WO2025250906A1

WO2025250906A1 - Process for preparation of targeted lipid nanoparticles

Info

Publication number: WO2025250906A1
Application number: PCT/US2025/031602
Authority: WO
Inventors: Robert DORKIN; Jingyuan TIAN; Mengmeng ZHENG
Original assignee: Aera Therapeutics Inc
Current assignee: Aera Therapeutics Inc
Priority date: 2024-05-30
Filing date: 2025-05-30
Publication date: 2025-12-04
Anticipated expiration: 2026-11-30

Abstract

The present invention relates to processes for preparing lipid nanoparticles having an antibody or fragment antibody binding region.

Description

Process for preparation of targeted lipid nanoparticles

[0001] This application claims the benefit of U.S. Provisional Application No. 63/653,684, filed May 30, 2024, which is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to continuous in-line processes for preparing lipid nanoparticles having an antibody or fragment antibody binding region.

BACKGROUND OF THE INVENTION

[0003] Current methods of forming lipid nanoparticle that comprise a targeting moiety have many deficiencies. The two most common approaches are post-formulation conjugation where the chemical conjugation of mAb/Fab to a chemically activated PEG-lipid takes place after formulation of the lipid nanoparticles. The second approach is post-insertion wherein the mAb/Fab -PEG-lipid is inserted into preformed lipid nanoparticles.

[0004] Problems associated with post-formulation conjugation include reproducibility of conjugation, possibility of unreacted PEG-lipid being included in the final product of lipid nanoparticles, and difficulties in scale-up to larger production of lipid nanoparticles. Problems associated with the post-insertion method include reproducibility of conjugated PEG-lipid insertion, an extra process step to remove undesired formation of lipid micelles, and difficulties in scale-up to larger production of lipid nanoparticles.

[0005] There is a continuing need for improved processes for preparing lipid nanoparticles having targeting moieties. SUMMARY OF THE INVENTION

[0006] One embodiment of the invention is a continuous in-line process for producing lipid nanoparticles having a targeting moiety (such as an antibody or fragment antibody binding region (Fab) targeting moiety) and carrying a therapeutic agent, such as a therapeutic nucleic acid, comprising: (a) in-line mixing an aqueous solution with an organic solution in a mixing connector to form a first solution, where (i) the aqueous solution comprises a therapeutic nucleic acid and optionally a weak acid and (ii) the organic solution comprises a solvent (such as ethanol), an ionizable lipid, a non-cationic lipid (such as DSPC), and sterol (such as cholesterol); (b) in-line mixing the first solution with a dilution solution to form the lipid nanoparticles, wherein the dilution solution comprises (i) a targeted PEG-lipid comprising a PEG-lipid (such as DSPE-PEG) conjugated to a monoclonal antibody or Fab, (ii) a buffer solution, and (iii) optionally, a non-targeted PEG-lipid. The organic solution may further comprise a PEG-lipid (such as DSPE-PEG or DMG-PEG) (also referred to herein as a non-targeted PEG-lipid). This process is beneficially reproducible, continuous, and scalable.

[0007] In some embodiments, the targeted PEG-lipid comprises a DSPE-PEG conjugated to a fragment antibody binding region (Fab).

[0008] In some embodiments, the targeted PEG-lipid comprises a DSPE-PEG conjugated ot a monoclonal antibody (mAb).

[0009] In some embodiments, the targeted PEG-lipid comprises a PEG-lipid conjugated to a targeting moiety (e.g. a mAb or Fab) through a crosslinking agent.

[0010] In some embodiments, the weak acid has a pH of 3 to 6. In some embodiments, the weak acid is citric acid or a salt thereof. In some embodiments, the weak acid is a citrate buffer at a pH of about 4. In some embodiments, the weak acid is an acetate buffer at a pH of about 5.

[0011] In some embodiments, the flow rate of the aqueous solution ranges from about 5 mL/min to about 100 mL/min, such as from about 20 mL/min to about 100 mL/min. In some embodiments, the flow rate of the aqueous solution ranges from about 5 mL/min to about 50 mL/min.

[0012] In some embodiments, the flow rate of the organic solution ranges from about 1 mL/min to about 50 mL/min, such as from about 5 mL/min to about 50 mL/min. In some embodiments, the flow rate of the organic solution ranges from about 1 mL/min to about 5 mL/min.

[0013] In some embodiments, the total lipid concentration in the organic solution ranges from about 5 mg/mL to about 30 mg/mL.

[0014] In some embodiments, the volume ratio or flow rate ratio of the aqueous solution to organic solution is from about 1 : 1 to about 5:1.

[0015] In some embodiments, the flow ratio of organic solution to aqueous solution to dilution solution is about 1 :3:3 to about 1 :3:5, such as about 1 :3:4.

[0016] In some embodiments, the flow rate of the dilution solution ranges from about 10 mL/min to about 120 mL/min, such as from about 40 mL/min to about 120 mL/min. In some embodiments, the flow rate of the dilution solution ranges from about 10 mL/min to about 15 mL/min.

[0017] In some embodiments, the concentration of the targeted PEG-lipid, such as conjugated DSPE-PEG, ranges from about 15 pg/mL to about 400 pg/mL.

[0018] In some embodiments, the dilution solution further comprises a non-targeted PEG-lipid. In one embodiment, the non-targeted PEG-lipid is DMG-PEG2000. In some instances, the concentration of the non-targeted PEG-lipid, such as DMG-PEG2000, ranges from about 0.1 pg/mL to about 200 pg/mL.

[0019] In some embodiments, the solutions are mixed through microfluidics. [0020] In some embodiments, the buffer solution of the dilution solution is phosphate buffered saline (PBS). In some instances, the buffer solution of the dilution solution is Tris buffer saline (TBS).

[0021] In some embodiments, the process further comprises step (c) diluting the lipid nanoparticle solution, for example, with a bulk dilution buffer (such as TBS).

[0022] In some embodiments, the lipid nanoparticles are isolated via dialysis. In some instances, the lipid nanoparticles are isolated or concentrated via tangential flow filtration (TFF). TFF can also be performed to exchange the buffer.

[0023] The lipid nanoparticles may be purified to remove any targeted PEG-lipid which was not incorporated into the lipid nanoparticles, such as by size exclusion chromatography. This may be done before or after isolation of the lipid nanoparticles.

[0024] In one embodiment, the lipid nanoparticles (or a composition of the lipid nanoparticles) formed by the processes described herein contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5% (area under the curve) of targeted PEG-lipid micelles (such as micelles of a PEG-lipid conjugated to a Fab or micelles of a PEG-lipid conjugated to an antibody), as measured by size exclusion chromatography-fluorescence detection (SEC-FLD). These micelles are not incorporated into the lipid nanoparticles. In one embodiment, the lipid nanoparticles (or a composition of the lipid nanoparticles) formed by the processes described herein contain more than 0% but less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5% (area under the curve) of targeted PEG- lipid micelles (such as micelles of a PEG-lipid conjugated to a Fab or micelles of a PEG-lipid conjugated to an antibody), as measured by SEC-FLD. In other words, in this embodiment, targeted PEG-lipid micelles are present with the lipid nanoparticles. In another embodiment, the lipid nanoparticles (or a composition of the lipid nanoparticles) formed by the processes described herein contain less than 5% (area under the curve) of targeted PEG-lipid micelles (such as micelles of a PEG-lipid conjugated to a Fab or micelles of a PEG-lipid conjugated to an antibody), as measured by SEC-FLD. In yet another embodiment, the lipid nanoparticles (or a composition of the lipid nanoparticles) formed by the proesses described herein contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5% (area under the curve) of micelles of a PEG-lipid conjugated to a Fab, as measured by size exclusion chromatography-fluorescence detection (SEC-FLD). Tn yet another embodiment, the lipid nanoparticles (or a composition of the lipid nanoparticles) formed by the processes described herein contain less than 5% (area under the curve) of micelles of a PEG-lipid conjugated to a Fab, as measured by SEC-FLD. In one embodiment of any embodiment described herein, the lipid nanoparticles (or a composition of the lipid nanoparticles) contain more than 0% of targeted PEG-lipid micelles (such as micelles of a PEG- lipid conjugated to a Fab or micelles of a PEG-lipid conjugated to an antibody), as measured by size exclusion chromatography-fluorescence detection (SEC-FLD).

[0025] In an alternative embodiment, the dilution solution does not contain a targeted PEG-lipid, and the organic solution contains a PEG-lipid having a coupling group. The in-line mixing of a dilution solution containing a targeting moiety (such as an antibody or Fab targeting moiety) causes conjugation of the targeting moiety to the PEG lipid in the lipid nanoparticle (which was formed from the mixing of the aqueous and organic solutions). One embodiment of the invention is a process for producing lipid nanoparticles having a targeting moiety (such as an antibody or fragment antibody binding region (Fab) targeting moiety) and carrying a therapeutic agent, such as a therapeutic nucleic acid, comprising: (a) in-line mixing an aqueous solution with an organic solution in a mixing connector to form a first solution, where (i) the aqueous solution comprises a therapeutic nucleic acid and optionally a weak acid and (ii) the organic solution comprises a solvent (such as ethanol), an ionizable lipid, a non-cationic lipid (such as DSPC), a PEG-lipid, and sterol (such as cholesterol); (b) in-line mixing the first solution with a dilution solution to form the lipid nanoparticles, wherein the dilution solution comprises (i) a targeting moiety which can conjugate with the PEG-lipid to form a targeted PEG-lipid, (ii) a buffer solution, and (iii) optionally, a non-targeted PEG-lipid. In one embodiment, the PEG-lipid (prior to conjugation to the targeting moiety) includes a coupling group. In some embodiments, the coupling group is selected from maleimides, N- hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha- haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure l is a flow chart of the process of the invention in which an aqueous stream (aqueous solution) and organic stream (organic solution) are mixed together in microfluidics before a dilution stream (dilution solution) with a conjugated PEG solution (targeted PEG-lipid) is added in.

[0027] Figure 2 is a flow chart of the process of the invention in which an aqueous RNA stream (aqueous solution) and organic lipid stream (organic solution) are mixed together to form a first solution, which is then mixed with a dilution stream containing the targeted PEG-lipid.

[0028] Figure 3 is a bar graph of GFP expression on CD4, CD8, and total T cells, as described in Example 2, with various targeted lipid nanoparticle (tLNP) formulations prepared by the process of the present invention. The ratios recited in the legend are the flow rates (mL/min) for the organic, aqueous, and dilution solutions (e.g., 10:30:40 refers to flow rate for the organic, aqueous, and dilution solutions of 10, 30, and 40 mL/min, respectively). The concentrations recited in the legend (e.g., 60, 90, or 120 pg/mL) are the RNA concentrations in the lipid nanoparticle.

[0029] Figure 4A are size exclusion chromatography-fluorescence detection (SEC-FLD) chromatograms for (i) DSPE-PEG-Fab having micelles and monomers of DSPE-PEG-Fab and (ii) lipid nanoparticles prepared by the process of the present invention, as described in Example 3.

[0030] Figure 4B are SEC-FLD chromatograms for (i) DSPE-PEG-Fab having micelles and monomers of DSPE-PEG-Fab and (ii) lipid nanoparticles prepared by the process of the present invention, as described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0031] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.

[0032] Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0033] In this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. In certain aspects, the term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "multiple."

[0034] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0035] The terms "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. [0036] Unless the context requires otherwise, the terms "comprise," "comprises," and "comprising" are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that Applicant intends each of those words to be so interpreted in construing this patent, including the claims below.

[0037] The term "targeting moiety" includes any type of molecule capable of specifically recognizing and interacting or binding with cell surface antigens whose expression is restricted to or enriched on a specific cell(s). In some embodiments, the targeting moiety is selected from antibodies, peptides, ligands, ligand-mimic, agonists and/or antagonists. In some embodiments, the targeting moiety may be any type of antibody, or a fragment thereof. In some embodiments, the targeting moiety is a monoclonal antibody. In some embodiments, the targeting moiety is a fragment antibody binding region (Fab).

[0038] The term “Fab handle” refers to a Fab having a leaving or coupling group which permits the Fab to conjugate to a PEG-lipid. Analogously, the term “antibody handle” refers to an antibody having a leaving or coupling group which permits the Fab to conjugate to a PEG- lipid.

[0039] One embodiment is a continuous in-line process for producing lipid nanoparticles having a targeting moiety (such as an antibody or fragment antibody binding region (Fab) targeting moiety) and carrying a therapeutic nucleic acid. The process comprises (a) in-line mixing an aqueous solution with an organic solution in a mixing connector to form a first solution, where (i) the aqueous solution comprises a therapeutic nucleic acid and optionally a weak acid and (ii) the organic solution comprises a solvent (such as ethanol), an ionizable lipid, a non-cationic lipid, and sterol (such as cholesterol), and (b) in-line mixing a dilution solution into the first solution in a mixing connector to form lipid nanoparticles, where the dilution solution comprises (i) a targeted PEG-lipid (such as a PEG-lipid (e.g., DSPE-PEG) conjugated to an antibody or Fab), (ii) a buffer solution, and (iii) optionally, a non-targeted PEG-lipid. This process is exemplified in Figures 1 and 2.

[0040] In some embodiments, the targeted PEG-lipid comprises a targeting moiety. In some instances, the targeting moiety is a fragment antibody binding region (Fab). In some instances, the Fab is aza-dibenzocyclooctyne (DBCO)-Fab. In some instances, the targeting moiety structure is an antibody. In some instances, the targeting moiety is a monoclonal antibody (mAb). In some instances, the targeting moiety is a Fab’. In some instances, the targeting moiety is a F(ab’)2. In some instances, the targeting moiety is an ApoE protein, such as an ApoE mutant protein. In some instances, the targeting moiety is an ApoE peptide sequence. In some instances, the targeting moiety is a binding protein. In some instances, the binding protein is ApoAl. In some instances, the targeting moiety is a ligand, antibody against cell surface receptor, peptide, lipoprotein, glycoprotein, hormone, vitamin, or any combination of any of the foregoing.

[0041] Also, under certain circumstances, a targeting moiety cannot directly bind to the PEG-lipid. In these circumstances, a molecular bridge in the form of a crosslinking agent may be used to facilitate the interaction. In certain embodiments, it is advantageous to use a crosslinking agent if steric restrictions of the targeting moiety directly conjugated to the PEG-lipid prevents sufficient interaction with the intended physiological target. Additionally, if the targeting moiety structure is only functional under certain orientations (e.g., monoclonal antibody), linking to a PEG-lipid via crosslinking agent is beneficial. Traditional processes of bioconjugation may be used to link the targeting moiety to the PEG-lipid. Reducible or hydrolysable linkages may be applied to prevent accumulation of the formulation in vivo and subsequent cytotoxicity.

[0042] In some instances, the PEG-lipid is DSPE-PEG, such as DSPE-PEG2000 or DSPE- PEG400. In some instances, the PEG-lipid is DSPC-PEG, such as DSPC-PEG2000 or DSPC-PEG- 400. In some instances, the PEG-lipid is DSG-PEG.

[0043] In some instances, the PEG-lipid to be conjugated to a targeting moiety is DSPE- PEG, such as DSPE-PEG2000, DSPE-PEG400, DSPE-PEG2000- Azide, DSPE-PEG₄oo-Azide, DSPE-PEG2ooo-maleimide, or DSPE-PEG4oo-maleimide. In some instances, the PEG-lipid to be conjugated to a targeting moiety is DSPC-PEG, such as DSPC-PEG2000, DSPC-PEG400, DSPC- PEG2000- Azide, DSPC-PEG4oo-Azide, DSPC-PEG2ooo-maleimide, or DSPC-PEG4oo-maleimide. In some instances, the PEG-lipid to be conjugated to a targeting moiety is DSG-PEG, such as DSG-PEG2000, DSG-PEG400, DSG-PEG₂ooo-Azide, DSG-PEG₄oo-Azide, DSG-PEG2000- maleimide, or DSG-PEG4oo-maleimide. [0044] In one embodiment of any of the processes described herein, the PEG-lipid in any of the components described herein can be selected from the PEG-lipids described in International Publication No. WO 2007/012191 and WO 2015/048020, which is hereby incorporated by reference.

[0045] In some embodiments, the buffer solution in the dilution solution is phosphate- buffered saline (PBS). In some instances, the buffer solution is Tris-buffered saline (TBS).

[0046] In some embodiments, the dilution solution may comprise a non-targeted PEG lipid. In some instances, the non-targeted PEG-lipid is DSPE-PEG, such as DSPE-PEG2000 or DSPE-PEG400. In some instances, the non-targeted PEG-lipid is DSPC-PEG, such as DSPC- PEG2000 or DSPC-PEG400. In some instances, the non-targeted PEG-lipid is DSG-PEG, such as DSG-PEG2000 or DSG-PEG400.

[0047] In one embodiment of any of the embodiments described herein, the PEG in the PEG-lipid, targeted PEG-lipid, or non-targeted PEG-lipid, has a molecular weight ranging from 200 to 4000, such as from 300 to 3000, or 400 to 2000 daltons.

[0048] In some embodiments, the concentration of the targeted PEG-lipid ranges from about 10 ng/ml. to about 400 pg/mL. In some instances, the concentration of the targeted PEG- lipid ranges from about 15 pg/mL to about 250 pg/mL

[0049] The aqueous solution comprises water, a therapeutic nucleic acid, and optionally a weak acid. In some instances, the weak acid is citric acid, acetic acid, formic acid, oxalic acid, nitrous acid, sulphurous acid, phosphoric acid, benzoic acid, a pharmaceutically acceptable salt thereof, or any combination of any of the foregoing.

[0050] The process and apparatus disclosed herein are useful in the preparation and manufacture of lipid nanoparticles carrying a therapeutic payload (e.g., a therapeutic agent), such as a nucleic acid payload. Therapeutic agent payloads include proteins, peptides, plasmids, nucleic acids (such as a gene editor, mRNA, siRNA, antisense, aptamer, ribozyme, tRNA, snRNA, siRNA, shRNA, or ncRNA), small molecules, large molecules, antibodies, and fragment antibody binding regions (Fab’s). [0051] In one embodiment, the nucleic acid has a length of 9 to 30 or 13 to 25 nucleobases, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases.

[0052] The nucleic acid payload may include mRNA and RNAi agents (e.g. siRNA, dsRNA, and miRNA) as well as antisense molecules, ribozymes, and plasmid-based constructs or any nucleic acid based molecules. Suitable nucleic acid payloads and RNAi agents are described in International Publication No. WO 2007/012191, WO 2015/048020, and WO 2023/178167 which are hereby incorporated by reference. As used herein a "therapeutic payload" is any compound, substance or molecule which has a therapeutic benefit and which can be incorporated into or encapsulated within a lipid nanoparticle made by the processes described herein.

[0053] As used herein, the term "RNAi agent" refers to an agent that contains RNA, and which mediates the targeted cleavage of an RNA transcript or target sequence via an RNA- induced silencing complex (RISC) pathway. The RNA may be expressed or found in nature, or alternatively by an analog or derivative thereof.

[0054] The term "double-stranded RNA" or "dsRNA," as used herein, refers to an RNA (such as an RNAi agent) that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having "sense" and "antisense" orientations with respect to a target RNA.

[0055] The term "antisense strand" or "antisense molecule" refers to the strand of an RNAi agent, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus. [0056] The term "sense strand" as used herein, refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

[0057] The duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs.

[0058] The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a "hairpin loop") between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a "linker." The term "siRNA" is also used herein to refer to a dsRNA as described above. [0059] In one aspect, an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to first the cleavage of the target RNA.

[0060] In yet another embodiment, the RNA of an RNAi agent, e.g., a dsRNA or siRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by processes well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. eta! . (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3' end modifications ( conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases ( abasic nucleotides), or conjugated bases, ( c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages.

[0061] The nucleic acid, such as RNA or RNAi agent, may chemically linked one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the nucleic acid. Such moieties include but are not limited to lipid moieties such as a sterol moiety (such as a cholesterol moiety), peptides, peptidomimetics, and vitamins.

[0062] In some embodiments, the concentration of the therapeutic payload, such as a therapeutic nucleic acid, ranges from about 1 pg/mL to about 200 pg/mL. In some instances, the concentration of the therapeutic payload, such as a therapeutic nucleic acid, ranges from about 40 pg/mL to about 200 pg/mL. In some instances, the concentration of the therapeutic payload, such as a therapeutic nucleic acid, ranges from about 50 pg/mL to about 180 pg/mL.

[0063] In some embodiments, the nucleic acid may be mRNA, which may be synthesized as unmodified or modified mRNA. The term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. Modified mRNA comprise nucleotide modifications in the RNA. A modified mRNA can include nucleotide modification that are, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g., 1 -methyl -adenine, 2-methyl-adenine, 2-methylthio-N-6- isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl- cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl- guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5- uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-u racil, 5-fluoro-u racil, 5-bromo-uracil, 5-ca rboxymethyla minomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'- methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5- oxyacetic acid (v), 1 -methylpseudouracil, queuosine, beta-D-mannosyl-queuosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g., from the U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.

[0064] In some embodiments, the organic solution comprises a solvent (such as ethanol), an ionizable lipid, a non-cationic lipid, and sterol (such as cholesterol). In some instances, the non-cationic lipid may be an anionic lipid. In some instances, the non-cationic lipid may be a neutral lipid. In some instances, the non-cationic lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), 20 palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl-25 phosphatidy ethanolamine (SOPE), cholesterol, or any combination of any of the foregoing. In some instances, the non-cationic lipid may be distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl- phosphatidylethanolamine (POPE) and di oleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-0- monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1 -stearoyl- 2-oleoyl- phosphatidy ethanolamine (SOPE), 1 ,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), or any combination of any of the foregoing. In some instances, the non-cationic lipid is selected from phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids. In some instances, the non-cationic lipid is DSPC. Suitable non-cationic lipids are described in International Publication No. WO 2007/012191 and WO 2015/048020, which is hereby incorporated by reference.

[0065] In some embodiments, the organic solution comprises an ionizable lipid. In some instances, the ionizable lipid is ALC-0315, C12-200, cKK-E12, Dlin-MC3-DMA, SM-102, LpO l, CL1, TCL053, or any combination of any of the foregoing. In some instances, the ionizable lipid is a multi-chargeable lipid. In some instances, the ionizable lipid is selected from DLinDMA, DLin-MC3-DMA, DLin-KC2-DMA, Di-ol eyl-succinyl-serinyltobramycin, Di-ol eyl- adipyl-tobramycin, Di-oleyl-suberyl-tobramycin, Di-oleyl-sebacyl-tobramycin, N,N-dimethyl- N',N'-di[(9Z, 12Z)-octadeca-9,12-dien-l -yl] ethane- 1,2-diamine, Di-oleyl-dithioglycolyl- tobramycin, or any combination of any of the foregoing. Other suitable ionizable agents (such as cationic lipids) are described in International Publication No. WO 2007/012191 and WO 2015/048020, which is hereby incorporated by reference.

[0066] In some embodiments, the total lipid concentration of the organic solution ranges from about 1 mg/mL to about 30 mg/mL. In some instances, the total lipid concentration of the organic solution ranges from about 7.5 mg/mL to about 26 mg/mL.

[0067] In some embodiments, the aqueous solution and organic solution are mixed together in a mixing connector to form a first solution. In some instances, the flow rate of the aqueous solution ranges from about 5 mL/min to about 10 mL/min. In some instances, the flow rate of the aqueous solution ranges from about 40 mL/min to about to about 60 mL/min. In some instances, the flow rate of the aqueous solution ranges from about 1 mL/min to about 70 mL/min. In some instances, the flow rate of the organic solution ranges from about 1 mL/min to about 5 mL/min. In some instances, the flow rate of the organic solution ranges from about 1 mL/min to about 20 mL/min. In some instances, the flow rate of the organic solution ranges from about 10 mL/min to about 20 mL/min.

[0068] A dilution solution is mixed in with the first solution in a mixing connector. In some instances, the flow rate of the of the first solution ranges from about 1 mL/min to about 100 mL/min. In some instances, the flow rate of first solution ranges from about 50 mL/min to about 75 mL/min. In some instances, the flow rate of the dilution solution ranges from about 1 mL/min to about 100 mL/min. In some instances, the flow rate of the dilution solution ranges from about 50 mL/min to about 75 mL/min. In some instances, the combined solution of the dilution solution and first solution exits the mixing connector at a flow rate ranging from about 1 mL/min to about 200 mL/min. In some instances, the combined solution of the dilution solution and first solution exits the mixing connector at a flow rate ranging from about 100 mL/min to about 150 mL/min.

[0069] In some embodiments, solutions are mixed in a mixing connector. In some instances, the solutions are mixed in a staggard T-junction, such as a Tee mixer (for example, having a mixing bore of about 0.01 to about 0.05 inches, such as about 0.02 to about 0.04 inches). In this instance, it is preferred that the fluid lines, and hence fluid flows, meet in a narrow aperture within the "T"-connector as opposing flows at approximately 180° relative to each other. Other mixing chambers or connectors having shallower relative introduction angles may be used, such as for example between 27° and 90° and between 90° and 180°. Upon meeting and mixing of the solution flows in the mixing environment, lipid vesicles are substantially instantaneously formed. Lipid vesicles are formed when an organic solution including dissolved lipid and an aqueous solution (e.g., buffer) are simultaneously and continuously mixed. Advantageously, and surprisingly, by mixing the dilution solution with the mixed product from the organic and aqueous solutions, a lipid nanoparticle incorporating the targeted PEG-lipid is formed with good uniformity of the target PEG-lipid in the lipid nanoparticles and without material degradation of the targeting moiety of the targeted PEG-lipid. The pump mechanism(s) can be configured to provide equivalent or different flow rates of the organic solution, aqueous solution, first solution, and dilution solution into the mixing environment which creates lipid nanoparticles having targeted PEG-lipids. Each mixing step may include stirring.

[0070] In one embodiment, the aqueous solution and organic solution are introduced into a mixing connector as opposing flows. The mixing connector may be a T-connector or a Y- connector. The mixing connector may include herringbone structures and/or structures that generate dean vortices.

[0071] In one embodiment, the first solution and dilution solution are introduced into a mixing connector as opposing flows. The mixing connector may be a T-connector or a Y- connector. The mixing connector may include herringbone structures and/or structures that generate dean vortices.

[0072] The mixing steps may be performed as described in International Publication No. WO 2022/194615, which is hereby incorporated by reference. The mixing systems described in WO 2022/194615 may be used in the process described herein.

[0073] The solution including the lipid nanoparticles encapsulating the therapeutic payload may have a concentration of about 20% v/v to about 55% v/v buffer. The solution including the lipid nanoparticles encapsulating the therapeutic agent may have a concentration of about 20% v/v to about 55% v/v buffer. The lipid nanoparticle solution may have a concentration of less than about 25% v/v buffer. The lipid nanoparticles may have a diameter less than about 150 nm. The lipid nanoparticles may have a diameter greater than 20 nm.

[0074] The appartus may include a continuous flow microfluidic platform, such as a NanoAssemblr.

[0075] In some embodiments, the lipid nanoparticles are isolated after the dilution solution is mixed with the first solution. In some instances, the lipid nanoparticles are isolated via dialysis. In some instances, the lipid nanoparticles are isolated via tangential flow filtration.

[0076] In one embodiment, the lipid nanoparticles may include about 30 to about 60 mol % ionizable lipid, about 10 to about 50 mol % non-cationic lipid, about 30 to about 60 mol %, about 10 to about 50 mol % sterol, and about 1 to about 10 mol % PEG-lipid (in total for both targeted PEG-lipid and non-targeted PEG-lipid).

Example 1

[0077] Lipid nanoparticles having a targeting moiety are prepared as follows. An organic solution is prepared by dissolving an ionizable lipid, DSPC, and cholesterol in ethanol. An aqueous solution is prepared by mixing a nucleic acid (such as an RNA) in a lOmM citrate solution (pH 4.0). The aqueous and organic solutions are combined at a 3 : 1 flow ratio using a microfluidics device, with a combined flow rate of 12 mL/min to form a first solution. A dilution solution was prepared by diluting DMG-PEG2000, DSPE-PEG2000-Azide-DBCO-Fab, and DSPE-PEG2000-Azide in TBS (or PBS). The dilution stream is added to the first solution at a flow rate of 12 mL/min for a 1 : 1 volume ratio with the first solution, combining immediately after passing through the microfluidics device.

Example 2 [0078] Targeted lipid nanoparticles (tl.NPs) were prepared by a process analogous to that described in Example 1, but with Fab mCD5. The flow rate of the organic, aqueous, and dilution solutions were varied as shown in the legend of Figure 3. The ratios recited in the legend are the flow rates (mL/min) for the organic, aqueous, and dilution solutions (e.g., 10:30:40 refers to flow rate for the organic, aqueous, and dilution solutions of 10, 30, and 40 mL/min, respectively). The concentrations recited in the legend (e.g., 60, 90, or 120 pg/mL) are the RNA concentrations in the lipid nanoparticle. The formulations were tested in CD4+ T-cells, CD8+ T-cells, and total T-cells from mice. The percentage of GFP+ cells is shown in Figure 3 and compared to vehicle (TBS). All the lipid nanoparticles resulted in delivery of the RNA cargo.

Example 3

[0079] Targeted lipid nanoparticles (tLNPs) were prepared by an in-line dilution process analogous to that described in Example 1, but with DSPE-PEG-Fab. Size exclusion chromatography-fluorescence detection (SEC-FLD) was performed on DSPE-PEG-Fab, which included DSPE-PEG-Fab micelles and DSPE-PEG-Fab monomers. SEC-FLD was also performed on the tLNPs. The results are shown in Figure 4A. The tlNPs prepared included a small amount of DSPE-PEG-Fab micelles (which are not incorporated into the tLNPs) and a very small amount of DSPE-PEG-Fab monomers which were not incorporated into the tLNPs. The DSPE-PEG-Fab monomers can be removed from the tLNP by size exclusion chromatography.

[0080] Additional targeted lipid nanoparticles were prepared by a post-conjugation process, that is, lipid nanoparticles were prepared and then Fabs (through a handle of the Fab) were conjugated to the PEG-lipid in the lipid nanoparticles. SEC-FLD was performed on these post-conjugation tLNPs. The results are shown in Figure 4B. Figure 4B also shows the SEC- FLD for DSPE-PEG-Fab.

[0081] The post-conjugation tLNPs exhibit a significant Fab-handle peak (i.e., Fab which was not conjugated to the tLNP) in Figure 4B at a retention time of approximately 19 minutes. In contrast, the tLNPs prepared by the in-line dilution process do not exhibit such a peak (see Figure 4A). The present inventors have discovered that even after purification, the postconjugation tLNPs still exhibit a significant Fab-handle peak (e.g., at least 5% (area under the curve) by SEC-FLD).

[0082] All references and patent publications cited herein are hereby incorporated by reference.

Claims

Claims:

1. A continuous in-line process for producing lipid nanoparticles having an antibody or fragment antibody binding region (Fab) targeting moiety and carrying a therapeutic nucleic acid comprising:

(a) in-line mixing an aqueous solution with an organic solution in a mixing connector to form a first solution, wherein

(i) the aqueous solution comprises a therapeutic nucleic acid and optionally a weak acid; and

(ii) the organic solution comprises (A) a solvent (such as ethanol), (B) an ionizable lipid, (C) a non-cationic lipid (such as DSPC), (D) sterol (such as cholesterol), and (E) optionally, a non-targeted PEG-lipid; and

(b) in-line mixing the first solution with a dilution solution to form the lipid nanoparticles, wherein the dilution solution comprises (i) a targeted PEG-lipid comprising a PEG-lipid (such as DSPE-PEG) conjugated to a monoclonal antibody or Fab, (ii) a buffer solution, and (iii) optionally, a non-targeted PEG-lipid.

2. The process of claim 1, wherein the targeted PEG-lipid comprises a DSPE-PEG conjugated to a fragment antibody binding region (Fab).

3. The process of claim 1, wherein the targeted PEG-lipid comprises a DSPE-PEG conjugated to a monoclonal antibody (mAb).

4. The process of any one of the preceding claims, wherein the weak acid is citric acid or a salt thereof.

5. The process of any one of the preceding claims, wherein the flow rate of the aqueous solution ranges from about 5 mL/min to about 50 mL/min.

6. The process of any one of the preceding claims, wherein the flow rate of the organic solution ranges from about 1 mL/min to about 5 mL/min.

7. The process of any one of the preceding claims, wherein the total lipid concentration in the organic solution ranges from about 5 mg/mL to about 30 mg/mL.

8. The process of any one of the preceding claims, wherein the flow rate of the dilution solution ranges from about 10 mL/min to about 15 mL/min.

9. The process of any one of the preceding claims, wherein the concentration of the conjugated DSPE-PEG ranges from about 15 ug/mL to about 400 ug/mL.

10. The process of any one of the preceding claims, wherein the dilution solution further comprises a non-targeted PEG-lipid, wherein the non-targeted PEG-lipid is DMG- PEG2000.

11. The process of claim 10, wherein the concentration of DMG-PEG2000 ranges from about 0.1 ug/mL to about 200 ug/mL.

12. The process of any one of the preceding claims, wherein the organic solution further comprises a non-targeted PEG-lipid, wherein the non-targeted PEG-lipid is DMG- PEG2000.

13. The process of claim 12, wherein the concentration of DMG-PEG2000 ranges from about 0.1 ug/mL to about 200 ug/mL.

14. The process of any one of the preceding claims, wherein the solutions are mixed through microfluidics.

15. The process of any one of the preceding claims, wherein the buffer solution of the dilution solution is phosphate buffered saline (PBS).

16. The process of any one of claims 1-14, wherein the buffer solution of the dilution solution is Tris buffer saline (TBS).

17. The process of any one of the preceding claims, further comprising isolating the lipid nanoparticles via dialysis.

18. The process of any one of the preceding claims, further comprising isolating the lipid nanoparticles via tangential flow filtration (TFF).

19. The process of any one of the preceding claims, wherein the lipid nanoparticles contain less than 5% (area under the curve) of targeted PEG-lipid micelles which are not incorporated into the lipid nanoparticles, as measured by size exclusion chromatographyfluorescence detection (SEC-FLD).