WO2025230906A1

WO2025230906A1 - Lipid nanoparticle (lnp) formulations

Info

Publication number: WO2025230906A1
Application number: PCT/US2025/026663
Authority: WO
Inventors: Shashank Shukla; Kevin Love; Vishwesh PATIL; Madison MCKINNEY; Shifalika TANGUTOORI
Original assignee: Saliogen Therapeutics Inc
Current assignee: Saliogen Therapeutics Inc
Priority date: 2024-04-29
Filing date: 2025-04-28
Publication date: 2025-11-06
Anticipated expiration: 2026-10-29

Abstract

The present disclosure relates to lipid nanoparticles and uses thereof for, e.g. delivery to the liver and/or treating liver-related disorders or diseases.

Description

LIPID NANOPARTICLE (LNP) FORMULATIONS

FIELD

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/639,937, filed April 29, 2024, which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The Sequence Listing for this application is labeled “SAL-045_SequenceListing”, which was created on April 28, 2025, and is 2,349 bytes in size.

BACKGROUND

Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a lipid core. These vesicles are used in, e.g., nucleic acid delivery.

Messenger RNA (mRNA) is inherently unstable and prone to degradation by nucleases and self-hydrolysis. Encapsulation of mRNA within LNPs protects the mRNA from extracellular ribonucleases and assists with intracellular mRNA delivery.

A variety of lipids have been developed for use in LNPs, yet there remains a need for specific formulations for nucleic acid delivery, especially complicated nucleic acid cargoes involving mRNA, that can effectively be used in specific tissues or organs (e.g., liver).

SUMMARY

Accordingly, the present disclosure describes, in part, various lipid nanoparticles, e.g., for delivering one or more nucleic acid-based agents to the liver.

In aspects, the present compositions provide one or more lipids in association with one or more nucleic acidbased agents. In embodiments the one or more lipids associate into an aggregate. In embodiments the one or more lipids associate into a lipid nanoparticle (LNP). In embodiments the one or more lipids, e.g., LNP, encapsulate the one or more nucleic acid-based agents. In embodiments the LNP comprising the one or more nucleic acid-based agents is suitable for delivery of the one or more nucleic acid-based agents in a subject. In embodiments the LNP comprising the one or more nucleic acid-based agents is formulated with a pharmaceutically acceptable carrier or excipient.

In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme in the form of modified mRNA (mmRNA) and a donor DNA or transgene or mobile element in the form of plasmid DNA. In embodiments, the weight to weight ratio of plasmid DNA : mmRNA is about 5 : about 1 , or about 2 : about 1, or about 1 : about 1, or about 1 : about 2, or about 1 : about 5.

In aspects, there is provided a method for introducing a nucleic acid-based agent into a cell, the method comprising contacting the cell with a composition described herein.

In aspects, there is provided a method for the in vivo delivery of a nucleic acid-based agent, the method comprising administering to a subject a composition described herein.

In aspects, there is provided a method for the delivery of a nucleic acid-based agent delivery to the liver, the method comprising administering to a subject a composition described herein

In aspects, there is provided a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition described herein.

In aspects, there is provided a method for treating a liver disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition described herein.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows encapsulation efficiencies of compositions of the disclosure with components of the same.

FIG. 2 shows formulations of MC3 and C12-200 mixed LNPs and physical properties thereof.

FIG. 3A and FIG. 3B show alteration of the amount of PEG in compositions. For each condition in FIG. 3A, the left bar refers to size and the right bar refers to polydispersity index (PDI). FIG. 4A and FIG. 4B show the effects of different PEG identities and conjugated lipid length. For each condition in FIG. 4A, the left bar refers to size and the right bar refers to PDI.

FIG. 5A and FIG. 5B show the effects of different pHs on %EE. For each condition in FIG. 5A, the left bar refers to size and the right bar refers to PDI.

FIG. 6 shows SS-OP LNPs and physical properties thereof.

FIG. 7 shows SS-OP LNPs used in vivo in mouse via intrahepatic administration.

FIG. 8A and FIG. 8B show MC3 LNPs delivering both DNA and mRNA together in vivo (intrahepatic injection).

FIG. 9 shows bioluminescence data in mouse summarizing intrahepatic administration with SS-OP formulations.

FIG. 10 also shows characteristics of compositions of the disclosure tested for use in the liver. These compositions include MC3, and SS-OP formulations.

FIG. 11 shows the effects of altering the amount of Cationic/I onizable lipid in formulations of the disclosure.

FIG. 12 shows the screening of various compositions of MC3 LNPs with a method suitable for use in certain organs or tissues (e.g., the liver). In the various compositions four components remained same, but the molar % relative to each other varied.

FIG. 13 shows the selection of beneficial formulations for mRNA delivery with a luciferase reporter.

FIG. 14 shows the relative increase in mRNA protein expression with ALC LNPs compared to MC3 LNPs. The LNPs were encapsulated with Flue mRNA and injected in to LDLR KO mice. The in-life MS imaging was done at 6h after injection and the total flue from the bioluminescence was plotted.

FIG. 15 is a histogram showing the amount of nucleic acid (NA) delivery with various MC3 and ALC LNPs 2% - 5% PEG formulations of the disclosure.

FIG. 16 is a histogram showing mRNA expression with squaramide (SQAM) LNPs.

FIG. 17 is a histogram showing DNA + mRNA expression with SQAM LNPs.

DETAILED DESCRIPTION

In aspects, the present compositions (also referred to as “formulations” herein) provide one or more lipids in association with one or more nucleic acid-based agents. In embodiments the one or more lipids associate into an aggregate. In embodiments the one or more lipids associate into a lipid nanoparticle (LNP). In embodiments, the one or more lipids, e.g., LNP, encapsulate the one or more nucleic acid-based agents. In embodiments, the composition or LNP comprising the one or more nucleic acid-based agents is suitable for delivery of the one or more nucleic acid-based agents in a subject. In embodiments, the composition or LNP comprising the one or more nucleic acid-based agents is formulated with a pharmaceutically acceptable carrier or excipient.

In embodiments, the composition or LNP comprising the one or more nucleic acid-based agents is suitable for delivery of the one or more nucleic acid-based agents to the liver of a subject

Lipids

In embodiments, the one or more lipids compact the one or more nucleic acid-based agents to be delivered.

In embodiments, the one or more lipids protect the one or more nucleic acid-based agents to be delivered from nuclease degradation.

In embodiments, the one or more lipids cause, promote or enhance receptor-mediated uptake of the one or more nucleic acid-based agents, e.g., by a cell.

In embodiments, the one or more lipids, e.g., when contacted with a cell, cause, promote or enhance association with negatively charged cellular membranes.

In embodiments, the one or more lipids, e.g., when contacted with a cell, cause, promote or enhance fusion with endosomal membranes, without wishing to be bound by theory, facilitating the release of complexes from endosomal compartments.

In embodiments, the one or more lipids, e.g., when contacted with a cell, cause, promote or enhance transport of the one or more nucleic acid-based agents from the cytoplasm to the nucleus.

In embodiments, the one or more lipids comprise cationic lipids; anionic lipids; neutral lipids; multi-valent charged lipids; and zwitterionic lipids.

In embodiments, the one or more lipids include a delivery lipid. In embodiments, the delivery lipid is or comprises a cationic lipid. In embodiments, the cationic lipid is ionizable and/or hydrolysable. In embodiments, the delivery lipid encapsulates, in whole or in part, the one or more nucleic acid-based agents. In embodiments, the delivery lipid, e.g., when contacted with a cell, promotes or increases endosomal escape. In embodiments, the delivery lipid is or comprises MC3 (DLin-MC3-DMA), C12-200 (1, T-((2-(4-(2- ((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-1- yl)ethyl)azanediyl)bis(dodecan-2-ol)), SM102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}octanoate), squaramide (3, 4-diaminocyclobut-3-ene-1, 2-dione; SQAM), SS-EC (ssPalmE-P4C2), SS-OP (ssPalmO-Phe), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2- hexyldecanoate), KC2 (DLin-KC2-DMA), DOTAP (Dioleoyl-3-trimethylammonium propane), cKK-E12 (3,6- bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2, 5-dione), ATX-001 (di((Z)-non-2-en-1-yl) 8,8'-((2-((2- (dimethylamino)ethyl)thio)acetyl)azanediyl)dioctanoate), ATX-002 (di((Z)-non-2-en-1-yl) 8,8'-((((2- (dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate), ATX-012, ATX-61, ATX-0081 , ATX-0095, Lipid 5 (8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1 -octylnonyl ester), and/or ALC-0315 (2- hexyl-decanoic acid, 1 ,1 '-[[(4-hydroxybutyl)imino]di-6,1 -hexanediyl] ester, [(4- Hydroxybutyl)azanediyl]di(hexane-6,1 -diyl) bis(2-hexyldecanoate)). Various ATX lipids that are in embodiments delivery lipids of the disclosure are described in WO2018222890 and WO2015074085, each of which is hearby incorporate by reference in its entirety. In embodiments, the delivery lipid is or comprises DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200.

In embodiments, the one or more lipids include one type of delivery lipid. In embodiments, the one or more lipids include more than one type of delivery lipid. In embodiments, the one or more lipids include two types of delivery lipid. In embodiments, the one or more lipids include both MC3 and C12-200.

In embodiments, the one or more lipids include cholesterol. In embodiments, the cholesterol provides increased or enhanced LNP stability.

In embodiments, the one or more lipids include a helper lipid. In embodiments, the helper lipid encapsulates, in whole or in part, the one or more nucleic acid-based agents. In embodiments, the helper lipid, e.g., when contacted with a cell, promotes or increases endosomal escape. In embodiments, the helper lipid is a phospholipid. In embodiments the helper lipid is a neutral lipid. In embodiments, the neutral lipid is or comprises dioleoylphosphatidylethanolamine (DOPE) and/or 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In embodiments, the helper lipid is a cationic lipid. In embodiments, the cationic lipid is or comprises N-[1 - (2 , 3-d ioleoyloxy) p ropy I]- N, N, N-trimethylammonium chloride (DOTMA), 1 ,2-bis(oleoyloxy)-3-3- (trimethylammonia) propane (DOTAP), or 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP). In embodiments, the helper lipid is or comprises DOPE, DOPC, and/or Distearoylphosphatidylcholine (DSPC). In embodiments, the helper lipid is or comprises both DOPE and DSPC. In embodiments, the helper lipid is or comprises both DOPE and DOPC. In embodiments, the helper lipid is or comprises all of DOPE, DSPC, and DOPC. In embodiments, the one or more lipids include a PEGylated lipid. In embodiments, the PEGylated lipid shields the LNP and reduces or prevents degradation of the LNP, e.g., in the bloodstream. In embodiments, the PEGylated lipid has a PEG molecule covalently attached to it, where the PEG has an average molecular weight of from about 1 kDa to about 50 kDa, or about 1 kDa to about 30 kDa, or from about 1 kDa to about 10 kDa, or about 1 kDa, or about 2 kDa, or about 3 kDa, or about 4 kDa, or about 5 kDa, or about 10 kDa. In embodiments, the PEG is a linear, a branched PEG, a star PEG, or a comb PEG. In embodiments the PEG is selected from PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, and PEG4000. In embodiments, the PEG is PEG2000. In embodiments the PEGylated lipid is or comprises 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). In embodiments, the DMG- PEG is or comprises 1 , 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-2k- DMG) and/or a PEG conjugated to a ceramide, e.g., C8 PEG 2k Ceramide, C16 PEG 2k Ceramide. In embodiments the PEGylated lipid is or comprises PEG-dimyristyloxypropyl (PEG-DMA), a PEG-distearyloxypropyl (PEG-DSA), PEG-diacylglycerol (PEG-DAG), or PEG-dialkyloxypropyl (PEG-DAA).

In embodiments, the one or more lipids do not include a PEGylated lipid. In embodiments, the one or more lipids do not include a PEGylated lipid, e.g., when being formulated for local administration.

In embodiments, the composition comprises a mixture of PEGylated lipids and free PEG chains.

Compositions/Fomiulations

In embodiments, there is provided a composition comprising:

(a) one or more nucleic acid-based agents, and

(b) one or more lipids, the one or more lipids comprising:

(i) a delivery lipid,

(ii) cholesterol,

(iii) a helper lipid, and

(iv) a PEGylated lipid.

In embodiments, the delivery lipid is selected from squaramide (SQAM), and ALC-0315.

In embodiments, the helper lipid is selected from distearoylphosphatidylcholine (DSPC), 1,2-Dioleoyl-sn- glycero-3-phosphocholine (DOPC), and dioleoylphosphatidylethanolamine (DOPE). In embodiments, the PEGylated lipid is selected from PEG-2K-DMG, C8 PEG 2K, and C16 PEG 2K. In embodiments, the one or more lipids do not comprise a PEGylated lipid.

In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acidbased agents of about 7:1, or about 8:1 , or about 9:1 , or about 10:1 , or about 11 :1 , or about 12:1, or about 13:1 , and/or a has a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 14:1 , or about 15:1, or about 16:1 , or about 17:1 , or about 18:1, or about 19:1, or about 20:1 , or about 21:1 , or about 22: 1 , or about 23: 1 , or about 24: 1 , or about 25: 1 , or about 26:1.

In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acidbased agents from about 7:1 to about 13:1 and/or a has a weight to weight ratio of total lipid to one or more nucleic acid-based agents from about 14:1 to about 26:1 .

In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acidbased agents of about 7:1 and/or a has a weight to weight ratio of total lipid to one or more nucleic acidbased agents of about 25:1.

In embodiments, the

(i) a delivery lipid comprising from about 15 mol % to about 75 mol % of the total lipid present,

(ii) cholesterol comprising from about 25 mol % to about 65 mol % of the total lipid present,

(iii) a helper lipid comprising from about 5 mol % to about 40 mol % of the total lipid present, and

(iv) a PEGylated lipid comprising from about 0.5 mol % to about 7 mol % of the total lipid present.

In embodiments, the

(i) a delivery lipid comprising from about 25 mol % to about 75 mol % of the total lipid present,

(iv) a PEGylated lipid comprising from about 0.5 mol % to about 3 mol % of the total lipid present.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 30 mol % to about 60 mol % of the total lipid present, the delivery lipid being MC3,

(ii) cholesterol comprising from about 30 mol % to about 60 mol % of the total lipid present, (iii) a helper lipid comprising from about 5 mol % to about 35 mol % of the total lipid present, optionally being one or more of:

DSPC from about 8 mol % to about 12 mol % of the total lipid present,

DOPC from about 8 mol % to about 10 mol % of the total lipid present, and DOPE from about 7.5 mol % to about 25 mol % of the total lipid present, and

(iv) a PEGylated lipid comprising from about 0.5 mol % to about 3 mol % of the total lipid present, optionally being one or more of:

PEG-2K-DMG from about about 0.5 mol % to about 3 mol % of the total lipid present,

C8 PEG 2K ceramide from about about 0.5 mol % to about 2.5 mol % of the total lipid present, and

C16 PEG 2K ceramide from about about 0.5 mol % to about 2.5 mol % of the total lipid present.

In embodiments, the one or more lipids comprises: In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising about 45 mol % of the total lipid present, the delivery lipid being MC3,

(ii) cholesterol comprising about 45 mol % of the total lipid present,

(iii) a helper lipid comprising about 10 mol % of the total lipid present, the helper lipid being DOPE, and

(iv) a PEGylated lipid comprising about 2 mol % of the total lipid present, the PEGylated lipid being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 45 mol % of the total lipid present,

(iv) a PEGylated lipid comprising about 2 mol % of the total lipid present, the PEGylated lipid being PEG-2K-DMG and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 13: 1 and/or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 25 mol % to about 75 mol % of the total lipid present, the delivery lipid being SS-OP,

(iii) a helper lipid comprising from about 5 mol % to about 30 mol % of the total lipid present, optionally being one or more of:

DOPC from about 5 mol % to about 30 mol % of the total lipid present, and

DOPE from about 2.5 mol % to about 30 mol % of the total lipid present, and (iv) a PEGylated lipid comprising from about 0.5 mol % to about 3 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about

31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, or about 45 mol % of the total lipid present, the delivery lipid being SS- OP, (ii) cholesterol comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about

39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol %, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol % of the total lipid present, (iii) a helper lipid comprising about 5 mol %, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, or about 35 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

(iv) a PEGylated lipid comprising from about 3 mol %, about 4 mol%, about 5 mol%, about 6 mol%, or about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 15 mol % to about 45 mol % of the total lipid present, the delivery lipid being SS-OP,

(ii) cholesterol comprising from about 35 mol % to about 60 mol % of the total lipid present,

(iii) a helper lipid comprising from about 5 mol % to about 35 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

(iv) a PEGylated lipid comprising from about 3 mol % to about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 22 mol % to about 39 mol % of the total lipid present, the delivery lipid being SS-OP,

(ii) cholesterol comprising from about 46 mol % to about 53 mol % of the total lipid present,

(iii) a helper lipid comprising from about 7 mol % to about 26 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

(iv) a PEGylated lipid comprising from about 4.5 mol % to about 5.5 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises: (i) a delivery lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, the delivery lipid being SS-OP,

(ii) cholesterol comprising from about 46 mol % to about 50 mol % of the total lipid present,

(iii) a helper lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

(iv) a PEGylated lipid comprising from about 4 mol % to about 6 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 34 mol % to about 38 mol % of the total lipid present, the delivery lipid being SS-OP,

(ii) cholesterol comprising from about 48 mol % to about 52 mol % of the total lipid present,

(iii) a helper lipid comprising from about 8 mol % to about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising about 36 mol % of the total lipid present, the delivery lipid being SS- OP,

(ii) cholesterol comprising about 51 mol % of the total lipid present,

(iii) a helper lipid comprising about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

(iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG.

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 51 mol % of the total lipid present, (iii) a helper lipid comprising about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

(iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 and/or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- OP,

(ii) cholesterol comprising about 48 mol % of the total lipid present,

(iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 48 mol % of the total lipid present,

(iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 and/or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 . In embodiments, the one or more lipids comprises:

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol % of the total lipid present, the delivery lipid being ALC-0315,

(ii) cholesterol comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol %, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol % of the total lipid present,

(iii) a helper lipid comprising about 5 mol %, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol % of the total lipid present, optionally being DOPC, and

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 35 mol % to about 55 mol % of the total lipid present, the delivery lipid being ALC-0315,

(ii) cholesterol comprising from about 35 mol % to about 55 mol % of the total lipid present,

(iii) a helper lipid comprising from about 5 mol % to about 15 mol % of the total lipid present, optionally being DOPC, and (iv) a PEGylated lipid comprising from about 3 mol % to about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 43 mol % to about 47 mol % of the total lipid present, the delivery lipid being ALC-0315,

(ii) cholesterol comprising from about 42 mol % to about 46 mol % of the total lipid present,

(iii) a helper lipid comprising from about 7 mol % to about 11 mol % of the total lipid present, optionally being DOPC, and

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising about 45 mol % of the total lipid present, the delivery lipid being ALC- 0315,

(ii) cholesterol comprising about 44 mol % of the total lipid present,

(iii) a helper lipid comprising about 9 mol % of the total lipid present, optionally being DOPC, and

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 44 mol % of the total lipid present,

(iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 , and/or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 . In embodiments, the one or more lipids comprises:

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol % of the total lipid present, the delivery lipid being squaramide,

(ii) cholesterol comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, 55 mol % of the total lipid present,

(iv) a PEGylated lipid comprising from about 1 mol %, about 2 mol%, about 3 mol%, about 4 mol%, or about 5 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 35 mol % to about 55 mol % of the total lipid present, the delivery lipid being squaramide,

(iii) a helper lipid comprising from about 5 mol % to about 15 mol % of the total lipid present, optionally being DOPC, and (iv) a PEGylated lipid comprising from about 1 mol % to about 5 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising from about 43 mol % to about 47 mol % of the total lipid present, the delivery lipid being squaramide,

(iii) a helper lipid comprising from about 7 mol % to about 11 mol % of the total lipid present, optionally being DOPE, and

(iv) a PEGylated lipid comprising from about 2.5 mol % to about 3.5 mol % of the total lipid present, optionally being PEG-2K-DMG.

In embodiments, the one or more lipids comprises:

(i) a delivery lipid comprising about 45 mol % of the total lipid present, the delivery lipid being squaramide,

(ii) cholesterol comprising about 44 mol % of the total lipid present,

(iii) a helper lipid comprising about 9 mol % of the total lipid present, and

(iv) a PEGylated lipid comprising about 3 mol % of the total lipid present, optionally being PEG-2K- DMG.

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 44 mol % of the total lipid present,

(iv) a PEGylated lipid comprising about 3 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 13: 1 , and/or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 . In embodiments, the composition comprises one of: F5, and F6 of FIG. 1.

In embodiments, the composition comprises one of: F1 , and F8 of FIG. 2.

In embodiments, the composition comprises one of the formulations of FIG. 3A and FIG. 3B.

In embodiments, the composition comprises one of the formulations of FIG. 4A and FIG. 4B.

In embodiments, the composition comprises one of the formulations of FIG. 5A and FIG. 5B.

In embodiments, the composition comprises one of the formulations of FIG. 6.

In embodiments, the composition comprises one of the formulations of FIG. 7.

In embodiments, the composition comprises one of the formulations of FIG. 8B.

In embodiments, the composition comprises one of the formulations of FIG. 9.

In embodiments, the composition comprises one of the formulations of FIG. 10.

In embodiments, the composition comprises one of the formulations: F1, F2, F3, F4, F5, and F6 of FIG. 11.

In embodiments, the composition comprises one of the formulations: F1 , F2, F3, F4, F5, F6, and F7 of FIG. 12.

In embodiments, the composition comprises one of the formulations from the groups: 2, 3, 4, 5, 6, 7, and 8 of FIG. 13.

In embodiments, the composition comprises one of the formulations of FIG. 15.

In embodiments, the composition comprises one of the formulations of FIG. 16.

In embodiments, the composition comprises one of the formulations of FIG. 17.

In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acidbased agents of about 5: 1 to about 15: 1 , or about 7: 1 to about 13: 1 , or about 8: 1 to about 12: 1 , or about 5: 1 , or about 6: 1 , or about 7: 1 , or about 8: 1 , or about 9: 1 , or about 10: 1 , or about 11 : 1 , or about 12: 1 , or about 13:1 , or about 14:1 , or about 15:1. In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents from about 7:1 to about 13:1 . In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1, or of about 13:1. In embodiments, the composition has a weight to weight ratio of total lipid to one or more nucleic acid-based agents about 10:1 to about 35:1, about 15:1 to about 30:1 , about 20:1 to about 25:1 , or about 15:1 , or about 20:1 , or about 25:1 , or about 30:1. In embodiments, the composition has a weight to weight ratio of total lipid to one or more nucleic acid-based agents from about 14:1 to about 26:1. In embodiments, the composition has a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

In embodiments, the composition forms a nucleic acid/lipid particle.

In embodiments, the one or more lipids associate into a lipid nanoparticle (LNP).

In embodiments, the LNP encapsulates the one or more nucleic acid-based agents.

Nucleic Acid-Based Agents : Mobile Element Enzyme

In embodiments, the one or more nucleic acid-based agents comprise a nucleic acid which encodes a mobile element enzyme.

In embodiments, the mobile element enzyme has the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence having at least about 80%, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

SEQ ID NO: 1 : Myotis lucifugus mobile element enzyme protein (572 amino acids, positions 2, 8, 13, and 125, shown in bold and underline)

MAQHSDYSDDEFCADKLSNYSCDSDLENASTSDEDSSDDEVMVRPRTLRRRRISSSSSDSESDIEGGREEW SHVDNPPVLEDFLGHQGLNTDAVINNIEDAVKLFIGDDFFEFLVEESNRYYNQNRNNFKLSKKSLKWKDIT PQEMKKFLGLIVLMGQVRKDRRDDYWTTEPWTETPYFGKTMTRDRFRQIWKAWHFNNNADIVNESDRLCKV RPVLDYFVPKFINIYKPHQQLSLDEGIVPWRGRLFFRVYNAGKIVKYGILVRLLCESDTGYICNMEIYCGE GKRLLETIQTWSPYTDSWYHIYMDNYYNSVANCEALMKNKFRICGTIRKNRGIPKDFQTISLKKGETKFI RKNDILLQVWQSKKPVYLISSIHSAEMEESQNIDRTSKKKIVKPNALIDYNKHMKGVDRADQYLSYYSILR RTVKWTKRLAMYMINCALFNSYAVYKSVRQRKMGFKMFLKQTAIHWLTDDIPEDMDIVPDLQPVPSTSGMR AKPPTSDPPCRLSMDMRKHTLQAIVGSGKKKNILRRCRVCSVHKLRSETRYMCKFCNIPLHKGACFEKYHT LKNY

In embodiments, the mobile element enzyme comprises an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 1. In embodiments, the amino acid is a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I, and P, optionally A. In embodiments, the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 1. In embodiments, the enzyme has one or more mutations which confer hyperactivity. In embodiments, the enzyme has one or more amino acid substitutions selected from S8X1, C13X2, and N 125X3, or positions corresponding thereto relative to SEQ ID NO: 1 . In embodiments, the enzyme has S8X1 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the enzyme has C13X2 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1 . In embodiments, the enzyme has N125X3 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, Xi is selected from G, A, V, L, I, and P. In embodiments, X2 is selected from K, R, and H. In embodiments, X3 is selected K, R, and H. In embodiments, Xi is P and X2 is R.

In embodiments, the enzyme is capable of inserting a donor DNA at a TA dinucleotide site.

In embodiments, the enzyme is capable of inserting a donor DNA at a TTAA tetranucleotide site. In embodiments, the enzyme comprises: (a) a targeting element, and (b) an enzyme that is capable of inserting the donor DNA (e.g., a mobile element) comprising a gene, optionally at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS).

In embodiments, the targeting element targets the enzyme to a locus of interest.

In embodiments, the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive or a transcription activator-like effector (TALE).

In embodiments, the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD) and zinc fingers (ZF) DBD. In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG.

In embodiments, the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof. A CRISPR/Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity. See Jinek et al. (2012) Science 337, 816-821; Chylinski et al. (2014) Nucleic Acids Res 42, 6091-6105. The inactivated form of Cas9, which is a nuclease-deficient (or inactive, or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity. Qi, L. S. et al. (2013). Cell 152, 1173-1183. CRISPR/dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences. See Dominguez et al., Nat Rev Mol Cell Biol. 2016;17:5-15; Wang et al., Annu Rev Biochem. 2016;85:227-64. dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and/or locus in a genome. When the dCas9 protein is coupled to guide RNA (gRNA) to create dCas9 guide RNA complex, dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. Thus, dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription.

In embodiments, the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9”) guide RNA complex.

In embodiments, the targeting element comprises a Cas9 enzyme guide RNA complex. In embodiments, the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.

Embodiments of the present disclosure make use of the ability of TALE or Cas or dCas9/gRNA DBDs to target specific sites in a host genome. The DNA targeting ability of a TALE or Cas DBD or dCas9/gRNA DBD is provided by TALE repeat sequences (e.g., modular arrays) or gRNA which are linked together to recognize flanking DNA sequences. Each TALE or gRNA can recognize certain base pair(s) or residue(s).

In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.

In embodiments, the enzyme and the targeting element are connected. In embodiments, the enzyme and the targeting element are fused to one another or linked via a linker to one another. In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)_n, where n is from about 1 to about 12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. Nucleic Acid-Based Agents: Donor DNA or Trans ene or Mobile Element

In embodiments, the one or more nucleic acid-based agents comprise a donor DNA or transgene or mobile element.

In embodiments, the donor DNA or transgene or mobile element comprises a gene encoding a complete polypeptide. In embodiments, the donor DNA or transgene or mobile element comprises a gene which is defective or substantially absent in a disease state.

In embodiments, the donor DNA or transgene or mobile element is flanked by one or more ends. In embodiments, the mobile element enzyme recognizes the one or more ends.

In embodiments, the donor DNA or transgene or mobile element has a size of up to about 10 kb, or up to about 12 kb, up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb.

RNA/DNA and Ratios Thereof

In embodiments, the one or more nucleic acid-based agents is in the form of RNA. In embodiments, the RNA is or comprises mRNA. In embodiments, the mRNA is or comprises mmRNA. In embodiments, the mmRNA comprises one or more of a 5'-m7G cap (capO, cap1, or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length.

In embodiments, the one or more nucleic acid-based agents is in the form of DNA. In embodiments, the DNA is or comprises plasmid DNA or miniplasmid DNA. In embodiments, the plasmid DNA has a size of up to about 10 kb, or up to about 12 kb, up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb.

In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme and a donor DNA or transgene or mobile element.

In embodiments, the nucleic acid-based agent comprises both RNA and DNA. In embodiments, the nucleic acid-based agent comprises both mRNA and DNA. In embodiments, the weight to weight ratio of DNA : mRNA is from about 10:1 to about 1 :10, or about 10 : about 1 , or about 9 : about 1, or about 8 : about 1 , or about 7 : about 1 , or about 6 : about 1, or about 5 : about 1 , or about 4 : about 1 , or about 3 : about 1, or about 2 : about 1 , or about 1 : about 1 , or about 1 : about 2, or about 1 : about 3, or about 1 : about 4, or about 1 : about 5, or about 1 : about 6, or about 1 : about 7, or about 1 : about 8, or about 1 : about 9, or about 1 : about 10.

In embodiments, the nucleic acid-based agent comprises both mmRNA and plasmid DNA. In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA.

In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme of SEQ ID NO: 1 in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA.

In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA and the weight to weight ratio of plasmid DNA : mmRNA is about 5 : about 1 , or about 2 : about 1 , or about 1 : about 1, or about 1 : about 2, or about 1 : about 5.

In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA and the weight to weight ratio of plasmid DNA : mmRNA about 2 : about 1 .

Features of the LNP

In embodiments, the composition provides high encapsulation of the nucleic acid-based agent, e.g., compared a different collection of lipids, e.g., a different LNP.

In embodiments, the composition provides an encapsulation efficiency of greater than about 90%, greater than about 95%, greater than about 98%. Encapsulation efficiency can be measured as is known in the art, e.g., using fluorescent dye that is used in the detection and quantification of nucleic acids, e.g., using the RiboGreen assay (THERMO), or a similar assay.

In embodiments, the composition provides low polydispersity, e.g., compared a different collection of lipids, e.g., a different LNP. Assays/calculations used for the determination of polydispersity are known in the art, e.g., using parameters are defined in the ISO standard documents 13321:1996 E and ISO 22412:2008.

In embodiments, the composition is an LNP having a particle size of about 60 to about 200 nm, or about 100 to about 150 nm, or about 60 to about 120 nm. Assays/calculations used for the determination of particle size are known in the art, e.g., using parameters are defined in the ISO standard documents 13321 :1996 E and ISO 22412:2008.

Administration In embodiments, the composition is suitable for systemic delivery in a subject. In embodiments, the composition is suitable for intravenous, subcutaneous, or intraperitoneal delivery in a subject.

In embodiments, the composition is suitable for local delivery in a subject. In embodiments, the composition is suitable for intrahepatic delivery in a subject.

Methods

In aspects, there is provided a method for introducing a nucleic acid into a cell, the method comprising contacting the cell with a composition described herein.

In aspects, there is provided a method for the in vivo delivery of a nucleic acid, the method comprising administering to a subject a composition described herein.

In aspects, there is provided a method fortreating a metabolic disease or disorder in a subject in need thereof, the method comprising administering to the subject’s liver a therapeutically effective amount of a composition described herein.

In aspects, there is provided a method for treating a hepatic disease or disorder in a subject in need thereof, the method comprising administering to the subject’s liver a therapeutically effective amount of a composition described herein.

In embodiments, the method for treating a hepatic disease or disorder in the subject in need thereof comprises administering to the subject’s liver a therapeutically effective amount of a composition comprising:

(a) one or more nucleic acid-based agents, and

(b) one or more lipids, the one or more lipids comprising:

(i) a delivery lipid,

(ii) cholesterol,

(iii) a helper lipid, and (iv) a PEGylated lipid.

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 44 mol % of the total lipid present, (iii) a helper lipid comprising about 9 mol % of the total lipid present, optionally being DOPC, and

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 44 mol % of the total lipid present,

(iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 , and/or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

In embodiments, the one or more lipids comprises:

(iii) a helper lipid comprising about 5 mol %, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about

15 mol % of the total lipid present, optionally being DOPC, and

In embodiments, the one or more lipids comprises: (i) a delivery lipid comprising from about 43 mol % to about 47 mol % of the total lipid present, the delivery lipid being squaramide,

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 44 mol % of the total lipid present,

In embodiments, the one or more lipids comprises:

(ii) cholesterol comprising about 44 mol % of the total lipid present,

(iii) a helper lipid comprising about 9 mol % of the total lipid present, optionally being DOPC,

(iv) a PEGylated lipid comprising about 3 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 13: 1 , and/or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1.

Kits In embodiments, the compositions, or one or more lipids, optionally with the nucleic acid-based agents, is included in a container, kit, pack, or dispenser together with instructions for administration and/or association (e.g., to form an LNP).

In embodiments, the kit includes instructions for administration and/or association (e.g., to form an LNP) in accordance with the method of the present disclosure.

Definitions

The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.

An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.

As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and/or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.

As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.” As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

SELECTED SEQUENCES

In embodiments, the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

SEQ ID NO: 1 : Myotis lucifugus mobile element enzyme protein (572 amino acids, positions 2, 8, 13, and 125, described in the text, reshown in bold and underline)

This invention is further illustrated by the following non-limiting examples.

EXAMPLES

Hereinafter, the present disclosure will be described in further detail with reference to examples. These examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. In addition, various modifications and variations can be made without departing from the technical scope of the present invention.

Example 1: Formulation Development

FIG. 1 shows formulations load DNA/RNA. Lipid content is given in % mol and Cationic/Total lipid:NA is in mg:mg. FIG. 2 shows formulations of MC3 and C12-200 mixed LNPs. Mixed lipids improved %EE (keeping cationic lipid constant) gave excellent results.

A higher MC3 Cationic lipid : nucleic acid (NA) also allowed for reduction of %PEGs as shown in FIG. 3A and FIG. 3B (showing size, polydispersity index (PDI), and %EE).

FIG. 4A and FIG. 4B show MC3 compositions also allowed changing PEG anchors of different lengths with high %EE. The composition was 9% DOPE + 45% MC3 + 44% CHOLESTEROL + 2% PEG. PEG studied were DMG and Ceramide-based. Further, C8 and C16, length of lipids conjugated to PEG, were analyzed.

FIG. 5A and FIG. 5B show effects of different pH on %EE. Different pH buffers had similar profiles (high %EE). The composition was 9% DOPE + 45% MC3 + 44% CHOLESTEROL + 2% PEG.

FIG. 6 shows that SS-OP LNPs can be formulated with robust size and encapsulation. The sizes were very small (<100nm) for all formulations. Nearly all particles resulted in precipitation but high N:P PEG non- MW corrected resulted in few precipitates.

Example 2: In Vivo Studies with Various Routes of Administration

FIG. 7 shows SS-OP LNPs formulation composed of 9.65 DOPE/36.28 SS-OP/50.72 cholesterol/3.25 PEG- DMG 2k had criteria acceptable for in vivo testing. Mouse injection with this SS-OP LNPs formulation delivering DNA demonstrated acceptable DNA expression (10⁷ total flux).

FIG. 8A and FIG. 8B show MC3 LNPs delivering both DNA and mRNA together in vivo (PBS subtracted) locally (intrahepatic injection). Nucleic acids- CAG-Luciferase + nucleic acid encoding the mobile element enzyme of SEQ ID NO: 1 . Durable expression was observed.

FIG. 15 shows MC3 and ALC-0315 LNPs 2% - 5% PEG delivering nucleic acids when injected intrahepatically. MC3 and ALC-0315 LNPs with dual cargo (Flue mRNA + SalioB) with various PEG amounts were formulated and injected into LDLR KO mice. 6h after injection the in-life images were taken to capture the luciferase signal. The total flux with MC3 LNPs both 2% and 5% PEG was lower than the ALC-0315 LNPs. Thus, ALC-0315 LNPs were chosen for the subsequent studies. The term “SalioB” refers to the mobile element enzyme of SEQ ID NO: 1 .

FIG. 9 shows bioluminescence data in mouse summarizing intrahepatic administration with SS-OP formulations. FIG. 10 shows characteristics of compositions of the disclosure in the liver. These compositions include MC3, and SS-OP.

Example 3: Formulation Engineering

Without wishing to be bound by theory, to decrease the toxicity of cationic lipids and ionizable lipids in formulations and to widen the therapeutic window, various amount (mg) of cationic lipids and ionizable lipids were assessed.

In prior experiments, the amount of cationic lipids and ionizable lipids remained constant in formulations and the toxicity profile did not change. As shown in FIG. 11 , additional experiments focused, inter alia, on altering the amount of cationic lipid and ionizable lipid in formulations. These experiments helped, inter alia, assessing C12-200 and led to consider other compositions.

As shown in FIG. 12, additional LNP screenings and new engineering methods were established. The physico-chemical characteristics such as DLS size (where DLS relates to dynamic light scattering measuring the hydrodynamic radius of the nanoparticle), PDI (where PDI, polydispersity index, relates to the homogeneity in the size of the particles, lower is more equally sized particles), and percentage of encapsulation were reported for new MC3 LNPs compositions.

FIG. 13 shows the selection of specific formulation for mRNA delivery with a luciferase reporter. LNPs with dual cargos (mVLDLR + Flue mRNA) were made at various ratios as shown in FIG. 13 and injected into BalbB/C mice. The luciferase signal (total flux) was measured in I VIS at 6hr and 24hrs after injections. The 1-5-5 ratio with the MC3 lipid LNPs showed the highest mRNA delivery with respect to the luciferase signal from Flue mRNA. The MC3 formulation with 1-5-5 composition was selected to be used in inherited liver diseases (ILD).

FIG. 14 shows the selection of the mRNA-LNPs formulations of particular interest for MC3. LNPs with dual cargos were made at various ratios as shown in FIG. 14 and injected into BalbB/C mice. Mice were euthanized on Day 8 and livers were harvested. The cryosections of the liver tissue were stained with anti- VLDLR antibody for the VLDLR. The 1-1-2 ratio formulation with the MC3 lipid LNPs showed strong DNA expression with respect to the mVLDLR staining in hepatocytes. The MC3 formulation with 1-1-2 ratio was selected to be used in ILD and I RD formulations. Squaramide LNPs formulations were tested in the liver (FIG. 16 and FIG. 17). Squaramide LNPs were formulated with different ionizable lipid. Nucleic acid ratios were computed with either Flue mRNA or CAG- Luc-NP + Saliogase B mRNA with the following composition .

(a) 9 DOPC / 45 SQAM/ 44 Cholesterol/ 3% PEG-DMG (without GalNac);

(b) 9 DOPC/ 45 SQAM/ 44 Cholesterol/2.5% PEG-DMG/0.5% GalNac

Various ratios (4:1 ; 7:1 ; and 13:1) of Ionizable lipid: SQAM were assessed.

Animal Studies: SQAM formulations were made using Precision Nanosystems Inc.'s (PNI) technology and the formulations passed the criteria for in-vivo use in the liver. The WT mice were injected with the above formulations. The luciferase protein expression signal was reported in terms of total flux with I VIS imaging.

EQUIVALENTS

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

CLAIMS What is claimed is:

1 . A composition comprising:

(a) one or more nucleic acid-based agents, and

(b) one or more lipids, the one or more lipids comprising:

(i) a delivery lipid,

(ii) cholesterol,

(iii) a helper lipid, and

(iv) optionally, a PEGylated lipid.

2. The composition of claim 1 , wherein the delivery lipid is selected from squaramide (SQAM), and ALC- 0315 and any combination thereof.

3. The composition of claim 1 or 2, wherein the helper lipid is selected from distearoylphosphatidylcholine (DSPC), 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and any combination thereof.

4. The composition of any one of claims 1-3, wherein the PEGylated lipid is selected from PEG-2K-DMG, C8 PEG 2K, and C16 PEG 2K.

5. The composition of any one of claims 1-4, comprising:

6. The composition of any one of claims 1-5, wherein the one or more lipids comprises:

(ii) cholesterol comprising from about 35 mol % to about 55 mol % of the total lipid present, (iii) a helper lipid comprising from about 5 mol % to about 15 mol % of the total lipid present, and

7. The composition of any one of claims 1-5, wherein the one or more lipids comprises:

(iii) a helper lipid comprising from about 5 mol % to about 15 mol % of the total lipid present, and

(iv) a PEGylated lipid comprising from about 1 mol % to about 5 mol % of the total lipid present, optionally being PEG-2K-DMG.

8. The composition of any one of claims 1 -7, wherein the composition has a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 5:1 to about 15:1 , or about 7:1 to about 13:1 , or about 8:1 to about 12:1, or about 5:1, or about 6:1 , or about 7: 1 , or about 8:1 , or about 9:1, or about 10:1 , or about 11 :1 , or about 12:1 , or about 13:1 , or about 14:1, or about 15:1.

9. The composition of any one of claims 1-8, wherein the composition has a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 10:1 to about 35:1 , or about 15:1 to about 30:1, or about 20:1 to about 25:1 , or about 15:1 , or about 20:1 , or about 25:1, or about 30:1.

10. The composition of any one of claims 1-9, wherein the composition forms a nucleic acid/lipid particle.

11. The composition of any one of claims 1-10, wherein the one or more lipids associate into a lipid nanoparticle (LNP).

12. The composition of claim 1-11, wherein the LNP encapsulates the one or more nucleic acid-based agents.

13. The composition of any one of claims 1-12, wherein the one or more nucleic acid-based agents comprise a nucleic acid which encodes a mobile element enzyme.

14. The composition of claim 1-13, wherein the mobile element enzyme has the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence having at least about 80%, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

15. The method of any one of claims 1-14, wherein the mobile element enzyme comprises an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 1 .

16. The method of any one of claims 1-15, wherein the amino acid is a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I, and P, optionally A.

17. The method of any one of claims 1-16, wherein the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 1 .

18. The method of any one of claims 1-17, wherein the enzyme has one or more mutations which confer hyperactivity.

19. The method of any one of claims 1-18, wherein the enzyme has one or more amino acid substitutions selected from S8X1, 013X2, and N125X3, or positions corresponding thereto relative to SEQ ID NO: 1.

20. The method of claim 1-19, wherein the enzyme has S8X1 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1 .

21. The method of claim 1-20, wherein the enzyme has C13X2 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1 .

22. The method of claim 1-21 , wherein the enzyme has N125X3 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1 .

23. The method of any one of claims 1-22, wherein Xi is selected from G, A, V, L, I, and P.

24. The method of any one of claims 1-23, wherein X2 is selected from K, R, and H.

25. The method of any one of claims 1-24, wherein X3 is selected K, R, and H.

26. The method of claim 1-25, wherein: Xi is P and X2 is R.

27. The method of any one of claims 1-26, wherein the enzyme is capable of inserting a donor DNA at a TA dinucleotide site.

28. The method of any one of claims 1-27, wherein the enzyme is capable of inserting a donor DNA at a TTAA tetranucleotide site.

29. The method of any one of claims 1-28, wherein the enzyme comprises: a targeting element and an enzyme that is capable of inserting the donor DNA (e.g., a mobile element) comprising a gene, optionally at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS).

30. The method of any one of claims 1 -29, wherein the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive or a transcription activator-like effector (TALE).

31. The method of any one of claims 1 -30, wherein the targeting element comprises a transcription activatorlike effector (TALE) DNA binding domain (DBD) and zinc fingers (ZF) DBD.

32. The method of any one of claims 1-31 , wherein the targeting element comprises a Cas9 enzyme guide RNA complex.

33. The method of claim 1 -32, wherein the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.

34. The method of any one of claims 1-33, wherein the GSHS is in an open chromatin location in a chromosome.

35. The method of any one of claims 1-34, wherein the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.

36. The method of any one of claims 1-35, wherein the enzyme and the targeting element are connected.

37. The method of any one of claims 1-36, wherein the enzyme and the targeting element are fused to one another or linked via a linker to one another.

38. The method of claim 1 -37, wherein the linker is a flexible linker.

39. The method of claim 1-38, wherein the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)_n, where n is from about 1 to about 12.

40. The method of claim 1-39, wherein the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.

41. The composition of any one of claims 1-40, wherein the one or more nucleic acid-based agents comprise a donor DNA or transgene or mobile element.

42. The method of any one of claims 1 -41 , wherein the donor DNA or transgene or mobile element comprises a gene encoding a complete polypeptide.

43. The method of any one of claims 1 -42, wherein the donor DNA or transgene or mobile element comprises a gene which is defective or substantially absent in a disease state.

44. The method of any one of claims 1 -43, wherein the donor DNA or transgene or mobile element is flanked by one or more ends.

45. The method of any one of claims 1-44, wherein the donor DNA or transgene or mobile element has a size of up to about 10 kb, or up to about 12 kb, or up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb.

46. The composition of any one of claims 1-45, wherein the one or more nucleic acid-based agents is in the form of RNA.

47. The composition of claim 1-46, wherein the RNA is or comprises messenger RNA (mRNA).

48. The composition of claim 1-47, wherein the mRNA is or comprises modified mRNA (mmRNA).

49. The composition of claim 1-48, wherein the mmRNA comprises one or more of a 5’-m7G cap (capO, cap 1 , or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or of about 50, or of about 100, or of about 150 nucleotides in length.

50. The composition of any one of claims 1-49, wherein the one or more nucleic acid-based agents is in the form of DNA.

51. The composition of claim 1-50, wherein the DNA is or comprises plasmid DNA or miniplasmid DNA.

52. The composition of claim 1-51 , wherein the plasmid DNA has a size of up to about 10 kb, or up to about 12 kb, or up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb.

53. The composition of any one of claims 1-52, wherein the nucleic acid-based agent comprises both: a nucleic acid encoding a mobile element enzyme and a donor DNA or transgene or mobile element.

54. The composition of any one of claims 1-53, wherein the nucleic acid-based agent comprises both RNA and DNA.

55. The composition of any one of claims 1-54, wherein the nucleic acid-based agent comprises both mRNA and DNA.

56. The composition of claim 1-55, wherein the weight to weight ratio of DNA : mRNA is from about 10:1 to about 1 : 10, or about 10 : about 1 , or about 9 : about 1 , or about 8 : about 1 , or about 7 : about 1 , or about

6 : about 1 , or about 5 : about 1 , or about 4 : about 1 , or about 3 : about 1 , or about 2 : about 1 , or about

1 : about 1, or about 1 : about 2, or about 1 : about 3, or about 1 : about 4, or about 1 : about 5, or about

1 : about 6, or about 1 : about 7, or about 1 : about 8, or about 1 : about 9, or about 1 : about 10.

57. The composition of any one of claims 1-56, wherein the nucleic acid-based agent comprises both mmRNA and plasmid DNA.

58. The composition of any one of claims 1-57, wherein the nucleic acid-based agent comprises both: a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA.

59. The composition of any one of claims 1-58, wherein the nucleic acid-based agent comprises both: a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA and the weight to weight ratio of plasmid DNA : mmRNA is about 5 : about 1 , or about 2 : about 1 , or about 1 : about 1 , or about 1 : about 2, or about 1 : about 5.

60. The composition of any one of claims 1-59, wherein the nucleic acid-based agent comprises both: a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA and the weight to weight ratio of plasmid DNA : mmRNA is about 2 : about 1 .

61. The composition of any one of claims 1 -60, wherein the composition provides high encapsulation of the nucleic acid-based agents.

62. The composition of any one of claims 1 -61 , wherein the composition provides an encapsulation efficiency of greater than about 90%, or greater than about 95%, or greater than about 98%.

63. The composition of any one of claims 1-62, wherein the composition provides low polydispersity.

64. The composition of any one of claims 1-63, wherein the composition is an LNP having a particle size of about 60 to about 200 nm, or about 100 to about 150 nm, or about 60 to about 120 nm.

65. The composition of any one of claims 1-64, wherein the composition is suitable for systemic delivery in a subject.

66. The composition of any one of claims 1-65, wherein the composition is suitable for intravenous, subcutaneous, or intraperitoneal delivery in a subject.

67. The composition of any one of claims 1-66, wherein the composition is suitable for local delivery in a subject.

68. The composition of any one of claims 1-67, wherein the composition is suitable for intrahepatic delivery in a subject.

69. A method for introducing a nucleic acid into a cell, the method comprising contacting the cell with a composition of any one of claims 1-68.

70. A method for the in vivo delivery of a nucleic acid, the method comprising administering to a subject a composition of any one of claims 1-68.

71. A method for delivery of a nucleic acid to the liver of a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 1-68.

72. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 1-68.

73. A method for treating a hepatic disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 1-