Modified RNA and lipid nanoparticles comprising same

Abstract

Provided herein is a modified RNA comprising a terminal nucleic acid shoe sequence, which terminal nucleic acid shoe sequence is hybridized to a complementary nucleic acid shoelace sequence, the nucleic acid shoe sequence and complementary nucleic acid shoelace sequence forming a stabilizing hybridized structure and wherein the complementary nucleic acid shoelace sequence is neutral at physiological pH. Further provided is an LNP with elevated neutral lipid content and having an encapsulated RNA with a stabilizing shoe / shoelace sequence as described herein.

Description

Modified RNA and Lipid Nanoparticles Comprising SameTechnical field

[0001] The present disclosure relates to modifications of RNA to improve stability or expression properties.Background

[0002] Current approaches to modulate the properties of therapeutic mRNAs involve the use of chemical 5' cap analogs, introducing unnatural nucleosides into the mRNA coding region (typically pseudouridine) or optimizing codons. The 5’ cap and the poly(A) tail of mRNA are known to regulate translation and improve stability of the mRNA transcript. In a therapeutic context, most mRNA modifications investigated to date involve modifications of the 5’ cap. Cap analogs improve both mRNA stability and translation efficiency. One key finding was that 3’-O- methylation of the ribose sugar of 7-methylguanosine of the cap prevents reverse orientation of the cap and thereby avoids capped structures that are incorporated in the incorrect orientation (Stepinski et al, RNA, 10: 1486). A further key discovery was that the replacement of uridine with pseudouridine reduces the immunogenicity of mRNA in addition to stabilizing it and protecting the RNA against degradation by nucleases (Morais et al., 2021, Front Cell Dev. Biol., 9: 789427). Comparatively fewer groups have investigated modification of the 3’ poly(A) tail to improve the properties of therapeutic mRNA.

[0003] Naturally occurring RNA triple helices are known to stabilize the 3 ’ end of noncoding RNAs that lack a poly(A) tail. (Wilusz et al., 2012, Genes & Development, 26:2392-2407). Chemical modifications at the 3’ end of mRNA including structural motifs and nuclease resistant modifications (Aditham et al, ACS Chem Biol, 17: 3352, Anhauser et al, Nucleic Acids Research, 47: e42, WO 2025 / 147660), loop elements (Oh et al Npj Vaccines, 10: 234) and multiple branches in the polyA tail (WO 2023 / 141474) have been shown to stabilize mRNA. Furthermore, phosphorothioate modifications to poly(A) tails have been examined with a view to decrease susceptibility to enzymatic deadenylation (Strzelecka et al., 2020, RNA, 26: 1815-1837). Replacement of the poly(A) tail with RNA binding protein aptamers was used to render translation of the mRNA responsive to the presence of RNA binding proteins that conditionally associate with translation initiation factors (Shao et al., 2023, Cell Research, 34:31-46). Terminal RNA hairpinloops at both the 5 ’ and 3 ’ ends of mRNA have also been investigated for their ability to improve mRNA expression in mRNAs lacking polyA tails (Solodushko and Fouty, 2023, Gene Therapy, 30:620-627), although studies suggest that its inclusion is required to facilitate translation efficiency of mRNA. More recently, groups have investigated the stabilization of prime editor guide RNA (pegRNA) by modifying the RNA at its 3’ end to introduce various structural motifs. One such strategy involved fusing the 3' terminal end of pegRNA with a noncanonical secondary structure formed by G-rich stretches of nucleic acids with a central monovalent cation stabilizing the quartet (G-quadruplex pegRNA). It was found that G-quadruplex modified pegRNA provided an >80% increase in the editing of endogenous targets (Li et al., 2022, J. Mol. Cell Biol., 14(4)). The inclusion of a stabilizing RNA / DNA hybrid structure linked to the 3 ’ end of a poly(A) tail was found to increase mRNA protein expression via reducing immunogenicity (WO 2024 / 237657 and Son et al., 2024, Adv. Sci, 11:2307541 (1 to 17)). Another approach involved engineering an mRNA with a DNA / RNA hybrid linked 3 ’ to a poly(A) tail, further linked to a terminal 3 ’ RNAi sequence (Lee et al., 2020, Journal of Controlled Release, 327:225-234). Cleavage of the DNA / RNA hybrid by RNase H results in separation of the mRNA and RNAi sequences, which in turn each exert their effects in vivo. Addition of microRNA cleavage sequences and translation inhibitory elements downstream of the polyA tail have been used to enable cell specific upregulation of mRNA expression (WO 2018 / 003779 Al). Hybridization of a DNA oligonucleotide complementary to the 5’-UTR and polyA tail has been used to protect the mRNA from degradation (Choi et al Angewandte Chem Inti Ed, 2025, el6389).

[0004] While protein expression from mRNA has been significantly improved using the foregoing approaches, non-specific mRNA expression can lead to unwanted off-target effects. To this end, various groups are developing delivery vehicles for targeted mRNA delivery in specific tissues and organs of interest. Lipid nanoparticles show promise for achieving this goal but they often lead to delivery to the liver. The ability to selectively target mRNA expression in organs and tissues beyond the liver would greatly expand the clinical utility of these delivery systems.

[0005] Thus, despite these efforts, there is an ongoing need to develop RNA expression systems with improved properties, such as stability against RNase activity and / or improved tissue or organ selectivity in vivo.Summary

[0006] The present disclosure addresses one or more of the foregoing problems in the prior art and / or provides useful alternatives to known compositions for enhancing RNA stability and / or expression.

[0007] In some embodiments, the present disclosure is based on the observation that an mRNA that is modified to include a 3’ terminal sequence (“shoe”) downstream of the poly(A) tail and hybridized to a chemically modified oligonucleotide (“shoelace”) to form a heteroplex, such as a double stranded 3’ terminal stabilizing nucleotide sequences referred to herein as a “shoe / shoelace” is capable of providing improved mRNA stability and / or expression profiles. Without being limited by theory, the improved stability and / or expression profiles may be due to modifications to the shoelace backbone (complementary to the shoe) to improve binding affinity of the duplex thereby formed. As described herein, such modified shoelace may include a backbone modified nucleotide sequence or nucleic analogue that is complementary to the terminal shoe sequence.

[0008] According to one aspect of the disclosure, there is provided a modified RNA comprising a terminal nucleic acid shoe sequence, which terminal nucleic acid shoe sequence is hybridized to a complementary nucleic acid shoelace sequence, the nucleic acid shoe sequence and complementary nucleic acid shoelace sequence forming a stabilizing shoe-shoelace heteroplex wherein the complementary nucleic acid shoelace sequence is a nucleic acid analogue with a modified backbone.

[0009] According to some examples of the foregoing aspect of the disclosure, the terminal nucleic acid shoe sequence is 10 to 50 nucleotides in length. According to some embodiments of the foregoing aspect, the terminal nucleic acid shoe sequence is 15 to 35 or 20 to 30 nucleotides in length.

[0010] According to some examples of the foregoing aspect of the disclosure, the complementary nucleic acid shoelace sequence is 10 to 50 nucleotides in length. According to some embodiments of the foregoing aspect, the complementary nucleic acid shoelace sequence is 15 to 35 or 20 to 30 nucleotides in length.

[0011] In some embodiments, the terminal nucleic acid shoe sequence and the complementary nucleic acid shoelace sequence are of the same length. In other embodiments, the terminal nucleic acid shoe sequence and the complementary nucleic acid shoelace sequence are of different lengths.

[0012] In some examples of the foregoing aspect, the modified RNA is an mRNA. In some embodiments, the mRNA comprises: (i) a 5’ untranslated region; (ii) a coding region encoding a protein or polypeptide; (iii) a 3’ untranslated region; and (iv) a 3’ tailing region downstream of the 3’ untranslated region, wherein the stabilizing shoe-shoelace is 3’ of the tailing region.

[0013] In some embodiments of the foregoing aspect, the complementary nucleic acid shoelace is formed of a nucleic acid analogue such as a peptide nucleic acid (PNA). In other embodiments, the complementary nucleic acid shoelace is a locked nucleic acid. In a further embodiment of the foregoing aspect, the complementary nucleic acid shoelace is a glycol nucleic acid (GNA).

[0014] In one embodiment, the terminal nucleic acid shoe sequence forms a duplex with the complementary shoelace sequence. In yet another embodiment, the terminal nucleic acid shoe sequence forms a triplex with the shoelace. In a further embodiment, the terminal nucleic acid sequence forms a quadruplex with the shoelace. In some examples of the foregoing aspect, the mRNA shoe forms a duplex with a PNA shoelace. In some embodiments, the duplex includes a terminal stem loop. In some other embodiments, the duplex includes at least two stem loops. In some other embodiments, the duplex includes at least three stem loops. In one embodiment, the terminal nucleic acid shoe sequence forms a stem loop. In another embodiment, the complementary shoelace sequence forms a stem loop. In some embodiments, the shoe sequence and the shoelace sequence form at least one stem loop. In one embodiment, the duplex has a terminal stem loop structure. In some other embodiments, the duplex does not form a stem loop.

[0015] In some embodiments of the foregoing aspect, at least 50% of the stabilizing shoe-shoelace is in a double stranded form at 55 °C. In some other embodiments, at least 45% of the stabilizing shoe-shoelace is in a double stranded form at 55°C. In yet other embodiments, at least 60% of the stabilizing shoe-shoelace is in a double stranded form at 55°C. In further embodiments, at least 65% of the stabilizing shoe-shoelace is in a double stranded form at 55°C. In yet further embodiments at least 40% of the stabilizing shoe-shoelace is in a double stranded form at 55°C.In one embodiment, at least 70% of the stabilizing shoe-shoelace is in a double stranded form at 55°C. In another embodiment, at least 80% of the stabilizing shoe-shoelace is in a double stranded form at 55°C. In yet another embodiment, at least 90% of the stabilizing shoe-shoelace is in a double stranded form at 55°C.

[0016] In some embodiments of the foregoing aspect of the disclosure, the 3’ tailing region is a poly(A) tail that is 3’ to the 3’ untranslated region and 5’ to the terminal nucleic acid shoe sequence.

[0017] In one embodiment, the modified RNA further comprises a micro RNA target.

[0018] In some embodiments of the forgoing aspect, the modified RNA further comprises a 5’ terminal cap.

[0019] In some embodiments of the foregoing aspect, the nucleic acid shoelace sequence is uncharged. In some other embodiments, at least 60% of the shoelace sequence lacks a charge. In some embodiments, at least 70% of the shoelace sequence lacks a charge. In yet other embodiments, at least 80% of the shoelace sequence lacks a charge. In yet further embodiments, at least 90% of the shoelace sequence lacks a charge. In some embodiments, at least 95% of the shoelace sequence lacks a charge.

[0020] In some embodiments of the foregoing aspect of the disclosure, the backbone of the shoelace sequence is acyclic.

[0021] In a second aspect of the present disclosure, there is provided a lipid nanoparticle for extrahepatic delivery, the lipid nanoparticle comprising: a neutral lipid having at least two tails and a head group, the neutral lipid being present at a content of at least 20 mol%; and an ionizable lipid content of from 15 mol% to 45 mol%, wherein the nucleic acid is a modified RNA comprising a stabilizing sequence comprising at least two strands, a first of said strands being a terminal nucleic acid shoe sequence, and a second of said strands being a complementary nucleic acid shoelace sequence hybridized to the nucleic acid shoe sequence. In another embodiment, the lipid nanoparticle comprises at least 25 mol% of neutral lipid content. In a yet another embodiment, the lipid nanoparticle comprises at least 30 mol% of neutral lipid content. In another embodiment, the lipid nanoparticle comprises at least 35 mol% of neutral lipid content. In yet another embodiment,the lipid nanoparticle comprises at least 40 mol% of neutral lipid content. In a further embodiment, the lipid nanoparticle comprises at least 45 mol% of neutral lipid content. In some embodiments, the lipid nanoparticles comprise at least 50 mol% of neutral lipid content. In some other embodiments, the lipid nanoparticles comprise at least 55 mol% of neutral lipid content.

[0022] In some examples of the foregoing aspect, the lipid nanoparticle comprises at least 20 mol% of ionizable lipid. In some other examples of the foregoing aspect, the lipid nanoparticle comprises at least 25 mol% of ionizable lipid content. In several embodiments, the lipid nanoparticles comprise at least 27 mol% of ionizable lipid content. In some embodiments of the foregoing aspect, the lipid nanoparticle comprises an ionizable lipid content of 27.4 mol%.

[0023] According to some examples of the foregoing aspect of the disclosure, the lipid nanoparticle comprises modified mRNA bearing a terminal nucleic acid shoe sequence that is 10 to 50 nucleotides in length. According to some embodiments of the foregoing aspect, the lipid nanoparticle comprises modified mRNA bearing terminal nucleic acid shoe sequence that is 20 to 30 nucleotides in length.

[0024] In some embodiments of the foregoing aspect of the disclosure, the modified mRNA encapsulated in the lipid nanoparticle comprises a complementary nucleic acid shoelace sequence that is 10 to 50 nucleotides in length. According to some embodiments, the lipid nanoparticle encapsulates modified mRNA bearing a complementary nucleic acid shoelace sequence that is 20 to 30 nucleotides in length.

[0025] In some embodiments, the mRNA encapsulated in the lipid nanoparticle has terminal nucleic acid shoe sequence and the complementary nucleic acid shoelace sequence that are of the same length. In other embodiments of the mRNA encapsulated lipid nanoparticle, the terminal nucleic acid shoe sequence and the complementary nucleic acid shoelace sequence are of different lengths.

[0026] In some examples of the foregoing aspect, the modified mRNA encapsulated in the lipid nanoparticle further comprises: (i) a 5 ’ untranslated region; (ii) a coding region encoding a protein or polypeptide; (iii) a 3 ’ untranslated region; and (iv) a 3 ’ tailing region downstream of the 3 ’ untranslated region, wherein the stabilizing shoe-shoelace is 3’ of the tailing region.

[0027] In some examples of the foregoing aspect of the disclosure, the modified mRNA encapsulated in the lipid nanoparticle comprises complementary nucleic acid shoelace sequence that is neutral or wherein at least 60%, 70%, 80%, 90% or 95% of said shoelace sequence lacks a charge.

[0028] In several examples of the foregoing aspect of disclosure, the mRNA encapsulated lipid nanoparticle comprises a complementary nucleic acid shoelace that is a peptide nucleic acid (PNA).

[0029] In some examples, the modified mRNA encapsulated in the lipid nanoparticle comprises terminal nucleic acid shoe sequence and complementary nucleic acid shoelace that form a duplex, triplex or quadruplex. In some examples of the foregoing aspect, the lipid nanoparticle comprises mRNA bearing a terminal nucleic acid shoe sequence that forms a duplex with a complementary nucleic acid shoelace. In some embodiments, the duplex includes a terminal stem loop structure.

[0030] In some embodiments of the lipid nanoparticle bearing encapsulated mRNA, at least 50% of the stabilizing shoe-shoelace is in a double -stranded form at 55°C. In some other embodiments, at least 40% of the stabilizing shoe-shoelace is in a double-stranded form at 55°C. In yet other embodiments, at least 60% of the stabilizing shoe-shoelace is in a double-stranded form at 55°C. In yet further embodiments, at least 70% of the stabilizing shoe-shoelace is in a double-stranded form at 55°C. In some embodiments, at least 80% of the stabilizing shoe-shoelace in the mRNA is in a double-stranded form at 55°C.

[0031] In some examples of the foregoing aspect of the disclosure, the lipid nanoparticle comprises modified mRNA wherein the 3’ tailing region is a poly(A) tail that is 3’ to the 3’ untranslated region and 5’ to the terminal nucleic acid shoe sequence.

[0032] In some embodiments of the foregoing aspect, the modified mRNA encapsulated in the lipid nanoparticle further comprises a microRNA target site.

[0033] In several examples of the foregoing aspect, the modified mRNA encapsulated in the lipid nanoparticle further comprises a 5’ terminal cap.

[0034] In some embodiments of the foregoing aspect of the disclosure, the lipid nanoparticle comprises a modified mRNA having a complementary shoelace sequence wherein the shoelace sequence has a modified backbone. In several embodiments, the backbone of the shoelace sequence is acyclic. In some embodiments, the shoelace sequence is a non -naturally occurring nucleic acid analogue.

[0035] In some embodiments, the shoelace nucleic acid is modified so that it is neutral at physiological pH. In such latter embodiments, the neutral shoelace enhances formulation in an LNP.

[0036] In additional or alternative embodiments, the disclosure is directed to an RNA with a shoe / shoelace that confers protection to the 3’ end of RNA towards RNase R degradation as measured on an agarose gel as described herein. In additional or alternative embodiments, when in isolated form and subj ected to heat treatment, at least 50% of the stabilizing RNA shoe / shoelace is in double-stranded form at 55 °C as determined using the method set forth herein.

[0037] Such RNA in any of the foregoing aspects or embodiments may be encapsulated in a lipid nanoparticle, including any one of the LNPs described herein.

[0038] In further embodiments, the mRNA having the stabilizing shoe / shoelace is encapsulated in a lipid nanoparticle having elevated neutral lipid content. The combination of mRNA having the terminal 3’ shoe / shoelace and lipid nanoparticle with elevated neutral lipid leads to unexpected increases in relative expression of mRNA in tissues of interest beyond the liver. In some embodiments, it has been observed that the normalized bone marrow / liver expression exceeds 1.5.

[0039] While the inventors have found unexpected increases in mRNA expression beyond the liver using the shoe / shoelace sequences described herein, the shoe / shoelace sequences could be used to stabilize a wide variety of RNA sequences, including siRNA or antisense oligonucleotides or hybrid molecules, such as mRNA / siRNA conjugates.Brief description of the drawings

[0040] Figure 1 depicts formation of a modified mRNA comprising a 5' untranslated region (UTR), a coding region, a 3' untranslated region, a poly(A) tail and a terminal 3' sequence, alsoreferred to herein as the terminal nucleic acid “shoe” sequence. A complementary nucleic acid, referred to herein as a “shoelace” or “lace” sequence hybridizes to the terminal nucleic acid shoe sequence to form a duplex.

[0041] Figure 2 shows the chemical structure of RNA and PNA. As can be seen, the RNA (structure on the left) has repeating pentose and phosphate units, whereas the PNA, structure on the right, has a modified acyclic backbone of repeating amino acid derived units.

[0042] Figure 3A depicts the mRNA terminal 3' sequence that is 3' to the poly(A) tail and various complementary DNA, RNA and PNA shoelace sequences that can hybridize to the terminal 3' sequence with various affinities.

[0043] Figure 3B depicts a method to test formation of the shoe-shoelace duplex terminal to the poly(A) tail of the mRNA based on RNase R degradation of the mRNA without a shoelace (top) and an mRNA in which the shoe is hybridized to the shoelace (bottom) to form a shoe / shoelace duplex that is resistant to RNase R degradation.

[0044] Figure 4 is a denatured 2% formaldehyde, 2% agarose with SYBRGold™ RNA gel showing lanes with samples of modified FLuc mRNA having a 3' terminal shoe sequence (FLuc- Shoe) loaded from left to right in the following order: untreated, treated with RNase R, treated without RNase R, hybridized to a complementary RNA shoelace sequence and treated with RNase R, hybridized to a complementary DNA shoelace sequence and treated with RNase R, and hybridized to a complementary peptide nucleic acid (PNA, from PNABio) shoelace sequence and treated with RNase R. The ladder (shown to the left) is a RiboRuler High Range RNA Ladder (#SM1821) from Thermo Scientific™.

[0045] Figure 5 depicts backbone differences of various nucleic acid and nucleic acid analogues used as part of the complementary shoelace sequence, from left to right: in top row, DNA (deoxyribonucleic acid) and PNA (peptide nucleic acid); in middle row, RNA (ribonucleic acid), 2’-M0E (2’-O-methoxyethyl-RNA) , PMO (morpholino); in bottom row, cET (locked 2’, d’constrained 2’-O-ethyl) and RNA with phosphorothiate backbone.

[0046] Figure 6 is a denatured 2% formaldehyde, 2% agarose with SYBRGold™ RNA gel showing lanes with samples of modified FLuc mRNA having a 3 ’terminal shoe sequence (FLuc-Shoe) loaded from left to right in the following order: control treated with RNase R, control FLuc- Shoe mRNA treated without RNase R, hybridized to a complementary RNA shoelace sequence and treated with RNase R, hybridized to a complementary DNA shoelace sequence and treated with RNase R, hybridized to a complementary peptide nucleic acid (PNA, from PNABio) shoelace sequence and treated with RNase R, hybridized to a complementary 2’, 4’ constrained 2’-O-Ethyl (cET) shoelace sequence and treated with RNase R, hybridized to a complementary morpholino (PMO) shoelace sequence and treated with RNase R, hybridized to a complementary 2’-O- methoxyethyl-RNA (2’ -MOE) shoelace sequence and treated with RNase R, and hybridized to a complementary phosphorothioate (P=S) shoelace sequence and treated with RNase R. The ladder (shown in the centre having various bands) is a RiboRuler High Range RNA Ladder (#SM1821) from Thermo Scientific™.

[0047] Figure 7A provides a comparison of protein expression in mice liver tissue as measured by luminescence intensity 24 hours after intravenous injection with lipid nanoparticles encapsulating the following: wildype FLuc (FLuc WT) mRNA, modified FLuc mRNA having a 3’ terminal shoe sequence (FLuc-Shoe), modified FLuc-Shoe mRNAs with complementary 3’ RNA shoelace sequence, and modified FLuc-Shoe mRNAs with complementary 3 ’ PNA shoelace sequences. The LNP formulation used had ionizable lipid 18 (details in Table 4) / DSPC / cholesterol / PEG2ooo-DMG at 27.4:50:21.1 : 1.5 (mol / mol). Phosphate buffered saline was used as the control. The error bars were calculated by mean with standard deviation.

[0048] Figure 7B provides a comparison of protein expression in mice spleen tissue as measured by luminescence intensity 24 hours after intravenous injection with lipid nanoparticles encapsulating the following: wildype FLuc (FLuc WT) mRNA, modified FLuc mRNA having a 3’ terminal shoe sequence (FLuc-Shoe), modified FLuc-Shoe mRNAs with complementary 3’ RNA shoelace sequence, and modified FLuc-Shoe mRNAs with complementary 3 ’ PNA shoelace sequences. The LNP formulation used had ionizable lipid 18 (details in Table 4) / DSPC / cholesterol / PEG2ooo-DMG at 27.4:50:21.1 : 1.5 (mol / mol). Phosphate buffered saline was used as the control. The error bars were calculated by mean with standard deviation.

[0049] Figure 7C provides a comparison of protein expression in mice bone marrow tissue as measured by luminescence intensity 24 hours after intravenous injection with lipid nanoparticlesencapsulating the following: wildype FLuc (FLuc WT) mRNA, modified FLuc mRNA having a 3’ terminal shoe sequence (FLuc-Shoe), modified FLuc-Shoe mRNAs with complementary 3’ RNA shoelace sequence, and modified FLuc-Shoe mRNAs with complementary 3 ’ PNA shoelace sequences. The LNP formulation used had ionizable lipid 18 (details in Table 4) / DSPC / cholesterol / PEG2ooo-DMG at 27.4:50:21.1 : 1.5 (mol / mol). Phosphate buffered saline was used as the control. The error bars were calculated by mean with standard deviation.

[0050] Figure 7D provides a comparison of protein expression in mice abdominal skin as measured by luminescence intensity 24 hours after intravenous injection with lipid nanoparticles encapsulating the following: wildype FLuc (FLuc WT) mRNA, modified FLuc mRNA having a 3’ terminal shoe sequence (FLuc-Shoe), modified FLuc-Shoe mRNAs with complementary 3’ RNA shoelace sequence, and modified FLuc-Shoe mRNAs with complementary 3 ’ PNA shoelace sequences. The LNP formulation used had ionizable lipid 18 (details in Table 4) / DSPC / cholesterol / PEG2ooo-DMG at 27.4:50:21.1 : 1.5 (mol / mol). Phosphate buffered saline was used as the control. The error bars were calculated by mean with standard deviation.

[0051] Figure 7E shows spleen-to-liver selectivity of protein expression for (i) FLuc-Shoe mRNA hybridized with a complementary RNA shoelace and (ii) FLuc-Shoe mRNA hybridized with a complementary PNA shoelace. Both measurements are normalized to wild-type FLuc (FLuc WT) mRNA that does not contain a 3’ terminal shoe or a complementary shoelace sequence.

[0052] Figure 7F shows bone marrow-to-liver selectivity of protein expression for (i) FLuc-Shoe mRNA hybridized with a complementary RNA shoelace and (ii) FLuc-Shoe mRNA hybridized with a complementary PNA shoelace. Both measurements are normalized to wild-type FLuc (FLuc WT) mRNA that does not contain a 3’ terminal shoe or a complementary shoelace sequence.

[0053] Figure 7G shows abdominal skin-to-liver selectivity of protein expression for (i) FLuc- Shoe mRNA hybridized with a complementary RNA shoelace and (ii) FLuc-Shoe mRNA hybridized with a complementary PNA shoelace. Both measurements are normalized to wild-type FLuc (FLuc WT) mRNA that does not contain a 3 ’ terminal shoe or a complementary shoelace sequence.

[0054] Figure 8 shows the scrambled mRNA 3' terminal shoe sequence that is 3' to the poly(A) tail in the modified mRNA (on top) and its complementary scrambled peptide nucleic acid (PNA, from PNABio) shoelace sequence (below).

[0055] Figure 9 is a denatured 2% formaldehyde, 2% agarose with SYBRGold™ RNA gel showing lanes with samples of modified FLuc mRNA having a 3 ’terminal scrambled shoe sequence (FLuc-Scrambled Shoe) loaded from left to right in the following order: control treated with RNase R, hybridized to a complementary peptide nucleic acid (PNA, from PNABio) scrambled shoelace sequence and treated with RNase R. The ladder (shown in the centre having various bands) is a RiboRuler High Range RNA Ladder (#SM1821) from Thermo Scientific™.

[0056] Figure 10A provides a comparison of protein expression in mice liver tissue as measured by luminescence intensity 24 hours after intravenous injection with lipid nanoparticles encapsulating the following: wildype FLuc (FLuc WT) mRNA, modified FLuc mRNA having a 3’ terminal scrambled shoe sequence (FLuc-Scrambled Shoe), modified FLuc-Scrambled Shoe mRNAs with complementary 3’ PNA Scrambled shoelace sequence. The LNP formulation used had ionizable lipid 19 (details in Table 4) / DSPC / cholesterol / PEG2ooo-DMG at 27.4:50:21.1: 1.5 (mol / mol). Phosphate buffered saline was used as the control. The error bars were calculated by mean with standard deviation.

[0057] Figure 10B provides a comparison of protein expression in mice spleen tissue as measured by luminescence intensity 24 hours after intravenous injection with lipid nanoparticles encapsulating the following: wildype FLuc (FLuc WT) mRNA, modified FLuc mRNA having a 3’ terminal scrambled shoe sequence (FLuc-Scrambled Shoe), modified FLuc-Scrambled Shoe mRNAs with complementary 3’ PNA Scrambled shoelace sequence. The LNP formulation used had ionizable lipid 19 (details in Table 4) / DSPC / cholesterol / PEG2ooo-DMG at 27.4:50:21.1: 1.5 (mol / mol). Phosphate buffered saline was used as the control. The error bars were calculated by mean with standard deviation.

[0058] Figure IOC provides a comparison of protein expression in mice bone marrow tissue as measured by luminescence intensity 24 hours after intravenous injection with lipid nanoparticles encapsulating the following: wildype FLuc (FLuc WT) mRNA, modified FLuc mRNA having a 3’ terminal scrambled shoe sequence (FLuc-Scrambled Shoe), modified FLuc-Scrambled ShoemRNAs with complementary 3’ PNA Scrambled shoelace sequence. The LNP formulation used had ionizable lipid 19 (details in Table 4) / DSPC / cholesterol / PEG2ooo-DMG at 27.4:50:21.1: 1.5 (mol / mol). Phosphate buffered saline was used as the control. The error bars were calculated by mean with standard deviation.

[0059] Figure 10D provides a comparison of protein expression in mice abdominal skin tissue as measured by luminescence intensity 24 hours after intravenous injection with lipid nanoparticles encapsulating the following: wildype FLuc (FLuc WT) mRNA, modified FLuc mRNA having a 3’ terminal scrambled shoe sequence (FLuc-Scrambled Shoe), modified FLuc-Scrambled Shoe mRNAs with complementary 3’ PNA Scrambled shoelace sequence. The LNP formulation used had ionizable lipid 19 (details in Table 4) / DSPC / cholesterol / PEG2ooo-DMG at 27.4:50:21.1: 1.5 (mol / mol). Phosphate buffered saline was used as the control. The error bars were calculated by mean with standard deviation.

[0060] Figure 10E shows spleen-to-liver selectivity of protein expression for FLuc-Scrambled Shoe mRNA hybridized with a complementary PNA Scrambled shoelace. The measurement is normalized to wild-type FLuc (FLuc WT) mRNA that does not contain a 3’ terminal shoe or a complementary shoelace sequence.

[0061] Figure 10F shows bone marrow -to-liver selectivity of protein expression for FLuc- Scrambled Shoe mRNA hybridized with a complementary PNA Scrambled shoelace. The measurement is normalized to wild-type FLuc (FLuc WT) mRNA that does not contain a 3’ terminal shoe or a complementary shoelace sequence.

[0062] Figure 10G shows abdominal skin-to-liver selectivity of protein expression for FLuc- Scrambled Shoe mRNA hybridized with a complementary PNA Scrambled shoelace. The measurement is normalized to wild-type FLuc (FLuc WT) mRNA that does not contain a 3’ terminal shoe or a complementary shoelace sequence.Detailed description

[0063] Figure 1 depicts a representative schematic of a modified RNA 10 of the disclosure. The modified RNA in some embodiments is a messenger RNA (mRNA). The RNA 10 of such nonlimiting example comprises a 5’ untranslated region (5’ UTR) 12, a coding region 14 that encodesa polypeptide or protein, a 3’ untranslated region (3’ UTR) 16, a polyadenosine (poly(A)) tail 18 and a terminal 3’ RNA shoe sequence 20 (shoe). The terminal 3’ RNA shoe sequence 20 comprises a 3’ terminal nucleotide sequence that hybridizes with a complementary nucleic acid sequence 22, such as a nucleic acid with a modified backbone. The complementary nucleic acid sequence 22 is also referred to herein as a shoelace. A non-limiting example of such a shoelace sequence 22 is a peptide nucleic acid (PNA) sequence. The addition 28 of the shoelace sequence 22 to the modified mRNA 10 results in a modified mRNA 24 comprising a terminal stabilizing duplex 26. While PNA shoelace sequences are described, as would be appreciated by those of skill in the art, other shoelaces with modified backbones could be used to hybridize the shoe to form the stabilizing duplex 26. Further, as would be understood by those of ordinary skill in the art, the modified mRNA may further comprise a 5’ terminal cap and / or a microRNA targeting site.

[0064] Turning now to Figure 2, there is shown a comparison of an RNA 30 and a non-limiting example of a PNA 32 sequence, the latter of which is an example of the complementary shoelace sequence 22 that hybridizes to the terminal 3’ RNA sequence (“shoe”) of the modified mRNA 10 to form the heteroduplex 26. The PNA 32 sequence comprises a backbone of repeating amino acid derived units, such as repeating N-(2-aminoethyl)-glycine units as depicted in the PNA 32 sequence of Figure 2. The RNA 30 sequence has a backbone of repeating sugar-phosphate units.

[0065] Figure 3A shows an example of a terminal 3’ RNA shoe sequence 20 linked to the poly(A) tail, labelled in the figure as 3’ mRNA substrate. Figure 3 A also shows non-limiting examples of complementary DNA, RNA and PNA shoelace sequences. The complementary nucleic acid based DNA and RNA shoelace sequences are shown from 5 ’ to 3 ’ . The PNA shoelace sequence is shown with the C-terminal end on the left and the N-terminal end (N’) on the right-side of the drawing. Without being limited by theory, Figure 3B depicts a possible mechanism whereby the complementary shoelace sequence stabilizes the modified mRNA against degradation by exonucleases such as RNAse R. As shown, in the absence of any hybridized complementary shoelace sequence forming a duplex with the terminal 3 ’ RNA shoe sequence, an RNase R enzyme can degrade the terminal end of the mRNA by hydrolyzing the nucleotides. By contrast, the addition of a complementary shoelace such as a PNA sequence to the modified-mRNA results in hybridization of the PNA shoelace sequence to the terminal 3 ’ RNA sequence, thereby forming acomplex such as a duplex. Without limiting, it is believed by the inventors that such duplex causes the 3’ end of the mRNA to be resistant to RNase R degradation in vitro (see Example 1 herein).

[0066] Figure 8 shows a non-limiting example of a 3’ terminal scrambled RNA shoe sequence linked to the poly(A) tail, and the complementary scrambled PNA shoelace with the C-terminal end on the left and the N-terminal end on the right-side of the drawing. The modified RNA and the 3’ terminal scrambled shoe and complementary shoelace sequence are described in more detail below.Modified RNA

[0067] The modified RNA includes a variety of RNA molecules, including small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), guide RNA (gRNA), including single guide RNA (sgRNA) and prime editing guide RNA (pegRNA), messenger RNA (mRNA), small activating RNA (saRNA), self-replicating RNA (srRNA), transamplifying RNA (taRNA), long noncoding RNA (IncRNA), and transfer RNA (tRNA). The RNA length can vary and may include nucleic acid of 1-50,000 nucleotides in length. The RNA can be in any form, including single stranded RNA, double stranded RNA, including hybrids such as RNA and DNA complexes.

[0068] In some embodiments, the RNA is an mRNA that codes a protein, polypeptide, or peptide. mRNA

[0069] As used herein, the term “messenger RNA” or “mRNA” refers to a polynucleotide comprising an RNA sequence that encodes and expresses at least one protein, polypeptide or peptide.

[0070] 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.

[0071] The mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / or backbone 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, C5-bromouridine, C5- fluorouridine, C5 -iodouridine, 5-methyl uridine, 5-methoxy uridine, 5-formyl uridine, 5-carboxy methyl uridine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, O(6)-methylguanine, 2-thiocytidine and pseudouridine, such as N1 -methyl pseudouridine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, 2’-O-methylribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N- phosphoramidite linkages).

[0072] The mRNA of the disclosure may be synthesized according to any 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.

[0073] In those embodiments in which the mRNA is encapsulated in a lipid nanoparticle, in vitro synthesized mRNA may be purified before encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0074] 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 1-20 kb, about 1-15 kb, about 1-10 kb, about 2-20 kb, about 2-15 kb, about 2-10 kb, about 3-20 kb, about 3-15 kb, about 3-10 kb, about 3-7 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.

[0075] In some embodiments, the mRNA includes a “cap” on the 5' end, and a “tail” on the 3' end. In some non-limiting examples, the cap comprises a guanine nucleotide connected to the mRNA by a 5’ to 5’ triphosphate linkage. 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. The terminally optimized mRNA described herein may comprise at least one 5' cap structure such as, but not limited to, CapO, Cap 1 , ARCA, inosine, N 1 -methyl-guanosine, 2 '-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido- guanosine, Cap2, Cap4, and CAP-003 -CAP-225.

[0076] 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 the stability or translation of mRNA, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length or longer.

[0077] In some embodiments, a 3' untranslated region includes one or more of a tailing sequence, such as 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.

[0078] A “poly(A) tail” is a region of mRNA that is 3’ from the 3' UTR and comprises adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, a poly(A) tail may comprise 10 to 300 or 50 to 250 adenosine monophosphates. A poly(A) tail generally functions to protect mRNA from enzymatic degradation by RNases. However, in certain embodiments, the mRNA of the disclosure lacks a poly(A) tail (i.e., the mRNA is “tailless”). The poly(A) tail may be modified, such as at the alpha-phosphate position. An example of such a modification is described in Strzelecka et al., 2020, RNA, 26: 1815-1837 (incorporated herein by reference) in which the backbone is modified to include a phosphorothioate or boranophosphate moiety. Such modifications may further reduce degradation by exonucleases.

[0079] While mRNA provided 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.

[0080] In some non-limiting examples, 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.Terminal shoe / shoelace stabilizing sequences

[0081] As discussed, the RNA of the disclosure also includes a non-coding sequence, referred to herein as a shoe at a 3 ’ terminal region of the RNA that hybridizes with a complementary nucleic acid shoelace sequence, thereby stabilizing one or both ends of the RNA. In some embodiments, the shoe / shoelace sequence is at the 3’ terminal end of the RNA. In further embodiments, the shoe / shoelace sequence is not at the terminal end of the RNA, in which case, the modified RNA has one or more additional sequences terminal to the shoe / shoelace.

[0082] As used herein, the term “shoe” refers to a nucleotide sequence at a terminal region of the RNA that is hybridized to a complementary nucleotide shoelace sequence to thereby form a stabilizing structure. The shoe is operably linked to a 3’ terminal region of the RNA, such as covalently bonded to a tailing sequence (e.g., poly(A) tail) or directly to the last nucleotide of the mRNA. The shoe can include any nucleotide sequence but is typically an RNA sequence.

[0083] As used herein, the term “shoelace” refers to a nucleotide sequence that is complementary to at least the shoe to thereby form a stabilizing hybridized structure.

[0084] As used herein, the term “shoe / shoelace” refers to a hybridized structure that comprises a duplex, triplex or quadruplex of stabilizing shoe and shoelace sequences. The shoe / shoelace may include one or more additional secondary structures, such as a terminal stem loop in one or both of their sequences.

[0085] As used herein, the term “nucleic acid analogue with a modified backbone” with reference to a shoelace refers to a synthetic analogue of DNA or RNA nucleic acid in which the sugarphosphate backbone of the nucleic acid is modified or replaced with a modified polymeric structure with repeating subunits. The term encompasses a chemical modification of sugar and / or modification of phosphate. In some embodiments, the sugar-phosphate backbone is replaced by a polymer of repeating monomeric backbone units lacking a phosphate or sugar moiety and to which RNA or DNA nucleobases are linked. The latter embodiment includes without limitation repeating subunits that are uncharged (e.g., lacking a phosphate group that imparts a negative charge to the shoelace). In some non -limiting examples, the polymer of repeating monomeric backbone units is partially charged.

[0086] In some embodiments, the shoelace is "non-naturally occurring” meaning that it presently has no known natural counterparts found in nature.

[0087] As used herein, the term “complementary” with reference to the shoelace strands refers to sufficient Watson -Crick base pairing between the 3’ terminal shoe and corresponding complementary shoelace sequence to form a stabilizing hybridized structure such as a duplex, triplex or quadruplex. The stabilizing duplex might also contain a loop without base pairing. In some non-limiting embodiments, the stabilizing duplex might also contain a stem loop element in the shoelace or shoe sequence. In some embodiments, the stabilizing duplex might contain a stem loop element in the shoe and shoelace sequence. In some embodiments, the stabilizing duplex might also contain at least two stem loop elements in the shoelace or shoe sequence. Without limiting, in some embodiments, at least 80% of the bases of the two strands are paired. In further embodiments, at least 85% of the bases of the two strands are paired. In yet further embodiments, at least 90% of the bases of the two strands are paired.

[0088] The shoelace may additionally comprise nucleotides that are complementary to the 3 ’ UTR or a tailing sequence at the 3 ’ end of the mRNA. For example, the shoelace may comprise a polyT sequence that hybridizes to an upstream poly(A) tail. In some non-limiting examples, 3’ terminal shoe and / or the complementary shoelace sequence has regions that do not form base pairing.

[0089] In some embodiments, the shoe contains 5 to 100 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 10 to 28 nucleotides, 10 to 27 nucleotides, 10 to 26 nucleotides, 10 to 25 nucleotides, 10 to 24 nucleotides, 10 to 23 nucleotides, 10 to 22 nucleotides, 10 to 21 nucleotides, 10 to 20 nucleotides, 10 to 18 nucleotides, 10 to 16 nucleotides, or 10 to 15 nucleotides. In some non-limiting embodiments, the shoe contains 20 to 30 nucleotides. In some embodiments, the 3’ terminal shoe of the disclosure is a nucleic acid that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID No: 1 or 9, using local alignment and using the most current version of BLASTn available online at NIH.

[0090] As discussed, in one embodiment, the complementary shoelace nucleic acid is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by a backbone that is modified. The inventors have found that certain chemically modified shoelaces may provideenhanced shoe / shoelace stability relative to non-modified shoelaces, such as DNA or RNA shoelaces. A non -limiting example of a modified shoelace that can increase stability of the shoe / shoelace duplex is a neutral nucleic acid, such as a peptide nucleic acid (PNA).

[0091] In some embodiments, the complementary shoelace sequence is at least substantially uncharged at physiological pH. For example, in some embodiments, at least 60% of the nucleotides lack a charge on the backbone (e.g., a phosphate), at least 65% of the nucleotides lack a charge on the backbone, at least 70% of the nucleotides lack a charge on the backbone, at least 75% of the nucleotides lack a charge on the backbone, at least 80% of the nucleotides lack a charge on the backbone, at least 85% of the nucleotides lack a charge on the backbone, or at least 90% of the nucleotides lack a charge on the backbone.

[0092] In some embodiments, the shoelace backbone lacks phosphate groups. In alternative embodiments, the shoelace backbone lacks cyclic moieties. For example, the shoelace backbone may lack a pentose sugar. In some embodiments, the shoelace is a non-DNA sequence or a non- RNA sequence.

[0093] Non-limiting examples of modified shoelaces are peptide nucleic acids (PNA), morpholino (PMO) sequences, locked 2’, 4 ’-constrained 2’-O-ethyl (cET) sequences, a 2’-O-methoxyethyl- RNA (2 ’-MOE) sequences, and phosphorothioate (P=S) sequences wherein the phosphate backbone is modified.

[0094] Further examples of modified shoelaces include locked nucleic acid (LNA), unlocked nucleic acid (UNA), and triazole-linked nucleic acid sequences.

[0095] In some embodiments, the shoelace comprises natural bases (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, C5 -bromouridine, C5 -fluorouridine, C5-iodouridine, C5-methylcytidine, 5-methyl uridine, 5- methoxy uridine, 5-formyl uridine, 5-carboxy methyl uridine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2- thiocytidine and pseudouridine, such as Nl-methyl pseudouridine); non-naturally occurring bases;chemically modified bases; biologically modified bases (e.g., methylated bases); and / or intercalated bases.

[0096] In some embodiments, the shoelace contains 5 to 100 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 10 to 28 nucleotides, 10 to 27 nucleotides, 10 to 26 nucleotides, 10 to 25 nucleotides, 10 to 24 nucleotides, 10 to 23 nucleotides, 10 to 22 nucleotides, 10 to 21 nucleotides, 10 to 20 nucleotides, 10 to 18 nucleotides, 10 to 16 nucleotides, or 10 to 15 nucleotides. In some embodiments, the shoelace contains 20 to 30 nucleotides.

[0097] In some embodiments, when in isolated form and subjected to heat treatment, at least 50% of the stabilizing RNA shoe-shoelace is in double-stranded form at 55°C. In some embodiments, when in isolated form and subjected to heat treatment, at least 55% of the stabilizing RNA shoeshoelace is in double-stranded form at 55°C. In some embodiments, when in isolated form and subjected to heat treatment, at least 60% of the stabilizing RNA shoe-shoelace is in doublestranded form at 55°C. In some embodiments, when in isolated form and subjected to heat treatment, at least 65% of the stabilizing RNA shoe-shoelace is in double-stranded form at 55°C. In some embodiments, when in isolated form and subjected to heat treatment, at least 70% of the stabilizing RNA shoe-shoelace is in double-stranded form at 55°C. In some embodiments, when in isolated form and subj ected to heat treatment, at least 80% of the stabilizing RNA shoe / shoelace is in double -stranded form at 55°C. The measurement of the percentage of RNA shoe / shoelace in double-stranded is determined by the methods described in Jasinski et al., 2019, J Phys Chem B., 123(39): 8168-8177 as set forth therein in Figure 4 (incorporated herein by reference). The method described by Janinski et al., 2019, comprises UV-monitored melting at 260 nm. Complementary strands are mixed at a concentration of 4 pM in 10 mM phosphate buffer with 20 mM NaCl at pH 7. The absorbance curves are fitted with sloping baselines from which the fraction of base paired molecules as a function of temperature is calculated (see Mergny J-L; Lacroix L Analysis of thermal melting curves. Oligonucleotides 2003, 13, 515-537, cited in Janinski and incorporated herein by reference). Each melting experiment should be repeated at least 5 times. It should be understood that these measurements refer to the hybridized structure formed by the shoe sequence alone (when not linked to the terminal end of the mRNA) and the shoelace.

[0098] In some embodiments, the modified RNA with the shoe / shoelace is resistant to RNase R degradation as measured on an agarose gel (see Examples herein). In some embodiments, the band intensity is at least 90%, 85%, 80%, 75%, 70%, 65% or 60% of a control without RNase R addition but otherwise identical and as determined using ImageJ™ software. The method for quantifying band intensity by ImageJ™ software is described in Kenji Ohgane, Hiromasa Oshioka, 2019, Quantification of Gel Bands by an Image J Macro, Band / Peak Quantifiation Tool. Protocols. io https: / / dx.doi.org / 10.17504 / protocols.io.7vgh3w, incorporated herein by reference.

[0099] In some embodiments, mRNA sequences without a 5 ’-Cap or a poly(A) tail may still provide for expression of protein / peptide / polypeptide. For example, the 5’-Cap of mRNA and / or the poly(A) tail can be replaced by hairpin structures (see Solodushko and Fouty, 2023, Gene Therapy, 30:620-627, incorporated herein by reference).

[0100] In some embodiments, the mRNA has one of the following structures: (i) 5'-UTR - ORF - 3'-UTR - Poly (A) tail - shoe / shoelace structure; (ii) 5 '-Cap - 5'-UTR - ORF - 3'-UTR - Poly (A) tail - shoe / shoelace structure; (iii) 5'-UTR - ORF - 3'-UTR - shoe / shoelace structure; and (iv) 5'- Cap - 5'-UTR - ORF - 3'-UTR - shoe / shoelace structure. In some embodiments, the mRNA has a poly(A) tail and is selected from structure (i) or (ii) as described above.

[0101] In some embodiments, the mRNA with the stabilizing shoe / shoelace may include a cleavable, terminal siRNA.Lipid nanoparticles

[0102] In some embodiments, the modified mRNA is encapsulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle is specifically designed for extrahepatic delivery. This includes lipid nanoparticles with elevated neutral lipid content as described below.Neutral lipid

[0103] The LNP includes “a neutral lipid”, which includes one or more neutral lipids other than sterols. The neutral lipid is any lipid that bears no charge at physiological pH, including zwitterionic lipids having at least substantially no net charge at physiological pH. In one embodiment, the neutral lipid is amphipathic and has at least two tails and a polar region, such asa head group. The term includes lipids with choline head groups (e.g., phosphatidylcholine), meaning amphipathic lipids that have a choline head group, and that bear no or substantially no net charge at physiological pH. The neutral lipid also includes phospholipids conjugated to sterols. In some embodiments, the lipid with a choline head group is a phosphatidylcholine lipid, such as a lipid that is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), l-palmitoyl-2 -oleoyl -phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin with a phosphocholine head group.

[0104] As used herein “head group”, means a moiety that imparts polarity to the lipid and comprises one or more electronegative atoms, such as nitrogen, oxygen and / or phosphorus. Generally, the electronegative atom or atoms are part of one or more functional groups. In some embodiments, the head group is zwitterionic. In some embodiments, the head group comprises a phosphate group and a nitrogen atom. In some embodiments, the head group is zwitterionic and has no net charge at a pH that is physiological (pH 7.4).

[0105] The phosphatidylcholine lipid content may include mixtures of two or more types of different phosphatidylcholine lipids. In one embodiment, the phosphatidylcholine lipid content is a mixture of two or more of the phosphatidylcholine lipids selected from DSPC, DPPC, DMPC, DOPC and POPC. In some embodiments, the phosphatidylcholine lipid content is primarily DSPC or DMPC or primarily DSPC.

[0106] In such embodiments, the neutral lipid mixture may have a DSPC content of at least 20, 22, 30, 35, 40, 45, 47, or 50 mol% based on the total lipid content of the lipid nanoparticle with the balance of the phosphatidylcholine lipid content being another phosphatidylcholine lipid(s). In another embodiment, the phosphatidylcholine content is made up of at least 40 or 50 mol% DSPC relative to the total phosphatidylcholine content of the lipid nanoparticle.

[0107] In alternative embodiments, the neutral lipid mixture may have a DMPC content of at least 20, 30, 35, 40, 45, 47, or 50 mol% based on the total lipid content of the lipid nanoparticle. In another embodiment, the phosphatidylcholine content is made up of at least 40 mol% DMPC relative to the total phosphatidylcholine content of the lipid nanoparticle. In another embodimentthe phospholipid content of the lipid nanoparticle has less than 10, or less than 5 mol% of nonphosphatidylcholine lipids, such as DOPE.

[0108] In some embodiments, the transition temperature of the lipid (e.g., phospholipid) having a choline head group is at least 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. In some embodiments, the transition temperature of the lipid (e.g., phospholipid) having a choline head group is at least 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. In some embodiments, the transition temperature of the lipid (e.g., phospholipid) having a choline head group is between 20°C and 60°C or between 42°C and 60°C. The phase transition temperature of the lipid is measured using differential scanning calorimetry (DSC) using techniques known to those of skill in the art and is the main phase transition temperature.

[0109] Without intending to be limited by any particular theory, it is believed that fusion and agglomeration of lipid nanoparticles with no hydrophilic polymer lipid conjugate (or low levels thereof) during particle formation using the mixing method described herein could be avoided by selecting a phospholipid having a choline head group that is in the gel phase rather than in the disordered liquid crystalline phase at room temperature and above. This inclusion of such phosphatidylcholine lipids in the lipid nanoparticle may also improve blood stability after injection.

[0110] In some embodiments, the phosphatidylcholine lipid is a phosphatidylcholine-sterol conjugate, such as an SPC-cholesterol, OPC-cholesterol or PPC-cholesterol conjugate. Additional phospholipid-sterol conjugates are described in US2011 / 0177156, which is incorporated herein by reference.

[0111] In some embodiments, the phosphatidylcholine lipid is a sphingolipid. As used herein the term “sphingolipid”, means a lipid comprising a sphingosine backbone and that is suitable for formulation in the LNPs herein. The sphingolipid includes a ceramide, a sphingomyelin, a cerebroside, a ganglioside, or derivatives, such as but not limited to reduced analogues thereof, that lack a double bond in the sphingosine unit. The sphingolipid has a phosphocholine head group and includes sphingomyelin.

[0112] The neutral lipid content in some embodiments (excluding sterol) is greater than 20 mol%, greater than 22 mol%, greater than 22.5 mol%, greater than 25 mol%, greater than 27.5 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 than 44 mol%, or greaterthan 46 mol% based on total lipid content of the lipid nanoparticle. In some embodiments, the upper limit of phosphatidylcholine 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.

[0113] The content of neutral lipid having acholine head group in some embodiments (excluding sterol) is greater than 20 mol%, greater than 22 mol%, greater than 22.5 mol%, greater than 25 mol%, greaterthan 27.5 mol% greater than 30 mol%, greater than 32 mol%, greaterthan 34 mol%, greater than 36 mol%, greaterthan 38 mol%, greaterthan 40 mol%, greaterthan 42 mol%, greater than 44 mol%, or greater than 46 mol% In some embodiments, the upper limit of phosphatidylcholine 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.

[0114] The content of neutral lipid having at least two tails and a polar region (e.g., head group) in some embodiments (excluding sterol) is greater than 20 mol%, greater than 22 mol%, greater than 22.5 mol%, greaterthan 25 mol%, greaterthan 27.5 mol% greaterthan 30 mol%, greaterthan 32 mol%, greater than 34 mol%, greater than 36 mol%, greater than 38 mol%, greater than 40 mol%, greater than 42 mol%, greater than 44 mol%, or greater than 46 mol% In some embodiments, the upper limit of phosphatidylcholine 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.

[0115] The phosphatidylcholine lipid content in some embodiments is greater than 20 mol%, greater than 22 mol%, greater than 22.5 mol%, greater than 25 mol%, greater than 27.5 mol% greaterthan 30 mol%, greaterthan 32 mol%, greaterthan 34 mol%, greaterthan 36 mol%, greater than 38 mol%, greater than 40 mol%, greater than 42 mol%, greater than 44 mol%, or greater than 46 mol% In some embodiments, the upper limit of phosphatidylcholine 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.

[0116] For example, in certain embodiments, the neutral lipid content is from 20 mol% to 60 mol%, 22 mol% to 60 mol%, 25 mol% to 60 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 42 mol% to 58 mol%, 43 mol% to 57 mol%, 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0117] For example, in certain embodiments, the content of neutral lipid having a choline head group is from 20 mol% to 60 mol%, 22 mol% to 60 mol%, 25 mol% to 60 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 42 mol% to 58 mol%, 43 mol% to 57 mol%,44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0118] For example, in certain embodiments, the content of neutral lipid having at least two tails and a polar region (e.g., head group) in some embodiments (excluding sterol) is from 20 mol% to 60 mol%, 22 mol% to 60 mol%, 25 mol% to 60 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 42 mol% to 58 mol%, 43 mol% to 57 mol%, 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0119] For example, in certain embodiments, the phosphatidylcholine lipid content is from 22 mol% to 60 mol%, 25 mol% to 60 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 42 mol% to 58 mol%, 43 mol% to 57 mol%, 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0120] In certain embodiments, the DSPC lipid content is from 20 mol% to 60 mol%, 25 mol% to 65 mol%, 30 mol% to 65 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 42 mol% to 58 mol%, 43 mol% to 57 mol%, 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0121] In certain embodiments, the DMPC lipid content is from 25 mol% to 65 mol%, 30 mol% to 65 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 42 mol% to 58 mol%, 43 mol% to 57 mol%, 44 mol%to 56 mol% or 45 mol%to 55 mol% of total lipid present in the lipid nanoparticle.

[0122] In certain embodiments, the sphingolipid or sphingomyelin lipid content is from 25 mol% to 65 mol%, 30 mol% to 65 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 42 mol% to 58 mol%, 43 mol% to 57 mol%, 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0123] The phosphatidylcholine lipid content is determined based on the total amount of lipid in the lipid nanoparticle, including the sterol and any amount of additional lipid components that are optionally present.

[0124] In alternative embodiments, the DMPC content is less than 22 mol%, 20 mol%, 18 mol%, 16 mol%, 14 mol%, 12 mol%, 10 mol%, 8 mol%, 6 mol%, 4 mol% or 2 mol% based on the total lipid content of the lipid nanoparticle. In alternative embodiments, the DPPC content is less than 22 mol%, 20 mol%, 18 mol%, 16 mol%, 14 mol%, 12 mol%, 10 mol%, 8 mol%, 6 mol%, 4 mol% or 2 mol% based on the total lipid content of the lipid nanoparticle. In alternative embodiments, the DOPE content is less than 22 mol%, 20 mol%, 18 mol%, 16 mol%, 14 mol%, 12 mol%, 10 mol%, 8 mol%, 6 mol%, 4 mol% or 2 mol% mol% based on the total lipid content of the lipid nanoparticle.Sterol

[0125] As set forth herein, the LNPs of the disclosure comprise a sterol.

[0126] The term “sterol” refers to steroids that are naturally occurring or synthetic. The term includes sterol derivatives such as phytosterols, zoosterols and derivatives thereof.

[0127] The term “sterol derivatives” refers to modified sterols or precursors thereof, including triterpenes.

[0128] The term “cholesterol” 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.

[0129] The LNP may alternatively or additionally comprise a “cholesterol derivative”. The cholesterol derivative may be naturally occurring or man-made and includes but is not limited to a cholesterol molecule having a gonane structure and one or more additional functional groups.

[0130] In another embodiment, the LNP may comprise a triterpene. Non-limiting examples include squalene, achilleol A, polypodatetraene, malabaricane, lanostane, cucuribitacin, hopane, oleanane, and urosolic acid.

[0131] The sterol or cholesterol derivative may be conjugated to another moiety, such as an amino acid or an alkyl group.

[0132] In one embodiment, the sterol (which encompasses a derivative thereof) is present at from 12 to 40 mol%, 12 to 35 mol%, 14 to 32 mol%, 15 to 28 mol% or 15 to 26 mol% based on the total lipid present in the lipid nanoparticle. In a further embodiment, the sterol is present at 21 to 40 mol%.

[0133] In another embodiment, the sterol (which encompasses a derivative thereof) is present at greaterthan 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol% or 22 mol%. The upper limit of the sterol or sterol derivative content may be 46 mol%, 44 mol%, 42 mol%, 40 mol%, 38 mol%, 36 mol%, 34 mol%, 32 mol% or 30 mol%. The sterol content may include any combination of the foregoing lower and upper limits and may include mixtures of different kinds of sterols.

[0134] In one embodiment, the cholesterol (encompassing derivatives thereof) is present at from 12 to 40 mol%, 12 to 35 mol%, 14 to 32 mol%, 15 to 28 mol% or 15 to 26 mol% based on the total lipid present in the lipid nanoparticle. In a further embodiment, the cholesterol is present at 21 to 40 mol%.

[0135] In another embodiment, the cholesterol is present at greater than 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol% or 22 mol%. The upper limit of the cholesterol or cholesterol derivative content may be 46 mol%, 44 mol%, 42 mol%, 40 mol%, 38 mol%, 36 mol%, 34 mol%, 32 mol%, or 30 mol%. The cholesterol content may include any combination of the foregoing lower and upper limits.

[0136] In some embodiments, the combined neutral lipid and sterol content (e.g., phosphatidylcholine, sterol and any additional neutral lipids, if present) is at least 50, 55, 60, 65 or 70 mol% relative to the total lipid content of the lipid nanoparticle. As used herein, the neutral lipid content encompasses non-sterol helper lipids.Ionizable cationic lipid

[0137] The LNP of the disclosure has an ionizable cationic lipid, which includes one or a combination of two or more of such lipids.

[0138] As used herein, the term "ionizable cationic lipid" refers to a lipid that, at a given pH, such as physiological pH, is in an electrostatically neutral form and that accepts protons, thereby becoming electrostatically positively charged at a pH below its pKa. 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 an apparent pKa (determined using the method described herein) that is between 5.0 and 7.5 or between 6.0 and 7.5 when formulated in the LNP.

[0139] Accordingly, the ionizable cationic lipid is charged at low pH and bears 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. Without being limited by theory, 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 acid-based therapeutics into the cytoplasm to exert their effects. Since the LNPs are non-sterically stabilized, it is believed that fusion with the endosomal membrane is enhanced relative to the same particle having 1.5 mol% PEG-lipid (e.g., PEG2000-DMG).

[0140] In some embodiments, the LNP has an apparent pKa of between 5.0 and 7.5, between 6.5 and 7.5 or between 6.8 and 7.3. The apparent pKa is measured using a 6-(p-Toluidino)-2- naphthalenesulfonic acid (TNS) assay adapted from previous studies from other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; and Jayaraman et al., 2012, Angew. Chem Int. Ed., 51:8529-8533, which are incorporated herein by reference for the purposes of determining apparent pKa). According to the method, a series of buffers are prepared spanning a pH range of 2-11 in 0.5 pH unit increments consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino) ethane sulfonic acid (MES), and 10 mM HEPES. 0.15-0.2 mM of the LNP. A solution of 0.06 mM of TNS is subsequently mixed with 175 pL of the LNP at eachbuffered pH in triplicate in a black, polysterene 96-well plate, to yield a final concentration of 6.25 and 6 pM of lipid and TNS in each well, respectively. Fluorescence is subsequently measured using an SpectraMax™ M5 microplate reader at Xex=321 nm, Zcm=445 nm. The fluorescence is then plotted against pH using a sigmoidal curve fit through Prism™, in which the pKa is determined to be the pH value with 50% of maximal fluorescent intensity.

[0141] In some embodiments, it is desirable to include less than 40 mol% ionizable lipids in the LNP. 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 lipids present in the lipid nanoparticle.

[0142] In certain embodiments, the ionizable lipid content may be less than 48 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%. In certain embodiments, the lower limit of the ionizable lipid content may be greater than 5 mol%, greater than 8 mol%, greater than 10 mol%, greater than 12 mol%, greater than 14 mol%, greater than 15 mol%, greater than 16 mol%, greater than 18 mol% or greater than 20 mol%. Any one of the upper limits may be combined with any one of the lower limits to arrive at a suitable ionizable lipid content in the LNP.

[0143] In some embodiments, the ionizable cationic lipid has an amino group. In another embodiment, the ionizable cationic lipid has a single amino group that is ionizable. In some embodiments, the ionizable cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group. Such lipids include, but are not limited to sulfur lipids, such as MF019 described herein 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.2024 / 0294462, each incorporated herein by reference.

[0144] In one embodiment, the ionizable cationic lipid comprises an ionizable amino head group and at least two lipophilic groups that converge to a central carbon or nitrogen atom and wherein the central carbon or nitrogen atom is bonded to a head group moiety. At least one or more typicallyboth of the lipophilic chains may contain a biodegradable group, such as an ester, and / or one or more sulfur atoms. In some embodiments, at least one lipophilic group comprises alkyl branching disposed between the head group and the biodegradable group and / or one or more cyclic alkyl groups distal to the biodegradable group. Examples of ionizable cationic lipids comprising an ionizable amino head group and two lipophilic 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 / 065043, which are incorporated herein by reference. Functional groups comprising one or more heteroatoms may be biodegradable in vivo.

[0145] In one embodiment, the lipid nanoparticle comprises a “sulfur-containing amino lipid”, which is an ionizable lipid having at least one ionizable nitrogen (tertiary nitrogen) and one or more sulfur atoms in at least one of its lipophilic chains. In one embodiment, the sulfur-containing amino lipid further comprises a biodegradable group, such as an ester moiety, in one or more of its lipophilic chains. Non-limiting examples of sulfur-containing lipids are provided in Table 3 and in the foregoing co-owned PCT applications. The incorporation of the biodegradable group(s) into the lipid increases metabolism post-administration and improves clearance of the lipid from the body following delivery of the active agent to a target area. Generally, such lipids have decreased toxicity when compared to similar lipids without biodegradable groups.

[0146] Sulfur-containing ionizable lipids and / or ionizable lipids with one or more biodegradable groups (e.g., esters, groups comprising an ester or a disulfide) in one or more of their lipophilic chains have been shown to be particularly efficacious when formulated in LNPs.

[0147] Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) in the lipophilic chains of the ionizable cationic lipid include cycloalkyl groups (mono or polycyclic); — O— ; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O)— ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ; and — S(O)XNR' — , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0148] By the term “biodegradable group”, with reference to a group in a lipophilic chain of an ionizable lipid, includes a functional group with one or more electronegative atoms (e.g., O, N, S or P) that is metabolized in vivo by an enzyme, thereby increasing its metabolism relative to an otherwise identical ionizable lipid that does not contain such group. Non-limiting examples include — O— ; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ; and — S(O)XNR'— , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0149] Non-limiting examples of atoms or substituents that may replace a hydrogen atom in the ionizable cationic lipid include halogen; deuterium, an alkyl group; a cycloalkyl group (mono or polycyclic); an oxo group (=0); a hydroxyl group (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; O(C=O)OR'-; —OR'; — S(O)XR'; —SR', — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=O)R'; — C(=O)NR'R'; — NR'C(=O)NR'R'; — OC(=O)NR'R'; — NR'C(=O)OR'; — NR'S(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0150] In certain embodiments, the ionizable cationic lipid content is from 15 mol%to 40 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 lipids present in the lipid nanoparticle.

[0151] In certain embodiments, the ionizable amino cationic lipid content is from 15 mol%to 40 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 lipids present in the lipid nanoparticle.

[0152] In some embodiments, the ionizable cationic lipid is not a lipidoid structure, including but not limited to C 12-200 (see Khare et al., 2022, AAPS Journal, 24:8, incorporated by reference) and related structures known to those of skill in the art. In some embodiments, the ionizable cationic lipid is not a dendrimer (termed herein “non -dendrimer”).Hydrophilic polymer-lipid conjugate

[0153] 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.

[0154] 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.

[0155] 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.

[0156] In one embodiment, the lipid nanoparticle has “substantially no hydrophilic polymer-lipid conjugate” or is “non-sterically stabilized” or “uncoated”, meaning the lipid nanoparticle has less than 0.8 mol% total hydrophilic-polymer lipid conjugate content as measured based on the total lipid content of the nanoparticle. In some embodiments, the 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, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments,the 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% or 0 and 0.10 mol%.

[0157] In another embodiment, the lipid nanoparticle is “PEG-less”, meaning that the lipid nanoparticle has no detectable amounts of polyethylene-glycol lipid conjugate.

[0158] Examples of lipid nanoparticles with low levels or no hydrophilic polymer lipid conjugate that can be used in the practice of the disclosure are described in co-owned and co-pending U.S. patent No. 12,343,429, which is incorporated herein by reference.Additional components

[0159] The LNP may comprise additional lipid components or modifications thereof. For example, the surface of the LNP may be grafted to comprise a targeting ligand. The targeting ligand may be conjugated to cholesterol. The targeting ligand may be conjugated to the distal end of a hydrophilic polymer-lipid conjugate when present (typically at less than 0.5 mol%). In some embodiments, the targeting ligand may be used to target receptors on cells in vivo. In some embodiments, the targeting ligand may be conjugated to a phospholipid.

[0160] The ligand includes peptides, polypeptides or proteins and includes antibodies or fragments thereof. In one embodiment, the ligand may be a single -chain antibody fragment. Examples of ligands are described in WO 2024 / 119279, which is incorporated herein by reference.Nanoparticle preparation and morphology

[0161] Lipid nanoparticles can be prepared using any of a variety of suitable methods, such as a rapid mixing / ethanol dilution process. Examples of preparation methods are described in Jeffs, L.B., et al., Pharm Res, 2005, 22(3):362-72; and Leung, A.K., et al., The Journal of Physical Chemistry. C, Nanomaterials and Interfaces, 2012, 116(34): 18440-18450, each of which is incorporated herein by reference in its entirety.

[0162] For example, the method of preparing the lipid nanoparticles may comprise dissolving lipid components (e.g., ionizable lipid, phosphatidylcholine and a sterol or derivative thereof) atappropriate ratios in an organic solvent (e.g., ethanol). An aqueous buffer comprising nucleic acid is prepared separately at a suitable pH to ensure that the head group (e.g., amino group) of an ionizable lipid is protonated to facilitate electrostatic interaction with the negatively charged cargo, and the positively charged ionizable lipid. Such charge interaction improves nucleic acid encapsulation.

[0163] In some embodiments, the aqueous phase comprising the nucleic acid is subsequently combined with the organic solvent-lipid mixture comprising the lipids. Combining of the aqueous phase and the organic-solvent-lipid mixture may be carried out in a mixing device (e.g., in-line mixer), such as a T-junction mixer with pumps (e.g., a T-tube mixer), a herringbone micromixer, a toroidal mixer, a multi -inlet vortex mixer or other suitable mixing devices known to those of skill in the art. In some embodiments, the mixing device refers to a device comprising two or more inlets meeting in a central mixing region and an outlet through which the mixture exits the device. The LNP formation may occur upon mixture of the aqueous phase and organic solvent-lipid mixture and / or subsequent to such mixing. (Kulkami et al., 2019, Nanoscale, 11 ( 18):9023-9031, which is incorporated herein by reference).

[0164] The aqueous phase typically comprises a buffer. Non-limiting examples of suitable buffers include one or more of sodium acetate, phosphate buffered saline (PBS), sodium formate and sodium succinate. Examples of suitable solvents to prepare the organic solvent-lipid mixture are organic solvents including ethanol, isopropanol, methanol and acetone. In one embodiment, the organic solvent-lipid mixture comprises ethanol.

[0165] The aqueous phase and organic-solvent lipid mixture may be introduced to the mixer as two separate respective streams via pumps. The volumetric flow rate of each stream may be the same or different and the respective flow rates of each stream may be adjusted to achieve optimal mixing and / or LNP formation.

[0166] The lipid nanoparticles may have an average size of between 40 and 200 nm or between 40 and 170 nm or between 45 and 130 nm or any range therebetween. In another embodiment, the lipid nanoparticle has a PDI of less than 0.30, or less than 0.20 or less than 0.15 or less than 0.12 or less than 0.10.

[0167] The nitrogen-to-phosphate ratio of the lipid nanoparticle may be between 1 and 12. In another embodiment, the nitrogen-to-phosphate ratio of the lipid nanoparticle may be between 1 and 9.

[0168] The LNP generally comprises a “core” region, which may be characterized as being electron dense as visualized by cryo-TEM microscopy or having an electron dense region and an aqueous region within the core. The morphology is assessed visually by cryo-TEM microscopy as set forth in the Materials and Methods herein. Figure 18 shows particles that display the latter morphology.

[0169] Without being limiting, the electron dense region within the core may be partially surrounded by an aqueous portion within the enclosed space, entirely surrounded or enveloped by an aqueous portion within the core or may have a solid core without an aqueous portion as observed by cryo-TEM. The core may comprise nucleic acid and ionizable lipid. In one embodiment, the phosphatidylcholine and sterol or derivative thereof is present primarily in an outer lipid layer and the ionizable cationic lipid and nucleic acid cargo is present in the core of the LNP.

[0170] In one embodiment, at least one about fifth of the core (trapped volume) contains the aqueous portion or compartment, and in which the electron dense region within the core is partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In another embodiment, at least one about quarter of the core contains the aqueous portion or compartment, and in which the electron dense core is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In a further embodiment, at least one about one third of the core contains the aqueous portion or compartment, and in which the electron dense region is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In another embodiment, at least one about one half of the core contains the aqueous portion or compartment, and in which the electron dense core is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM.

[0171] In another embodiment, the electron dense region of the LNP surprisingly appears to be completely surrounded by the aqueous portion of the core as visualized by cryo-TEM microscopy. This morphology is observed in a single plane and a portion of the electron dense region asobserved is contiguous with the lipid layer (e.g., bilayer) but cannot be seen since this portion is not within the plane that can be visualized.

[0172] In some embodiments, the LNP comprises at least a bilayer surrounding the core. In some embodiments, the LNP comprises a continuous or discontinuous bilayer surrounding an aqueous region of the core. In some embodiments, the LNP comprises a bilayer surrounding at least the aqueous region of the core. In some examples, the electron dense region is surrounded by a monolayer and the aqueous region is surrounded by a bilayer as visualized by cryo-TEM.

[0173] In one embodiment, the electron dense region is generally spherical in shape. In another embodiment, the electron dense region is hydrophobic.

[0174] In some embodiments, the density of the electron dense region has an intensity as measured by imaging software that is at least 10%, at least 20%, at least 30%, at least 40% greater or at least 50% greater than that of the aqueous region. The density of the electron dense region relative to the aqueous region of the core is assessed by analysis of the cryo-TEM image of the LNP using Image J™ software available at https: / / imagej.net / ij / (incorporated herein by reference), which is a Java-based image processing program developed by the National Institute of Health to analyze images. The cryo-TEM image is first focused and subsequently defocused to enhance the contrast for ease of visualization. The level of defocusing can be determined by a person of ordinary skill in the art. A suitable amount of defocus is 0.5-2 pm. After the desired level of contrast is achieved, a line is drawn through the LNP particle image bisecting the aqueous region and electron dense region. The image intensity of the line is measured along its length using Image J™ software available at https: / / imagej.net / ij / , which is a Java-based image processing program developed by the National Institute of Health to analyze images. The image intensity of two equal areas is measured, one of which is measured at the aqueous region and the other at the electron dense region. After the image intensities are quantified, the increase in intensity of the electron dense region relative to the aqueous portion is determined and expressed as a percentage.

[0175] In some embodiments, the electron dense region in the core has an image intensity that is similar to that of the periphery of the particle, which in this case is a lipid layer (bilayer or monolayer). For example, in some embodiments, the lipid layer image intensity as measured byImageJ™ does not deviate by more than 20% or 10% relative to that of the electron dense region within the core. The relative intensities are measured in equal areas as described earlier and expressed as a percentage.

[0176] In some embodiments, the LNP is not a lipoplex. Lipoplexes are prepared by mixing preformed cationic liposomes with nucleic acid in an aqueous solution and may exhibit undesirable properties such as localization of the cargo on the particle surface. Lipoplexes lack the abovedescribed core of the LNP particle. Further, LNPs have a defined size, shape and morphology whereas lipoplexes lack such defined physical characteristics. (See Kubota et al., 2017, Int. J. Nanomedicine, 12:5121-5133 and Kulkami et al., 2018, Nucleic Acid Therapeutics, 28(3): 146- 157, which are each incorporated herein by reference).

[0177] Further, it should be understood that the morphology observed results when particles are prepared with the molar ratios of lipids as described and using T-junction mixing or similar methods. This morphology is not observed with standard methods used to prepare liposomes, such as thin layer evaporation and extrusion.

[0178] As used herein, the term “encapsulation,” with reference to incorporating the nucleic acid cargo within an LNP refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle.Clinical and non-clinical uses of the unshielded LNP hereinEditing of genetic material

[0179] The LNP may comprise a nucleic acid that encodes for a protein or peptide that forms part of a “genetic editor” or “editor”, which includes without limitation products or compositions that edit genetic material, e.g., nucleic acid is inserted, deleted, modified (e.g., epigenetic editing) or replaced in the genetic material of an organism at a site-specific location.

[0180] The editor may be used for ex vivo or in vivo genetic modification of a hepatic cell and includes post-translational modifications.

[0181] The genetic editor includes, without limitation, Cas-based (e.g., CRISPR or non- CRISPR), transcription activator-like effector nuclease (TALEN), megaTALs, zinc fingernuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editors, base editors, epigenetic, transposase, meganuclease, ARCUS gene editing systems or any variant or combination thereof. These editors are exemplary, however, and the disclosure includes any product or composition that can modify genetic material (including RNA transcripts and noncoding regions) of a hepatic cell to treat, prevent or ameliorate a disorder or disease. Without limitation, the editor may include those that are designed by a process referred to as Directed Nuclease Editor (DNE), which is known to those of skill in the art.

[0182] Cas-based editors comprise CRISPR and non-CRISPRgene editing systems. In addition, the editor includes those that cut DNA as well as epigenetic editing systems that modify nucleic acid markers, as discussed below.

[0183] The CRISPR nucleic acid editor most advantageously comprises nucleic acid (e.g., 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 nuclease and guide RNA form a complex referred to as a ribonucleoprotein (RNP). In some embodiments, the nuclease is a Class II CRISPR enzyme, which is further subdivided into Types II, V, and VI. According to one embodiment, the mRNA encodes a Cas protein that is part of a Type II CRISPR / Cas system, such as a Cas9 protein or a Cpfl protein.

[0184] In another embodiment, the mRNA encodes a Cas protein that is part of a Type V CRISPR / Cas system, such as Cas 12a. In another embodiment, the mRNA encodes a Cas protein that is a Cas 13a, which is an RNA endonuclease and cleaves single-stranded RNA.

[0185] 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.

[0186] 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. ThecrRNA 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.

[0187] The guide RNA of the RNP can target any sequence of interest through the targeting sequence of a 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.

[0188] The length of the targeting sequence may depend on the RNP system and components used. For example, different Cas proteins from different bacterial species have various optimal targeting sequence lengths. Thus, the targeting sequences are: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides in length can be included. In some embodiments, the targeting sequence can comprise a length of 18 to 24 nucleotides. In some embodiments, the targeting sequence can comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence can comprise a length of 20 nucleotides.

[0189] In some embodiments, the nucleic acid editor 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 l, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.

[0190] As noted, non-CRISPR, Cas-based nucleic acid editors are encompassed by embodiments of the disclosure as well. A Cas-based editor may include a Cas enzyme fused to deaminase (Luo et al., 2020, Microbial Cell Factories, 19(93), incorporated herein by reference). An example is a cytosine base editor, or an adenine base editor produced by fusing endonuclease Cas to cytosine deaminase pmCDAl or heterodimer adenine deaminase TadA-TadA. A further non-limitingexample is Cas fused to reverse transcriptase (Mohr et al., 2018, Mol Cell., 72(4):700-714, incorporated herein by reference).

[0191] Fanzor is a eukaryotic RNA-guided endonuclease that could function as a nucleic acid editor. (See Saito et al., 2023, Nature 620:660-668, which is incorporated herein by reference). In some embodiments, Fanzor proteins use RNA as a guide to target DNA precisely and can be modified to edit a hepatic cell using the LNPs described herein. In some examples, the compact Fanzor systems may have the ability to facilitate more improved delivery than CRISPR-Cas systems.

[0192] In those embodiments in which the editor is a TALEN, the LNP comprises a nucleic acid encoding a peptide having a Transcription Activator-Like (TAL) effector DNA binding domain, a fragment or a variant thereof. In an embodiment, the editor comprises a nucleic acid encoding a peptide having nuclease activity, e.g., endonuclease activity. In an embodiment, the peptide having nuclease activity is atype-II restriction 1-like endonuclease, e.g., a Fokl endonuclease.

[0193] In those embodiments in which the nucleic acid editor is a zinc finger nuclease (ZFN), the nucleic acid may encode a peptide having: a Zinc finger DNA binding domain, a fragment or a variant thereof; and / or nuclease activity, e.g., endonuclease activity. In an embodiment, the Zn finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8 or more Zinc fingers. In an embodiment, the peptide having nuclease activity is a type-II restriction 1-like endonuclease, e.g., a Fokl endonuclease.

[0194] Adenosine Deaminase Acting on RNA (ADAR) is another editor encompassed by embodiments of the disclosure that may be used for post-transcriptional modification of RNA. Examples include AD ARI and ADAR2. AD ARI may catalyze post-transcriptional deamination of C6 of adenosines in dsRNA, converting them to inosines (see Song et al., 2022, PMC, 13(1): el665, incorporated herein by reference).

[0195] Meganucleases are enzymes in the endonuclease family that may induce homologous recombination, generate mutations, and alter reading frames. The meganuclease includes homing endonucleases that are intron or intein endonucleases. In one embodiment, the meganuclease is from the LAGLID ADG family, a GIY -YIG endonuclease, an HNH endonuclease, a His-Cys boxendonuclease or a PD-(D / E) XK endonuclease. Meganucleases may be combined with components of other editors. In one embodiment, a DNA binding domain from a transcription activator-like (TAL) effector is combined with a meganuclease to produce a “megaTAL”. In another embodiment, a meganuclease may be fused to a DNA end-processing enzyme to promote an error-prone non-homologous end joining.

[0196] ARCUS nuclease is an editor based on I-Crel, which is a kind of homing endonuclease that evolved in the algae Chlamydomonas reinhardtii . In some embodiments, the nuclease can deactivate itself after gene editing, thereby reducing off-targeting. ARCUS nucleases in some embodiments can generate a unique cleavage site that is a four-base-pair, 3’ overhang and may be able to carry out gene insertion, gene excision, gene repair or a combination thereof.

[0197] Epigenetic editing is also encompassed by examples of the disclosure. Such editing of genetic material does not cut nucleic acid but rather alters epigenomic marks “adorning” DNA. Changing the epigenic signature of a hepatic cell can serve to modify an epigenetic signature of the cell and change its transcriptional profile. In some embodiments, the epigenetic editing system may target and edit one or more methylation sites of a nucleic acid sequence. In some embodiments, genome homing proteins with engineered or naturally occurring nuclease functions for gene editing, can be mutated and adapted to function as only delivery systems. In one embodiment, an epigenetic modifying enzyme or domain can be fused to the homing protein, and local epigenetic modifications can be altered upon protein recruitment. A targeting protein that recognizes DNA sequences may be linked to an effector protein that alters epigenomic marks, such as methylation. Examples of targeting proteins include Transcription Activator-Like Effector (TALE), zinc finger proteins, and Cas systems, including but not limited to CRISPR-Cas. Nonlimiting examples of effector proteins include TET1, which induces demethylation of cytosine at CpG sites; LSD1, which induces demethylation of H3K4mel / 2, which also causes an indirect effect of deacetylation on H3K27; and CIB1 / CRY2, which is a cryptochrome / blue light activated complex allowing chromatin to be modified upon illumination.

[0198] Further examples of effector proteins include DNA methyltransferase, a fragment (e.g., a biologically active fragment) or variant thereof (e.g., DNMT1, DNMT2 DNMT3A, DNMT3B, DNMT3L, or CpG methyltransferase (M. Sssl)); or a poly comb repressive complex or acomponent thereof, e.g., PRC1 or PRC2, or PR-DUB, or a fragment (e.g., biologically active fragment) or a variant thereof.

[0199] In an embodiment, the epigenetic editor comprises a molecule that modifies chromatin architecture and / or modifies a histone. In an embodiment, the epigenetic modulator is a molecule that modifies chromatin architecture, e.g., a SWI / SNF remodeling complex or a component thereof. In an embodiment, the epigenetic modulator is a molecule that modifies a histone, e.g., methylates and / or acetylates a histone, e.g., a histone modifying enzyme or a fragment (e.g., biologically active fragment) or a variant thereof, e.g., HMT, HDM, HAT, or HD AC.Pharmaceutical formulations

[0200] The lipid nanoparticles described herein resulted in high ratios of bone marrow / liver expression. In some embodiments, the bone marrow-to-liver ratio is between 1.2 and 30, between 1.5 and 10 or between 5 and 20. The bone marrow-to-liver ratios are measured at 24 hours as set forth in Example 3 herein.

[0201] In some embodiments, the lipid nanoparticle comprising nucleic acid cargo is part of a pharmaceutical composition. The treatment may provide a prophylactic (preventative), ameliorative or a therapeutic benefit to treat any undesirable condition, such as a disease condition or disorder. In other embodiments, the unshielded lipid nanoparticle encapsulates a diagnostic agent. The pharmaceutical composition will be administered at any suitable dosage.

[0202] 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.

[0203] 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.

[0204] Non-limiting examples of other diseases, disorders or conditions that may be treated by the nucleic acid-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.

[0205] The LNP may be part of a vaccine pharmaceutical formulation.

[0206] 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.

[0207] 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 another embodiment, the pharmaceutical compositions are administered intranasally, intravitreally, subretinally, intrathecally or via other local routes.

[0208] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.

[0209] The compositions described herein may be administered to a patient. The term "patient” used herein includes a human or a non-human subject.

[0210] The examples below are intended to illustrate the preparation of specific lipid nanoparticle preparations and properties thereof but are in no way intended to limit the scope of the invention.

[0211] The article “a” or “an” as used herein is meant to include both singular and plural, unless otherwise indicated.ExamplesMaterials and MethodsLNP preparation

[0212] The LNPs were prepared by dissolving mRNA in 25 mM sodium acetate, pH 4.0 (or other buffer as indicated), while the lipid components at the mole % specified were dissolved in absolute ethanol. The lipids in ethanol and the luciferase mRNA in buffer were combined in a 1:3 volume by volume ratio using a t-junction with dual-syringe pumps. 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 at least -100 volumes of 1 x phosphate buffered saline, pH 7.4 using Spectro / Por dialysis membranes (molecular weight cut-off 12,000-14,000 Da). The LNPs were concentrated as required with an Amicon Ultra™ 100,000 MWCO (molecular weight cut-off), regenerated cellulose concentrator to achieve a final concentration of 0.1 mg / mL of mRNA.Tissue homogenate assay

[0213] The LNPs at a luciferase mRNA of 0.1 mg / mL were injected intravenously (i.v.) in mice at 1 mg / kg. Organs were harvested at 24 hours after the LNP injections.

[0214] Tissues were removed from the mice and placed in 2 mL tubes and snap frozen in liquid nitrogen. The tissues were subsequently stored at -80°C. 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 for three rounds. The homogenized samples were spun down for 10 minutes at 12,000 rpm at 4°C 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.Example 1: Modified mRNA with 3’ terminal shoe and complementary shoelace sequences prevent degradation of mRNA by RNase R in vitro

[0215] This example investigates the degradation of modified mRNA encoding for firefly luciferase (FLuc) comprising a 3 ’ terminal shoe sequence hybridized to various complementary shoelace sequences. The modified FLuc mRNA was capped with CleanCap AG 3 ’OMe ™ and has a poly(A) tail that is located 5’ to the 3’ terminal shoe sequence. The FLuc-Shoe mRNA was prepared by in-vitro transcription using a plasmid DNA template linearized using a restriction enzyme which cut the DNA post the poly(A) tail and shoe sequence.

[0216] The modified mRNA with the 3’ terminal shoe was subsequently hybridized with a complementary strand of a RNA, DNA, or PNA shoelace sequence.

[0217] The hybridization of the FLuc mRNA shoe sequences to the shoelace sequences was carried out by heating the mRNA: shoelace mix (in a 1: 10 molar ratio in water) at 65 °C for 2 minutes, followed by cooling at room temperature. The excess unhybridized shoelace was removed by purification of the mixture using NEB Monarch™ RNA purification columns, and the hybridized mRNA-shoelace was eluted at a concentration of 1 mg / mL.

[0218] The 3’ terminal mRNA shoe sequence (SEQ ID No: 1) and hybridized shoelace sequences are set forth in Figure 3A and Table 1 below.Table 1: Sequences of 3’ terminal shoe and complementary shoelace sequences

[0219] After hybridization, the mRNA sequences were incubated with an RNase R enzyme mixture for 30 min at 37°C at the concentrations indicated in Table 2 below.Table 2: RNase R reaction composition

[0220] The samples were loaded and run on a denaturing RNA gel (2% formaldehyde, 2% agarose with SYBRGold™). The results in Figure 4 show a distinct band for the modified FLuc- Shoe mRNA (with the 3’ terminal shoe sequence, SEQ ID No. 1, 3’ to the poly(A) tail) control lacking the shoelace (lane 1 , from left to right) and without RNase R treatment, while the same mRNA sample, treated with RNase R, was degraded (as seen in lane 2). The same sample, FLuc- Shoe mRNA, treated with the reaction mix shown in Table 2 but lacking RNase R resulted in a band having the same molecular weight as the untreated control sample (lane 3). Lanes 1 and 3 were used as controls for comparison. The FLuc-Shoe mRNA and the complementary RNA shoelace sequence (SEQ ID No. 3) in the presence of RNase R appeared as a distinct band and similar results were observed with the DNA shoelace sequence (SEQ ID No. 2), indicating a degree of protection from degradation by RNase R. The band corresponding to the mRNA FLuc-Shoe and complementary PNA shoelace (SEQ ID 4, lane 6) after treatment with RNase R was of similar intensity as the control bands, indicating that the PNA sequence also conferred protection to RNase R degradation.Example 2: A variety of shoelaces confer protection to 3’ end RNase R degradation

[0221] Given the positive results with PNA, DNA, and RNA shoe / shoelace duplexes, the ability of other complementary shoelaces with modified backbones to protect the mRNA from degradation was examined. The modified FLuc mRNA with an RNA shoe was hybridized with a complementary RNA shoelace, a DNA shoelace, a PNA shoelace, a locked 2’, 4 ’-constrained 2’- O-ethyl (cET) RNA shoelace, a morpholino (PMO) shoelace, a 2’-O-methoxyethyl-RNA (2’- MOE) shoelace, and a phosphorothioate (P=S) RNA shoelace. The structures of monomers comprising the backbone of the shoelaces are depicted in Figure 5. The reaction conditions are disclosed in Table 2.

[0222] The sequence details of each complementary shoelace are provided in Table 3 below.Table 3: 3’ terminal Shoe and complementary shoelace sequences investigated for protection against RNase.

[0223] An enzyme mixture of RNase R was prepared as described in Example 1. After incubation with the RNase R, each sample was loaded onto a denaturing RNA gel (2% formaldehyde, 2% agarose with SYBRGold™), and the gel was run for a sufficient time to provide for resolution of the bands. The results in Figure 6 confirm that all shoelaces confer protection from 3’ end degradation by RNase R.Example 3: mRNA modified with 3’ terminal shoe and complementary shoelace sequences provide tissue-specific expression

[0224] The following samples were prepared as described above in Examples 1 and 2: (i) modified FLuc mRNA with a 3’ terminal shoe sequence (FLuc-Shoe), (ii) modified FLuc-Shoe mRNA with a complementary RNA shoelace sequence, (iii) modified FLuc-Shoe mRNA with a complementary PNA shoelace sequence. The modified FLuc mRNAs as well as a control using wildtype FLuc (FLuc WT) mRNA, without a shoe, were formulated in lipid nanoparticles as described in the Materials and Methods section. The LNP compositions were as follows: ionizable lipid / DSPC / cholesterol / PEG2000-DMG at 27.4:50:21. 1: 1.5 (mol / mol). The ionizable lipid used in the LNP composition for subsequent studies is disclosed in Table 4 below. Ionizable lipid 18 was used in all lipid nanoparticle compositions for this study.Table 4: Ionizable Cationic Lipid used in the lipid nanoparticle

[0225] The LNPs were intravenously injected into mice as per the Materials and Methods and luminescence was measured in the harvested liver, spleen, bone marrow, and abdominal skin tissue at 24 hours after injection. As can be seen in Figures 7A-D, the protein expression of (i) the modified FLuc-Shoe mRNA without any hybridized shoelace and (ii) modified FLuc mRNA with a complementary RNA shoelace sequence were consistently low across all tissues. However, the protein expression of the modified FLuc-Shoe mRNA with a complementary PNA shoelace was low in liver and spleen but was elevated in bone marrow and abdominal skin tissues. The selectivity of expression of the modified mRNAs containing shoe-shoelace duplexes towards organs (e.g., bone marrow, BM) other than the liver can be quantified by the ratio of the average luminescence value for the shoe with shoelace mRNA (shoe-shoelace mRNA) normalized by the average luminescence of the wildtype mRNA without the shoe (no shoe mRNA) i.e., selectivity (BM relative to liver) using the following formula:where lumAVGdenotes the average luminescence values over all mice in the group. In the example above, the selectivity is greater, equal to, or less than one, for a shoe-shoelace mRNA which is more, equally as, or less selective, respectively, for expression in the bone marrow (BM) relative to the liver. As can be seen in Figures 7E-G, modified FLuc-Shoe mRNA with complementary RNA shoelace remains comparable to the wildtype FLuc (FLuc WT) mRNA, whereas, surprisingly, the FLuc-Shoe mRNA with complementary PNA shoelace shows at least 1.5-3 times more selective protein expression to tissues other than the liver in comparison to the FLuc WT mRNA. These results are unexpected in that the selectivity of mRNA expression may be increased towards organs other than the liver when using the 3 ’ terminal shoe with complementray PNA shoelace sequences.

[0226] This example supports a trend showing that a modified FLuc mRNA stabilized with a complementary peptide nucleic acid shoelace and formulated in a lipid nanoparticle having high neutral lipid (e.g., DSPC) composition exhibits tissue-specific expression.Example 4: Modified FLuc mRNA with a scrambled shoe sequence and complementary scrambled shoelace sequence retains tissue selective protein expression

[0227] Given the positive findings from Example 3 above, the inventors further evaluated the protein expression using a modified FLuc mRNA bearing a scrambled 3’ terminal shoe and a complementary scrambled shoelace sequence. The modified FLuc mRNA samples were prepared as described in Examples 1 and 2. The scrambled shoe sequence (SEQ ID No: 9) and a complementary scrambled PNA shoelace sequence (SEQ ID No: 10) are disclosed in Figure 8 and Table 5 below.Table 5: Scrambled shoe and complementary scrambled shoelace sequences investigated

[0228] The samples were loaded and run on a denaturing RNA gel (2% formaldehyde, 2% agarose with SYBRGold ™). The results in Figure 9 show, from left to right, a degraded band for the FLuc-Scrambled Shoe mRNA control sample lacking the complementary shoelace upon RNase R treatment (lane 1), which demonstrates the lack of protection against RNase. However, a distinct band is seen for the modified FLuc-Scrambled Shoe mRNA with the hybridized complementary scrambled PNA shoelace sequence upon treatment with RNase R (lane 2), indicating that the scrambled PNA sequence confers protection against RNase R degradation.

[0229] The above-mentioned mRNAs as well as control FLuc mRNA (without shoe) were formulated in LNPs with high neutral lipid content (e.g., DSPC) as per the Materials and Methods. The LNP composition used for this study was as follows: ionizable lipid 19 / DSPC / cholesterol / PEG2000-DMG at 27.4:50:21.1: 1.5 (mol / mol). The details of the ionizable lipid used are provided in Table 4 under Example 3.

[0230] LNPs were intravenously injected into mice as per the Materials and Methods and luminescence was measured in the liver, spleen, bone marrow, and abdominal skin at 24 hours after injection. As can be seen in Figures 10A-D, the protein expression of FLuc-Scrambled Shoe mRNA is low across all tissues. The expression of modified FLuc-Scrambled Shoe mRNA with complementary scrambled PNA shoelace was low in the liver, but was either restored or elevated in the spleen, bone marrow, and abdominal skin. The selectivity of expression for the mRNAs containing shoe-shoelace duplexes towards organs (e.g., bone marrow, BM) was quantified as described above in Example 3.

[0231] As can be seen in Figures 10E-G, FLuc-Scrambled Shoe mRNA with its complementary scrambled PNA shoelace shows at least 3-7 times more selectivity for tissues other than the liver when normalized to wildtype FLuc (FLuc WT) mRNA. These results further confirm that tissue selectivity of mRNA expression may be increased towards organs other than the liver using varied shoe sequence and complementary shoelace sequences as demonstrated with scrambled shoe and shoelace sequences described herein.

[0232] This example further demonstrates that modified FLuc mRNA stabilized with a 3’ terminal scrambled shoe sequence and complementary scrambled PNA shoelace when formulated with a lipid nanoparticle having high neutral lipid (e.g., DSPC) content exhibits tissue-specific expression.

[0233] 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.

[0234] The article "a" or "an" as used herein is meant to include both singular and plural, unless otherwise indicated.

Claims

CLAIMS:

1. A modified RNA comprising a terminal nucleic acid shoe sequence, which terminal nucleic acid shoe sequence is hybridized to a complementary nucleic acid shoelace sequence, the nucleic acid shoe sequence and complementary nucleic acid shoelace sequence forming a stabilizing shoe-shoelace heteroplex; and wherein the complementary nucleic acid shoelace sequence is a nucleic acid analogue with a modified backbone.

2. The modified RNA of claim 1, wherein the terminal nucleic acid shoe sequence and the complementary nucleic acid shoelace sequence each, independently, is 10 to 50 nucleotides in length.

3. The modified RNA of claim 1 or 2, wherein the terminal nucleic acid shoe sequence and the complementary nucleic acid shoelace sequence each, independently, is 20 to 30 nucleotides in length.

4. The modified RNA of any one of claims 1 to 3, wherein the RNA is an mRNA, the mRNA comprising:(i) a 5 ’ untranslated region;(ii) a coding region encoding a protein or polypeptide;(iii) a 3 ’ untranslated region; and(iv) a 3 ’ tailing region downstream of the 3 ’ untranslated region, wherein the stabilizing shoe-shoelace is 3’ of the tailing region.

5. The modified RNA of any one of claims 1 to 4, wherein the complementary nucleic acid shoelace is selected from a peptide nucleic acid (PNA), a locked nucleic acid and a glycol nucleic acid (GNA).

6. The modified RNA of claim 5, wherein the nucleic acid shoelace is the PNA.

7. The modified RNA of any one of claims 1 to 6, wherein the terminal nucleic acid shoe sequence and the complementary nucleic acid shoelace form a duplex, triplex or quadruplex.

8. The modified RNA of claim 7, wherein the terminal nucleic acid shoe sequence forms a duplex with the complementary nucleic acid shoelace.

9. The modified RNA of claim 8, wherein the duplex includes a terminal stem loop structure.

10. The modified RNA of any one of claims 1 to 9, wherein at least 50% of the stabilizing shoe-shoelace is in a double-stranded form at 55°C.

11. The modified mRNA of claim 4, wherein the 3 ’ tailing region is a poly(A) tail that is 3 ’ to the 3 ’ untranslated region and 5’ to the terminal nucleic acid shoe sequence.

12. The modified RNA of any one of claims I to 11, further comprising a microRNA target site.

13. The modified RNA of any one of claims 1 to 12 further comprising a 5’ terminal cap.

14. The modified RNA of any one of claims 1 to 13, wherein the nucleic acid shoelace sequence is uncharged or wherein at least 60%, 70%, 80%, 90% or 95% of said sequence lacks a charge.

15. The modified RNA of any one of claims 1 to 14, wherein a backbone of the shoelace sequence is acyclic.

16. A lipid nanoparticle for extrahepatic nucleic acid delivery, the lipid nanoparticle comprising: a neutral lipid having at least two tails and a head group, the neutral lipid being present at a content of at least 20 mol%; and an ionizable lipid content of from 15 mol% to 45 mol%, wherein the nucleic acid is a modified RNA comprising a stabilizing sequence comprising at least two strands, a first of said strands being a terminal nucleic acid shoe sequence, and a second of said strands being a complementary nucleic acid shoelace sequence hybridized to the nucleic acid shoe sequence.

17. The lipid nanoparticle of claim 16, wherein the nucleic acid shoe and complementary shoelace sequences each, independently, is 10 to 50 nucleotides in length.

18. The lipid nanoparticle of claim 16 or 17, wherein the nucleic acid shoe and complementary shoelace sequences each, independently, is 20 to 30 nucleotides in length.

19. The lipid nanoparticle of any one of claims 16 to 18, wherein the modified mRNA further comprises:(i) a 5 ’ untranslated region;(ii) a coding region encoding a protein or polypeptide;(iii) a 3 ’ untranslated region; and(iv) a 3 ’ tailing region downstream of the 3 ’ untranslated region, wherein the stabilizing shoe-shoelace is 3’ of the tailing region.

20. The lipid nanoparticle of any one of claims 16 to 19, wherein the complementary nucleic acid shoelace sequence is neutral or wherein at least 60%, 70%, 80%, 90% or 95% of said sequence lacks a charge.

21. The lipid nanoparticle of claim 20, wherein the nucleic acid shoelace is a peptide nucleic acid (PNA).

22. The lipid nanoparticle of any one of claims 16 to 21, wherein the terminal nucleic acid shoe sequence and the complementary nucleic acid shoelace form a duplex, triplex or quadruplex.

23. The lipid nanoparticle of claim 22, wherein the terminal nucleic acid shoe sequence forms a duplex with the complementary nucleic acid shoelace.

24. The lipid nanoparticle of claim 23, wherein the duplex includes a terminal stem loop structure.

25. The lipid nanoparticle of any one of claims 16 to 24, wherein at least 50% of the stabilizing shoe-shoelace is in double-stranded form at 55°C.

26. The lipid nanoparticle of claim 19, wherein the 3’ tailing region is a poly(A) tail that is 3’ to the 3 ’ untranslated region and 5’ to the terminal nucleic acid sequence.

27. The lipid nanoparticle of any one of claims 16 to 26, wherein the modified mRNA further comprises a microRNA target site.

28. The lipid nanoparticle of any one of claims 16 to 27, wherein the modified mRNA further comprises a 5’ terminal cap.

29. The lipid nanoparticle of any one of claims 16 to 28, wherein the shoelace sequence has a modified backbone.

30. The lipid nanoparticle of claim 29, wherein the backbone of the shoelace sequence is acyclic.

31. The lipid nanoparticle of claim 16, wherein the complementary shoelace sequence is a non-naturally occurring nucleic acid analogue with a modified backbone.

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