosure provides a lipid nanoparticle comprising encapsulated mRNA with a 5’ cap having a methylated guanosine with a 3’ modified ribose at nucleoside position 0 and a guanosine at nucleoside position 1 of the cap, wherein the guanosine at position 1 is modified at a 2’ carbon of its ribose, 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle, an ionizable cationic lipid and a sterol, the lipid nanoparticle having at least 1.2-fold increase in expression in extrahepatic tissue at 24 hours relative to the same lipid nanoparticle with an mRNA cap lacking the 2’carbon modification, wherein the expression is measured by firefly luciferase protein levels.

[0054] In yet another aspect, there is provided a lipid nanoparticle for extrahepatic expression of a polypeptide in a tissue or organ of a subject comprising administering to the subject ffithe lipidnanoparticle encapsulating a 5 ’capped mRNA having an N7-methylated guanosine at a position 0 with a modification at a 3’ carbon of its ribose and a guanosine at a position 1 with its ribose modified at a 2’ carbon, the lipid nanoparticle having 30 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle, an ionizable cationic lipid and a sterol, the lipid nanoparticle having at least 1.2-fold increase in expression in extrahepatic tissue at 24 hours relative to the same lipid nanoparticle with an mRNA cap lacking the ribose modifications, wherein the expression is measured by firefly luciferase protein levels.

[0055] In one embodiment, the mRNA is capped co-transcriptionally in vitro.

[0056] In another embodiment, the mRNA is capped post-transcriptionally in vitro. In a further embodiment, the capping is done enzymatically.

[0057] In yet a further embodiment, the capping enzyme is selected from one of vaccinia virus and faustovirus.

[0058] In another aspect, the present disclosure provides a method for extrahepatic expression of a polypeptide in a tissue or organ of a subject, the method comprising administering to the subject a lipid nanoparticle encapsulating a 5 ’capped mRNA having an N7-methylated guanosine at position 1 with a modification at a 3’ carbon of its ribose and a guanosine at position 2 with its ribose modified at a 2’ carbon, the lipid nanoparticle having 30 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle, an ionizable cationic lipid and a sterol, the lipid nanoparticle having at least 10% increase in expression in extrahepatic tissue at 24 hours relative to a baseline LNP formulation of lipid nanoparticle comprising less than 20% of a neutral lipid encapsulating the same capped mRNA, but otherwise measured under identical conditions, wherein the expression is measured by firefly luciferase protein levels.

[0059] In some embodiments, the extrahepatic tissue or organ is selected from spleen, bone marrow, lungs, kidney, heart, abdominal skin, back skin and / or ear.

[0060] In some embodiment, the lipid nanoparticle provides a prophylactic, ameliorative and / or a therapeutic benefit.

[0061] In one embodiment, the lipid nanoparticle is used to treat a disease or disorder that is an autoimmune disorder.

[0062] In another embodiment, the lipid nanoparticle is used to treat a disease or disorder that is an infectious disease.

[0063] In one embodiment, the lipid nanoparticle is used to treat a disease or disorder that is cancer.

[0064] In a further embodiment, there is provided use of the lipid nanoparticle for in vivo or in vitro delivery and expression of the mRNA to mammalian cells.

[0065] In another embodiment, there is provided use of the lipid nanoparticle for the manufacture of a medicament for in vivo or in vitro delivery of the mRNA to mammalian cells.

[0066] In one embodiment, the mRNA is used to target an autoimmune disorder in vivo. In another embodiment, the lipid nanoparticle is used to target an infectious disease in vivo.

[0067] In one embodiment, the mRNA is used to target a cancer in vivo. In another embodiment, the lipid nanoparticle is used to manufacture a vaccine composition.

[0068] In one aspect, the present disclosure provides a method of preparing a lipid nanoparticle for extrahepatic mRNA delivery, the method comprising: (i) synthesizing a capped mRNA in vitro using a dinucleotide cap comprising an N7-modified guanosine at a position 0, and a nucleoside at a position 1 linked to the N7-modified guanosine by a 5’ to 5’ bridge, wherein the N7— modified guanosine at position 0 has a modification at a 3’ carbon and / or a 2’ carbon of its ribose; (ii) labelling a 2’ OH of a nucleoside at a position 1 of the dinucleotide cap with a methyl group enzymatically to form a 2’-O-methylated ribose; (iv) purifying the mRNA bearing the modified cap; and (v) encapsulating the purified mRNA in a lipid nanoparticle; wherein the lipid nanoparticle comprises 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid.

[0069] In another aspect, there is provided a method of preparing a lipid nanoparticle for extrahepatic mRNA expression, the method comprising (i) synthesizing an uncapped mRNA in vitro; (ii) incubating the uncapped mRNA in vitro with one or more capping enzymes that bear N7-methyl transferase activity and / or guanylyl transferase activity and modified guanosine nucleotides bearing modifications at a 3’ carbon and / or 2’ carbon positions to allow capping of mRNA; (iii) labelling the capped mRNA enzymatically at a 2 ’OH of a nucleoside at a position 1 of the capped mRNA with a methyl group to form a 2’ -O-m ethylated ribose; (iv) purifying thelabelled mRNA; and (v) encapsulating the purified mRNA in a lipid nanoparticle; wherein the lipid nanoparticle comprises 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid.

[0070] In one embodiment, the labelling step is done with a 2’O-methyl transferase.

[0071] In one embodiment, the incubating step is performed with vaccinia capping enzyme.

[0072] In another embodiment, the incubating is performed with faustovirus capping enzyme.

[0073] In one aspect, the present disclosure provides a lipid nanoparticle for extrahepatic expression of a polypeptide in a subject comprising an encapsulated mRNA comprising a 5’ cap comprising an N7-methylated guanosine at a position 0, and a nucleoside at a position 1 linked to the N7-methylated guanosine by a 5’ to 5’ bridge, wherein the N7-methylated guanosine has a modification at a 3’ carbon of its ribose and / or the nucleoside at the position 1 has a modification at a 2’ carbon of its ribose, 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid, the lipid nanoparticle having at least 10% increase in protein expression in extrahepatic tissue at 24 hours post injection as compared to a baseline lipid nanoparticle formulation of ionizable lipid / neutral lipid / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mokmol encapsulating the mRNA, but otherwise measured under identical conditions, wherein the expression is measured by firefly luciferase protein levels.

[0074] In one embodiment, the N7-m ethylated guanosine comprises a further modification at a 2’ carbon of its ribose.

[0075] In one embodiment, the nucleoside at position 1 is a guanosine. In another embodiment, the nucleoside at position 1 is an adenosine.

[0076] In one embodiment, the nucleoside at position l is a modified nucleoside.

[0077] In one embodiment, the lipid nanoparticle further comprises a sterol or a derivative thereof.in a further embodiment, the sterol is cholesterol or a derivative thereof.

[0078] In one embodiment, the lipid nanoparticle further comprises a hydrophilic polymer-lipid conjugate.

[0079] In yet a further embodiment, the hydrophilic polymer-lipid conjugate is a polyethylene glycol-lipid conjugate.

[0080] In one embodiment, the 3’ carbon of N7-methylated guanosine at position 0 is modified with an H group.

[0081] In another embodiment, the 3’ carbon of N7-methylated guanosine is modified with an OCH3 group.

[0082] In one embodiment, the 3’ carbon ofN7-methylated guanosine is modified with an OC2H5 group.

[0083] In another embodiment, the 3’ carbon of N7-methylated guanosine is modified with an OC3H7 group.

[0084] In one embodiment, the ribose of nucleoside at position 1 has an O-alkylation modification.

[0085] In a further embodiment, the ribose of nucleoside at position 1 has an O-methylation modification.

[0086] In another embodiment, the ribose of nucleoside at position 1 has an O-ethylation modification.Brief description of the drawings

[0087] Figure 1 A is a depiction of the chemical structure of a trinucleotide N7-methylguanosine mRNA cap with no further modifications on the N7-methylguanosine at position 0, bearing a 2’ ribose modification at position 1, and an unmodified guanosine at position 2 (m7GpppA2 OMepG).

[0088] Figure IB is a depiction of the chemical structure of a trinucleotide N7-methylguanosine mRNA cap bearing a 3’ ribose modification on the N7-methylguanosine at position 0, a 2’ ribose modification on the adenosine at position 1 and an unmodified guanosine at position 2 (m7G3’OMePPpA2’OMepG).

[0089] Figure 2A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC Cholesterol :PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with a trinucleotide N7-methylguanosine mRNA cap lacking a 3’ ribose modification on its N7- methylguanosine at position 0 (m7GpppA2 OMepG); and (ii) a lipid nanoparticle comprising 50mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with a trinucleotide N7-methylguanosine mRNA cap bearing a 3’ ribose modification on its N7- methylguanosine at position 0 (m7G3’OMepppA2’OMepG) in bone marrow tissue at 4 hours and 24 hours post intravenous injection.

[0090] Figure 2B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC Cholesterol :PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with a trinucleotide N7-methylguanosine mRNA cap lacking a 3’ ribose modification on its N7- methylguanosine at position 0 (m7GpppA2 OMepG); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with a trinucleotide N7-methylguanosine mRNA cap bearing a 3’ ribose modification on its N7- methylguanosine at position 0 (m7G3’OMepppA2’OMepG) in spleen tissue at 4 hours and 24 hours post intravenous injection.

[0091] Figure 2C provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 10 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC Cholesterol :PEG-lipid (50: 10:38.5: 1.5 mol:mol) (baseline LNP) encapsulating an FLuc mRNA co-transcriptionally capped with a trinucleotide N7-methylguanosine mRNA cap lacking a 3’ ribose modification on its N7- methylguanosine at position 0 (m7GpppA2 OMepG); and (ii) a lipid nanoparticle comprising 10 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (50: 10:38.5: 1.5 mokmol) (baseline LNP) encapsulating an FLuc mRNA co-transcriptionally capped with a trinucleotide N7-methylguanosine mRNA cap bearing a 3’ ribose modification on its N7- methylguanosine at position 0 (m7G3’OMepppA2’OMepG) in bone marrow tissue at 4 hours and 24 hours post intravenous injection.

[0092] Figure 2D provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 10 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC Cholesterol :PEG-lipid (50: 10:38.5: 1.5mol:mol) (baseline LNP) encapsulating an FLuc mRNA co-transcriptionally capped with a trinucleotide N7-methylguanosine mRNA cap lacking a 3’ ribose modification on its N7- methylguanosine at position 0 (m7GpppA2 OMepG); and (ii) a lipid nanoparticle comprising 10 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (50: 10:38.5: 1.5 mol:mol) (baseline LNP) encapsulating an FLuc mRNA co-transcriptionally capped with a trinucleotide N7-methylguanosine mRNA cap bearing a 3’ ribose modification on its N7- methylguanosine at position 0 (m7G3’OMepppA2’OMepG) in spleen tissue at 4 hours and 24 hours post intravenous injection.

[0093] Figure 3A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3 OMepppA2 OMepG in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a negative control.

[0094] Figure 3B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2’OMepG in spleen tissue at 24 hours post intravenous injection. PBS is used as a negative control.

[0095] Figure 3C provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulatingan FLuc mRNA co-transcriptionally capped with m7G3 OMepppA2 OMepG in abdominal skin tissue at 24 hours post intravenous injection. PBS is used as a negative control.

[0096] Figure 3D provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mokmol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2’OMepG in lung tissue at 24 hours post intravenous injection. PBS is used as a negative control.

[0097] Figure 3E provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mokmol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2’OMepG in heart tissue at 24 hours post intravenous injection. PBS is used as a negative control.

[0098] Figure 4A is a diagrammatic illustration of the chemical structure of a dinucleotide mRNA cap bearing an N7-methylguanosine at position 0 and an unmodified guanosine at position71 (m GpppG) with no ribose modifications.

[0099] Figure 4B is a diagrammatic illustration of the chemical structure of a dinucleotide mRNA cap bearing a N7-methylguanosine at position 0 and a guanosine at position 1 bearing a 2’ ribose7O-methylation (m GpppG2,OMe).[000100] Figure 4C is a diagrammatic illustration of the chemical structure of a dinucleotide mRNA cap bearing a N7-m ethylguanosine at position 0 additionally having a 3’ ribose modification and an unmodified guanosine at position 1 (m7G3 OMepppG).[000101] Figure 4D is a diagrammatic illustration of the chemical structure of a dinucleotide mRNA cap bearing a N7-m ethylguanosine at position 0 additionally having a 3’ ribosemodification and a modified guanosine bearing a 2’ ribose O-methylation at position 1 (m7G3’OMePPpG2’OMe).[000102] Figure 5 A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppG subsequently enzymatically-labelled at the 2’ribose O-position with a methyl group (m7GpppG2 OMe) in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a control.[000103] Figure 5B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7GpppG subsequently enzymatically-labelled at the 2’ribose O-position with a methyl group (m7GpppG2 OMe) in spleen tissue at 24 hours post intravenous injection. PBS is used as a control.[000104] Figure 6A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3 OMepppG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppG subsequently enzymatically- labelled at the 2’ribose O-position with a methyl group (m7G3 OMepppG2 OMe) in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a control.[000105] Figure 6B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3 OMepppG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3 OMepppG subsequently enzymatically- labelled at the 2’ribose O-position with a methyl group (m7G3’OMepppG2’OMe) in spleen tissue at 24 hours post intravenous injection. PBS is used as a control.[000106] Figure 7A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe) in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a control.[000107] Figure 7B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe) in spleen tissue at 24 hours post intravenous injection. PBS is used as a control.[000108] Figure 7C provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe) in abdominal skin tissue at 24 hours post intravenous injection. PBS is used as a control.[000109] Figure 7D provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe) in lung tissue at 24 hours post intravenous injection. PBS is used as a control.[000110] Figure 7E provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose ofadenosine at position 1 (m7GpppA2 OMe) in heart tissue at 24 hours post intravenous injection. PBS is used as a control.[000111] Figure 8A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTPs oMe, followed by enzymatic N7-methylation of guanosine at position 0 (m7G3 OMepppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7G3 OMepppA2 OMe) in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a control.[000112] Figure 8B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mokmol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTPs oMe, followed by enzymatic N7-methylation of guanosine at position 0 (m7G3 OMepppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7G3 OMepppA2 OMe) in spleen tissue at 24 hours post intravenous injection. PBS is used as a control.[000113] Figure 8C provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mokmol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP,followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’0-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTPs oMe, followed by enzymatic N7-methylation of guanosine at position 0 (m7G3 OMepppA) and subsequent enzymatic 2’0-methylation of the ribose of adenosine at position 1 (m7G3 OMepppA2 OMe) in abdominal skin tissue at 24 hours post intravenous injection. PBS is used as a control.[000114] Figure 8D provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mokmol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe) (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTPs oMe, followed by enzymatic N7-methylation of guanosine at position 0 (m7G3 OMepppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7G3 OMepppA2 OMe) in lungs tissue at 24 hours post intravenous injection. PBS is used as a control.[000115] Figure 8E provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor-MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mokmol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP, followed by enzymatic N7-methylation of guanosine at position 0 (m7GpppA) and subsequent enzymatic 2’O-methylation of the ribose of adenosine at position 1 (m7GpppA2 OMe); and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid (nor- MC3):DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTPs oMe, followed by enzymatic N7-methylation of guanosine at position 0 (m7G3 OMepppA) and subsequent enzymatic 2’O-methylation of theribose of adenosine at position 1 (m7G3 OMepppA2 OMe) in heart tissue at 24 hours post intravenous injection. PBS is used as a control.[000116] Figure 9 is a diagrammatic illustration of the chemical structure of a trinucleotide N7- methylguanosine mRNA cap bearing a 3’0-methyl ribose modification on the N7- methylguanosine at position 0, a 2’0-ethyl ribose modification on the adenosine at position 1 and an unmodified guanosine at position 2 (m7G3 OMepppA2 OEtpG).[000117] Figure 10A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2’OEtpG_in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a negative control.[000118] Figure 10B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2’OEtpG_in spleen tissue at 24 hours post intravenous injection. PBS is used as a negative control.[000119] Figure 11A is a diagrammatic illustration of the chemical structure of a dinucleotide mRNA cap bearing a N7-m ethylguanosine at position 0 with a 2’ ribose O-methylation modification and a guanosine at position 1 bearing a 2’ ribose O-methylation (m7G2’OMePPpG2’OMe).[000120] Figure 1 IB is a diagrammatic illustration of the chemical structure of a dinucleotide mRNA cap bearing a N7-m ethylguanosine at position 0 with 2’ and 3’ ribose O-methylation modifications and a guanosine at position 1 bearing a 2’ ribose O-methylation (m7G2’,3’OMePPpG2’OMe).[000121] Figure 11C is a diagrammatic illustration of the chemical structure of a dinucleotide mRNA cap bearing a N7-m ethylguanosine at position 0 with a deoxygenated 2’ ribose modification and guanosine at position 1 bearing a 2’ ribose O-methylation (m7G2 deoxyPPpG2 OMe).[000122] Figure 1 ID is a diagrammatic illustration of the chemical structure of a dinucleotide mRNA cap bearing a N7-m ethylguanosine at position 0 with a deoxygenated 3’ ribose modification and guanosine at position 1 bearing a 2’ ribose O-methylation (m7G3 deoxyPPpG2 OMe).[000123] Figure 12A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3 OMepppG subsequently enzymatically-labelled at the 2’ ribose in position 1 with a methyl group (m7G3’OMepppG2 OMe); (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G2 OMepppG subsequently enzymatically-labelled at the 2’ ribose in position 1 with a methyl group (m7G2 OMepppG2 OMe); and (iii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G2 ,3 OMepppG subsequently enzymatically-labelled at the 2’ ribose in position 1 with a methyl group (m7G2,3’OMepppG2’OMe) in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a negative control and m7G3 OMepppG2 OMe is used as a positive control.[000124] Figure 12B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3 OMepppG subsequently enzymatically-labelled at the 2’ ribose in position 1 with a methyl group (m7G3’OMepppG2 OMe); (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G2 OMepppG subsequently enzymatically-labelled at the 2’ ribose in position 1 with a methyl group (m7G2 OMepppG2 OMe); and (iii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid(27.4:50:21.1 :1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G2 ,3 OMepppG subsequently enzymatically-labelled at the 2’ ribose in position 1 with a methyl group (m7G2,3’OMepppG2’OMe) in spleen tissue at 24 hours post intravenous injection. PBS is used as a negative control and m7G3 OMepppG2 OMe is used as a positive control.[000125] Figure 12C provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3 OMepppG subsequently enzymatically-labelled at the 2’ ribose in position 1 with a methyl group (m7G3’OMepppG2 OMe); (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP2’deoxy, subsequently enzymatically N7-methylated (m7GTP2deoxy) and labelled at the 2’ ribose in position 1 with a methyl group (m7G2’deo yPPpG2 OMe); and (iii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTPs’deoxy, subsequently enzymatically N7- methylated (m7GTP3 deoxy) and labelled at the 2’ ribose in position 1 with a methyl group (m7G3’deoxyPPpG2 OMe) in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a negative control and m7G3 OMepppG2 OMe is used as a positive control.[000126] Figure 12D provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3 OMepppG subsequently enzymatically-labelled at the 2’ ribose in position 1 with a methyl group (m7G3’OMepppG2 OMe); (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTP2’deoxy, subsequently enzymatically N7-methylated (m7GTP2deoxy) and labelled at the 2’ ribose in position 1 with a methyl group (m7G2’deoxyPPpG2 OMe); and (iii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 7:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA post-transcriptionally capped with GTPs’deoxy, subsequently enzymatically N7-methylated (m7GTP3 deoxy) and labelled at the 2’ ribose in position 1 with a methyl group (m7G3’deo yPPpG2 OMe) in spleen tissue at 24 hours post intravenous injection. PBS is used as a negative control and m7G3 OMepppG2 OMe is used as a positive control.[000127] Figure 13 is a diagrammatic illustration of the chemical structure of a trinucleotide N7- methylguanosine mRNA cap bearing a 3’ O-methyl ribose modification on the N7- methylguanosine at position 0, a 2’ O-methyl ribose modification and an N6-methyl base modification on the adenosine at position 1 and an unmodified guanosine at position 2 (m7G3’OMePPpM6A2’OMepG).[000128] Figure 14A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppM6A2’OMepG in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a negative control.[000129] Figure 14B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppM6A2’OMepG in spleen tissue at 24 hours post intravenous injection. PBS is used as a negative control.[000130] Figure 15 is a diagrammatic illustration of the chemical structure of a trinucleotide N7- methylguanosine mRNA cap bearing a 3’ O-methyl ribose modification on the N7- methylguanosine at position 0, a vinyl phosphonate modified bridge linked to N7-methyl guanosine, a 2’ O-methyl ribose modification on the adenosine at position 1 and an unmodified guanosine at position 2 (m7G3’OMevpppA2’OMepG).[000131] Figure 16A provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMevpppA2’OMepG in bone marrow tissue at 24 hours post intravenous injection. PBS is used as a negative control.[000132] Figure 16B provides a comparison of protein expression in mice as measured by luminescence intensity between the following delivery systems: (i) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMepppA2 OMepG; and (ii) a lipid nanoparticle comprising 50 mol% DSPC LNP (Ionizable lipid 4:DSPC:Cholesterol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”) encapsulating an FLuc mRNA co-transcriptionally capped with m7G3’OMevpppA2’OMepG in spleen tissue at 24 hours post intravenous injection. PBS is used as a negative control.Detailed Description[000133] The lipid nanoparticle as described herein contains elevated neutral lipid content mol% beyond what is used in conventional formulations for nucleic acid delivery and provides improved mRNA delivery to and protein expression in extrahepatic tissues relative to the benchmark baseline LNP formulation.[000134] As used herein, the term "ionizable lipid" refers to a lipid that, at a given pH, is in an electrostatically neutral form and that may either accept or donate protons, thereby becoming electrostatically charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1 -octanol (i.e., a cLogP) that is greater than 8.[000135] As used herein, the term "cationic ionizable lipid" refers to an ionizable lipid that, at a given pH, is in an electrostatically neutral form, but that at an appropriately lower pH accepts a proton, thereby becoming electrostatically positively charged. The terms “ionizable lipid” and “cationic ionizable lipid” are used interchangeably.[000136] The term “neutral lipid” refers to any of a number of polar lipid species, including vesicle-forming lipids, that exist either in an uncharged or neutral zwitterionic form at physiological pH.[000137] Optionally, the lipid nanoparticle further comprises one or more sterols or derivatives thereof. The term “sterol derivatives” refers to modified sterols or precursors thereof, including triterpenes. The term “cholesterol” refers to a naturally-occurring or synthetic compound having a gonane skeleton and that has a hydroxyl bonded to one of its rings, typically the A-ring.[000138] The lipid nanoparticle as described herein comprises a cargo that is messenger RNA. As used herein, the term “messenger RNA” or “mRNA”, refers to a polynucleotide that encodes and expresses at least one peptide, polypeptide or protein. The term is meant to include, but is not limited to, small activating RNA (saRNA), circular RNA (circRNA) and trans-amplifying RNA (taRNA).[000139] The lipid nanoparticle as described herein may further comprise a cargo that is guide RNA. In some embodiments, the lipid nanoparticle encapsulates a cargo that includes CRISPR gene editing systems, most advantageously mRNA encoding for one or more of a Class II Cas nuclease family of proteins and a guide RNA. The nucleases encoded by the nucleic acid are enzymes with DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by an appropriate guide RNA. The guide RNA can direct the Cas nuclease to the target sequence on a target nucleic acid molecule, where the guide RNA hybridizes to the target sequence and the Cas nuclease cleaves or modulates the sequence. In some embodiments, the guide RNA binds to a class 2 nuclease, thereby providing specificity of cleavage.[000140] Guide RNAs for the CRISPR / Cas9 nuclease system include CRISPR RNA (crRNA) or tracr RNA (tracr). In some embodiments, the crRNA can include a targeting sequence that is complementary to and hybridizes to a target sequence on a target nucleic acid molecule. The crRNA can also include a flagpole that is complementary to, and hybridize to, a portion of tracrRNA. In some embodiments, the crRNA can correspond to the structure of a naturally- occurring crRNA transcribed from a bacterial CRISPR locus, wherein the targeting sequence acts as a spacer for the CRISPR / Cas9 system. The flagpole corresponds to the part of the repetitive sequence adjacent to the spacer above the CRISPR locus.[000141] The guide RNA of the RNP can target any sequence of interest through the targeting sequence of crRNA. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can comprise at least one mismatch.[000142] As used herein, the term “encapsulation,” with reference to incorporating the mRNA within a lipid nanoparticle refers to any association of the mRNA with any lipid component or compartment of the lipid nanoparticle. In one example of the disclosure, the mRNA is present in the core of the LNP.[000143] “Position 0” and “Position 1” refer to the positions of the two nucleosides connected by the bridge in the mRNA cap. Position 1 refers to the first nucleobase in the mRNA. As used herein, the term “guanosine at position 0”, is a guanosine or any analogue thereof that may further comprise modifications such as alkylation (e.g., methylation) on its purine ring and / or its ribose ring. In one example of the disclosure, the guanosine at position 0 has an N7-m ethylation, referred to herein interchangeably as “N7-methylguanosine” or “N7-methylated guanosine”.[000144] As used herein, the modification of the “2’ carbon” and modification of the “3’ carbon” refer to one or more suitable chemical modifications on the ribose carbon atom so that the ribose carbon atoms are linked to moieties other than hydroxyl groups. In some examples, such moieties include optionally substituted O-alkyl and / or O-aryl groups bonded to the respective ribose carbon atoms and may be introduced via modification of a 2’ and / or 3’ hydroxyl group of the ribose ring by known organic synthesis methods. However, it should be understood that the term “modification” as used herein is not limited by the method of synthesis or by the use of any particular starting material for such synthesis. That is, the modification is used to denote the chemical structure of the ribose sugars and may be pre-existing, inherent or designed and is not limited to carrying out a specific physical or transformative act or step.[000145] As used herein, the terms “nucleoside”, “ribonucleoside”, “nucleotide” and “ribonucleotide” are used interchangeably.[000146] As used herein, the terms “cap”, “mRNA cap” and “5’cap” are used interchangeably.[000147] As used herein, the terms “chain initiation”, “transcription”, “zw vitro transcription” are used interchangeably and refer to the addition of a ribonucleotide to the 3 ’OH group of a ribonucleotide.[000148] As used herein, the terms “m7”, “m7”, “m7”, and “m7” are used interchangeably and refer to 7-m ethylguanosine, meaning that a methyl group (-CH3) is attached to the nitrogen at position 7 of the guanine base in guanosine.[000149] Without being bound by theory, the 3’ ribose modification of the guanosine at position 0 blocks incorrect chain initiation during in vitro transcription. In one embodiment, the modification of 3’ ribose of the guanosine at position 0 improves mRNA translation. In another embodiment, the 3’ modification of the guanosine at position 0 confers stability to mRNA. Without being bound by theory, the 2’ ribose modification of the guanosine at position 0 blocks chain initiation in in vitro transcription. In one embodiment, the modification of 2’ ribose of the guanosine at position 0 improves mRNA translation. In another embodiment, the 2’ modification of the guanosine at position 0 confers stability to mRNA. In one embodiment, the 3’ ribose modification at position 0 prevents translation repressor proteins from binding to the cap. In one embodiment, the 2’ ribose modification of the guanosine at position 0 prevents translation repressor proteins from binding to the cap. In one embodiment, the 3’ ribose modification of the guanosine at position 0 blocks translation repressor proteins from binding to the cap. In one embodiment, the 2’ ribose modification of the guanosine at position 0 blocks translation repressor proteins from binding to the cap. In one embodiment, the 3’ ribose modification of the guanosine at position 0 blocks decapping enzymes from binding to the mRNA cap. In one embodiment, the 2’ ribose modification of the guanosine at position 0 blocks decapping enzymes from binding to the mRNA cap.[000150] The term “modification” as used herein with reference to the mRNA cap structure includes substitution of suitable atoms, molecules, groups, and / or moieties of one or more atoms of the mRNA cap and that provides a desired level of expression from mRNA. “Substituted” means that one or more atoms such as carbon or hydrogen, preferably up to 5 atoms, more preferably 1 to 3 hydrogen atoms in a group, are independently substituted by one or more substituents. As used herein, “alkylation” refers to an “alkyl” group. “Alkyl” means a C1-C20 comprising or consisting of a linear, cyclic and / or branched aliphatic hydrocarbon group,preferably a Cl -CIO alkyl group, more preferably a C1-C6 alkyl group, or a C1-C4 alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1 -dimethylpropyl, 1,2-dimethylpropyl, 2,2- dimethylpropyl, 1 -ethylpropyl, 2-m ethylbutyl, 3 -methylbutyl, n-hexyl, 1 -ethyl -2-m ethylpropyl, 1,1,2-tri methylpropyl, 1,1 -dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 - dimethylbutyl, 2-ethylbutyl, 2-m ethylpentyl, 3 -methylpentyl, 4-m ethylpentyl and 2,3- dimethylbutyl. Alkyl groups may be substituted or unsubstituted.[000151] In some non-limiting examples, an alkyl group is added to a hydroxyl group to form an O-alkyl group. “O-alkyl” refers to the group (alkyl-O-), and the alkyl is as defined herein. In some embodiments, the alkyl is C1-C6 or C1-C4. Examples of O-alkyl include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy and tert-butoxy. O-alkyl groups may be substituted or unsubstituted.[000152] As used herein, “arylation” refers to an “aryl” group. Aryl refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be linked together in a fused manner. The term “aryl” includes monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, and tetrahydronaphthyl. Preferred aryl groups are C6-C10 aryl groups, and more preferred aryl groups are phenyl and naphthyl. Aryl groups may be substituted or unsubstituted.[000153] As used herein, “E” or “Z” configuration refers to “cis” or “trans” configurations in a monovalent hydrocarbon radical containing at least one carbon-carbon double bond and may have either an E- or Z-steric configuration.[000154] The term “optionally substituted” with reference to an aliphatic group (e.g., alkyl or alkyl group) or an aryl group means that at least one hydrogen atom of the aliphatic group or aryl group can be replaced by a non-hydrogen atom or group of atoms (i.e., a “substituent”), and / or the aliphatic or aryl group is interrupted (e.g., a -(CH)2- group replaced) by a non-carbon atom or one or more substituents. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino,sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.[000155] As used herein, the terms “wavy line”,, and refer to a point of attachment to an atom of the remainder of the molecule, and each of these terms may be used interchangeably.[000156] As used herein, the term “dashed line” indicates an optional bond. Unless chemically or structurally required, no directionality is indicated or implied by the order in which a chemical group is written or the point at which it is attached to the remainder of the molecule.[000157] The term “purine” is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. Similarly, the term “pyrimidine” is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. A non-limiting list of optionally substituted purine-bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g. 7-methylguanine), theobromine, caffeine, uric acid and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5 -alkylcytosine (e.g., 5-methylcytosine). Non-limiting examples include a 9-adeninyl, a N6-methyl 9-adeninyl, a 9-guaninyl, a 1-cytosinyl, a 1 -uracilyl modifications to the purine or pyrimidine bases.[000158] The term “triazole moiety”, as used herein, refers to a structural unit of a five-membered ring containing 1,2, 3 -triazole.[000159] As used herein, the term “terminal moiety” refers to the moiety connecting the 5’ cap to the rest of the mRNA molecule. In case of a dinucleotide cap, the terminal moiety is the nucleotide at position 1. In the case of a trinucleotide cap, the terminal moiety is the nucleotide at position 2. In the case of a tetranucleotide, the terminal moiety is the nucleotide at position 3.[000160] As used herein, the term “oligonucleotide” refers to nucleotides of various lengths: dinucleotide, trinucleotide, tetranucleotide and pentanucleotide.[000161] The term “halogenation” as used herein refers to the addition of fluorine, chlorine, bromine, iodine, astatine, and / or tennessine.[000162] Unless otherwise indicated, the structures described / illustrated herein further include all isomers (e.g., diastereomeric, enantiomeric, atropisomeric and geometrically (conformationally) isomeric forms) of the structure; and certain salts of the compound which can maintain its original biological activity.[000163] “Administering” and similar terms used herein (grammatical variations) refer to the introduction of a lipid nanoparticle and / or compositions thereof to the body of a subject, using any of one of various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include an intravenous, intramuscular, buccal, epidermal, epidural, intraarterial, intraarticular, intracapsular, intracardiac, intracoronary, intradermal, intralesional, intralymphatic, intranasal, intraorbital, intraperitoneal, intraspinal, intrasterna, intrathecal, mucosal, oral, rectal, subarachnoid, subcapsular, subcutaneous, subcuticular, sublingual, topical, transtracheal, or vaginal route of administration or any combination thereof. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.[000164] As used herein, “labelling” refers to the enzymatic O-m ethylation of the hydroxyl group in a nucleotide. The term is meant to include both in vitro and in vivo O-methylation of a nucleotide and / or nucleoside.[000165] As used herein, "synthesizing" with respect to mRNA refers to the process of incorporation of nucleotides in the 5’ to 3’ direction into a polynucleotide chain of variable length and nucleotide composition.[000166] As used herein, “purifying” refers to the process of removing contaminants from the mRNA preparation post synthesis and / or post capping and / or post labelling.[000167] The concentration of mRNA in the LNP may be between 0.01 and 20 mg / mL or between 0.01 and 10 mg / mL or between 0.05 and 5 mg / mL or between 0. 075 and 4 mg / mL.[000168] The mRNA as used herein encompasses both modified and unmodified mRNA. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or may be chemically synthesized.[000169] In those embodiments in which an mRNA is chemically synthesized, the mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / orbackbone modifications. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl- cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5 -propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaad enosine, 7-deazaguanosine, 8 -oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2- thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e g., methylated bases); intercalated bases; modified sugars (e g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).[000170] The mRNAs of the disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs in certain embodiments may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.[000171] In some embodiments, in vitro synthesized mRNA may be purified before encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.[000172] The present disclosure may be used to formulate mRNAs of a variety of lengths. In some embodiments, the present disclosure may be used to formulate and encapsulate in vitro synthesized mRNA ranging from about 250 bp to 20 kb, about 500 bp -20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length.[000173] Typically, mRNA synthesis includes the addition of a “cap” on the 5' end, and a “tail” on the 3' end. The cap may include modifications to its nucleobase and / or its ribose ring. The presence of the cap provides resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.[000174] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA'sstability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.[000175] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.[000176] While mRNA provided from in vitro transcription reactions may be desirable in certain embodiments, other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.[000177] The mRNA sequence may comprise a reporter gene sequence, although the inclusion of a reporter gene sequence in pharmaceutical formulations for administration is optional. Such sequences are incorporated into mRNA for in vivo studies in animal models to assess biodistribution.[000178] The mRNA sequence may comprise a gene editing system. In some embodiments, the editing system includes Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbll 1, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. mRNA cap[000179] The 5' cap structure of an mRNA is a key feature of every nuclear-encoded mRNA in mammals and plays important cellular roles in nuclear export, mRNA stability and mRNA translation. The 5’ cap interacts with several distinct cap-binding proteins that modulate the above- mentioned cellular functions.[000180] In mammals, natural endogenous mRNA molecules are generally 5 '-capped with a N7- methylated guanosine (at position 0) connected with the first-transcribed nucleobase (at position 1) of the mRNA through an inverted 5'- 5' triphosphate bridge. The N7-methyl-guanylate at position 0 is referred to, interchangeably, as the guanosine at position 0 throughout this document. The ribose sugar of the N7-methylated guanosine at position 0 may be artificially further modifiedat the 3’ carbon and / or optionally, naturally and or artificially, the ribose sugar of the nucleoside at position 1 may also be modified at the 2’ carbon.[000181] In one embodiment, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be artificially modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group. Other non-limiting examples include modifications with one or more of the following groups and / or moieties: S, CH3, NH2.[000182] Additional artificial modifications of the 5’ - 5’ triphosphate bridge may be incorporated using a-methyl-phosphonate and / or seleno-phosphate modifications to reduce mRNA decapping.[000183] Additional natural and / or artificial modifications include, but are not limited to, 2'-O- methylation of the ribose sugars of the N7-methylguanosine (at position 0), 2'-O-methylation of the ribose sugars of the first nucleoside (at position 1) and / or the second nucleoside (at position 2).[000184] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5 '-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and / linked to a nucleic acid molecule. Cap analogs may also be enzymatically labelled and / or modified after the capping process is completed, such as the modification of the nucleoside at position 2 through the process of enzymatic methylation.[000185] In another embodiment, the cap analog is a N7-(4-chlorophenoxy ethyl) substituted dinucleotide form of a cap analog known in the art and / or described herein. Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4- chlorophenoxyethyl)-G(5')ppp(5')G and a N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analog (e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 2013 21 :4570-4574; the contents of which are herein incorporated by reference in its entirety). In another embodiment, a cap analog of the present invention is a 4-chloro / bromophenoxy ethyl analog.[000186] The mRNA may be capped co- or post-transcriptionally. mRNA may be capped co- transcriptionally using natural or artificial dinucleotides, trinucleotides or oligonucleotides. mRNA may be capped post-transcriptionally using natural or artificial mononucleotides.[000187] Modified nucleic acids may be capped post-transcriptionally, using enzymes with dual guanyl-transferase and methyl-transferase activities, in order to generate naturally occurring or artificial 5 'cap structures. Non-limiting examples of such 5 'cap structures of the present invention are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5' decapping, as compared to synthetic 5 '-cap structures known in the art (or to a wild-type, natural or physiological 5 '-cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O- methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5'- terminal nucleotide of an mRNA at position 1 and a guanine cap nucleotide at position 0 wherein the cap guanine contains an N7 methylation and the 5 '-first nucleotide of the mRNA at position 1 contains a 2'-O-methyl group. Because the modified nucleic acids may be capped post- transcriptionally, and because this process is more efficient, nearly 100% of the modified nucleic acids may be efficiently capped. This is in contrast to when a mRNA cap dinucleotide or trinucleotide is used to cap mRNA co-transcriptionally, i.e. in the course of an in vitro transcription reaction, where only 70-95% of the modified nucleic acids may be efficiently capped.Neutral Lipid[000188] The term “neutral lipid” refers to a lipid that exists either in an uncharged or neutral zwitterionic form at physiological pH. In some embodiments, the neutral lipid includes an amphipathic lipid that allows for the formation of particles. In alternative embodiments, the lipid nanoparticle comprises elevated levels (>20 mol%, 25 mol%, 30 mol% or 35 mol%) of a structural lipid that is a non-cationic lipid.[000189] In some embodiments, the neutral lipid is selected so that the lipid nanoparticle has “substantially no net charge” at physiological pH, which means a net surface charge of about zero, or near neutral at physiological pH, such as without limitation about -2.5 mV to about 2.5 mV.[000190] Neutral lipids include, but are not limited to, phosphotidylcholines such as 1,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), l-Palmitoyl-2-oleoyl-sn- glycero-3 -phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phophatidylethanolamines such as l,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE),sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.[000191] The neutral lipid content may include mixtures of two or more types of different structural lipids. In one embodiment, the phosphatidylcholine lipid content is a mixture of DSPC and DOPC and POPC. In such embodiments, the mixture may have a DSPC content of at least 20 or 30 mol%.[000192] The neutral lipid content in some embodiments is present at greater than 20 mol%, greater than 25 mol%, greater than 30 mol%, greater than 32 mol%, greater than 34 mol%, greater than 36 mol%, greater than 38 mol%, greater than 40 mol%, greater than 42 mol%, greater than44 mol%, greater than 46 mol%, greater than 48 mol% or greater than 50 mol%. In some embodiments, the upper limit of helper lipid content is 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol% or 45 mol%. The disclosure also encompasses sub-ranges of any combination of the foregoing numerical upper and lower limits.[000193] For example, in certain embodiments, the phosphatidylcholine lipid content is from 20 mol% to 80 mol% or 25 mol% to 60 mol% or 30 mol% to 60 mol% or 35 mol% to 60 mol% or 40 mol% to 60 mol% or 42 mol% to 58 mol%, or 43 mol% to 57 mol% or 44 mol% to 56 mol% or45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.[000194] In one embodiment, the neutral lipid is DSPC. For example, in certain embodiments, the DSPC lipid content is from 20 mol% to 80 mol% or 25 mol% to 60 mol% or 30 mol% to 60 mol% or 35 mol% to 60 mol% or 40 mol% to 60 mol% or 42 mol% to 58 mol%, or 43 mol% to 57 mol% or 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.Ionizable lipid[000195] The LNP of the disclosure has an ionizable lipid. The ionizable lipid may be charged at low pH and have substantially no net charge at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid cargo during initial formulation. Since the ionizable lipid is near neutral at physiological pH, toxicity and renal clearance is reduced. After cellular uptake by endocytosis, the acidic environment of the endosome leads to an increase in the net positive charge of the ionizable amino lipids, which promotes fusionwith the anionic lipids of the endosomal membrane and subsequent membrane destabilization and release of the nucleic acid-based therapeutics into the cytoplasm to exert their effects.[000196] In some embodiments, it is desirable to include less than 50 mol% ionizable lipid. That is, the ionizable lipid content may be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol% or less than 5 mol%.[000197] In certain embodiments, the ionizable lipid content is from 5 mol% to 50 mol% or 8 mol% to 47 mol% or 10 mol% to 50 mol% or 15 mol% to 45 mol% or 15 mol% to 35 mol% of total lipid present in the lipid nanoparticle.[000198] As used herein, the term "cationic lipid" refers to a lipid that, at a given pH, such as physiological pH, is in an electrostatically neutral form and that may either accept or donate protons, thereby becoming electrostatically positively charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1 -octanol (i.e., a cLogP) greater than 8. In some embodiments, the cationic lipid has a pKa that is between 5.0 and 8.0 when formulated in an LNP.[000199] In some embodiments, the cationic lipid has an amino group. In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, C16 to C18 alkyl chains, optionally ether linkages between its head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, but are not limited to sulfur lipids, such as MF019 described in WO 2022 / 155728A1 and DODMA. Other lipids that may be used in the practice of the disclosure include MC3- and KC2-type lipids, which are well-known to those of skill in the art. In further embodiments, the ionizable lipid is selected from one or more lipids set forth in WO 2022 / 246555; WO 2022 / 246568; WO 2022 / 246571; WO 2023 / 147657; WO2022 / 155728; WO 2023 / 215989; WO 2024 / 065041; WO 2024 / 065042; WO 2024 / 130421; WO 2024 / 065043; and U.S. patent Publication No. 2024 / 0294462, each incorporated herein by reference.[000200] In one embodiment, the ionizable cationic lipid comprises an ionizable amino head group and at least two lipophilic groups, at least one of which comprises a heteroatom or a moiety comprising one or more heteroatoms, such as an ester or one or more sulfur atoms. In some embodiments, at least one lipophilic group comprises distal branching and / or one or more cyclic groups. Examples of ionizable cationic lipids comprising an ionizable amino head group and twolipophilic chains, at least one chain comprising one or more sulfur atoms and / or ester groups are described in co-owned and co-pending WO 2023 / 215989; WO 2024 / 065041; WO 2024 / 065042; WO 2024 / 130421; and WO 2024 / 06504. Functional groups comprising one or more heteroatoms may be biodegradable in vivo.[000201] In one embodiment, the ionizable cationic lipid has a protonatable amino head group; at least two lipophilic moieties, wherein the amino head group has a central nitrogen atom or carbon atom to which each of the two lipophilic moieties are directly bonded; each lipophilic chain has between 15 and 40 carbon atoms in total; and wherein the lipid has (i) an apparent pKaof between 6 and 7.5 when formulated in the LNP; and (ii) a ClogP of at least 11. The apparent pKais measured as set forth in WO 2025 / 035202, which is incorporated herein by reference.[000202] Optionally, at least one of the lipophilic moieties bonded to the head group has a biodegradable group, including but not limited to an ester group or a moiety comprising an ester (in any orientation). In one embodiment, at least one of the lipophilic moieties comprises a sulfur atom. In another embodiment, at least one of the lipophilic moieties comprises an ester or a moiety comprising an ester, one or more sulfur atoms or any combination thereof.[000203] In one non-limiting example, at least one of the lipophilic moieties has the formula:amino acid or a sarcosine group.The ester is present in either orientation.[000204] In some embodiments, R1and R2in the lipophilic moieties are, independently, linear, cyclic and / or branched optionally substituted C3-C20 alkyl and optionally with varying degrees of unsaturation; and n is 4 to 8.[000205] In some embodiments, it is desirable to include less than 50 mol% ionizable cationic lipid in the LNP. That is, the ionizable cationic lipid content may be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol% or less than 5 mol%.[000206] In certain embodiments, the ionizable cationic lipid content is from 5 mol% to 50 mol% or 8 mol% to 47 mol% or 10 mol% to 50 mol% or 15 mol% to 45 mol% or 15 mol% to 35 mol% of total lipid present in the lipid nanoparticle.[000207] The ionizable lipid component may include an ionizable anionic lipid as part of the ionizable lipid content. An example of such a lipid is cholesteryl hemisuccinate (CHEMS). Further examples of ionizable anionic lipids are described in co-pending and co-owned PCT / CA2024 / 050347 filed on March 22, 2024, which is incorporated herein by reference in its entirety.Sterol[000208] The LNP further includes a sterol in some embodiments. The term “sterol” refers to a naturally-occurring or synthetic compound having a gonane skeleton and that has a hydroxyl moiety attached to one of its rings, typically the A-ring.[000209] Examples of sterols include cholesterol, or a cholesterol derivative, the latter referring to a cholesterol molecule having a gonane structure and one or more additional functional groups.[000210] The cholesterol derivative includes P-sitosterol, 3 -sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, 3P[N-(N'N'-dimethylaminoethyl)carbamoyl cholesterol (DC-Chol), 24(S)- hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23- oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7- hydroxy cholesterol, 19-hydroxy cholesterol, 22-hydroxy cholesterol, 25-hydroxycholesterol, 7- dehydrocholesterol, 5a-cholest-7-en-3P-ol, 3,6,9-trioxaoctan-l-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, sitocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22- dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxy cholic acid, zymosterol, diosgenin, fucosterol, fecosterol or a salt or ester thereof.[000211] In one embodiment, the sterol is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.[000212] In another embodiment, the sterol is cholesterol and is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.[000213] In another embodiment, the sterol is a cholesterol derivative and is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.[000214] In one embodiment, the combined (i) sterol content (e.g., cholesterol or cholesterol derivative thereof); and (ii) neutral lipid content is at least 50 mol%; at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol% or at least 85 mol% based on the total lipid present in the lipid nanoparticle.Hydrophilic polymer-lipid conjugate[000215] In one embodiment, the lipid nanoparticle comprises a hydrophilic-polymer lipid conjugate capable of incorporation into the LNP. The conjugate includes a lipid or lipophilic moiety covalently attached to a polymer chain that is hydrophilic, optionally via a linker region. Examples of hydrophilic polymers include polyethyleneglycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropyl methacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine and polyaspartamide. In one embodiment, the hydrophilic-polymer lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer lipid conjugate may also be a naturally occurring or synthesized oligosaccharide-containing molecule, such as monosialoganglioside (GMI). The ability of a given hydrophilic-polymer lipid conjugate to enhance the circulation longevity of the LNPs herein could be readily determined by those of skill in the art using known methodologies.[000216] The hydrophilic polymer lipid conjugate may be present in the nanoparticle at 0.5 mol% to 5 mol%, or at 0.5 mol% to 3 mol%, or at 0.5 mol% to 2.5 mol% or at 0.5 mol% to 2.0 mol% or at 0.5 mol% to 1.8 mol% of total lipid. In certain embodiments, the hydrophilic polymer lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or at 0 mol% to 3 mol%, or at 0 mol% to 2.5 mol% or at 0 mol% to 2.0 mol% or at 0 mol% to 1.8 mol% of total lipid.[000217] In another embodiment, the PEG-lipid conjugate is present in the nanoparticle at 0.5 mol% to 5 mol%, or at 0.5 mol% to 3 mol% or at 0.5 mol% to 2.5 mol% or at 0.5 mol% to 2.0 mol% or at 0.5 mol% to 1.8 mol% of total lipid. In certain embodiments, the PEG-lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or at 0 mol% to 3 mol%, or at 0 mol% to 2.5 mol% or at 0 mol% to 2.0 mol% or at 0 mol% to 1.8 mol% of total lipid.[000218] In one embodiment, the lipid nanoparticle has “substantially no hydrophilic polymerlipid conjugate” or is “non-sterically stabilized”, “unshielded” or “uncoated”, meaning the lipid nanoparticle has less than 0.8 mol% total hydrophilic-polymer lipid conjugate content or other surface stabilizer content as measured based on the total lipid content of the nanoparticle as measured based on the total lipid content of the nanoparticle. In some embodiments, the hydrophilic-polymer lipid conjugate or other surface stabilizer content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the hydrophilic-polymer lipid conjugate or other surface stabilizer mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%.[000219] As used herein, the term “surface stabilizer” is a macromolecule, including a protein, polysaccharide or polymer, including a block copolymer, that is used to stabilize a lipid nanoparticle, and in which at least a portion (e.g., hydrophilic) is present on the surface of the lipid nanoparticle. Such molecules are employed by those of skill in the art to prevent aggregation, improve shelf life and / or improve the stability of the particle after administration, such as the circulation lifetime of the lipid nanoparticle. The term includes surface stabilizers that are known to control the size of lipid nanoparticles, such as amphiphilic polymers (e.g., block co-polymer). As would be appreciated by those of skill in the art, a hydrophobic portion of the surface stabilizer may partition in a lipophilic portion of the lipid nanoparticle.[000220] In some embodiments, the surface stabilizer is present at less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the lipid nanoparticle. In further embodiments, the surface stabilizer mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%,0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%.[000221] In some embodiments, the hydrophilic-polymer conjugate (e.g., a hydrophilic-polymer lipid conjugate) content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the hydrophilic-polymer conjugate (e.g., a hydrophilic-polymer lipid conjugate) mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol%, 0 and 0.10 mol% or 0 and 0.05 mol%.[000222] In some embodiments, the amphipathic polymer content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the amphipathic polymer mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol%, 0 and 0.10 mol% or 0 and 0.05 mol%. Examples of amphipathic polymers are provided in US 2021 / 0046192, which is incorporated herein by reference.[000223] In some embodiments, the poloxamer content is less than 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the poloxamer mol% content is between 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol%, 0 and 0.10 mol% or 0 and 0.05 mol%.[000224] In further embodiments, the LNP lacks a surface stabilizer that is a protein, referred to as a protein stabilizer. This includes an apolipoprotein stabilizer, derivative or mimetic thereof (see e.g., WO 2023 / 233042, which is incorporated herein by reference). Such apolipoprotein may be selected from one or a combination of apo Al, apo Al- Milano, apo A2, apo A4, apo A5, apo B48, apo Bl 00, apo C-l, apo C-l I, apo C-lll, apo C-IV, apo D, apo E, apo F, apo H, apo L and apo M. In some embodiments, the protein stabilizer content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipidcontent of the nanoparticle. In some embodiments, the protein stabilizer content is less than 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the protein stabilizer content mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%. Since lipid nanoparticles can adsorb proteins after administration, the protein content is measured in vitro prior to administration.[000225] Alternatively, in some embodiments a lipid nanoparticle preparation lacks or has low levels thereof of one or more stabilizing agents, which includes a surface stabilizer as described above and a cryoprotectant. In some embodiments, the lipid nanoparticle preparation has less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v of one or more cryoprotectants in the preparation. In some embodiments, a lipid nanoparticle preparation having a plurality of LNPs has low levels or lacks glycerol and / or propylene glycol as a cryoprotectant, such as at concentration levels less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v in the preparation.[000226] In another embodiment, the lipid nanoparticle is “PEG-less”, meaning that the lipid nanoparticle has no detectable amounts of polyethylene-glycol lipid conjugate.[000227] Examples of lipid nanoparticles with low levels or no hydrophilic polymer lipid conjugate or other surface stabilizer that can be used in the practice of the disclosure are described in co-owned U.S. Patent No. 12,343,429, which is incorporated herein by reference. The lipid nanoparticles herein may exhibit particularly high encapsulation efficiencies of nucleic acid. As used herein, the term “encapsulation,” with reference to incorporating the nucleic acid within a lipid nanoparticle refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle, including a lipophilic or the aqueous portion. In one embodiment, the nucleic acid is present at least in the core of the LNP.[000228] In one embodiment, the encapsulation efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85, 90% or 92%. The encapsulation efficiency of the nucleic acid is determined as set forth in the Materials and Methods section in the Examples herein.[000229] Embodiments of the present disclosure also provide lipid nanoparticles described according to the molar ratio between the positively charged amine groups of the amine lipid (N)and the negatively charged phosphate groups (P) of the oligonucleotide to be encapsulated. This may be mathematically represented by the equation N / P. In one embodiment, the N / P ratio of the lipid nanoparticle is between 4 and 15 or between 4.5 and 10 or between 5 and 10 or between 5.5 and 8.[000230] In one embodiment, the N / P ratio of the lipid nanoparticle is at least 4, 4.25, 4.50, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0 or 6.25. The upper limit may be 15, 14, 13, 12, 11, 10, 9 or 8. The disclosure also encompasses a combination of any two of the upper and lower limits.[000231] In one embodiment, the lipid nanoparticle has a weight nucleic acid / micromole of total lipid that is 0.05: 1 to 1 : 1. In one embodiment, the lower limit is 0.06: 1, 0.08: 1, 0.10: 1, 0.12: 1, 0.14:1, 0.16: 1, 0.18: 1, 0.20: 1, 0.22: 1, 0.24: 1, 0.26: 1, 0.28: 1, 0.30: 1, 0.32: 1, 0.34: 1, 0.36: 1, 0.38:1 or 0.40: 1 weight nucleic acid / micromole of total lipid. In another embodiment, the upper limit is 0.80: 1, 0.82: 1, 0.84: 1, 0.86: 1, 0.88: 1, 0.90: 1, 0.92: 1, 0.94: 1, 0.96: 1 or 0.98: 1 weight nucleic acid / micromole of total lipid. The disclosure also encompasses a combination of any two of the upper and lower limits.[000232] In one embodiment, the mRNA copy number / LNP is 1-10 or 4-8.Improved gene expression in extrahepatic organs or tissues[000233] As used herein, “expression” of an mRNA refers to translation of an mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme) and also can include, as indicated by context, the post-translational modification of the peptide, polypeptide or fully assembled protein (e.g., enzyme).[000234] In one embodiment, the lipid nanoparticle exhibits at least a 10% increase in gene expression of the mRNA in vivo as measured in an extrahepatic tissue or organ.[000235] In another embodiment, the extrahepatic tissue or organ is spleen, bone marrow, lungs, kidney, heart, abdominal skin, back skin and / or ear.[000236] Whether or not a lipid particle exhibits such enhanced delivery to a given tissue or organ can be determined by biodistribution studies in an in vivo mouse model. In such embodiments, the biodistribution may be studied using firefly luciferase (FLuc) expression levels in extrahepatic tissue or organ.[000237] To assess whether a given mRNA-lipid nanoparticle exhibits an increase in gene expression in a relevant tissue or organ at 4 hours, 12 hours, 24 hours, 48 hours or 3 days postinjection, the mRNA-LNP of the disclosure is evaluated using cap analogues bearing one or more modifications.[000238] In one embodiment, the mRNA-lipid nanoparticle exhibits at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290% or 300% increase in gene expression of an encapsulated mRNA encoding FLuc as measured in vivo in spleen and / or bone marrow tissue at 4 hours, 24 hours and / or 3 days postinjection as compared to a lipid nanoparticle encapsulating FLuc mRNA with an unmodified cap analogue, wherein the gene expression is measured in a mouse model by FLuc translated from the mRNA. The measurement is carried out using luminescence intensity. The cap analogues include ribose modifications at the 2’ and / or 3’ carbon positions in the nucleosides at position 1 and / or position 0.[000239] In one embodiment, the extrahepatic tissue and / or organs selected are from the spleen and determined at 4 hours, 24 hours and / or 3 days post-injection. In another embodiment, the extrahepatic tissue and / or organs selected are from bone marrow tissue and determined at 4 hours, 24 hours and / or 3 days post-injection. In one embodiment, the extrahepatic tissue and / or organs selected are from abdominal skin tissue and determined at 4 hours, 24 hours and / or 3 days postinjection. In another embodiment, the extrahepatic tissue and / or organs selected are from lung tissue and determined at 4 hours, 24 hours and / or 3 days post-injection. In one embodiment, the extrahepatic tissue and / or organs selected are from heart tissue and determined at 4 hours, 24 hours and / or 3 days post-injection.Clinical and non-clinical uses of the LNP herein[000240] In some embodiments, the lipid nanoparticle comprising mRNA is part of a pharmaceutical composition and is administered to treat and / or prevent a disease condition. The treatment may provide a prophylactic (preventive), ameliorative or a therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage.[000241] The LNPs described herein may be used to treat and / or prevent any disease, disorder or condition in a mammalian subject. This includes a disease, disorder or condition, such as cancer, infectious diseases such as bacterial, viral, fungal or parasitic infections, inflammatory and / or autoimmune disorders, including treatments that induce immune tolerance and cardiovascular diseases such as hypertension, cardiac arrhythmia and restenosis.[000242] Examples of cancers include lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach (gastric) cancer, esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, renal cancer (e.g., renal cell carcinoma), cancer of the central nervous system, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head and neck cancers, osteogenic sarcomas, and blood cancers. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., caused by metastasis of some other non-liver cancer cell type), and hepatoblastoma.[000243] Non-limiting examples of other diseases, disorders or conditions that may be treated by the mRNA-LNPs herein and that may be attributed at least in part to an immunological disorder include colitis, Crohn's disease, allergic encephalitis, allograft transplant / graft vs. host disease (GVHD), diabetes and multiple sclerosis.[000244] The LNPs herein may also be used in other applications besides the treatment and / or prevention of a disease or disorder. The LNPs may be used to treat conditions such as aging, preventative medicine and / or as part of a personalized medicine regime. In further embodiments, the LNP is used in a diagnostic application.[000245] In one embodiment, the LNP is part of a pharmaceutical composition administered parenterally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, the pharmaceutical compositions are for intra- tumoral administration. In anotherembodiment, the pharmaceutical compositions are administered intranasally, intravitreally, subretinally, intrathecally or via other local routes.[000246] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.[000247] The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non -human subject.[000248] The examples below are intended to illustrate the preparation of specific lipid nanoparticle mRNA preparations and properties thereof but are in no way intended to limit the scope of the invention.Materials and Methods mRNA synthesis (in vitro transcription)[000249] Firefly luciferase (FLuc) mRNA encoding 5’-beta globin and 3’-beta globin untranslated regions was prepared by in vitro transcription by using the bacteriophage T7 RNA polymerase (ON-004-C200-G, Hongene Biotech™, China) or Codex HiCap™ RNA Polymerase (E014-B032A-250uL, Aldevron, US). In brief, 100 pL template DNA was digested with 500 units of Bbsl enzyme (R3539M, New England Biolabs™, US) overnight at 37°C. Capping of the mRNA was performed either co- or post-transcriptionally. Trinucleotide cap analogs (CleanCap™ AG (m7GpppA2’OMeG, N-7113-100), CleanCap™3’OMe (m7G3’OMepppA2’OMeG, N-7413-100) or CleanCap™M6 (m7G3’OMepppM6A2’OMeG, N-7453-100) TriLink™ Biotechnologies, US; m7G3’OMepppA2’OEtG Hongene Biotech™, China; m7G3’OMevpppA2’OMeG CAP2811 Areterna-A Synthegene™ Company) or dinucleotide caps (anti-reverse cap analog [ARCA], Cat. No. ON- 121, m7G2’OMepppA2’OMeG and m7G2’,3’OMepppA2’OMeG, Hongene Biotech™, China; m7GpppG (S1404L) New England Biolabs™) were used for co-transcriptional capping of the mRNA. Guanosine triphosphate (GTP; R2331, Hongene Biotech™, China) or 3 ’O-m ethylated GTP (GTP3’OMe; R2-127, Hongene Biotech™, China) (both purchased from Hongene Biotech™, China) were used for post-transcriptional capping of FLuc mRNA, along with 2’-dGTP (NU- 1003L) and 3’-dGTP (NU-1145L)(both purchased from Jena Biosciences™). For one-step Cap- 1 mRNA synthesis, the procedure involves incubating uncapped RNA with Vaccinia Capping Enzyme™ (Cat. No M2080, New England Biolabs™, US) and mRNA Cap 2 -O-Methyltransferase (M0366S, New England Biolabs™, US) in the presence of GTP (or GTPs oMe) and S-adenosylmethionine (SAM; B9003S, New England Biolabs™, US). The uncapped RNA and nuclease-free water are combined to a final volume of 14.0 pL, heated at 65°C for 5 minutes, then placed on ice for 5 minutes. The following components were added: 14.0 pL denatured uncapped RNA, 2.0 pL 10X Capping Buffer, 1.0 pL GTP (10 mM), 1.0 pL SAM (4 mM, diluted from 32 mM stock), 1.0 pL Vaccinia Capping™ Enzyme (10 U / pL), and 1.0 pL mRNA Cap 2'- O-Methyltransferase (50 U / pL) to make a total volume of 20.0 pL. RNase inhibitor is recommended to enhance RNA stability in the reaction, and involves adding 0.5 pL (e.g., Murine RNase Inhibitor™ NEB #M0314) and adjusting the H2O volume accordingly. The mixture is incubated at 37°C for 60 minutes (or 2 hours for RNA less than 200 nucleotides), followed by purification of the RNA for downstream applications. mRNA purification[000250] RNA was purified using the Monarch RNA Cleanup™ Kit (T2050L, New England Biolabs™, US). The in vitro transcription (IVT) reaction was stopped by purifying with either a 500 pg or 50 pg Monarch RNA Cleanup Kit™ according to the manufacturer's procedures. For the 100 pL IVT reaction, 242 pL of binding buffer (T2041-2, New England Biolabs™, US) was added to the sample, followed by addition of 363 pL of 100% ethanol. The mixture was loaded onto the respective columns and centrifuged at maximum speed for 1 minute, with repeated twice repeated loading.[000251] The column was washed with 500 pL of wash buffer (T2042-2, New England Biolabs™, US) twice, each time centrifuged at maximum speed for 1 minute. After washing, the column was transferred to a 1.7 mL microfuge tube, and 100 pL of RNase-free water was added directly onto the column. The sample was incubated at room temperature for 5 minutes and centrifuged at maximum speed for 1 minute to elute the RNA. This elution step was repeated to achieve a final elution volume of 200 pL. The RNA concentration was quantified using UV spectroscopy or a Nanodrop™ device and adjusted to 1 pg / uL before storing at -80°C freezer. mRNA quality control[000252] RNA gel: Agarose (16500500, Invitrogen™, US) was added to a flask according to the following proportions: for a 2% denaturing gel, 1g of agarose was added to 35 mL of water, 5 mL of lOx MOPS-EDTA-Sodium Acetate (MESA) buffer (M5755, Sigma-Aldrich™, US), and 10 mLof 10% formalin solution, neutral buffered, 10% (HT501128-4L, Sigma-Aldrich™, US). The mixture was swirled before heating. After the agarose was fully dissolved, 2 pL Sybr Gold™ lOOOOx (SI 1494, Invitrogen™, US) was added to the mixture. The solution was mixed by swirling and then poured into the tray. Once solidified, the gel was placed in a gel tank and submerged in 1 x MESA buffer. 500 ng of RNA samples were loaded per well. The gel was run at 130 V for 35 minutes. A BioRad™ V3 Western Workfl ow™Imager (BioRad™, US) was used to image the gel to ensure the RNA integrity.Splint-Ligation Capping Efficiency Assay[000253] The splint-ligation capping efficiency assay (see also Blewett et al., 2011, 17(3):535— 543) was carried out as described in the steps below.Step 1: Phosphatase Treatment[000254] To remove uncapped triphosphates, the moles corresponding to 10 pg of mRNA were calculated. The reaction scales were adjusted as follows: for 1 pmol of mRNA, the reaction contained 2 pL of rCutSmart™ (10*) buffer (B6004S, New England Biolabs™, US), 1 pL of calf intestinal phosphatase (Quick CIP™) (M0525L, New England Biolabs™, US), and water up to a total volume of 20 pL. For 17 pmol of mRNA, the reaction contained 34 pL of rCutSmart™ (10*) buffer (B6004S, New England Biolabs™, US), 17 pL of calf intestinal phosphatase (Quick CIP™) (M0525L, New England Biolabs™, US), and water up to a total volume of 340 pL. The reaction was started with 10 pg of mRNA, incubated at 37°C for 10 minutes, then transferred to a 0.5 mL tube and heat-inactivated at 80°C for 2 minutes.Step 2: Kinase Treatment[000255] The reaction from the previous step was used for the kinase treatment. The reagents added were as follows: for the FLuc example, 340 pL of the reaction mix was combined with 40.9 pL of kinase (10*) buffer (M0201L, New England Biolabs™, US), 40.9 pL of adenosine triphosphate (ATP, 10 mM) (R1331, Hongene Biotech™, China), and 8.2 pL of T4 polynucleotide kinase (PNK) (M0201L, New England Biolabs™, US), resulting in a total volume of 430 pL. The mixture was heated at 37°C for 30 minutes. The reaction was then cleaned up using a Monarch™ 50 pg RNA cleanup column (T2047-1, New England Biolabs™, US), eluting with 20 pL of water.Step 3: RNA-DNA Splinted Ligation[000256] For the RNA-DNA splinted ligation, 2 pg of RNA from the previous cleanup was added to a PCR tube with the following reagents: For 2 pg of RNA, 0.4 pL of Splint DNA™ (20 pM) (Integrated DNA Technologies™), 0.6 pL of Anchor RNA™ (30 pM) (Integrated DNA Technologies™), and water up to a total volume of 20 pL were added. The mixture underwent a temperature gradient in a PCR machine: 70°C for 5 minutes, 60°C for 5 minutes, 42°C for 5 minutes, and 25°C for 5 minutes.[000257] For the ligation, 10 pL of the hybridized mixture was combined with 2 pL of T4 DNA ligase buffer (10*) New England Biolabs™, US), 1 pL of RNase inhibitor (ON-039-G, Hongene Biotech™, China), 1 pL of T4 DNA ligase (M0202S, New England Biolabs™, US), and 6 pL of water, resulting in a total volume of 20 pL. For the negative control, uncapped RNA was used, and water was added instead of T4 ligase. The mixture was incubated at 16°C overnight. The next day, 1 pL of DNase I (ON-109, Hongene Biotech™, China) and 2.2 pL of DNase I buffer (10*) (ON-077, Hongene Biotech™, China) were added, and the mixture was heated at 37°C for 30 minutes. The reaction was then cleaned up using a Monarch™ 50 pg RNA cleanup column (T2047-1, New England Biolabs™, US), eluting with 20 pL of water.Step 4: qRT-PCR[000258] The mRNA samples were diluted to 0.5 ng / pL. A sample with 50% capped mRNA was prepared by combining 5 pL of capped RNA (1 ng / pL), 5 pL of uncapped RNA (1 ng / pL), and 10 pL of water to achieve a final concentration of 0.5 ng / pL. Two master mixes were prepared: one with the anchor RNA forward primer (Integrated DNA Technologies™) and another with the internal Flue forward primer (Integrated DNA Technologies™). Each sample had three technical replicates. The master mix for each reaction was prepared with 5 pL of Luna™ universal probe one-step reaction mix (E3005L, New England Biolabs™, US), 0.5 pL of Luna™ RT enzyme mix (E3005L, New England Biolabs™, US), 0.4 pL of forward primer (10 pM), 0.4 pL of reverse primer (10 pM), 0.2 pL of probe (10 pM), and 1.5 pL of water, resulting in a total volume of 8 pL. Each well in the qPCR plate was loaded with 8 pL of the assay mix and 2 pL of RNA samples (0.5 ng / pL). The plates were sealed with optically transparent film and spun briefly for 1 minute at 3000 rpm. The real-time instrument was programmed with the indicated thermocycling protocol:Step 1 : 55°C for 10 min; Step 2: 95°C for 1 min; and Step 3: 95°C for 10 s followed by 60°C for 30 s (40-45 times).Step 5: Data Analysis[000259] Two cycle threshold (Ct) datasets were generated: one targeting uncapped RNA (qSL- RT-PCR) and another targeting total RNA (qRT-PCR). The Ct value from the qSL-RT-PCR (anchor) assay for each sample (target) was normalized to the Ct of total RNA detected by qRT- PCR (reference) in the same sample: ACt=Ctanchor_Cttotai. The Ct value from the anchor assay was then normalized to the 100% uncapped RNA sample: AACt=Cttest_CtUncapped. The percentage of uncapped RNA in each sample was calculated as: %uncapped=2A(-AACt). The percentage of capped RNA was calculated as: %capped=l-%uncapped.Double-strand RNA Assay[000260] The dsRNA content generated during in vitro transcription of mRNA was determined using the double-stranded RNA (dsRNA) ELISA kit (J2 based) (Cat No. 10613005(XS), Cedarlane™, Canada). For plate coating, 31.5 pL of coating antibody (J0170) was mixed with 10.5 mL phosphate buffered saline (PBS) and distributed into a 96-well plate at 100 pL / well. The plate was covered with aluminum foil and refrigerated overnight at 4°C. On day 2, the liquid was discarded, and 100 pL / well of 1% bovine serum albumin (BSA) in PBS (5x ELISA diluent buffer in water) was added. The plate was covered and incubated at 37°C for 2 hours.[000261] Standards and samples were prepared as follows: For standard preparations, 1 pL of dsRNA standard stock (1 pg / pL) (JO 171) was diluted with 999 pL RNase-free H2O to a final concentration of 1 ng / pl, followed by serial dilutions to create standard concentrations. For sample preparations, stock solutions were prepared with RNase-free H2O at a final concentration of 0.01 pg / pL in a total volume of 500 pL, and serial dilutions were performed to create sample concentrations of 50, 5, and 0.5 ng / pL.[000262] The antigen / sample addition involved washing the plate 4 times with wash buffer (200 pL per wash), adding standards and diluted samples to duplicated wells at 100 pL / well, covering the plate, and incubating at 37°C for 1 hour. The detector antibody (JO 172) was added at 100 pL / well after another round of washing, followed by a 1-hour incubation at 37°C. Secondary antibody (JO 173) was then added at 100 pl / well after dilution, with a final 1-hour incubation at37°C. For development, 100 pL of tetramethylbenzidine (TMB) substrate solution (J0005) was added to each well after washing, and the plate was incubated at room temperature for 1 hour. After adding 100 pL of STOP™ solution, the absorbance was read at 450 nm.LNP preparation[000263] The LNPs were prepared by dissolving mRNA in 25 mM sodium acetate, pH 4.0, while the lipid components at the mole % specified were dissolved in absolute ethanol. The lipids in ethanol and the Firefly luciferase (FLuc) mRNA in buffer were combined in a 1 :3 volume by volume ratio using a t-junction with dual-syringe. The solutions were pushed through the T- junction at a combined flow rate of 20 mL / min (5 mL / minute for the lipid-containing syringe, 15 mL / minute for the mRNA-containing syringe). The mixture was subsequently dialyzed overnight against -100 volumes of l x phosphate buffered saline, pH 7.4 using Spectro / Por dialysis membranes (molecular weight cut-off 12000-14000 Da). The LNPs were concentrated as required with an Amicon Ultra™ 10000 MWCO (molecular weight cut-off), regenerated cellulose concentrator.[000264] Encapsulation efficiency was calculated by determining unencapsulated mRNA content by measuring the fluorescence upon the addition of RiboGreen™ to the mRNA-LNP (F) and comparing this value to the total mRNA content that is obtained upon lysis of the LNP by 2% Triton X-100 (Ft): % encapsulation = (Ft -Fi)ZFt x 100.[000265] The particle size and poly dispersity index (PDI) were characterized using a Zetasizer Nano ZS™.In vivo Performance of the lipid nanoparticle[000266] The LNPs at an mRNA concentration of 1.0 mg / kg were injected intravenously (i.v.) in CD-I mice of 6-8 weeks upon arrival at a volume using the formula weight of the mouse (in grams) * 10 pL. At 24 hours after injection, mice were anesthetized in 5% isofluorane (set to 1% air flow) followed by carbon dioxide to induce asphyxiation until the animals lost their reflexes. Afterwards, the skin of animals was pinned back, and animals were cut from the bladder to the rib cage. Bone marrow, spleen, abdominal skin, lung and heart tissues were collected, placed in 2 mL tubes and snap frozen in liquid nitrogen. The tissues were subsequently stored at -80°C until further analysis.Tissue homogenate assay[000267] An appropriate volume of GLO™ lysis buffer from Promega™ was added to each of the tubes, ensuring that the samples remained frozen before addition of the lysis buffer. Samples were placed in a FastPrep™ homogenizer and the homogenizer was operated at a speed of 6 m / s for 20 seconds and repeated 2 times for a total of three rounds. The homogenized samples were spun down for 10 minutes at 12,000 rpm at room temperature and subsequently 50 pL of homogenate in duplicate was added to a black plate. The plate was transferred to a plate reader and the fluorescence was read at 640 nm excitation / 720 nm emissions. Luminescence was determined by adding 50 pL of Steady Gio™ substrate into the homogenate sample and a luciferase signal was read.EXAMPLESExample 1: 3’O-alkylation of the ribose in the guanosine at position 0 of the mRNA cap allows for sustained extrahepatic protein expression but only upon delivery of the mRNA using high neutral lipid containing LNPs.[000268] To assess the contribution of different mRNA cap modifications to extrahepatic protein expression levels in vivo, the inventors evaluated the effect of 3’ O-m ethylation of the ribose in the guanosine at position 0 of the mRNA cap against an unmodified mRNA cap using lipid nanoparticles containing variable levels neutral lipid (DSPC) content. To this end, the following trinucleotide mRNA cap structures: m7GpppA2’OMepG and m7G3’OMepppA2’OMepG were used by the inventors to prepare capped FLuc mRNA by in vitro transcription. Transcribed FLuc mRNA was subsequently formulated using a high DSPC-LNP delivery system (with the following formulation: ionizable lipid (norMC3): DSPC: Cholesterol: PEG2000 -DMG [27.4:50:21.1 : 1.5 mol%]) or a low DSPC-LNP delivery system (with the following formulation: ionizable lipid (norMC3): DSPC: Cholesterol, PEG2000 -DMG [50: 10:38.5: 1.5 mol%]) and injected intravenously into CD1 female mice. The extrahepatic expression levels for FLuc protein were analyzed at both 4 hours and 24 hours post-injection with high- and low-DSPC LNP-formulated mRNAs. All other features of the mRNA cap and the LNP formulation were kept unmodified. FIG. 1 A and FIG. IB show the chemical structure of each cap. Table 1 (below) shows the capping method employed and the efficiency of the process. Phosphate-buffered saline (PBS) was used as a negative control for expression.Table 1: Trinucleotide co-transcriptional mRNA capping efficiency examined in vitro[000269] Table 2 (below) shows formulation data for high neutral lipid content-containing (50 mol% DSPC) and low neutral lipid content-containing (10 mol% DSPC) formulations.Table 2: Formulations containing different levels of DSPC examined in vivo using FLuc mRNA[000270] The details of the high neutral lipid content-containing (50 mol% DSPC) LNP formulation for the mRNAs are as follows: (i)m7GpppA2’OMepG-capped mRNA LNP size, polydispersion index, encapsulation (57.88 nm, 0.063, 85.2%) and (ii)m7G3’OMepppA2’OMepG- capped mRNA LNP size, polydispersion index, encapsulation (57.51 nm, 0.077, 85.5%). The ionizable lipid, norMC3 is described in co-owned and co-pending WO 2022 / 246571, which is incorporated herein by reference.[000271] The low neutral lipid content-containing (10 mol% DSPC) mRNA-LNP formulation contained the following cargo and formulation characteristics: (i)m7GpppA2 OMepG-capped mRNA and LNP size, polydispersion index, encapsulation of 46.8 nm, 0.045 and 97.3% respectively; and (ii)m7G3’OMepppA2’OMepG-capped mRNA and LNP size, polydispersion index and encapsulation respectively 46.2 nm, 0.038 and 97.4%.[000272] FIG. 2 A, FIG. 2B, FIG. 2C and FIG. 2D show that at 4 hours post-injection, 3’0- methylation of the ribose in the guanosine at position 0 had an insignificant effect on extrahepatic FLuc protein expression, irrespective of the neutral lipid content used in the LNP for the delivery of the mRNA.[000273] At 24 hours post-injection, 3 ’O-m ethylation of the mRNA cap allows for high FLuc protein expression but, surprisingly, only upon mRNA delivery using a high neutral lipid contentcontaining LNP (50 mol% DSPC). The use of high neutral lipid content-containing formulations in combination with 3 ’O-m ethylated capped mRNA offers a significant advantage in achieving enhanced levels of extrahepatic protein expression for sustained periods of time. In particular, the mRNA bearing the 3’0 cap-methylation allowed for a 7.4-fold increase in FLuc protein expression in the bone marrow at 24 hours, using high neutral lipid content-containing (50 mol% DSPC) LNP. The observed increase in FLuc protein expression is statistically significant (p = 0.0226). In contrast, 3 ’O-m ethylation did not offer any advantage in FLuc protein expression in the bone marrow when a low neutral lipid content-containing (10 mol% DSPC) was used in the formulation of the LNP. No significant difference in expression was observed in this case.[000274] Similarly, in the spleen, a 9.6-fold increase in FLuc protein expression was observed at 24 hours, using high neutral lipid content-containing (50 mol% DSPC) LNP. The observed increase in FLuc protein expression is statistically significant (p = 0.0045). Again, 3’0- methylation did not offer any advantage in FLuc protein expression in the spleen when a low neutral lipid content-containing (10 mol% DSPC) was used in the formulation of the LNP.[000275] Taken together, these data demonstrate that 3’O-methylation of the ribose in the guanosine at position 0 of the mRNA cap allows for sustained extrahepatic protein expression but, surprisingly, only when high neutral lipid content-containing LNPs (50 mol% DSPC), not when low neutral lipid content-containing LNPs (10 mol% DSPC) are employed.[000276] The synergy between 3 ’O-m ethylation of the mRNA cap and high neutral lipid content containing-LNP for sustained protein expression was further evaluated in other extrahepatic tissues, namely: the abdominal skin, the lungs and the heart. To this end, the inventors used mRNAs capped with either of the following trinucleotide mRNA cap structures: m7GpppA2’OMepG and m7G3’OMepppA2’OMepG. Each mRNA was encapsulated using a high DSPC-LNP delivery system (with the following formulation: ionizable lipid (norMC3): DSPC: Cholesterol: PEG2000 - DMG [27.4:50:21.1 : 1.5 mol%]) and injected intravenously into CD1 female mice. Samples of bone marrow, spleen, abdominal skin, lungs, and heart tissues were collected at 24 hours postinjection and analyzed for FLuc protein expression.[000277] FIG. 3A - FIG. 3E show in vivo FLuc protein expression levels at 24 hours in female CD1 mice across a variety of extrahepatic tissues, namely bone marrow, spleen, abdominal skin, lungs and heart tissue. The data show that 3 ’O-m ethylation of the ribose ring in the N7- methylguanosine at position 0 of mRNA cap leads to increased FLuc protein expression (3.2- to 7.5-fold) compared to the non 3’-O-methylated mRNA cap, in all extrahepatic tissues examined, including: the bone marrow, the spleen, abdominal skin, lungs and the heart.Example 2: 2’O-methylation in the ribose of guanosine at position 1 of the mRNA cap moiety enhances extrahepatic protein expression in high neutral lipid containing LNP.[000278] To understand the contribution of different mRNA cap modifications to extrahepatic protein expression levels in vivo, the inventors evaluated the effect of 2’O-methylation of the ribose in the guanosine at position 1 of the mRNA cap against an unmodified mRNA cap. To this end, the following dinucleotide mRNA cap structures: m7GpppG and m7GpppG2 OMe (FIG. 4 A and FIG. 4B) were used to prepare FLuc mRNA by in vitro transcription. Additionally, to further evaluate the effect of 2’O-methylation of the ribose in the guanosine at position 1 of the mRNA cap against an unmodified mRNA cap, in the context of 3’O-methylation of the guanosine at position 0, the following dinucleotide mRNA cap structures: m7G3 OMepppG and m7G3’OMepppG2 OMe (FIG. 4C and FIG. 4D) were used to prepare FLuc mRNA by in vitro transcription. All four transcripts were encapsulated using a high DSPC-LNP delivery system (with the following formulation: ionizable lipid (norMC3): DSPC: Cholesterol: PEG2000 -DMG [27.4:50:21.1 :1.5 mol%]) and injected intravenously into CD1 female mice. Samples of bone marrow, spleen, abdominal skin, lungs, and heart tissues were collected at 24 hours post-injection and analyzed for FLuc protein expression.[000279] Tables 3 and 4 (below) show the capping method employed and the efficiency of the process. Phosphate-buffered saline (PBS) was used as a negative control for expression.Table 3: Dinucleotide co-transcriptional mRNA capping efficiency examined in vitro.Table 4: Dinucleotide co-transcriptional mRNA capping efficiency examined in vitro.[000280] FIG. 5 A and FIG. 5B show in vivo FLuc protein expression levels at 24 hours in female CD1 mice in bone marrow and spleen. The data show that 2’O-methylation of the ribose ring in the guanosine at position 1 of mRNA cap leads to increased FLuc protein expression (11.0- to 22.4-fold) compared to the non 2’-O-methylated mRNA cap. Similarly, FIG. 6A and FIG. 6B show in vivo FLuc protein expression levels at 24 hours in female CD1 mice in bone marrow and spleen. The data show that 2’O-methylation of the ribose ring in the guanosine at position 1 of mRNA cap(in the context of a 3’0-methylated N7-guanosine at position 0) also leads to increased FLuc protein expression (10.9- to 18.2-fold) compared to the non 2’-O-methylated mRNA cap.[000281] Furthermore, in the bone marrow, the mRNA cap with 2'0Me alone leads to a luminescence (intensity / mg bone marrow) of roughly 1,000 (FIG. 5A) while 3'0Me alone leads to a luminescence of approximately 150 (FIG. 6 A). However, when combined, a luminescence of roughly 2,800 (FIG. 6A) was observed. These results indicate that the combination of 2' OMe and 3’0Me have a synergistic effect on the extrahepatic protein expression.Example 3: Capping methodology utilized in the preparation of FLuc mRNA in vitro for formulation using high neutral lipid LNP does not alter extrahepatic protein expression levels in vivo.[000282] Tables 5 and 6 (below) show the capping method employed and the efficiency of the process. Phosphate-buffered saline (PBS) was used as a negative control for expression.Table 5: Mononucleotide post-transcriptional mRNA capping efficiency examined in vitro.Table 6: Mononucleotide post-transcriptional mRNA capping efficiency examined in vitro.[000283] To understand whether the capping methodology utilized in the preparation of FLuc mRNA in vitro for formulation using high neutral lipid LNP alters extrahepatic protein expression levels in vivo, the inventors compared the expression profile of co-transcriptionally with post- transcriptionally capped FLuc mRNA. As observed previously for co-transcriptionally capped mRNAs (in FIG. 3 A to FIG. 3E), post-transcriptionally capped mRNA also allows for extrahepatic expression in the bone marrow, spleen, abdominal skin, lungs and heart (FIG. 7A to FIG. 7E and FIG. 8A to FIG. 8E).[000284] Additionally, the inventors observed that 2’O-methylation of the ribose in the adenosine at position 1 enhances extrahepatic protein expression using a high DSCP LNP formulation. Both transcripts (2’O-methylated and non-2'O-methylated mRNAs) were encapsulated using a high DSPC-LNP delivery system (with the following formulation: ionizable lipid (norMC3): DSPC: Cholesterol: PEG2000 -DMG [27.4:50:21.1 :1.5 mol%]) and injected intravenously into CD1 female mice. Samples of bone marrow, spleen, abdominal skin, lungs and heart tissues were collected at 24 hours post-injection and analyzed for FLuc protein expression. Similarly, 2’O-methylation also enhanced extrahepatic FLuc protein expression using a high DSPC LNP delivery system. Notably, 2’O-methylation is advantageous to drive extrahepatic FLuc protein expression, while 3’0- methylation in this example has a contributive role in protein expression.Example 4: 2’O-ethylation in the ribose of adenosine at position 1 of the mRNA cap moiety provides slightly increased extrahepatic protein expression compared to 2’-O methylation at the same position in high neutral lipid containing LNP.[000285] To understand the contribution of different mRNA cap modifications in the ribose at position 1 to extrahepatic protein expression levels in vivo, inventors evaluated the effect of 2’0- ethylation of the ribose in the adenosine at position 1 of the mRNA cap against the corresponding 2’0-methyl modified mRNA cap. To this end, the following trinucleotide mRNA cap structures: m7G3’OMepppA2 OMepG (FIG. IB) and m7G3 OMepppA2 OEtpG (FIG. 9) were used to prepare FLuc mRNA by in vitro transcription. Both transcripts were encapsulated using a high DSPC-LNP delivery system (with the following formulation: Ionizable lipid 4: DSPC: Cholesterol: PEG2000 - DMG [27.4:50:21.1 : 1.5 mol%]) and injected intravenously into CD1 female mice. Samples of bone marrow and spleen were collected at 24 hours post-injection and analyzed for FLuc protein expression.[000286] Table 7 below lists the ionizable lipids used in the subsequent in vivo studies. High DSPC-LNP delivery system with the following formulation was used in the studies: ionizable cationic lipid: DSPC: cholesterol: PEG2ooo -DMG at 27.4:5O:21.1 : 1.5 mol%Table 7: Ionizable cationic lipids used in the high DSPC panel of formulations[000287] Table 8 shows the capping method employed and the efficiency of the process.Phosphate-buffered saline (PBS) was used as a negative control for expression.Table 8: Trinucleotide co-transcriptional mRNA capping efficiency examined in vitro.[000288] FIG. 10A and FIG. 10B show in vivo FLuc protein expression levels at 24 hours in female CD1 mice in bone marrow and spleen. The data show that 2’0-ethylation of the ribose ring in the adenosine at position 1 of mRNA cap leads to somewhat increased FLuc protein expression (1.6-fold) compared to the corresponding 2’-O-methylated mRNA cap.Example 5: Effect of O-methylation and deoxygenation at 2’ and 3’ of ribose in position 0 on extrahepatic protein expression using high neutral lipid containing LNP.[000289] To understand the contribution of O-methylation of the ribose at position 0 of the mRNA cap to extrahepatic protein expression levels in vivo, inventors evaluated the effect of 2’0- methylation of the ribose against the corresponding 3 ’O-m ethylated mRNA cap (m7G3’OMepppG2’OMe). To further examine whether there is synergy between both O-methyl modifications, the effect of combined 2’ and 3’ O-methylation of the ribose at position 0 was also evaluated.[000290] To understand the effect of deoxygenation of the ribose at position 0 of the mRNA cap on extrahepatic protein expression levels in vivo, the inventors introduced deoxygenation at 2’ and 3’ positions of ribose in position 0 and evaluated the resulting capped FLuc mRNAs against the 3 ’O-methyl modified (m7G3’OMepppG2’OMe) capped mRNA.[000291] To this end, the following dinucleotide mRNA cap structures: m7G3 OMepppG2 OMe (FIG. 4D), m7G2’OMepppG2’OMe (FIG. 11 A) and m7G2’,3'OMepppG2’OMe (FIG. 11B) were prepared by co- transcriptional capping of in vitro transcribed FLuc mRNA. In addition, the cap structures m7G2’deoxypppG2’OMe (FIG. 11C) and m7G3’deoxypppG2’OMe (FIG. 11D) were prepared by post-transcriptional capping of in vitro transcribed FLuc mRNA. All transcripts were encapsulated using a high DSPC-LNP delivery system (with the following formulation: ionizable lipid 7: DSPC: Cholesterol: PEG2000 -DMG [27.4:50:21.1 :1.5 mol%]) and injected intravenously into CD1 female mice. Samples of bone marrow and spleen were collected at 24 hours post-injection and analyzed for FLuc protein expression.[000292] Tables 9 shows the capping method employed and the efficiency of the process. Phosphate-buffered saline (PBS) was used as a negative control for expression.Table 9: Capping method and efficiency for mRNAs in vitro.[000293] FIG. 12A and FIG. 12B show in vivo FLuc protein expression levels at 24 hours in female CD1 mice in bone marrow and spleen. The data shows that 2’-0 methyl modification in the ribose at position 0 (m7G2’OMepppG2 OMe) supports high protein expression comparable to the 3’-0 methyl modified cap (m7G3 OMepppG2 OMe). Protein expression with combined 2’ and 3’ O methylation at position 0 (m7G2’,3’OMepPPG2’OMe) is also comparable to that with only the 3’ O methylated cap, suggesting that either of the two O-methylations is sufficient to sustain high extrahepatic protein expression in the context of high neutral lipid containing LNPs.[000294] FIG. 12C and 12D show in vivo FLuc protein expression levels at 24 hours in female CD1 mice in bone marrow and spleen.Example 6: N6-methylation of the adenosine base at position 1 of the mRNA cap moiety increases extrahepatic protein expression in high neutral lipid containing LNP.[000295] To understand the contribution of base modification at position 1 to extrahepatic protein expression levels in vivo, the inventors evaluated the effect of N6-m ethylation in the adenosine at position 1 of the mRNA cap in the context of 3’-0 methyl modification at position 0 and 2’-0 methyl modification at position 1. To this end, the following trinucleotide mRNA cap structures: m7G3’OMepppA2’OMepppG (FIG. IB) and m7G3’OMepppM6A2’OMepppG (FIG. 13) were used to prepare FLuc mRNA by in vitro transcription. Both transcripts were encapsulated using a high DSPC-LNP delivery system (with the following formulation: ionizable lipid 4: DSPC: cholesterol: PEG2000 -DMG [27.4:50:21.1 : 1.5 mol%]) and injected intravenously into CD1 female mice. Samples of bone marrow and spleen were collected at 24 hours post-injection and analyzed for FLuc protein expression.[000296] Table 10 shows the capping method employed and the efficiency of the process. Phosphate-buffered saline (PBS) was used as a negative control for expression.Table 10: Trinucleotide co-transcriptional mRNA capping efficiency examined in vitro.[000297] FIG. 14A and FIG. 14B show in vivo FLuc protein expression levels at 24 hours in female CD1 mice in bone marrow and spleen. The data shows that N6-methylation of adenosine at position 1 increases protein expression by 1.8-2.1 fold compared to unmodified adenosine, in the context of 3’-0 methyl modification at position 0 and 2’-0 methyl modification at position 1.Example 7: Vinyl bridge modified mRNA cap with O-alkylation of ribose at positions 0 and 1 sustains high extrahepatic protein expression in high neutral lipid containing LNPs.[000298] To understand the contribution of bridge modifications to extrahepatic protein expression levels in vivo, the inventors evaluated the effect of introduction of vinyl phosphonate bridge modification of the mRNA cap in the context of 3’-0 methyl modification at position 0 and 2’-0 methyl modification at position 1. To this end, the following trinucleotide mRNA cap structures: m7G3’OMepppA2’OMepppG (FIG. IB) and m7G3’OMevpppA2’OMepppG (FIG. 15) were used to prepare FLuc mRNA by in vitro transcription. Both transcripts were encapsulated using a high DSPC-LNP delivery system (with the following formulation: ionizable lipid 4: DSPC: Cholesterol: PEG2000 -DMG [27.4:50:21.1 :1.5 mol%]) and injected intravenously into CD1 female mice. Samples of bone marrow and spleen were collected at 24 hours post-injection and analyzed for FLuc protein expression.[000299] Table 11 shows the capping method employed and the efficiency of the process. Phosphate-buffered saline (PBS) was used as a negative control for expression.Table 11: Trinucleotide co-transcriptional mRNA capping efficiency examined in vitro.[000300] FIG. 16A and FIG. 16B show in vivo FLuc protein expression levels at 24 hours in female CD1 mice in bone marrow and spleen. The data shows that protein expression with vinyl bridge modification is comparable (0.81-0.88 fold) to the unmodified bridge, in the context of 3’- O methyl modification at position 0 and 2’-0 methyl modification at position 1.[000301] The examples are intended to illustrate the preparation of specific lipid nanoparticle mRNA preparations and properties thereof but are in no way intended to limit the scope of the invention.[000302] The article "a" or "an" as used herein is meant to include both singular and plural, unless otherwise indicated.

Claims

CLAIMS1. A lipid nanoparticle for extrahepatic delivery of mRNA, the lipid nanoparticle comprising:(i) a neutral lipid content of from 20 mol% to 70 mol%;(ii) an ionizable cationic lipid content of from 5 mol% to 50 mol%; wherein the mRNA is encapsulated in the lipid nanoparticle, the mRNA comprising a 5’ cap comprising a guanosine at a position 0, and a nucleoside at a position 1 linked to the guanosine by a 5’ to 5’ bridge, wherein the guanosine has a modification at a 3’ carbon of its ribose and / or the nucleoside at the position 1 has a modification at a 2’ carbon of its ribose; and wherein each mol% is measured relative to a total lipid content of the lipid nanoparticle.

2. The lipid nanoparticle of claim 1, wherein the guanosine at position 0 further comprises a modification at a 2’ carbon of its ribose.

3. The lipid nanoparticle of claim 1 or 2, wherein the 5’ cap is a mononucleotide cap.

4. The lipid nanoparticle of claim 1 or 2, wherein the 5’ cap is a dinucleotide cap.

5. The lipid nanoparticle of claim 1 or 2, wherein the 5’ cap is a trinucleotide cap.

6. The lipid nanoparticle of claim 1 or 2, wherein the 5’ cap is a tetranucleotide cap.

7. The lipid nanoparticle of claim 5 or 6, wherein the 5’ cap further comprises a 2’ carbon modification on a nucleoside at a position 2.

8. The lipid nanoparticle of claim 1 or 2, wherein the 5’ cap is an oligonucleotide.

9. The lipid nanoparticle of claim 1, wherein the guanosine at position 0 is modified.

10. The lipid nanoparticle of claim 9, wherein the modification is an alkylation.

11. The lipid nanoparticle of claim 9, wherein the modification is an arylation.

12. The lipid nanoparticle of claim 11, wherein the arylation is a cyclic modification.

13. The lipid nanoparticle of claim 1, wherein the 3’ carbon of guanosine at position 0 is modified with an H group.

14. The lipid nanoparticle of claim 1, wherein the 3’ carbon is modified with an OCH3 group.

15. The lipid nanoparticle of claim 1, wherein the 3’ carbon is modified with an OC2H5 group.

16. The lipid nanoparticle of claim 1, wherein the 3’ carbon is modified with an OC3H7 group.

17. The lipid nanoparticle of claim 1, wherein the nucleoside at position 1 is a guanosine.

18. The lipid nanoparticle of claim 1, wherein the nucleoside at position 1 is an adenosine.

19. The lipid nanoparticle of claim 1, wherein the ribose of nucleoside at position 1 has an O- alkylation.

20. The lipid nanoparticle of claim 19, wherein the ribose has an O-methylation.

21. The lipid nanoparticle of claim 19, wherein the ribose has an O-ethylation.

22. The lipid nanoparticle of claim 1, wherein the modification of the ribose of nucleoside at position l is a halogenation.

23. The lipid nanoparticle of claim 22, wherein the halogenation is done using a F, Cl, Br, or I.

24. The lipid nanoparticle of claim 1, wherein the 5’ to 5’ bridge is a triphosphate moiety.

25. The lipid nanoparticle of claim 1, wherein the 5’ to 5’ bridge is a tetraphosphate moiety.

26. The lipid nanoparticle of claim 1, wherein the 5’ to 5’ bridge is a pentaphosphate moiety.

27. The lipid nanoparticle of any one of claims 24 to 26, wherein the bridge comprises a BH3 group.

28. The lipid nanoparticle of any one of claims 24 to 26, wherein the bridge comprises an S group.

29. The lipid nanoparticle of any one of claims 24 to 26, wherein the bridge comprises a Se group.

30. The lipid nanoparticle of any one of claims 24 to 26, wherein the bridge comprises a CH2 group.

31. The lipid nanoparticle of any one of claims 24 to 26, wherein the bridge comprises an NH group.

32. The lipid nanoparticle of any one of claims 1 to 31, wherein the 5’ cap of the encapsulated mRNA has a structure of Formula A:Formula A or a pharmaceutically acceptable salt thereof, wherein the -R1moiety is a lone pair of electrons or the R1of the -R1moiety is an optionally substituted Ci-Cio alkyl, cycloalkyl or aryl, optionally a methyl;A2and A3are, independently, absent or present, and if A2is absent, then R2is H directly bonded to the 2’ carbon of the ribose, and if A2is present, then A2is O and R2is H or an optionally substituted alkyl, which alkyl is optionally C1-C3 alkyl; if A3is absent, then R3is H directly bonded to the 3’ carbon of the ribose, and if A3is present, then A3is O and R3is H or an optionally substituted alkyl, which alkyl is optionally C1-C3 alkyl; if A2and A3are both present, then both A2and A3are O and R2and R3are either not bonded to each other or bonded to each other, as indicated by a dashed bond, whereinif R2and R3are not bonded to each other, then R2and R3are, independently, H or optionally substituted C1-C3 alkyl, and if R2and R3are bonded to each other, then R2and R3together form a moiety ofFormula 1: o oLFormula 1 wherein L is an optionally substituted C1-C3 alkylidene, optionally methylene (CH2), 1,1 -ethylene (CH-CH3), 1,2-ethylene (CH2-CH2), 1,1 -propylene (CH-CH2- CH3), 1,2-propylene (CH2-CH-CH3), 1,3-propylene (CH2-CH2-CH2), 2,2- propylene (CH3-C-CH3), and derivatives thereof;X is O, S, CH2 or N-R5, wherein R5is optionally H or an optionally substituted C1-C3 alkyl,A1is OH, SH, SeH, BH3 or a salt thereof, wherein if A1is the salt, a counterion is present, which counterion is optionally selected from Na+, NH4+and Et3NH+; and n is 3 to 5, andY is O, S, CH2 or N-R6, wherein R6is H or an optionally substituted C1-C3 alkyl;B1is a purine or a pyrimidine, optionally 9-adeninyl, N6-methyl 9-adeninyl, 9-guaninyl, 1- cytosinyl, 1 -uracilyl, or variants of the purine or pyrimidine thereof;R4is H, OH or O-R6, wherein R6is an optionally substituted alkyl, which alkyl is optionally Ci- C3 alkyl; wherein if R2and R3are not bonded to one another, then at least one of A3-R3and R4is an O- alkyl,G1is H or a nucleotide having a structure of Formula 2:Formula 2 wherein theof Formula 2 represents a bond to the O atom to which G1is bonded;A4is as defined above for A1;Z is as defined above for Y;B2is as defined above for B1;R7is as defined above for R4; andG2is as defined above for G1, wherein the compound of Formula A comprises from 0 to 3 moi eties of Formula 2, and wherein if Formula 2 is present, a terminal moiety of Formula 2 is such that G2of the terminal moiety is H.

33. The lipid nanoparticle of any one of claims 1 to 31, wherein the 5’ cap of the encapsulated mRNA has a structure of Formula B:Formula B or a pharmaceutically acceptable salt thereof, whereinthe -R1moiety is a lone pair of electrons or R1is an optionally substituted C1-C10 alkyl, cycloalkyl or aryl, optionally a methyl;A2and A3are, independently, absent or present, and if A2is absent, then R2is H directly bonded to 2’ carbon of the ribose, and if A2is present, then A2is O and R2is H or an alkyl, which alkyl is optionally substituted C1-C3 alkyl; if A3is absent, then R3is H directly bonded to 3’ carbon of the ribose, and if A3is present, then A3is O and R3is H or an alkyl, which alkyl is optionally substituted C1-C3 alkyl; if A2and A3are both present, then both A2and A3are O and R2and R3are either not bonded to each other or bonded to each other, as indicated by a dashed bond, wherein if R2and R3are not bonded to each other, then R2and R3are, independently, H or optionally substituted C1-C3 alkyl, and if R2and R3are bonded to each other, then R2and R3together form a moiety of Formula 3: o oLFormula 3 wherein L is an optionally substituted C1-C3 alkylidene, optionally methylene (CH2), 1,1 -ethylene (CH-CH3), 1,2-ethylene (CH2-CH2), 1,1 -propylene (CH-CH2- CH3), 1,2-propylene (CH2-CH-CH3), 1,3-propylene (CH2-CH2-CH2), 2,2- propylene (CH3-C-CH3), and derivatives thereof;B1is a purine or a pyrimidine moiety, optionally 9-adeninyl, N6-methyl 9-adeninyl, 9-guaninyl, 1-cytosinyl, 1 -uracilyl, or variants of the purine or pyrimidine thereof;R4is H, OH or O-R6, wherein R6is an alkyl, which alkyl is optionally substituted C1-C3 alkyl; wherein if R2and R3are not bonded to one another, then at least one of A3-R3and R4is an O- alkyl,G1is H or a nucleotide having a structure of Formula 4:Formula 4 wherein theof Formula 4 represents a bond to the O atom to which the G1is bonded;A4is OH, SH, SeH, BH3 or a salt thereof, wherein if A4is the salt, a counterion is present optionally selected from Na+, NH4+or EtsNH ;Z is O, S, CH2, or N-R6, wherein R6is H or an optionally substituted C1-C3 alkyl;B2is as defined above for B1;R7is as defined above for R4; andG2is as defined above for G1; wherein Z2and Z3are independently present or absent; wherein Z1is: a moiety of Formula bla:Formula bla wherein the wavy line connected to the CH2 group of Formula bla represents a bond between the CH2 group and the ribose of the nucleotide at position 0, the wavy line connected to the O atom represents a bond to Z2, or to Z3if Z2is absent, or to Z4if both Z2and Z3are absent; p is 0-5, or a moiety of Formula bibFormula bib wherein L is CH=CH of either E or Z configuration or L is C=C, the wavy line connected to L represents a bond to the ribose of the nucleotide at position 0, and the wavy line connected to the O atom represents a bond to Z2, to Z3if Z2is absent, or to Z4if both Z2and Z3are absent, or a triazole moiety of Formula i or Formula ii:Formula ii wherein q, r and t are, independently, 1-5, s is 0-5, the wavy lines connecting to (CH2)qand to (CH2)Srepresent bonds to the ribose of the nucleotide at position 0, and the wavy lines connected to the O atoms of Formula i or Formula ii represent bonds between the O atoms and Z2, or to Z3if Z2is absent, or to Z4if both Z2and Z3are absent;Z2and Z3if present are, independently: a moiety of structurewherein u is 1-5, or a triazole moiety of Formula iii or Formula iv:Formula iiiFormula iv wherein the wavy lines connected to (CH2)qand to (CH2)Srepresent respective bonds to Z1when Z2is the triazole moiety or to Z2when Z3is the triazole moiety and the wavy lines connected to the respective O atoms of (CH2)r-0 and to (CH2)t-0 represent bonds between the respective O atoms and Z3when Z2is the triazole moiety or Z4when Z3is the triazole moiety;Z4is: a moiety of structurewherein the wavy line connected to the P atom represents a chemical bond to Z3if present or to Z2if present or to Z1and the wavy line connected to the CH2 group represents a chemical bond between the CH2 group and the ribose of the nucleotide at position 1; and wherein v is 0-5, or a moiety of structureO s / W P II-MJW 0 6 wherein M is CH=CH, of either E or Z configuration or M is C=C, the wavy line connected to M represents a chemical bond to the ribose of the nucleotide at position 1, and the wavy line connected to the P atom represents a chemical bond to Z1or to Z2if present or to Z3if present, or a triazole moiety of Formula v or Formula vi:Formula vFormula vi wherein the wavy lines connected to (CH2)Wand to (CH2)yrepresent respective bonds to Z1or to Z2if present or to Z3if present, the wavy lines connected to (CH2)Xand to (CH2)Zrepresent chemical bonds to the ribose of nucleotide at position 1; w is 2-5; x and y are, independently, 0-5 and z is 1-5; wherein none or at most one of Z1, Z2, Z3, Z4is the triazole moiety of the Formula i-vi as defined above; and wherein the compound of Formula B comprises from 0 to 3 moi eties of Formula 4, and wherein if Formula 4 is present, a terminal moiety of Formula 4 is such that G2of the terminal moiety is H.

34. The lipid nanoparticle of claim 33, wherein the 5’ cap of the encapsulated mRNA is selected from the group consisting of35. The lipid nanoparticle of any one of claims 1 to 34, further comprising a guide RNA.

36. The lipid nanoparticle of claim 1, wherein the neutral lipid is a phosphatidylcholine lipid selected from distearoylphosphatidylcholine (DSPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC).

37. The lipid nanoparticle of claim 36, wherein the neutral lipid is a mixture of two phosphatidylcholine lipids.

38. The lipid nanoparticle of claim 37, wherein the mixture comprises di stearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC).

39. The lipid nanoparticle of claims 37 or 38, wherein the phosphatidylcholine lipid content is between 40 mol% and 60 mol%.

40. The lipid nanoparticle of claim 39, wherein the phosphatidylcholine lipid content is between 43 mol% and 57 mol%.

41. The lipid nanoparticle of claims 39 or 40, wherein the phosphatidylcholine lipid content is between 44 mol% and 56 mol%.

42. The lipid nanoparticle of claim 1, wherein the ionizable cationic lipid is an amino lipid.

43. The lipid nanoparticle of claim 42, wherein the ionizable cationic lipid is present at from 5 mol% to 40 mol% based on the total lipid present in the lipid nanoparticle.

44. The lipid nanoparticle of claim 43, wherein the ionizable cationic lipid is present at from 10 mol% to 30 mol%.

45. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle further comprises sterol or a derivative thereof.

46. The lipid nanoparticle of claim 45, wherein the sterol is cholesterol or a derivative thereof.

47. The lipid nanoparticle of claim 45 or 46, wherein the sterol is present at from 17.5 mol% to 42.5 mol% based on the total lipid present in the lipid nanoparticle.

48. The lipid nanoparticle of claim 47, wherein the sterol is present at from 18.5 mol% to 39.5 mol% based on the total lipid present in the lipid nanoparticle.

49. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle further comprises a hydrophilic polymer-lipid conjugate.

50. The lipid nanoparticle of claim 46, wherein the hydrophilic polymer-lipid conjugate is a polyethylene glycol-lipid conjugate.

51. A lipid nanoparticle for extrahepatic expression comprising an encapsulated mRNA comprising a 5’ cap comprising a guanosine at position 0, and a nucleoside at position 1 linked to the guanosine by a 5’ to 5’ bridge, wherein the guanosine has a modification at a 3’ carbon of its ribose and / or the nucleoside at position 1 of the mRNA has a modification at a 2’ carbon of its ribose, 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid, the lipid nanoparticle having at least 1.2-fold increase in expression in extrahepatic tissue at 24 hours relative to the same lipid nanoparticle having an mRNA cap lacking the modifications, wherein the expression is measured by firefly luciferase protein levels.

52. A method for extrahepatic expression of a polypeptide in a tissue or organ of a subject, the method comprising administering to the subject a lipid nanoparticle encapsulating a 5’ cap comprising a guanosine at position 0, and a nucleoside at position 1 linked to the guanosine by a 5’ to 5’ bridge, wherein the guanosine has a modification at a 3’ carbon of its ribose and / or the nucleoside at position 1 of the mRNA has a modification at a 2’ carbon of its ribose, the lipid nanoparticle having 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid, the lipid nanoparticle having at least 1.2-fold increase in expression in extrahepatic tissue at 24 hours relative to the same lipid nanoparticle with an mRNA cap lacking the modifications, wherein the expression is measured by firefly luciferase protein levels.

53. The lipid nanoparticle of claim 51, wherein the extrahepatic tissue or organ is spleen, bone marrow, lungs, kidney, heart, abdominal skin, back skin and / or ear.

54. The lipid nanoparticle of claim 51 , wherein the extrahepatic organ is bone marrow or tissue thereof.

55. The lipid nanoparticle of any one of claims 1 to 51, 53 or 54, wherein the lipid nanoparticle provides a prophylactic, ameliorative or a therapeutic benefit.

56. The lipid nanoparticle of claim 55, wherein the lipid nanoparticle is used to treat a disease or disorder that is an autoimmune disorder.

57. The lipid nanoparticle of claim 56, wherein the lipid nanoparticle is used to treat a disease or disorder that is an infectious disease.

58. The lipid nanoparticle of claim 57, wherein the lipid nanoparticle is used to treat a disease or disorder that is cancer.

59. Use of the lipid nanoparticle of any one of claims 1 to 51 or 53 to 58 for in vivo or in vitro delivery and expression of the mRNA to mammalian cells.

60. Use of the lipid nanoparticle of any one of claims 1 to 51 or 53 to 58 for the manufacture of a medicament for in vivo or in vitro delivery of the mRNA to mammalian cells.

61. The use of the lipid nanoparticle of claim 59 or 60 to target an autoimmune disorder in vivo.

62. The use of the lipid nanoparticle of claim 59 or 60 to target an infectious disease in vivo.

63. The use of the lipid nanoparticle of claim 59 or 60 to target a cancer in vivo.

64. The use of the lipid nanoparticle of any one of claims 1 to 51 or 53 to 58 for the manufacture of a vaccine composition.

65. A method of preparing a lipid nanoparticle for extrahepatic mRNA delivery, the method comprising:(i) synthesizing a capped mRNA in vitro using a dinucleotide cap comprising an N7-modified guanosine at a position 0, and a nucleoside at a position 1 linked to the N7-modified guanosine by a 5’ to 5’ bridge, wherein the N7-modified guanosine at position 0 has a modification at a 3’ carbon and / or a 2’ carbon of its ribose;(ii) labelling a 2’ OH of a nucleoside at a position 1 of the dinucleotide cap with a methyl group enzymatically to form a 2’-O-methylated ribose;(iii) purifying the mRNA bearing the modified cap; and(iv) encapsulating the purified mRNA in a lipid nanoparticle; wherein the lipid nanoparticle comprises 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid.

66. A method of preparing a lipid nanoparticle for extrahepatic mRNA expression, the method comprising(i) synthesizing an uncapped mRNA in vitro;(ii) incubating the uncapped mRNA in vitro with one or more capping enzymes that bear N7- methyl transferase activity and / or guanylyl transferase activity and modified guanosine nucleotides bearing modifications at a 3’ carbon and / or 2’ carbon positions to allow capping of mRNA;(iii) labelling the capped mRNA enzymatically at a 2’OH of a nucleoside at a position 1 of the capped mRNA with a methyl group to form a 2’-O-methylated ribose;(iv) purifying the labelled mRNA; and(v) encapsulating the purified mRNA in a lipid nanoparticle; wherein the lipid nanoparticle comprises 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid.

67. The method of claim 65 or 66, wherein the labelling is performed with a 2’0-methyl transferase.

68. The method of claim 66, wherein the incubating is performed with vaccinia capping enzyme.

69. The method of claim 66, wherein the incubating is performed with faustovirus capping enzyme.

70. A lipid nanoparticle for extrahepatic expression of a polypeptide in a subject comprising an encapsulated mRNA comprising a 5’ cap comprising an N7-methylated guanosine at a position0, and a nucleoside at a position 1 linked to the N7-methylated guanosine by a 5’ to 5’ bridge, wherein the N7-methylated guanosine has a modification at a 3’ carbon of its ribose and / or the nucleoside at the position 1 has a modification at a 2’ carbon of its ribose, 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle and an ionizable cationic lipid, the lipid nanoparticle having at least 10% increase in protein expression in extrahepatic tissue at 24 hours post injection as compared to a baseline lipid nanoparticle formulation of ionizable lipid / neutral lipid / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mokmol encapsulating the mRNA, but otherwise measured under identical conditions, wherein the expression is measured by firefly luciferase protein levels.

71. The lipid nanoparticle of claim 70, wherein the N7-methylated guanosine comprises a further modification at a 2’ carbon of its ribose.

72. The lipid nanoparticle of claim 70, wherein the nucleoside at position 1 is a guanosine.

73. The lipid nanoparticle of claim 70, wherein the nucleoside at position 1 is an adenosine.

74. The lipid nanoparticle of claim 70, wherein the nucleoside at position 1 is a modified nucleoside.

75. The lipid nanoparticle of claim 70, further comprising a sterol or a derivative thereof.

76. The lipid nanoparticle of claim 75, wherein the sterol is cholesterol or a derivative thereof.

77. The lipid nanoparticle of claim 70, further comprising a hydrophilic polymer-lipid conjugate.

78. The lipid nanoparticle of claim 77, wherein the hydrophilic polymer-lipid conjugate is a polyethylene glycol-lipid conjugate.

79. The lipid nanoparticle of claim 70, wherein the 3’ carbon of N7-methylated guanosine at position 0 is modified with an H group.

80. The lipid nanoparticle of claim 70, wherein the 3’ carbon of N7-methylated guanosine is modified with an OCH3 group.

81. The lipid nanoparticle of claim 70, wherein the 3’ carbon of N7-methylated guanosine is modified with an OC2H5 group.

82. The lipid nanoparticle of claim 70, wherein the 3’ carbon of N7-methylated guanosine is modified with an OC3H7 group.

83. The lipid nanoparticle of claim 70, wherein the ribose of nucleoside at position 1 comprises an O-alkyl modification.

84. The lipid nanoparticle of claim 70, wherein the ribose of nucleoside at position 1 comprises an O-methyl modification.

85. The lipid nanoparticle of claim 71, wherein the ribose of nucleoside at position 1 comprises an O-ethyl modification.

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