Process for preparing oligosaccharide complexes

Abstract

The present disclosure provides methods and systems for preparing a pharmaceutical composition comprising a plurality of nucleic acid particles, the method comprising contacting a nucleic acid solution with an organic solution via a mixer configured to receive the nucleic acid solution and the organic solution, wherein: the organic solution comprises an organic solvent and an oligosaccharide comprising one or more cationic moieties.

Description

Attorney Docket No. 2013237-1455PROCESS FOR PREPARING OLIGOSACCHARIDE COMPLEXESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63 / 679,468, filed August 5, 2024, the entirety of which is incorporated herein by reference.BACKGROUND

[0002] Nucleic acid-based therapies have emerged as a promising treatment option for many diseases. For example, nucleic acids can be used to affect expression of particular cellular products, e.g., proteins, and have enormous potential in the fields of infectious diseases, oncology, and the like. Challenges associated with delivery of nucleic acid therapies remain, however, as nucleic acids rapidly degrade in the body before entering a target cell.

[0003] Recent focus around nucleic acid therapies has centered around methods of delivery, with much of the focus on cationic or ionizable polymers or lipids that are useful for forming polyplexes, lipoplexes, or (with the addition of other lipids), lipid nanoparticles. A new class of cationic or ionizable lipids was recently reported in PCT App. No. 2023 / 067121, having an oligosaccharide structure, and has shown particular promise in delivering nucleic acid agents.SUMMARY

[0004] The present disclosure provides, among other things, methods and systems for preparing cationic and / or ionizable oligosaccharide complexes that are prepared by a fluid-path process, and thereby imparts particular improvements over previous oligosaccharide complexes, such as improved colloidal and frozen stability.

[0005] In some embodiments, the present disclosure provides a method of preparing a pharmaceutical composition comprising a plurality of nucleic acid particles, the method comprising contacting a nucleic acid solution with an organic solution via a mixer configured to receive the nucleic acid solution and the organic solution, wherein: the organic solution comprises an organic solvent and an oligosaccharide comprising one or more cationic moieties.

[0006] In some embodiments, the present disclosure provides a drug production system for producing a pharmaceutical composition by a continuous flow manufacturing process, the drugPage 1 of 20712885786vlAttorney Docket No. 2013237-1455 production system comprising a first composition containment unit comprising a nucleic acid solution and a second composition containment unit comprising an organic solution, wherein: the first composition containment unit and the second composition containment unit are connected via a mixer unit configured to receive the nucleic acid solution and the organic solution.BRIEF DESCRIPTION OF THE DRAWING

[0007] FIG. 1 is a diagram of an example process for developing nucleic acid particles described herein.

[0008] FIG. 2A is a schematic representation of nucleic acid particles (referred to as CPLXs) manufacturing process using hand mixing process.

[0009] FIG. 2B is a bar graph illustrating size and PDI of nucleic acid particles (CPLXs) manufactured using hand mixing method and fluid path using DMSO and ethanol as the organic phase.

[0010] FIG. 3 is a schematic representation of nucleic acid particles (CPLXs) manufacturing process using fluid path and DMSO or ethanol as the organic phase.

[0011] FIG. 4 is a schematic representation of nucleic acid particles (CPLXs) manufacturing with fluid path process using Y and T mixer at different flow rates.

[0012] FIGs. 5 A and 5B are bar graphs illustrating size and PDI of raw nucleic acid particles (CPLXs) manufactured with fluid path process using (FIG. 5A) Y and (FIG. 5B) T mixers at different flow rates.

[0013] FIGs. 6A-6D are bar graphs illustrating size, PDI, pH and osmolality of nucleic acid particles (CPLXs) manufactured with fluid path process using Y (FIGs. 6A and 6B) and T (FIGs. 6C and 6D) mixer at different flow rates.

[0014] FIG. 7 is a schematic representation of nucleic acid particles (CPLXs) manufacturing with fluid path process using Y and T mixer at different total flow rates.

[0015] FIGs. 8A and 8B are bar graphs illustrating size and PDI of nucleic acid particles (CPLXs) manufactured using fluid path process with (FIG. 8A) Y and (FIG. 8B) T mixer at different total flow rates.

[0016] FIG. 9 is a schematic representation of nucleic acid particles (CPLXs) manufacturing with fluid path process using T mixer at different manufacturing concentrations (0.05-0.3 mg / ml).Page 2 of 20712885786vlAttorney Docket No. 2013237-1455

[0017] FIG. 10 is a bar graph illustrating size and PDI of nucleic acid particles (CPLXs) manufactured using fluid path process with T mixer, TFR 100 ml / min and 0.05-0.3 mg / mL RNA manufacturing concentration.

[0018] FIG. 11 is a schematic representation of nucleic acid particles (CPLXs) manufacturing with fluid path process using T mixer at different TFR (25-200 ml / min).

[0019] FIG. 12 is a bar graph illustrating size and PDI of nucleic acid particles (CPLXs) manufactured using fluid path process with T mixer at RNA manufacturing cone, of 0.1 mg / ml and different TFR.

[0020] FIG. 13 is a schematic representation of nucleic acid particles (CPLXs) manufacturing with fluid path process and dialyzed against lx MES buffer at different pH (5-6.1).

[0021] FIGs. 14A and 14B are bar graphs illustrating (in FIG. 14A) size, and PDI and (in FIG. 14B) pH and osmolality of nucleic acid particles dialyzed in lx MES buffer at different pH (5- 6.1).

[0022] FIGs. 15A-15E are bar graphs illustrating size and PDI of nucleic acid particles (CPLXs) dialyzed in different pH (FIG. 15A) 5, (FIG. 15B) 5.25, (FIG. 15C) 5.5, (FIG. 15D) 5.75, (FIG. 15E) 6.1 and subjected to 3FT cycles at -80 °C in absence of Tween 40.

[0023] FIGs. 16A-16E are bar graphs illustrating size and PDI of nucleic acid particles (CPLXs) dialyzed in different pH (FIG. 16A) 5, (FIG. 16B) 5.25, (FIG. 16C) 5.5, (FIG. 16D) 5.75, (FIG. 16E) 6.1 and subjected to 3FT cycles at -80 °C in presence of Tween 40.

[0024] FIGs. 17A-17D are bar graphs illustrating size and PDI of nucleic acid particles (CPLXs) subjected to 3FT cycles at -80 °C after spiking with different cone, of Tween 40, measured as a molar concentration relative to JLF99 (0 cone, of Tween in FIG. 17A, 0.125 cone, of Tween in FIG. 17B, 0.25 cone, of Tween in FIG. 17C, or 0.5 cone, of Tween in FIG. 17D).

[0025] FIGs. 18A- 18C are bar graphs illustrating in-vitro cell viability of nucleic acid particles (CPLXs) in three different cell lines (FIG. 18A) C2C12, (FIG. 18B) RAW, and (FIG. 18C) HepG2.

[0026] FIGs. 19A-19C are bar graphs illustrating in-vitro transfection efficiency of nucleic acid particles (CPLXs) in three different cell lines (FIG. 19A) C2C12, (FIG. 19B) RAW, and (FIG. 19C) HepG2.

[0027] FIG. 20 is a schematic representation of nucleic acid particles (CPLXs) manufacturing with fluid path and removal of organic phase using TFF process.Page 3 of 20712885786vlAttorney Docket No. 2013237-1455

[0028] FIGs. 21A and 21B are bar graphs illustrating size, PDI (FIG. 21A), pH, and osmolality (FIG. 2 IB) of nucleic acid particles (CPLXs) before and after TFF process.

[0029] FIGs. 22A-22D are bar graphs illustrating size and PDI of nucleic acid particles (CPLXs) subjected to 3FT cycles at -80 °C after spiking with different cone, of Tween 40, measured as a molar concentration relative to JLF99 (0 cone, of Tween in FIG. 22A, 0.125 cone, of Tween in FIG. 22B, 0.25 cone, of Tween in FIG. 22C, or 0.5 cone, of Tween in FIG. 22D).

[0030] FIGs. 23A-23C are bar graphs illustrating in-vitro cell viability of nucleic acid particles (CPLXs) after TFF with different Tween 40 amounts in three different cell lines (FIG. 23A) C2C12, (FIG. 23B) RAW and (FIG. 23C) HepG2.

[0031] FIGs. 24A-24C are bar graphs illustrating in-vitro transfection efficiency of nucleic acid particles (CPLXs) after TFF with different Tween 40 amounts in three different cell lines (FIG. 24A) C2C12, (FIG. 24B) RAW and (FIG. 24C) HepG2.

[0032] FIG. 25 is a bar graph illustrating particle size and PDI of CPLXs manufactured with JLF 218 after dialysis.

[0033] FIG. 26 is a visualization of agarose gel electrophoresis of CPLXs manufactured with JLF 218. The first and second well indicate CPLXs in absence and presence of heparin and triton X solution respectively. The last 5 wells (2-6) indicate RNA control in different concentrations ( 1 , 0.5, 0.25, 0.125 and 0.0625 mg / ml).

[0034] FIG. 27 is a bar graph illustrating size and PDI of CPLXs manufactured using JLF 218 and subjected to 3FT cycles at -80 °C.

[0035] FIG. 28 is a bar graph illustrating particle size and PDI of CPLXs manufactured with JLF 99 and DNA after dialysis in lOmM MES, pH 6.1.

[0036] FIG. 29 is a visualization of agarose gel electrophoresis of CPLXs manufactured with JLF 99 and DNA (dialysis in MES buffer, pH 6.1). The first and second well indicate CPLXs in absence and presence of heparin and triton X solution respectively. The last well (2) indicates DNA control at a concentration of 0.1 mg / ml.

[0037] FIG. 30 is a bar graph illustrating size and PDI of CPLXs manufactured using JLF 99 and DNA, dialyzed against lOmM MES, pH 6.1 and subjected to 3 freeze thaw cycles at -80 °C.

[0038] FIG. 31 is a bar graph illustrating particle size and PDI of CPLXs manufactured with JLF 99 and DNA after dialysis lOmM MES, pH 5.25.Page 4 of 20712885786vlAttorney Docket No. 2013237-1455

[0039] FIG. 32 is a visualization of agarose gel electrophoresis of CPLXs manufactured with JLF 99 and DNA (dialysis in MES buffer, pH 5.25). The first and second well indicate CPLXs in absence and presence of heparin and triton X solution respectively. The last well (2) indicates DNA control at a concentration of 0.1 mg / ml.

[0040] FIG. 33 is a bar graph illustrating size and PDI of CPLXs manufactured using JLF 99 and DNA, dialyzed against lOmM MES, pH 5.25 and subjected to 3 freeze thaw cycles at -80 °C.

[0041] FIGs. 34A-34C are line graphs showing long-term stability in terms of size (FIGs. 34A- 34B) and RNA integrity (FIG. 34C) of CPLXs manufactured using JLP99.

[0042] FIGs. 35A-35C are bar graphs showing size and PDI of CPLXs manufactured in presence of Tween 40 in upstream at JLF 218: Tween 40 molar ratio (FIG. 35A) 1:0.5, (FIG. 35B) 1:0.25, and (FIG. 35C) 1:0.125, and dialyzed against different buffer conditions - lOmM MES pH 5.25, lOmM HEPES pH 7.4, and IX PBS pH 7.4.

[0043] FIG 36. is a visualization of agarose gel electrophoresis of CPLXs manufactured using JLF 218 in presence of different amounts of Tween 40 in upstream. Wells 1 ,5, and 8 correspond to CPLXs dialyzed against lOmM MES pH 5.25. Wells 2, 6, and 9 correspond to CPLXs dialyzed against lOmM HEPES pH 7.4. Wells 3, 7, and 10 correspond to CPLXs dialyzed against IX PBS pH 7.4. Wells 11-15 indicate RNA control at different concentrations (1, 0.5, 0.25, 0.125 and 0.0625 mg / ml).

[0044] FIGs. 37A and 37B are bar graphs showing size and PDI of CPLXs manufactured using JLF 218 in presence of Tween 40 at 0.5, 0.25, and 0.125 molar ratios (FIG. 37A) after dialysis and (FIG. 37B) up-concentrated under different buffer conditions as final matrix (lOmM MES pH 5.25, lOmM HEPES pH 7.4 and IX PBS pH 7.4). In FIGs. 37A and 37B, x axis corresponds to different molar ratios of Tween 40 in upstream with respect to JLF 218. In FIG. 37B, values above each bar correspond to RNA cone, (in mg / ml) after up-concentration of CPLXs (measured from Ribogreen assay).

[0045] FIGs. 38A and 38B are bar graphs showing size and PDI of CPLXs manufactured using JLF 218 in presence of Tween 40 in upstream (0.5 mol ratio with respect to JLF 218), in final storage matrix of 10% sucrose and (FIG. 38A) lOmM MES pH 5.25 (FIG. 38B) IX PBS pH 7.4 and subjected to 3FT cycles at -80 °C at 0.76 mg / mL and 0.42 mg / mL, respectively.

[0046] FIGs. 39A-39C are bar graphs showing in vitro cell viability of CPLXs manufactured using JLF 218 in presence of different amounts of Tween in upstream (0-0.5 mol ratio with respectPage 5 of 20712885786vlAttorney Docket No. 2013237-1455 to JLF 218), dialyzed under three different buffer conditions ( lOmM MES pH 5.25, lOmM HEPES pH 7.4 and IX PBS pH 7.4) in three different cell lines (FIG. 39A) C2C12, (FIG. 39B) HepG2, and (FIG. 39C) RAW.

[0047] FIGs. 40A-40C are bar graphs showing in vitro transfection efficiency of CPEXs manufactured using JEF 218 in presence of different amounts of Tween in upstream (0-0.5 mol ratio with respect to JLF 218), in dialyzed under three different buffer conditions (lOmM MES pH 5.25, lOmM HEPES pH 7.4 and IX PBS pH 7.4) in three different cell lines (FIG. 40A) C2C12, (FIG. 40B) HepG2, and (FIG. 40C) RAW.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTSDefinitions

[0048] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75thEd. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5thEd., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0049] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, Tables 1 or 2 shows one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0050] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, Page 6 of 20712885786vlAttorney Docket No. 2013237-1455 compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by13C- or14C-enriched carbon are within the scope of this disclosure.

[0051] About or approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In general, those skilled in the art, familiar within the context, will appreciate the relevant degree of variance encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" may encompass a range of values that are within (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0052] Aliphatic: The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., Ci-e). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6 alkyl.

[0053] Alkyl: The term “alkyl”, used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., Ci-12, Ci-10, Ci-8, Ci-6, CM, Ci- 3, or C1-2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0054] Alkylene: The term “alkylene” refers to a bivalent alkyl group. In some embodiments, “alkylene” is a bivalent straight or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH2)n, wherein n is a positive integer, e.g., from 1 to 6,Page 7 of 20712885786vlAttorney Docket No. 2013237-1455 from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include those described in the specification herein. It will be appreciated that two substituents of the alkylene group may be taken together to form a ring system. In certain embodiments, two substituents can be taken together to form a 3- to 7-membered ring. The substituents can be on the same or different atoms. The suffix “-ene” or “-enyl” when appended to certain groups herein are intended to refer to a bifunctional moiety of said group. -For example, “-ene” or “-enyl”, when appended to “cyclopropyl” becomes “cyclopropylene” or “cyclopropylenyl” and is intended to refer to a bifunctional cyclopropyl group, e.g.,.

[0055] Alkenyl: The term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms(e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0056] Alkynyl: The term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0057] Aryl: The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an “aryl” group contains between six and twelve total ring members (e.g., C6-C12). The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are Page 8 of 20712885786vlAttorney Docket No. 2013237-1455 hydrocarbons. In some embodiments, an “aryl” ring system is an aromatic ring (e.g., phenyl) that is fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings include that are fused include

[0058] Biological sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0059] Carrier: As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal,Page 9 of 20712885786vlAttorney Docket No. 2013237-1455 vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are or include one or more solid components.

[0060] Comparable. As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0061] Composition: Those skilled in the art will appreciate that the term “composition” may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc.

[0062] Cycloaliphatic. As used herein, the term “cycloaliphatic” refers to a monocyclic C3-8 hydrocarbon or a bicyclic Ce-io hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.

[0063] Cycloalkyl. As used herein, the term “cycloalkyl” refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0064] Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example,Page 10 of 20712885786vlAttorney Docket No. 2013237-1455 starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0065] Heteroaliphatic. The term “heteroaliphatic” or “heteroaliphatic group”, as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2- CH2-O-CH2-CH2-O-CH3, and the like.

[0066] Heteroaryl: The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 ^-electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[l,2-a]pyrimidinyl, imidazo[l,2-a]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl,Page 11 of 20712885786vlAttorney Docket No. 2013237-1455 cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H quinolizinyl, carbazolyl, acridinyl, phenazinyl, pheno thiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-l,4-oxazin-3(4H)-one, 4H-thieno[3,2-b]pyrrole, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

[0067] Heteroatom: The term “heteroatom” as used herein refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.

[0068] Heterocycle: As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10-membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+(as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydro thienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl.Page 12 of 20712885786vlAttorney Docket No. 2013237-1455A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.

[0069] Parenteral: The phrases “parenteral administration” and “administered parenterally” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0070] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond between ring atoms. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (e.g., aryl or heteroaryl) moieties, as herein defined.

[0071] Patient or subject: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient or a subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient or subject displays one or more symptoms of a disorder or condition. In some embodiments, a patient or subject has been diagnosed with one or more disorders or conditions. In some embodiments, a patient or a subject is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition.

[0072] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic or dosing regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administrationPage 13 of 20712885786vlAttorney Docket No. 2013237-1455 in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0073] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0074] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).

[0075] Substituted or optionally substituted: As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogensPage 14 of 20712885786vlAttorney Docket No. 2013237-1455 otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes provided herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

[0076] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)o^R°; -(Cffcjo^OR0; -0(CH2)o-4R°, -O- (CH2)O^C(0)OR°; -(CH2)O-4CH(OR°)2; -(CH2)O^SR°; -(CH2)o^Ph, which may be substituted with R°; -(CH2)o^O(CH2)o-iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)O^O(CH2)O-I -pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)O^N(R°)2; -(CH2)O-4N(R°)C(0)R°; -N(R°)C(S)R°; -(CH2)O-4N(RO)C(O)NR°2; -N(RO)C(S)NR°2; -(CH2)O^N(R0)C(0)OR°;N(R°)N(R°)C(O)R°; -N(R°)N(RO)C(O)NRO2; -N(R°)N(R°)C(O)OR°; -(CH2)o^C(0)R°; C(S)R°; -(CH2)O^C(0)OR°; -(CH2)O^C(0)SR°; -(CH2)o^C(0)OSiR°3; -(CH2)o^OC(0)R°; -OC(0)(CH2)O-4SR°; -(CH2)O^SC(0)R°; -(CH2)O^C(0)NR02; -C(S)NRO2; -C(S)SR°; - SC(S)SR°, -(CH2)O^OC(0)NR02; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)RO; - C(NOR°)R°; -(CH2)O-4SSR°; -(CH2)O^S(0)2R0; -(CH2)O^S(0)2OR0; -(CH2)O^OS(0)2R0; - S(O)2NR°2; -(CH2)O^S(0)R°; -N(RO)S(O)2NR°2; -N(RO)S(O)2R°; -N(OR°)R°; -C(NH)NRO2; - P(O)2R°; -P(O)RO2; -OP(O)RO2; -OP(O)(ORO)2; SiR°3; -(C1-4 straight or branched alkylene)O- N(RO)3; or — (Ci— 4 straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH2PI1, -0(CH2)o- iPh, -CH2-(5- to 6-membered heteroaryl ring), or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken togetherPage 15 of 20712885786vlAttorney Docket No. 2013237-1455 with their intervening atom(s), form a 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0077] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o-2R*, -(haloR*), -(CH2)O-2OH, -(CH2)O-2OR*, -(CH2)O-2CH(OR*)2, -O(haloR’), -CN, -N3, -(CH2)o-2C(O)R*, -(CH2)O-2C(0)OH, -(CH2)O-2C(0)OR*, -(CH2)O-2SR*, -(CH2)O-2SH, -(CH2)O-2NH2, - (CH2)o-2NHR*, -(CH2)O-2NR*2, -NO2, -SiR*3, -OSiR*3, -C(O)SR*, -(Ci^ straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, - CH2PI1, -0(CH2)o-iPh, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0078] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0 (“oxo”), =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*,wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*2)2-3O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0079] Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.Page 16 of 20712885786vlAttorney Docket No. 2013237-1455

[0080] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -Rt, -NRf2, -C(O)Rf, -C(O)ORf, -C(O)C(O)Rt, C(O)CH2C(O)Rt, -S(O)2Rf, -S(O)2NRt2, -C(S)NRf2, -C(NH)NRf2, or -N(Rt)S(O)2Rt; wherein each R' is independently hydrogen, Ci-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R', taken together with their intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0081] Suitable substituents on the aliphatic group of R' are independently halogen, - R*, -(haloR*), -OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 3- to 6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0082] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer.

[0083] In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and / or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and / or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid.Page 17 of 20712885786vlAttorney Docket No. 2013237-1455

[0084] In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent.

[0085] Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms such as, for example, crystal forms (e.g., polymorphs, solvates, etc.), salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc.

[0086] Those of ordinary skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form.

[0087] Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms.

[0088] Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g.,2H or3H for H;nC,13C or14C for12C;13N or15N for14N;17O or18O for16O;36C1 for35C1 or37C1;18F for19F;131I for127I; etc.). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof.

[0089] In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form.

[0090] In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and / or utilized in accordance with the present disclosurePage 18 of 20712885786vlAttorney Docket No. 2013237-1455 in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[0091] Those skilled in the art will further appreciate that, in small molecule structures, the symbol •nnn' , as used herein, refers to a point of attachment between two atoms. Additionally, or alternatively, the symbol refers to a point of attachment ring in a spirocyclic manner.Methods for Preparing Particles Comprising Oligosaccharides and Nucleic Acids

[0092] The present disclosure provides, among other things, methods for preparing a pharmaceutical composition comprising a plurality of nucleic acid particles and an oligosaccharide. Methods provided herein represent improvements over previously provided processes, as the resulting particles (i.e., the complex of the oligosaccharide and the nucleic acid) exhibit improved properties relative to previously reported complexes, including improvements related to colloidal stability and frozen stability, which are demonstrated in Examples 8-14.

[0093] Oligosaccharide compounds useful for forming complexes with nucleic acid agents are reported in WO2023 / 067121, WO2023 / 067123, WO2023 / 067124, WO2023 / 067125, and WO2023 / 067126, each of which is incorporated by reference herein in its entirety. The present disclosure provides an improved process for preparing said particles relative to earlier work.Page 19 of 20712885786vlAttorney Docket No. 2013237-1455

[0094] As described herein, the present disclosure provides methods for preparing a pharmaceutical composition comprising a plurality of nucleic acid particles, the method comprising contacting a nucleic acid solution with an organic solution via a mixer configured to receive the nucleic acid solution and the organic solution, wherein: the organic solution comprises an organic solvent and an oligosaccharide comprising one or more cationic moieties.

[0095] Processes described herein yield a pharmaceutical composition comprising a plurality of nucleic acid particles, wherein said nucleic acid particles can also be referred to as “complexes” of oligosaccharide(s) and nucleic acid(s). Particles within a pharmaceutical composition can be characterized by the average features of the plurality of particles, e.g., a pharmaceutical composition may comprise particles having an average size, or an average charge ratio, and the like. A person of skill in the art will understand and appreciate that not every single particle within the pharmaceutical composition must have the same features, and instead the average features are used to describe the complete pharmaceutical composition.

[0096] For example, nucleic acid particles that are complexes of oligosaccharides and nucleic acids can be characterized by an “N / P ratio.” The N / P ratio is a molar ratio of cationic groups (or ionizable groups that can become cationic, referred to as “N” of N / P) in a composition comprising cationic oligosaccharide compounds relative to anionic groups in a composition comprising a nucleic acid (e.g., mRNA) (referred to as “P” of N / P). An N / P ratio can also be expressed as a single digit, e.g., 5, where it is understood to refer to the indicated number as a ratio of X: 1. For example, an N / P of 5, is intended to refer to an N / P of 5: 1. Similarly, an N / P of 10, is intended to refer to an N / P of 10: 1, etc.

[0097] In some embodiments, an N / P ratio of the organic solution and the nucleic acid solution, when mixed together via the mixer, is from about 2 to about 12. In some embodiments, an N / P ratio of the organic solution and the nucleic acid solution, when mixed together via the mixer is from about 4 to about 10. In some embodiments, an N / P ratio of the organic solution and the nucleic acid solution, when mixed together via the mixer is about 6. In some embodiments, an N / P ratio of the organic solution and the nucleic acid solution, when mixed together via the mixer, is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0098] In some embodiments, a concentration of the oligosaccharide in the organic solution is from about 10 mM to about 50 mM. In some embodiments, a concentration of the oligosaccharide in the organic solution is from about 15 mM to about 40 mM. In some embodiments, aPage 20 of 20712885786vlAttorney Docket No. 2013237-1455 concentration of the oligosaccharide in the organic solution is from about 15 mM to about 35 mM. In some embodiments, a concentration of the oligosaccharide in the organic solution is from about 20 mM to about 30 mM. In some embodiments, a concentration of the oligosaccharide in the organic solution is from about 20 mM to about 25 mM. In some embodiments, a concentration of the oligosaccharide in the organic solution is about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM.

[0099] In some embodiments, an organic solution further comprises a surfactant. In some embodiments, a surfactant is selected from a polysorbate (e.g., polysorbate 20 (Tween20), polysorbate 40 (Tween40), polysorbate 60 (Tween60), and polysorbate 80 (Tween80)), or combination thereof, poloxamers, and an amphiphilic group comprising a moiety selected from polyalkylene glycols (e.g., polyethylene glycol), poly(2-oxazoline), poly(2-methyl-2-oxazoline), polysarcosine, polyvinylpyrrolidone, and poly[N-(2-hydroxypropyl)methacrylamide, wherein the moiety is bonded to one or more C12-C20 aliphatic groups. In some embodiments a surfactant is selected from a polysorbate (e.g., polysorbate 20 (Tween20), polysorbate 40 (Tween40), polysorbate 60 (Tween60), and polysorbate 80 (Tween80)). In some embodiments, a surfactant is polysorbate 20. In some embodiments, a surfactant is polysorbate 40. In some embodiments, a surfactant is polysorbate 60. In some embodiments, a surfactant is polysorbate 80.

[0100] In some embodiments, a molar ratio of oligosaccharide to surfactant in the organic solution is from about 1:0.0075 to about 1:3. In some embodiments, a molar ratio of oligosaccharide to surfactant in the organic solution is from about 1:0.5 to about 1:0.01. In some embodiments, a molar ratio of oligosaccharide to surfactant is about 1:0.0075 to about 1: 1.5. In some embodiments, a molar ratio of oligosaccharide to surfactant is about 1: 1. In some embodiments, a molar ratio of oligosaccharide to surfactant is about 1:0.0075, about 1:0.01, about 1:0.1, about 1:0.5, about 1: 1, about 1:1.5, about 1:2, about 1:2.5. or about 1:3.

[0101] In some embodiments, a concentration of the surfactant in the organic solution is from about 50 mM to about 200 mM. In some embodiments, a concentration of the surfactant in the organic solution is from about 50 mM to about 125 mM. In some embodiments, a concentration of the surfactant in the organic solution is about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, aboutPage 21 of 20712885786vlAttorney Docket No. 2013237-1455100 mM, about 105 mM, about 110 mM, about 115 mM, or about 120 mM. In some embodiments, a concentration of the surfactant in the organic solution is about 100 mM.

[0102] In some embodiments, processes for preparing pharmaceutical compositions described herein are performed by a fluid path process. In some embodiments, a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is from about 25 ml / min to about 360 ml / min. In some embodiments, a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is from about 25 ml / min to about 250 ml / min. In some embodiments, a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is from about 25 ml / min to about 200 ml / min. In some embodiments, a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is from about 50 ml / min to about 150 ml / min. In some embodiments, a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is about 100 ml / min. In some embodiments, a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is about 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 55 ml / min, 60 ml / min, 65 ml / min, 70 ml / min, 75 ml / min, 80 ml / min, 85 ml / min, 90 ml / min, 100 ml / min, 105 ml / min, 110 ml / min, 115 ml / min, 120 ml / min, 125 ml / min, 130 ml / min, 135 ml / min, 140 ml / min, 145 ml / min, or 150 ml / min.

[0103] In some embodiments, a nucleic acid solution and an organic solution are contacted via a mixer at a ratio (e.g., volume ratio) of about 1:1 to about 5: 1. In some embodiments, a nucleic acid solution and an organic solution are contacted via a mixer at a ratio (e.g., volume ratio) of about 1: 1, 2: 1, 3: 1, 4:1, or 5: 1. In some embodiments, a nucleic acid solution and an organic solution are contacted via a mixer at a ratio (e.g., volume ratio) of about 3: 1.

[0104] In some embodiments, a mixer is a T-shaped mixer. In some embodiments, a mixer is a T-shaped mixer having a diameter of about 1 mm. In some embodiments, a mixer is a Y-shaped mixer. In some embodiments, a mixer is a Y-shaped mixer having a diameter that is about 1 mm.

[0105] In some embodiments, a pH of the nucleic acid solution is from about 4 to about 7. In some embodiments, a pH of the nucleic acid solution is from about 6 to about 7. In some embodiments, a pH of the nucleic acid solution is about 4, about 5, about 6, or about 7.

[0106] In some embodiments, an organic solvent is a polar solvent. In some embodiments, an organic solvent is an alcohol. In some embodiments, an organic solvent is methanol, ethanol, or isopropanol. In some embodiments, an organic solvent is methanol. In some embodiments, an organic solvent is ethanol. In some embodiments, an organic solvent is isopropanol.Page 22 of 20712885786vlAttorney Docket No. 2013237-1455

[0107] In some embodiments, an organic solvent is NMP, DMF, THF, or DMSO. In some embodiments, an organic solvent is NMP. In some embodiments, an organic solvent is DMF. In some embodiments, an organic solvent is THF. In some embodiments, an organic solvent is DMSO. In some embodiments, an organic solvent is ethanol or DMSO.

[0108] Pharmaceutical compositions described herein can be characterized by a concentration of nucleic acid as part of the complexes that make up said pharmaceutical composition. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is less than or equal to 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.025 mg / ml to 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.025 mg / ml to about 0.1 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.025 mg / ml to about 0.250 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.025 mg / ml to about 0.5 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.025 mg / ml to about 0.750 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.025 mg / ml to 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.05 mg / ml to about 0.1 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.05 mg / ml to about 0.250 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.05 mg / ml to about 0.5 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.05 mg / ml to about 0.750 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.05 mg / ml to about 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.1 mg / ml to about 0.250 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.1 mg / ml to about 0.5 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.1 mg / ml to about 0.750 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acidPage 23 of 20712885786vlAttorney Docket No. 2013237-1455 concentration that is from about 0.1 mg / ml to about 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.250 mg / ml to about 0.5 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.250 mg / ml to about 0.750 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.25 mg / ml to about 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.5 mg / ml to about 0.750 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.5 mg / ml to about 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is from about 0.750 mg / ml to about 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is about 0.025 mg / ml, about 0.05 mg / ml, about 0.075 mg / ml, about 0.1 mg / ml, about 0.125 mg / ml, about 0.150 mg / ml, about 0.175 mg / ml, about 0.2 mg / ml, about 0.225 mg / ml, about 0.250 mg / ml, about 0.275 mg / ml, about 0.3 mg / ml, about 0.325 mg / ml, about 0.350 mg / ml, about 0.375 mg / ml, about 0.4 mg / ml, about 0.425 mg / ml, about 0.450 mg / ml, about 0.475 mg / ml, about 0.5 mg / ml, about 0.525 mg / ml, about 0.550 mg / ml, about 0.575 mg / ml, about 0.6 mg / ml, about 0.625 mg / ml, about 0.650 mg / ml, about 0.675 mg / ml, about 0.7 mg / ml, about 0.725 mg / ml, about 0.750 mg / ml, about 0.775 mg / ml, about 0.8 mg / ml, about 0.825 mg / ml, about 0.850 mg / ml, about 0.875 mg / ml, about 0.9 mg / ml, about 0.925 mg / ml, about 0.950 mg / ml, about 0.975 mg / ml, or about 1.0 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is about 0.1 mg / ml. In some embodiments, a pharmaceutical composition described herein has a nucleic acid concentration that is about 0.9 mg / ml.

[0109] As described herein, a pharmaceutical composition comprising complexes (i.e., nucleic acid particles) of oligosaccharides and nucleic acids can be characterized by an average diameter of the complexes within the pharmaceutical composition comprising said complexes. In some embodiments, a complex described herein has a diameter of about 20 nm to about 500 nm. In some embodiments, a complex described herein has a diameter of about 30 nm to about 300 nm. In some embodiments a complex described herein has a diameter of about 50 nm to about 250 nm. In some embodiments, a complex described herein has a diameter that is about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. In some embodiments, a complexPage 24 of 20712885786vlAttorney Docket No. 2013237-1455 described herein has a diameter of about 30 nm to about 150 nm. In some embodiments, a complex described herein has a diameter of about 30 nm to about 100 nm. In some embodiments, a complex described herein has a diameter of about 100 nm. In some embodiments, a complex described herein has a diameter of about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nm.

[0110] A pharmaceutical composition comprising complexes of oligosaccharides and nucleic acids can be characterized by a polydispersity index (PDI) of the particle sizes in the pharmaceutical composition. In some embodiments, a pharmaceutical composition comprising particles described herein has a PDI of 0.4 or less. In some embodiments, a pharmaceutical composition comprising particles described herein has a PDI of 0.3 or less.

[0111] The present disclosure further encompasses the recognition that a fluid path process, as described herein, yields a pharmaceutical composition having improved properties relative to a hand-mixing process (as previously described).

[0112] In some embodiments, complexes of oligosaccharides and nucleic acids described herein exhibit improved stability relative to other nanoparticles and / or previously described complexes of oligosaccharides and nucleic acids. For example, in some embodiments, a plurality of complexes (i.e., nucleic acid particles) are stable after one or more freeze-thaw cycles. As described herein, particles that are “stable” with respect a freeze-thaw cycle is intended to refer to those particles that, on average across the pharmaceutical composition, have substantially the same physical characteristics, e.g., substantially the same size (on average) or PDI both before and after the freeze-thaw cycle. For example, in some embodiments, a stable composition is one where there is less than a 20% change in average particle size or PDI in the particles from before to after a freeze-thaw cycle. In some embodiments, a stable composition is one where there is less than a 10% change in average particle size or PDI in the particles from before to after a freeze-thaw cycle. In some embodiments, a stable composition is one where there is less than a 5% change in average particle size or PDI in the particles from before to after a freeze-thaw cycle. In some embodiments, a stable composition is one where there is less than a 1 % change in average particle size or PDI in the particles from before to after a freeze-thaw cycle.

[0113] In some embodiments, complexes described herein are stable across multiple (e.g., more than one) freeze-thaw cycles. For example, a complex described herein is stable after 2, 3, 4, or 5 freeze-thaw cycles.Page 25 of 20712885786vlAttorney Docket No. 2013237-1455

[0114] In some embodiments, a method described herein for preparing a pharmaceutical composition comprising particles of nucleic acids and oligosaccharides further comprises a step of purifying the pharmaceutical composition. In some embodiments, purification, as described herein (e.g., by dialysis or by tangential flow filtration) removes organic phase and up-concentrates the particles in the pharmaceutical composition.

[0115] In some embodiments, the step of purification comprises dialysis. In some embodiments, the pharmaceutical composition is dialyzed against a buffer having a pH of between about 5 and about 8. In some embodiments, the pharmaceutical composition is dialyzed against a buffer having a pH of between about 5.5 and about 7. In some embodiments, the pharmaceutical composition is dialyzed against a buffer having a pH of between about 6 and about 7. In some embodiments, the pharmaceutical composition is dialyzed against a buffer having a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the pharmaceutical composition is dialyzed against a buffer having a pH at about physiological pH (e.g., about 7 to about 7.5).

[0116] In some embodiments, the purification comprises tangential flow filtration (TFF).

[0117] In some embodiments, the present disclosure provides a method for preparing a composition comprising a plurality of nucleic acid particles, the method comprising contacting a nucleic acid solution with an organic solution via a mixer configured to receive the nucleic acid solution and the organic solution, wherein: the organic solution comprises an organic solvent and an oligosaccharide comprising one or more cationic moieties; a N / P ratio of the nucleic acid solution and the organic solution is from about 2 to about 12; and a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is about 25 ml / min to about 360 ml / min. In some embodiments, the method further comprises contacting the composition comprising a plurality of nucleic acid particles with a buffer. In some embodiments, the buffer is MES having 10% w / v sucrose. In some embodiments, the buffer is MES having 10% w / v sucrose and a pH of about 5 to about 6. In some embodiments, the buffer is MES having 10% w / v sucrose and the concentration of the nucleic acid in the composition is from about 0.25 to about 0.025 mg / ml. In some embodiments, the buffer is HEPES having 10% w / v sucrose and a pH of from about 7 to about 8. In some embodiments, the buffer is IX PBS having 10% w / v sucrose and a pH of from about 7 to about 8.Page 26 of 20712885786vlAttorney Docket No. 2013237-1455Oligosaccharides

[0118] Oligosaccharide compounds (e.g., cationic or ionizable oligosaccharide compounds) that can be used in the processes described herein are those that, in some embodiments, are useful for the delivery of nucleic acid therapeutic agents. Example oligosaccharide compounds include those described in WO2023 / 067121, WO2023 / 067123, WO2023 / 067124, WO2023 / 067125, and WO2023 / 067126, each of which is incorporated by reference in its entirety.

[0119] In some embodiments, a cationic oligosaccharide comprises a plurality of cationic moieties bonded to a trehalose, a sucrose, or a gluco-n-oligosaccharide moiety, where n is 2-6. In some embodiments, a trehalose moiety is of structure:where each G1moiety is independently a cationic moiety optionally connected to the trehalose moiety via a linker group, and each G2is independently H or an Ci-Ceo aliphatic group.

[0120] In some embodiments, a sucrose moiety is of structure:where each G1moiety is independently a cationic moiety optionally connected to the trehalose moiety via a linker group, and each G2is independently H or an Ci-Ceo aliphatic group.

[0121] In some embodiments, a gluco-n-oligosaccharide moiety is of structure:Page 27 of 20712885786vlAttorney Docket No. 2013237-1455 where each G1moiety is independently a cationic moiety optionally connected to the trehalose moiety via a linker group, each G2is independently H or an Ci-Ceo aliphatic group, and n is from 2-6 (e.g., 2, 3, 4, 5, or 6). In some embodiments, n is 2-5. In some embodiments, n is 3-6.

[0122] In some embodiments, each linker group is independently an optionally substituted Ci- 30 aliphatic group wherein one or more carbons are optionally and independently replaced by -Cy- , -NRY-, -NRYC(O)-, -C(O)NRY-, -NRYC(O)O-, -OC(O)NRY-, -NRYC(O)NRY-, -NRYC(S)NRY-, -C(S)NRY- -C(O)NRYSO2-, -SO2NRYC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O, -SO-, or -SO2-; each RYis independently H or optionally substituted Ci-Ce aliphatic; and each Cy is independently an optionally substituted C3-C14 cycloaliphatic, optionally substituted 5- to 14- membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or an optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S.

[0123] As used herein, a “cationic” moiety is a group having a net positive charge. As used herein, an “ionizable” moiety is a group that may have a neutral charge at a certain pH, but may become charged (e.g., cationic) at a different pH. For example, in some embodiments, an ionizable moiety becomes cationic (i.e., positively charged) at physiological pH (e.g., a pH of about 7.4). Example cationic moieties include, but are not limited to, ammonium, guanidinium, isothiouronium, amidinium, piperazinium, piperidinium, morpholinium, pyrrolidinium, imidazolium, pyrazolium, oxazolium, thiazolium, triazolium and the like. A person of skill in the art will also appreciate that an oligosaccharide described herein comprises a plurality (i.e., one or more) cationic moieties. A person of skill in the art will also appreciate that cationic moieties described herein can also exist as a salt (e.g., a pharmaceutically acceptable salt) that comprises a cationic moiety and one or more suitable counterions. For example, in some embodiments, a cationic group described herein comprises ammonium chloride. Suitable counterions include halogens (e.g., Br’, Cl’, I’, F ), acetates (e.g., C(O)O’), and the like. For additional examples, see the definition for pharmaceutically acceptable salts described herein. Reference to a particular counterion, e.g., a counterion as indicated in Table 1, is intended to encompass the charged moiety in isolation, as well as all chemically feasible counterions.Page 28 of 20712885786vlAttorney Docket No. 2013237-1455

[0124] In some embodiments, a cationic oligosaccharide is a compound of formula I:or a pharmaceutically acceptable salt thereof, wherein:A is A1, A2, or A3:each of R1and R2are independently selected, at each instance, from H, Ra, and -C(O)-Rawherein at least one instance of R1or R2is not H; each Rais independently selected from C1-C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein each Rais optionally substituted with one or more Rb; each Rbis independently selected from halogen, -N3, -Rc, -ORC, -SRC, -NHRC, -C(O)-RC, - OC(O)RC, -NHC(O)RC, -C(O)NHRC, and -NHC(O)NHRC; each Rcis independently selected from optionally substituted C1-C20 aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S;X1and X2are each independently selected from -S-, -S-S-, and -NH-;Page 29 of 20712885786vlAttorney Docket No. 2013237-1455Y1and Y2are each independently an optionally substituted Ci-30 aliphatic group wherein one or more carbons are optionally and independently replaced by Cy-, -NRY-, - NRYC(O)-, -C(O)NRY-, -NRYC(O)O-, -OC(O)NRY-, -NRYC(O)NRY-, - NRYC(S)NRY-, -C(S)NRY-, -C(O)NRYSO2-, -SO2NRYC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each RYis independently H or optionally substituted Ci-Ce aliphatic; each Cy is independently an optionally substituted C3-C14 cycloaliphatic, optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S;Z1and Z2are each independently a cationic or ionizable group selected from optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S, -N+(M)3,each M is independently -Co-Ce aliphatic-Rzor -Co-Ce aliphatic-N+(Rz)3; each Rzis independently selected from H, optionally substituted Ci-Ce aliphatic, optionally substituted C3-C2o cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; or two or more Rzcan come together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; and p is an integer selected from 1, 2, 3, 4, or 5.Page 30 of 20712885786vlAttorney Docket No. 2013237-1455

[0125] As described herein, A is A1, A2, or A3:

[0126] In some embodiments, A is A1or A2. In some embodiments, A is A1. In some embodiments, A is A2. In some embodiments, A is A3. In some embodiments, A is A1, and the oligosaccharide is a compound of formula la:la or a pharmaceutically acceptable salt thereof, wherein R1, R2, X1, X2, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0127] In some embodiments, an oligosaccharide is a compound of formula la:la or a pharmaceutically acceptable salt thereof, whereinPage 31 of 20712885786vlAttorney Docket No. 2013237-1455 each of R1and R2are independently selected, at each instance, from H, Ra, and -C(O)-Rawherein at least one instance of R1or R2is not H; each Rais independently selected from C1-C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein each Rais optionally substituted with one or more Rb; each Rbis independently selected from halogen, -N3, -Rc, -ORC, -SRC, -NHRC, -C(O)-RC, - OC(O)RC, -NHC(O)RC, -C(O)NHRC, and -NHC(O)NHRC; each Rcis independently selected from optionally substituted C1-C20 aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S;X1and X2are each independently selected from -S-, -S-S-, and -NH-;Y1and Y2are each independently an optionally substituted Ci-30 aliphatic group wherein one or more carbons are optionally and independently replaced by Cy-, -NRY-, - NRYC(O)-, -C(O)NRY-, -NRYC(O)O-, -OC(O)NRY-, -NRYC(O)NRY-, - NRYC(S)NRY-, -C(S)NRY-, -C(O)NRYSO2-, -SO2NRYC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each RYis independently H or optionally substituted Ci-Ce aliphatic; each Cy is independently an optionally substituted C3-C14 cycloaliphatic, optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S;Z1and Z2are each independently a cationic or ionizable group selected from optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S, -N+(M)3,each M is independently -Co-Ce aliphatic-Rzor -Co-Ce aliphatic-N+(Rz)3;Page 32 of 20712885786vlAttorney Docket No. 2013237-1455 each Rzis independently selected from H, optionally substituted Ci-Ce aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; or two or more Rzcan come together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S.

[0128] In some embodiments, A is A2, and an oligosaccharide is of formula lb:or a pharmaceutically acceptable salt thereof, wherein R1, R2, X1, X2, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0129] In some embodiments, an oligosaccharide is a compound of formula lb:or a pharmaceutically acceptable salt thereof, wherein each of R1and R2are independently selected, at each instance, from H, Ra, and -C(O)-Rawherein at least one instance of R1or R2is not H;Page 33 of 20712885786vlAttorney Docket No. 2013237-1455 each Rais independently selected from C1-C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein each Rais optionally substituted with one or more Rb; each Rbis independently selected from halogen, -N3, -Rc, -ORC, -SRC, -NHRC, -C(O)-RC, - OC(O)RC, -NHC(O)RC, -C(O)NHRC, and -NHC(O)NHRC; each Rcis independently selected from optionally substituted C1-C20 aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S;X1and X2are each independently selected from -S-, -S-S-, and -NH-;Y1and Y2are each independently an optionally substituted Ci-30 aliphatic group wherein one or more carbons are optionally and independently replaced by Cy-, -NRY-, - NRYC(O)-, -C(O)NRY-, -NRYC(O)O-, -OC(O)NRY-, -NRYC(O)NRY-, - NRYC(S)NRY-, -C(S)NRY-, -C(O)NRYSO2-, -SO2NRYC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each RYis independently H or optionally substituted Ci-Ce aliphatic; each Cy is independently an optionally substituted C3-C14 cycloaliphatic, optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S;Z1and Z2are each independently a cationic or ionizable group selected from optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S, -N+(M)3,each M is independently -Co-Ce aliphatic-Rzor -Co-Ce aliphatic-N+(Rz)3; each Rzis independently selected from H, optionally substituted Ci-Ce aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl,Page 34 of 20712885786vlAttorney Docket No. 2013237-1455 optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; or two or more Rzcan come together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S.

[0130] In some embodiments, A is A3, and an oligosaccharide is of formula Ic:Ic or a pharmaceutically acceptable salt thereof, wherein p, R1, R2, X1, X2, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0131] In some embodiments, an oligosaccharide is a compound of formula Ic:or a pharmaceutically acceptable salt thereof, wherein each of R1and R2are independently selected, at each instance, from H, Ra, and -C(O)-Rawherein at least one instance of R1or R2is not H;Page 35 of 20712885786vlAttorney Docket No. 2013237-1455 each Rais independently selected from C1-C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein each Rais optionally substituted with one or more Rb; each Rbis independently selected from halogen, -N3, -Rc, -ORC, -SRC, -NHRC, -C(O)-RC, - OC(O)RC, -NHC(O)RC, -C(O)NHRC, and -NHC(O)NHRC; each Rcis independently selected from optionally substituted C1-C20 aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S;X1and X2are each independently selected from -S-, -S-S-, and -NH-;Y1and Y2are each independently an optionally substituted Ci-30 aliphatic group wherein one or more carbons are optionally and independently replaced by Cy-, -NRY-, - NRYC(O)-, -C(O)NRY-, -NRYC(O)O-, -OC(O)NRY-, -NRYC(O)NRY-, - NRYC(S)NRY-, -C(S)NRY-, -C(O)NRYSO2-, -SO2NRYC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each RYis independently H or optionally substituted Ci-Ce aliphatic; each Cy is independently an optionally substituted C3-C14 cycloaliphatic, optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S;Z1and Z2are each independently a cationic or ionizable group selected from optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S, -N+(M)3,each M is independently -Co-Ce aliphatic-Rzor -Co-Ce aliphatic-N+(Rz)3; each Rzis independently selected from H, optionally substituted Ci-Ce aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl,Page 36 of 20712885786vlAttorney Docket No. 2013237-1455 optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; or two or more Rzcan come together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; and p is an integer selected from 1, 2, 3, 4, or 5.

[0132] In some embodiments, p is 1, and an oligosaccharide is of formula Ic-i:or a pharmaceutically acceptable salt thereof, wherein R1, R2, X1, X2, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0133] In some embodiments, p is 2, and an oligosaccharide is of formula Ic-ii:or a pharmaceutically acceptable salt thereof, wherein R1, R2, X1, X2, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.Page 37 of 20712885786vlAttorney Docket No. 2013237-1455

[0134] In some embodiments, p is 3, and an oligosaccharide is of formula Ic-iii:Ic-iii or a pharmaceutically acceptable salt thereof, wherein R1, R2, X1, X2, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0135] In some embodiments, p is 4, and an oligosaccharide is of formula Ic-iv:or a pharmaceutically acceptable salt thereof, wherein R1, R2, X1, X2, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0136] In some embodiments, p is 5, and an oligosaccharide is of formula Ic-v:Page 38 of 20712885786vlAttorney Docket No. 2013237-1455Ic-v or a pharmaceutically acceptable salt thereof, wherein R1, R2, X1, X2, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0137] As described generally herein, each of R1and R2are independently selected, at each instance, from H, Ra, and -C(O)-Ra, wherein at least one instance of R1or R2is not H. In some embodiments, R1and R2are each independently selected from Raand -C(O)-Ra. In some embodiments, R1and R2is Ra. In some embodiments, R1and R2is Ra, and each Rais independently selected from C1-C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12- membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein each Rais optionally substituted with one or more Rb.

[0138] In some embodiments, each of R1and R2is Ra, and each Rais independently selected from C1-C20 aliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is R , and each is independently selected from Ci~Ci4 aliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is Ra, and each Rais independently selected from C1-C10 aliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is Ra, and each Rais independently selected from C5-C10 aliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is Ra, and each Rais independently selected from C5-C10 alkyl optionally substituted with one or more Rb. In some embodiments, each of R1and R2is Ra, and each Rais independently selected from C5-C10 linear alkyl. In some embodiments, each of R1and R2is Ra, and each Rais independently selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and n-nonyl. In some embodiments, each R1and R2is Ra, and each Rais independently selected from;Page 39 of 20712885786vlAttorney Docket No. 2013237-1455

[0139] In some embodiments, each of R1and R2is Ra, and each Rais independently selected from C3-C20 cycloaliphatic, optionally substituted with one or more Rb. In some embodiments, each of R1and R2is Ra, and each Rais independently selected from C3-C6 cycloaliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is Ra, and each Rais independently selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0140] In some embodiments, each of R1and R2is Ra, and each Rais independently selected from C5-C6 aryl optionally substituted with one or more Rb. In some embodiments, each of R1and R2is Ra, and Rais phenyl.

[0141] In some embodiments, each of R1and R2is Ra, and Rais 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S optionally substituted with one or more Rb. In some embodiments, each of R1and R2is Ra, and Rais 3- to 6-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S optionally substituted with one or more Rb.

[0142] In some embodiments, each of R1and R2is -C(O)-Ra, and each Rais independently selected from C1-C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein each Rais optionally substituted with one or more Rb.

[0143] In some embodiments, each of R1and R2is -C(O)-Ra. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais C1-C20 aliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais C1-C14 aliphatic optionally substituted Page 40 of 20712885786vlAttorney Docket No. 2013237-1455 with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais C1-C10 aliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais C5-C10 aliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais C5-C10 alkyl optionally substituted with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais C5-C10 linear alkyl. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, or n-nonyl. In some embodiments, each of R1and R2is -C(O)-Ra, and each Rais independently selected from

[0144] In some embodiments, each of R1and R2is -C(O)-Ra, and Rais C3-C20 cycloaliphatic, optionally substituted with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais C3-C6 cycloaliphatic optionally substituted with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and each Rais independently selected from cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, each of R1and R2is -C(O)-Ra, and Rais polycyclic C10-C20 cycloaliphatic, optionally substituted with one or more Rb. In some embodiments, each of R1and R2isPage 41 of 20712885786vlAttorney Docket No. 2013237-1455

[0145] In some embodiments, each of R1and R2is -C(O)-Ra, and each Rais independently a Cs-Ce aryl optionally substituted with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and each Rais independently a phenyl.

[0146] In some embodiments, each of R1and R2is -C(O)-Ra, and each Rais independently a 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S optionally substituted with one or more Rb. In some embodiments, each of R1and R2is -C(O)-Ra, and each Rais independently a 3- to 6-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S optionally substituted with one or more Rb

[0147] In some embodiments, each of R1and R2is independently selected from:

[0148] As described herein, each Rbis independently selected from halogen, -N3, -Rc, -ORC, - SRC, -NHRC, -C(O)-RC, -OC(O)RC, -NHC(O)RC, -C(O)NHRC, and -NHC(O)NHRC. In some embodiments, Rbis halogen. In some embodiments, Rbis -N3. In some embodiments, Rbis Rc. In some embodiments, Rbis -ORC. In some embodiments, Rbis -SRC. In some embodiments, Rbis -NHRC. In some embodiments, Rbis -C(O)-RC. In some embodiments, Rbis -OC(O)RC. InPage 42 of 20712885786vlAttorney Docket No. 2013237-1455 some embodiments, Rbis -NHC(O)RC. In some embodiments, Rbis -C(O)NHRC. In some embodiments, Rbis -NHC(O)NHRC.

[0149] As described herein, each Rcis independently selected from optionally substituted Ci- C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, Rcis optionally substituted C1-C20 aliphatic. In some embodiments, Rcis optionally substituted C3-C20 cycloaliphatic. In some embodiments, Rcis optionally substituted C5-C6 aryl. In some embodiments, Rcis optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, Rcis optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S.

[0150] As described herein, X1and X2are each independently selected from -S-, -S-S-, and - NH-. In some embodiments, X1is -S-. In some embodiments, X1is -S-S-. In some embodiments, X1is -NH-. In some embodiments, X2is -S-. In some embodiments, X2is -S-S-. In some embodiments, X2is -NH-. In some embodiments, X1and X2are each -S-. In some embodiments, X1and X2are each -S-S-. In some embodiments, X1and X2are each -NH-. In some embodiments, X1is — S- and X2is -NH-. In some embodiments, X1is -NH- and X2is -S-.

[0151] As described herein, Y1and Y2are each independently an optionally substituted Ci-30 aliphatic group wherein one or more carbons of a Y1and / or Y2group are optionally and independently replaced by Cy-, -NRY-, -NRYC(O)-, -C(O)NRY-, -NRYC(O)O-, -OC(O)NRY-, - NRYC(O)NRY-, -NRYC(S)NRY-, -C(S)NRY- -C(O)NRYSO2-, -SO2NRYC(O)-, -OC(O)O-, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. In some embodiments, Y1and Y2are each independently an optionally substituted Ci-30 aliphatic group wherein one or more carbons of a Y1and / or Y2group are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH- -C(O)NHSO2-, -SO2NHC(O)- , -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. As described herein, it is understood that Y1and Y2are bivalent moieties, having one end bonded to a X1or X2group, and the other end bonded to a Z1or Z2group.

[0152] As described herein, each RYis independently selected from H and optionally substituted Ci-Ce aliphatic. In some embodiments, each RYis independently selected from H and C1-C3 alkyl. In some embodiments, each RYis independently selected from H and CH3.Page 43 of 20712885786vlAttorney Docket No. 2013237-1455

[0153] In some embodiments, Y1and Y2are each independently selected from C1-C10 aliphatic, Co-C4-aliphatic-NHC(0)NH-Co-C4 aliphatic, Co-C4-aliphatic-NHC(S)NH-Co-C4 aliphatic, Co-C4-aliphatic-C(0)NH-Co-C4 aliphatic, Co-C4-aliphatic-C(S)NH-Co-C4 aliphatic, Co- C4-aliphatic-NHC(0)-Co-C4 aliphatic, Co-C4-aliphatic-NHS02-Co-C4 aliphatic, and C0-C4- aliphatic-C(0)-Co-C4 aliphatic.

[0154] In some embodiments, Y1and Y2are each C1-C10 aliphatic. In some embodiments, Y1and Y2are each methylene, ethylene, propylene, or butylene. In some embodiments, Y1and Y2are each -CH2-CH2-.

[0155] In some embodiments, Y1and Y2are each Co-C4-aliphatic-NHC(0)NH-Co-C4 aliphatic. In some embodiments, Y1and Y2are each Ci-C4-aliphatic-NHC(O)NH-Ci-C4 aliphatic. In some embodiments, Y1and Y2are each -CH2-CH2-NHC(O)NH-CH2-CH2-.

[0156] In some embodiments, Y1and Y2are each Co-C4-aliphatic-NHC(S)NH-Co-C4 aliphatic. In some embodiments, Y1and Y2are each Ci-C4-aliphatic-NHC(S)NH-Ci-C4 aliphatic. In some embodiments, Y1and Y2are each -CH2-CH2-NHC(S)NH-CH2-CH2-.

[0157] In some embodiments, Y1and Y2are each Co-C4-aliphatic-C(0)NH-Co-C4 aliphatic. In some embodiments, Y1and Y2are each Ci-C4-aliphatic-C(O)NH-Ci-C4 aliphatic. In some embodiments, Y1and Y2are each -CH2-CH2-C(O)NH-CH2-CH2-.

[0158] In some embodiments, Y1and Y2are each Co-C4-aliphatic-NHC(0)-Co-C4 aliphatic. In some embodiments, Y1and Y2are each Ci-C4-aliphatic-NHC(O)-Ci-C4 aliphatic. In some embodiments, Y1and Y2are each -CH2-CH2-NHC(O)-CH2-CH2-.

[0159] In some embodiments, Y1and Y2are each Co-C4-aliphatic-NHS02-Co-C4 aliphatic. In some embodiments, Y1and Y2are each Ci-C4-aliphatic-NHSO2-Ci-C4 aliphatic. In some embodiments, Y1and Y2are each -CH2-CH2-NHSO2-CH2-CH2-.

[0160] In some embodiments, Y1is selected from C1-C10 aliphatic, Co-C4-aliphatic- NHC(0)NH-CO-C4 aliphatic, Co-C4-aliphatic-NHC(S)NH-Co-C4 aliphatic, Co-C4-aliphatic- C(0)NH-CO-C4 aliphatic, Co-C4-aliphatic-C(S)NH-Co-C4 aliphatic, Co-C4-aliphatic-C(0)NH-Co- C4 aliphatic, Co-C4-aliphatic-NHS02-Co-C4 aliphatic, and Co-C4-aliphatic-C(0)-Co-C4 aliphatic.

[0161] In some embodiments, embodiments, Y1is C1-C10 aliphatic. In some embodiments, Y1is methylene, ethylene, propylene, or butylene. In some embodiments, Y1is -CH2-CH2-.Page 44 of 20712885786vlAttorney Docket No. 2013237-1455

[0162] In some embodiments, Y1is Co-C4-aliphatic-NHC(0)NH-Co-C4 aliphatic. In some embodiments, Y1is Ci-C4-aliphatic-NHC(O)NH-Ci-C4 aliphatic. In some embodiments, Y1is - CH2-CH2-NHC(O)NH-CH2-CH2-.

[0163] In some embodiments, Y1is Co-C4-aliphatic-NHC(S)NH-Co-C4 aliphatic. In some embodiments, Y1is Ci-C4-aliphatic-NHC(S)NH-Ci-C4 aliphatic. In some embodiments, Y1is - CH2-CH2-NHC(S)NH-CH2-CH2-.

[0164] In some embodiments, Y1is Co-C4-aliphatic-C(0)NH-Co-C4 aliphatic. In some embodiments, Y1is Ci-C4-aliphatic-C(O)NH-Ci-C4 aliphatic. In some embodiments, Y1is -CH2- CH2-C(O)NH-CH2-CH2-.

[0165] In some embodiments, Y1is Co-C4-aliphatic-NHC(0)-Co-C4 aliphatic. In some embodiments, Y1is Ci-C4-aliphatic-NHC(O)-Ci-C4 aliphatic. In some embodiments, Y1is -CH2- CH2-NHC(O)-CH2-CH2-.

[0166] In some embodiments, Y1is Co-C4-aliphatic-NHS02-Co-C4 aliphatic. In some embodiments, Y1is Ci-C4-aliphatic-NHSO2-Ci-C4 aliphatic. In some embodiments, Y1is -CH2- CH2-NHSO2-CH2-CH2-.

[0167] In some embodiments, Y2is selected from Ci-Cio aliphatic, Co-C4-aliphatic- NHC(0)NH-CO-C4 aliphatic, Co-C4-aliphatic-NHC(S)NH-Co-C4 aliphatic, Co-C4-aliphatic- C(0)NH-CO-C4 aliphatic, Co-C4-aliphatic-C(S)NH-Co-C4 aliphatic, Co-C4-aliphatic-C(0)NH-Co- C4 aliphatic, Co-C4-aliphatic-NHS02-Co-C4 aliphatic, and Co-C4-aliphatic-C(0)-Co-C4 aliphatic.

[0168] In some embodiments, embodiments, Y2is C1-C10 aliphatic. In some embodiments, Y2is methylene, ethylene, propylene, or butylene. In some embodiments, Y2is -CH2-CH2-.

[0169] In some embodiments, Y2is Co-C4-aliphatic-NHC(0)NH-Co-C4 aliphatic. In some embodiments, Y2is Ci-C4-aliphatic-NHC(O)NH-Ci-C4 aliphatic. In some embodiments, Y2is - CH2-CH2-NHC(O)NH-CH2-CH2-.

[0170] In some embodiments, Y2is Co-C4-aliphatic-NHC(S)NH-Co-C4 aliphatic. In some embodiments, Y2is Ci-C4-aliphatic-NHC(S)NH-Ci-C4 aliphatic. In some embodiments, Y2is - CH2-CH2-NHC(S)NH-CH2-CH2-.

[0171] In some embodiments, Y2is Co-C4-aliphatic-C(0)NH-Co-C4 aliphatic. In some embodiments, Y2is Ci-C4-aliphatic-C(O)NH-Ci-C4 aliphatic. In some embodiments, Y2is -CH2- CH2-C(O)NH-CH2-CH2-.Page 45 of 20712885786vlAttorney Docket No. 2013237-1455

[0172] In some embodiments, Y2is Co-C4-aliphatic-NHC(0)-Co-C4 aliphatic. In some embodiments, Y2is Ci-C4-aliphatic-NHC(O)-Ci-C4 aliphatic. In some embodiments, Y2is -CH2- CH2-NHC(O)-CH2-CH2-.

[0173] In some embodiments, Y2is Co-C4-aliphatic-NHS02-Co-C4 aliphatic. In some embodiments, Y2is Ci-C4-aliphatic-NHSO2-Ci-C4 aliphatic. In some embodiments, Y2is -CH2- CH2-NHSO2-CH2-CH2-.

[0174] As described general herein, each Cy is independently an optionally substituted C3-C14 cycloaliphatic, 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O,S, 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S.

[0175] In some embodiments, a moiety X^Y^Z1is:

[0176] In some embodiments, a moiety X2-Y2-Z2is:

[0177] As described generally herein, Z1and Z2are each independently a cationic or ionizable group selected from optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S, -N+(M)3,

[0178] In some embodiments, Z1and Z2are each independently selected from optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, andS, optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected fromN, O, and S, -N+(M)3,

[0179] Page 46 of 20712885786vlAttorney Docket No. 2013237-1455

[0180] In some embodiments, Z1and Z2are each an optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z1and Z2are each an optionally substituted 5- to 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z1and Z2are each an optionally substituted 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z1and Z2are each selected from optionally substituted piperdinyl, piperazinyl, and morpholinyl.

[0181] In some embodiments, Z1and Z2are each an optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S.

[0182] In some embodiments, Z1and Z2are each independently selected from:

[0183] In some embodiments, Z1and Z2are each independently selected from:

[0185] In some embodiments, Z1and Z2are each -N+(M)3. In some embodiments, Z1and Z2are each -N+(Co-Ce aliphatic-Rz)3. In some embodiments, Z1and Z2are each -N+(RZ)3. In some embodiments, Z1and Z2are each -N+H3. In some embodiments, Z1and Z2are each -N+H(RZ)2. In some embodiments, Z1and Z2are each -N+H(Ci-Ce aliphatic)2. In some embodiments, Z1and Z2are each -N+(H)2CH3. In some embodiments, Z1and Z2are each -N+H(CH3)2. In some embodiments, Z1and Z2are each -N+(CH3)3.Page 47 of 20712885786vlAttorney Docket No. 2013237-1455

[0186] In some embodiments, Z1and Z2are each -N+(RZ)2-Ci-Ce aliphatic-N+(Rz)3. In some embodiments, Z1and Z2are each -N+(Rz)(Ci-Ce aliphatic-N+(Rz)3)2. In some embodiments, Z1and Z2are each:In some embodiments, Z1and Z2are each:

[0187] In some embodiments, Z1and Z2are eachRz

[0188] In some embodiments, Z1and Z2are each RZ©'RZ

[0189] In some embodiments, Z1and Z2are eachR izR iz

[0190] In some embodiments, Z1and Z2are each

[0191] In some embodiments, Z1is optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z1is each an optionally substituted 5- to 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z1is an optionally substituted 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z1is selected from optionally substituted piperdinyl, piperazinyl, and morpholinyl.Page 48 of 20712885786vlAttorney Docket No. 2013237-1455

[0192] In some embodiments, Z1is -N+(M)3. In some embodiments, Z1is-N+(Co-Ce aliphatic- RZ)3. In some embodiments, Z1is-N+(Rz)3. In some embodiments, Z1is -N+H3. In some embodiments, Z1is -N+H(RZ)2. In some embodiments, Z1is -N+H(Ci-Ce aliphatic)2. In some embodiments, Z1is -N+(H)2CH3. In some embodiments, Z1is -N+H(CH3)2. In some embodiments, Z1is -N+(CH3)3.

[0193] In some embodiments, Z1is -N+(RZ)2-Ci-Ce aliphatic-N+(Rz)3. In some embodiments, Z1is -N+(Rz)(Ci-Ce aliphatic-N+(Rz)3)2. In some embodiments, Z1is:

[0194] In some embodiments, Z1is:

[0195] In some embodiments, Z1is: some embodiments, Z1is:

[0197] In some embodiments, Z1is:

[0198] In some embodiments, Z1is:Page 49 of 20712885786vlAttorney Docket No. 2013237-1455

[0199] In some embodiments, Z2is optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z2is each an optionally substituted 5- to 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z2is an optionally substituted 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z2is selected from optionally substituted piperdinyl, piperazinyl, and morpholinyl.

[0200] In some embodiments, Z2is -N+(M)3. In some embodiments, Z2is-N+(Co-Ce aliphatic- RZ)3. In some embodiments, Z2is-N+(Rz)3. In some embodiments, Z2is -N+H3. In some embodiments, Z2is -N+H(RZ)2. In some embodiments, Z2is -N+H(Ci-Ce aliphatic)2. In some embodiments, Z2is -N+(H)2CH3. In some embodiments, Z2is -N+H(CH3)2. In some embodiments, Z2is -N+(CH3)3.

[0201] In some embodiments, Z2is -N+(RZ)2-Ci-Ce aliphatic-N+(Rz)3. In some embodiments, Z2is -N+(Rz)(Ci-Ce aliphatic-N+(Rz)3)2. In some embodiments, Z2is:Rz®N-RZR\® )RZx Rz

[0202] In some embodiments, Z2is:

[0203] In some embodiments, Z2is:

[0204] In some embodiments, Z2is:Page 50 of 20712885786vlAttorney Docket No. 2013237-1455

[0205] In some embodiments, Z2is:

[0206] In some embodiments, Z2is:

[0207] As described generally herein, each M is independently -Co-Ce aliphatic-Rzor -Co-Ce aliphatic-N+(Rz)3. In some embodiments, each M is Co-Ce aliphatic-Rz. In some embodiments, each M is -Co-Ce aliphatic-N+(Rz)3.

[0208] As described generally herein, each Rzis independently selected from H, optionally substituted Ci-Ce aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5- Ce aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; or two or more Rzcan come together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 12- membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S.

[0209] In some embodiments, each Rzis H. In some embodiments, each Rzis optionally substituted Ci-Ce aliphatic. In some embodiments, each Rzis optionally substituted C3-C20 cycloaliphatic. In some embodiments, each Rzis optionally substituted C5-C6 aryl. In some embodiments, each Rzis optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, each Rzis optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S.

[0210] In some embodiments, two or more Rzcan come together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, two or more Rzcan come togetherPage 51 of 20712885786vlAttorney Docket No. 2013237-1455 with the atoms to which they are attached to form an optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S.

[0211] In some embodiments, an Rzon a Z1moiety and an Rzon a Z2moiety can come together to form a 4- to 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from N, O, and S.

[0212] As used herein, a “cationic” moiety is a group having a net positive charge. As used herein, an “ionizable” moiety is a group that may have a neutral charge at a certain pH, but may become charged (e.g., cationic) at a different pH. For example, in some embodiments, an ionizable moiety becomes cationic (i.e., positively charged) at physiological pH (e.g., a pH of about 7.4). Moreover, in some embodiments, a Z1and Z2moiety, which is a cationic or ionizable moiety described herein, further comprises a counterion (e.g., an anion for each cationic or ionizable moiety). For example, in some embodiments, a Z1and Z2moiety comprises a counterion that is a halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In some embodiments, a Z1and Z2moiety comprises a counterion that is acetate (CH3COO ), chloride (Cl ), iodide (I ), bromide (Br ), or fluoride (F ).

[0213] In some embodiments, Z1and Z2are each independently selected from:Page 52 of 20712885786vlAttorney Docket No. 2013237-1455

[0214] In some embodiments, a compound of formula I is a compound of formula Id:or a pharmaceutically acceptable salt thereof, wherein n and m are each independently selected from 0, 1, 2, 3, 4, 5, or 6, and Ra, Y1, Y2, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0215] In some embodiments, a compound of formula Id is a compound of formula Id-i:Page 53 of 20712885786vlAttorney Docket No. 2013237-1455Id-i or a pharmaceutically acceptable salt thereof, wherein n and m are each independently selected from 0, 1, 2, 3, 4, 5, or 6, and Ra, Z1, and Z2are as described in classes and subclasses herein, both singly and in combination.

[0216] In some embodiments, a compound of formula I is selected from Table 1.Table 1Page 54 of 20712885786vlAttorney Docket No. 2013237-1455Page 55 of 20712885786vlAttorney Docket No. 2013237-1455Page 56 of 20712885786vlAttorney Docket No. 2013237-1455Page 57 of 20712885786vlAttorney Docket No. 2013237-1455Page 58 of 20712885786vlAttorney Docket No. 2013237-1455Page 59 of 20712885786vlAttorney Docket No. 2013237-1455Page 60 of 20712885786vlAttorney Docket No. 2013237-1455Page 61 of 20712885786vlAttorney Docket No. 2013237-1455Page 62 of 20712885786vlAttorney Docket No. 2013237-1455Page 63 of 20712885786vlAttorney Docket No. 2013237-1455Page 64 of 20712885786vlAttorney Docket No. 2013237-1455Page 65 of 20712885786vlAttorney Docket No. 2013237-1455Page 66 of 20712885786vlAttorney Docket No. 2013237-1455Page 67 of 20712885786vlAttorney Docket No. 2013237-1455Page 68 of 20712885786vlAttorney Docket No. 2013237-1455Page 69 of 20712885786vlAttorney Docket No. 2013237-1455Page 70 of 20712885786vlAttorney Docket No. 2013237-1455Page 71 of 20712885786vlAttorney Docket No. 2013237-1455Page 72 of 20712885786vlAttorney Docket No. 2013237-1455Page 73 of 20712885786vlAttorney Docket No. 2013237-1455Page 74 of 20712885786vlAttorney Docket No. 2013237-1455Page 75 of 20712885786vlAttorney Docket No. 2013237-1455Page 76 of 20712885786vlAttorney Docket No. 2013237-1455Page 77 of 20712885786vlAttorney Docket No. 2013237-1455Page 78 of 20712885786vlAttorney Docket No. 2013237-1455Page 79 of 20712885786vlAttorney Docket No. 2013237-1455Page 80 of 20712885786vlAttorney Docket No. 2013237-1455Page 81 of 20712885786vlAttorney Docket No. 2013237-1455Page 82 of 20712885786vlAttorney Docket No. 2013237-1455Page 83 of 20712885786vlAttorney Docket No. 2013237-1455Page 84 of 20712885786vlAttorney Docket No. 2013237-1455Page 85 of 20712885786vlAttorney Docket No. 2013237-1455Page 86 of 20712885786vlAttorney Docket No. 2013237-1455Page 87 of 20712885786vlAttorney Docket No. 2013237-1455Page 88 of 20712885786vlAttorney Docket No. 2013237-1455Page 89 of 20712885786vlAttorney Docket No. 2013237-1455Page 90 of 20712885786vlAttorney Docket No. 2013237-1455Page 91 of 20712885786vlAttorney Docket No. 2013237-1455Page 92 of 20712885786vlAttorney Docket No. 2013237-1455Page 93 of 20712885786vlAttorney Docket No. 2013237-1455Page 94 of 20712885786vlAttorney Docket No. 2013237-1455Page 95 of 20712885786vlAttorney Docket No. 2013237-1455Page 96 of 20712885786vlAttorney Docket No. 2013237-1455Page 97 of 20712885786vlAttorney Docket No. 2013237-1455Page 98 of 20712885786vlAttorney Docket No. 2013237-1455Page 99 of 20712885786vlAttorney Docket No. 2013237-1455Page 100 of 20712885786vlAttorney Docket No. 2013237-1455Page 101 of 20712885786vlAttorney Docket No. 2013237-1455Page 102 of 20712885786vlAttorney Docket No. 2013237-1455Page 103 of 20712885786vlAttorney Docket No. 2013237-1455Page 104 of 20712885786vlAttorney Docket No. 2013237-1455Page 105 of 20712885786vlAttorney Docket No. 2013237-1455Page 106 of 20712885786vlAttorney Docket No. 2013237-1455Page 107 of 20712885786vlAttorney Docket No. 2013237-1455Page 108 of 20712885786vlAttorney Docket No. 2013237-1455Page 109 of 20712885786vlAttorney Docket No. 2013237-1455Page 110 of 20712885786vlAttorney Docket No. 2013237-1455Page 111 of 20712885786vlAttorney Docket No. 2013237-1455Page 112 of 20712885786vlAttorney Docket No. 2013237-1455Page 113 of 20712885786vlAttorney Docket No. 2013237-1455Page 114 of 20712885786vlAttorney Docket No. 2013237-1455Page 115 of 20712885786vlAttorney Docket No. 2013237-1455Page 116 of 20712885786vlAttorney Docket No. 2013237-1455Page 117 of 20712885786vlAttorney Docket No. 2013237-1455Page 118 of 20712885786vlAttorney Docket No. 2013237-1455Page 119 of 20712885786vlAttorney Docket No. 2013237-1455Page 120 of 20712885786vlAttorney Docket No. 2013237-1455Page 121 of 20712885786vlAttorney Docket No. 2013237-1455Page 122 of 20712885786vlAttorney Docket No. 2013237-1455Page 123 of 20712885786vlAttorney Docket No. 2013237-1455Page 124 of 20712885786vlAttorney Docket No. 2013237-1455Page 125 of 20712885786vlAttorney Docket No. 2013237-1455Page 126 of 20712885786vlAttorney Docket No. 2013237-1455Page 127 of 20712885786vlAttorney Docket No. 2013237-1455Page 128 of 20712885786vlAttorney Docket No. 2013237-1455Page 129 of 20712885786vlAttorney Docket No. 2013237-1455Page 130 of 20712885786vlAttorney Docket No. 2013237-1455Page 131 of 20712885786vlAttorney Docket No. 2013237-1455Page 132 of 20712885786vlAttorney Docket No. 2013237-1455Page 133 of 20712885786vlAttorney Docket No. 2013237-1455Page 134 of 20712885786vlAttorney Docket No. 2013237-1455Page 135 of 20712885786vlAttorney Docket No. 2013237-1455

[0217] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated. Moreover, reference to particular salts herein, e.g., in Table 1, is also intended to include reference to a free base form of said compound.

[0218] It is understood that while the exemplary compounds in Table 1 are represented as particular salts (e.g, chloride salts, iodide salts, and the like) that comprise a counterion (e.g., CT, T, and the like), a person of skill in the art will appreciate that any suitable counterion can be used in conjunction with the cationic nitrogen groups in Table 1. Table 1, therefore, is intended to encompass any positively charged nitrogen group coupled with any chemically feasiblyPage 136 of 20712885786vlAttorney Docket No. 2013237-1455 counterion. Accordingly, the specific counterions provided above are provided by way of example and are not intended to be limiting.

[0219] In some embodiments, a compound of formula I is selected from Table 2:Table 2Page 137 of 20712885786vlAttorney Docket No. 2013237-1455Page 138 of 20712885786vlAttorney Docket No. 2013237-1455Page 139 of 20712885786vlAttorney Docket No. 2013237-1455Page 140 of 20712885786vlAttorney Docket No. 2013237-1455Page 141 of 20712885786vlAttorney Docket No. 2013237-1455Page 142 of 20712885786vlAttorney Docket No. 2013237-1455Page 143 of 20712885786vlAttorney Docket No. 2013237-1455Page 144 of 20712885786vlAttorney Docket No. 2013237-1455Page 145 of 20712885786vlAttorney Docket No. 2013237-1455Page 146 of 20712885786vlAttorney Docket No. 2013237-1455Page 147 of 20712885786vlAttorney Docket No. 2013237-1455Page 148 of 20712885786vlAttorney Docket No. 2013237-1455Page 149 of 20712885786vlAttorney Docket No. 2013237-1455Page 150 of 20712885786vlAttorney Docket No. 2013237-1455Page 151 of 20712885786vlAttorney Docket No. 2013237-1455Page 152 of 20712885786vlAttorney Docket No. 2013237-1455Page 153 of 20712885786vlAttorney Docket No. 2013237-1455Page 154 of 20712885786vlAttorney Docket No. 2013237-1455Page 155 of 20712885786vlAttorney Docket No. 2013237-1455Page 156 of 20712885786vlAttorney Docket No. 2013237-1455Page 157 of 20712885786vlAttorney Docket No. 2013237-1455Page 158 of 20712885786vlAttorney Docket No. 2013237-1455Page 159 of 20712885786vlAttorney Docket No. 2013237-1455Page 160 of 20712885786vlAttorney Docket No. 2013237-1455

[0220] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated. Moreover, reference to particular salts herein, e.g., in Table 2, is also intended to include reference to a free base form of said compound.

[0221] It is understood that while the exemplary compounds in Table 2 are represented as particular salts (e.g, chloride salts, iodide salts, and the like) that comprise a counterion (e.g., CT, T, and the like), a person of skill in the art will appreciate that any suitable counterion can be used in conjunction with the cationic nitrogen groups in Table 2. Table 2, therefore, is intended to encompass any positively charged nitrogen group coupled with any chemically feasibly counterion. Accordingly, the specific counterions provided above are provided by way of example and are not intended to be limiting.

[0222] Polycationic oligosaccharide complexes described herein can be prepared according to methods known to those of skill in the art. For example, provided oligosaccharide complexes can be prepared according to the methods provided in EP 21382958.3, which is incorporated herein by reference in its entirety.Page 161 of 20712885786vlAttorney Docket No. 2013237-1455

[0223] In some embodiments, the present disclosure provides a complex comprising a compound selected from:

[0224] In some embodiments, a complex described herein does not comprise JRL13 or JRL45.

[0225] In some embodiments, an oligosaccharide compound for use in preparing a complex described herein is JLF99.Page 162 of 20712885786vlAttorney Docket No. 2013237-1455Nucleic Acids

[0226] In some embodiments, a complex described herein comprises a nucleic acid. In some embodiments, a nucleic acid is RNA.

[0227] In some embodiments, an RNA amenable to technologies described herein is a singlestranded RNA. In some embodiments, an RNA as disclosed herein is a linear RNA. In some embodiments, a single-stranded RNA is a non-coding RNA in that its nucleotide sequence does not include an open reading frame (or complement thereof). In some embodiments, a singlestranded RNA has a nucleotide sequence that encodes (or is the complement of a sequence that encodes) a polypeptide or a plurality of polypeptides (e.g., epitopes) of the present disclosure.

[0228] In some embodiments, an RNA is or comprises an siRNA, an miRNA, or other noncoding RNA.

[0229] In many embodiments, a relevant RNA includes at least one open reading frame (ORF) (e.g., is an mRNA); in some embodiments, a relevant RNA includes a single ORF; in some embodiments, a relevant RNA includes more than one ORF.

[0230] In some embodiments, an RNA comprises an ORF, e.g., encoding a polypeptide of interest or encoding a plurality of polypeptides of interest. In some embodiments, an RNA produced in accordance with technologies provided herein comprises a plurality of ORFs (e.g., encoding a plurality of polypeptides). In some embodiments, an RNA produced in accordance with technologies herein comprises a single ORF that encodes a plurality of polypeptides. In some such embodiments, polypeptides are or comprise antigens or epitopes thereof (e.g., relevant antigens).

[0231] In some embodiments, an ORF for use in accordance with the present disclosure encodes a polypeptide that includes a signal sequence, e.g., that is functional in mammalian cells, such as an intrinsic signal sequence or a heterologous signal sequence. In some embodiments, a signal sequence directs secretion of an encoded polypeptide, in some embodiments, a signal sequence directs transport of an encoded polypeptide into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.

[0232] In some embodiments, an ORF encodes a polypeptide that includes a multimerization element (e.g., an intrinsic or heterologous multimerization element). In some embodiments, an ORF that encodes a surface polypeptide (e.g., that includes a signal sequence directing surface localization) includes a multimerization element.Page 163 of 20712885786vlAttorney Docket No. 2013237-1455

[0233] In some embodiments, an ORF encodes a polypeptide that includes a transmembrane element or domain.

[0234] In some embodiments, an ORF is codon -optimized for expression in cells of a particular host, e.g., a mammalian host, e.g., a human.

[0235] In some embodiments, an RNA includes unmodified uridine residues; in some embodiments, such RNA (e.g., an RNA that includes only unmodified uridine residues) may be referred to as a “uRNA”. In some embodiments, an RNA includes one or more modified uridine residues; in some embodiments, such an RNA (e.g., an RNA including entirely modified uridine residues) is referred to as a “modRNA”. In some embodiments, an RNA may be a self-amplifying RNA (saRNA). In some embodiments, an RNA may be a trans-amplifying RNA (taRNA) (see, for example, WO2017 / 162461).

[0236] In some embodiments, a relevant RNA includes a polypeptide-encoding portion or a plurality of polypeptide-encoding portions. In some particular embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or comprises a biologically active polypeptide or portion thereof (e.g., an enzyme or cytokine or therapeutic protein such as a replacement protein or antibody or portion thereof). In some particular embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or comprises an antigen (or an epitope thereof), a cytokine, an enzyme, etc. In some embodiments, an encoded polypeptide or polypeptides may be or include one or more neoantigens or neoepitopes associated with a tumor. In some embodiments, an encoded polypeptide or polypeptides may be or include one or more antigens (or epitopes thereof) of an infectious agent (e.g., a bacterium, fungus, virus, etc.). In certain embodiments, an encoded polypeptide may be a variant of a wild type polypeptide.

[0237] In some embodiments, a single-stranded RNA (e.g., mRNA) may comprise a secretion signal-encoding region (e.g., a secretion signal-encoding region that allows an encoded target entity or entities to be secreted upon translation by cells). In some embodiments, such a secretion signal-encoding region may be or comprise a non-human secretion signal. In some embodiments, such a secretion signal-encoding region may be or comprise a human secretion signal.

[0238] In some embodiments, a single-stranded RNA (e.g., mRNA) may comprise at least one non-coding element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non-coding elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, a cap structure (e.g., in some embodiments, an enzymatically-added cap; in some embodiments, aPage 164 of 20712885786vlAttorney Docket No. 2013237-1455 co-transcriptional cap), a poly adenine (poly A) tail (e.g., that, in some embodiments, may be or comprise 100 A residues or more, and / or in some embodiments may include one or more “interrupting” [i.e., non-A] sequence elements), and any combinations thereof. Exemplary embodiments of such non-coding elements may be found, for example, in WO2011015347, WO2017053297, US 10519189, US 10494399, W02007024708, W02007036366, W02017060314, W02016005324, W02005038030, WO2017036889, WO2017162266, and WO2017162461, each of which is incorporated herein by referenced in its entirety.

[0239] At least four formats useful for RNA pharmaceutical compositions (e.g., immunogenic compositions or vaccines) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleosidemodified mRNA (modRNA), self-amplifying mRNA (saRNA), and transamplifying RNAs.

[0240] Features of a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, good tolerability and safety, and strong antibody and T cell responses.

[0241] Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus augmented antigen expression, good tolerability and safety, and strong antibody and CD4-T cell responses. As noted herein, the present disclosure provides an insight that such strong antibody and CD4 T cell responses may be particularly useful for vaccination.

[0242] Features of self-amplifying platform may include, for example, long duration of polypeptide (e.g., protein) expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.

[0243] In some embodiments, a self-amplifying platform (e.g., RNA) comprises two nucleic acid molecules, wherein one nucleic acid molecule encodes a replicase (e.g., a viral replicase) and the other nucleic acid molecule is capable of being replicated (e.g., a replicon) by said replicase in trans (tro / rv- replication system). In some embodiments, a self-amplifying platform (e.g., RNA) comprises a plurality of nucleic acid molecules, wherein said nucleic acids encode a plurality of replicases and / or replicons.

[0244] In some embodiments, a Zran.s-replication system comprises the presence of both nucleic acid molecules in a single host cell.

[0245] In some such embodiments, a nucleic acid encoding a replicase (e.g., a viral replicase) is not capable of self -replication in a target cell and / or target organism. In some such embodiments,Page 165 of 20712885786vlAttorney Docket No. 2013237-1455 a nucleic acid encoding a replicase (e.g., a viral replicase) lacks at least one conserved sequence element important for (-) strand synthesis based on a (+) strand template and / or for (+) strand synthesis based on a (-) strand template.

[0246] In some embodiments, a self-amplifying RNA comprises a 5 ’-cap; in some transreplication systems, at least an RNA encoding a replicase is capped. Without wishing to be bound by any one theory, it has been found that a 5 ’-cap can be important for high level expression of a gene of interest in trans.

[0247] In some embodiments, a self-amplifying platform does not require propagation of virus particles (e.g., is not associated with undesired virus-particle formation). In some embodiments, a self-amplifying platform is not capable of forming virus particles.

[0248] In some embodiments, an RNA may comprise an Internal Ribosomal Entry Site (IRES) element. In some embodiments, an RNA does not comprise an IRES site; in particular, in some embodiments, an saRNA does not comprise an IRES site. In some such embodiments, translation of a gene of interest and / or replicase is not driven by an IRES element. In some embodiments, an IRES element is substituted by a 5 ’-cap. In some such embodiments, substitution by a 5 ’-cap does not affect the sequence of a polypeptide encoded by an RNA.

[0249] In some embodiments, a complex described herein comprises modRNA, saRNA, taRNA, or uRNA. In some embodiments, a complex comprises modRNA. In some embodiments, a complex comprises saRNA. In some embodiments, a complex comprises taRNA. In some embodiments, a complex comprises uRNA.Additional Components

[0250] In some embodiments, a complex described herein further comprises one or more additional lipids. In some embodiments, one or more additional lipids are selected from a helper lipid and a sterol.Helper Lipids

[0251] In some embodiments, a complex described herein further comprises a helper lipid. In some embodiments, a helper lipid is or comprises a lipid selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC),Page 166 of 20712885786vlAttorney Docket No. 2013237-1455 dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC) , dilignoceroylphatidy Icholine (DLPC), palmitoyloleoyl -phosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and combinations thereof.

[0252] In some embodiments, a helper lipid is DSPC and / or DMPC. In some embodiments, a helper lipid is DSPC. In some embodiments, a helper lipid is DMPC.

[0253] In some embodiments, a molar ratio of a cationic oligosaccharide to a helper lipid is from about 1:0.25 to about 1:1. In some embodiments, a molar ratio of a cationic oligosaccharide to a helper lipid is about 1:0.25, about 1:0.5, or about 1: 1. In some embodiments, a molar ratio of a cationic oligosaccharide to a helper lipid is about 1:0.5.

[0254] In some embodiments, a complex described herein comprises a cationic oligosaccharide, a surfactant, a sterol, and a helper lipid, and a molar ratio of a cationic oligosaccharide to a sterol to a helper lipid (i.e., a cationic oligosaccharide : a sterol : a helper lipid) is from about 1:0.25:0.25 to about 1: 1:1. In some embodiments, a molar ratio of a cationic oligosaccharide to a sterol to a helper lipid is from about l:0.5:0.5 to about 1: 1: 1. In some embodiments, a molar ratio of a cationic oligosaccharide to a sterol to a helper lipid is about 1:0.25:0.25, about 1:0.5:0.25, about 1:0.75:0.25, about 1:1:0.25, about 1:0.25:0.5, about 1:0.25:0.75, about 1:0.25:1, about 1:0.5:0.5, about 1:0.75:0.5, about l:l:0.5, about 1:0.5:0.75, about 1:0.5: 1, about 1:0.75:0.75, about 1: 1:0.75, about 1:0.75:1, or about 1: 1: 1. In some embodiments, a molar ratio of a cationic oligosaccharide to a sterol to a helper lipid is about l:0.5:0.5.Sterols

[0255] In some embodiments, a sterol is or comprises P-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid,Page 167 of 20712885786vlAttorney Docket No. 2013237-1455 cycloartenol, lanosterol, or a-tocopherol. In some embodiments, a sterol is or comprises an analogue of one or more of P-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol, bonded to an optionally substituted C1-C30 aliphatic moiety. In some embodiments, a sterol is P- sitosterol. In some embodiments, a sterol is stigmasterol. In some embodiments, a sterol is cholesterol. In some embodiments, a sterol is cholecalciferol. In some embodiments, a sterol is ergocalciferol. In some embodiments, a sterol is calcipotriol. In some embodiments, a sterol is botulin. In some embodiments, a sterol is lupeol. In some embodiments, a sterol is ursolic acid. In some embodiments, a sterol is oleanolic acid. In some embodiments, a sterol is cycloartenol. In some embodiments, a sterol is lanosterol. In some embodiments, a sterol is a-tocopherol.

[0256] In some embodiments, a molar ratio of a cationic oligosaccharide to a sterol is from about 1:0.25 to about 1: 1. In some embodiments, a molar ratio of cationic oligosaccharide to a sterol is about 1 :0.25, about 1 :0.5, or about 1: 1. In some embodiments, a molar ratio of a cationic oligosaccharide to a sterol is about 1:0.5.

[0257] In some embodiments a complex described herein comprises an oligosaccharide, a surfactant, a helper lipid, and a sterol.Systems

[0258] As described herein, the present disclosure describes, among other things, drug production systems for producing a pharmaceutical composition by a continuous flow manufacturing process using methods described herein according to configurations that would be readily apparent to a person of skill in the art. In some embodiments, the drug production system comprising a first composition containment unit comprising a nucleic acid solution and a second composition containment unit comprising an organic solution, wherein: the first composition containment unit and the second composition containment unit are connected via a mixer unit configured to receive the nucleic acid solution and the organic solution, wherein the organic solution comprises an organic solvent and an oligosaccharide comprising a plurality of cationic moieties.

[0259] An example drug production system 100 is provided in FIG. 1. In FIG. 1, the system 100 comprises a nucleic acid containment unit 101. Said nucleic acid containment unit 101 comprises a nucleic acid described herein, and a buffer. In some embodiments, the nucleic acidPage 168 of 20712885786vlAttorney Docket No. 2013237-1455 containment unit 101 is configured, to receive material (e.g., by piping) from a precursor containment unit 106. In some embodiments, multiple precursor containment units 106 are configured to deliver material to the nucleic acid containment unit 101. For example, in some embodiments, one precursor containment unit 106 comprises a precursor nucleic acid solution, and another precursor containment unit 106 comprises a buffer, both precursor containment units configured to deliver material to the nucleic acid containment unit, thereby providing the nucleic acid solution.

[0260] In the example embodiment of FIG. 1, the drug production system 100 further comprises an organic solution containment unit 102. As described herein, the organic solution containment unit 102 comprises an organic solvent and an oligosaccharide compound described herein. In some embodiments, an organic solvent is ethanol or DMSO.

[0261] The nucleic acid containment unit 101 and the organic solution containment unit 102 are configured to deliver a nucleic acid solution (via the nucleic acid containment unit 101) and an organic solution (via the organic solution containment unit 102) to a mixer unit 103. As described herein, a mixer unit 103 can be a T-shaped mixer unit, or a Y-shaped mixer unit. In some embodiments, a mixer unit 103 is a T-shaped mixer unit. In some embodiments, a mixer unit 103 is a Y -shaped mixer unit.

[0262] The mixer unit 103 is configured to deliver the mixed material to a receiving unit 104 that contains a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising nucleic acid particles, wherein said nucleic acid particles comprise a nucleic acid and an oligosaccharide compound described herein). Optionally, a drug delivery system 100 further comprises a purification unit 105. In some embodiments, a purification unit 105 is configured for dialysis or tangential flow filtration (TFF).EXEMPLARY EMBODIMENTS

[0263] The present disclosure provides the following non-limiting numbered embodiments.Embodiment 1. A method of preparing a pharmaceutical composition comprising a plurality of nucleic acid particles, the method comprising contacting a nucleic acid solution with an organic solution via a mixer configured to receive the nucleic acid solution and the organic solution, wherein: the organic solution comprises an organic solvent and an oligosaccharide comprising one or more cationic moieties.Page 169 of 20712885786vlAttorney Docket No. 2013237-1455Embodiment 2. The method of Embodiment 1 , wherein a N / P ratio of the nucleic acid solution and the organic solution is from about 2 to about 12.Embodiment 3. The method of Embodiments 1 or 2, wherein a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is about 25 ml / min to about 360 ml / min.Embodiment 4. The method of Embodiment 3, wherein the total flow rate of nucleic acid solution and the organic solution into the mixer is about 25 ml / min to about 250 ml / min.Embodiment 5. The method of any one of Embodiments 1-4, wherein the organic solution further comprises a surfactant.Embodiment 6. The method of Embodiment 5, wherein the surfactant is or comprises a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.Embodiment 7. The method of Embodiments 5 or 6, wherein a molar ratio of the oligosaccharide to the surfactant is from about 1:0.5 to about 1:0.01.Embodiment 8. The method of any one of Embodiments 5-7, wherein a concentration of the surfactant in the organic solution is about 50 to about 200 mM.Embodiment 9. The method of Embodiment 8, wherein a concentration of the surfactant in the organic solution is about 100 mM.Embodiment 10. The method of any one of Embodiments 1-9, wherein a concentration of the oligosaccharide in the organic solution is from about 10 to about 50 mM.Embodiment 11. The method of Embodiment 10, wherein a concentration of the oligosaccharide in the organic solution is from about 20 mM to about 25 mM.Embodiment 12. The method of any one of Embodiments 1-11, wherein the TFR is about 25 ml / min to about 200 ml / min.Embodiment 13. The method of any one of Embodiments 1-12, wherein the TFR is about100 ml / min.Embodiment 14. The method of any one of Embodiments 1-13, wherein the mixer is a T- shaped mixer.Embodiment 15. The method of any one of Embodiments 1-13, wherein the mixer is a Y- shaped mixer.Page 170 of 20712885786vlAttorney Docket No. 2013237-1455Embodiment 16. The method of any one of Embodiments 1-15, wherein a diameter of the mixer is about 1 mm.Embodiment 17. The method of any one of Embodiments 1-16, wherein a pH of the nucleic acid solution is from about 4 to about 7.Embodiment 18. The method of Embodiment 17, wherein a pH of the nucleic acid solution is about 6.Embodiment 19. The method of any one of Embodiments 1-18, wherein the organic solvent is a polar organic solvent.Embodiment 20. The method of Embodiment 19, wherein the organic solvent is selected from methanol, ethanol, isopropanol, and DMSO.Embodiment 21. The method of Embodiment 20, wherein the organic solvent is ethanol orDMSO.Embodiment 22. The method of Embodiment 21, wherein the organic solvent is ethanol.Embodiment 23. The method of any one of Embodiments 1 -22, wherein the pharmaceutical composition has a nucleic acid concentration from about 0.025 to about 1.0 mg / ml.Embodiment 24. The method of Embodiment 23, wherein the pharmaceutical composition has a nucleic acid concentration of about 0.1 mg / ml.Embodiment 25. The method of Embodiment 23, wherein the pharmaceutical composition has a nucleic acid concentration of about 0.9 mg / ml.Embodiment 26. The method of any one of Embodiments 1-25, wherein the plurality of nucleic acid particles in the pharmaceutical composition have a PDI of less than 0.4.Embodiment 27. The method of any one of Embodiments 1-26, wherein the plurality of nucleic acid particles in the pharmaceutical composition have an average particle size of about 20 nm to about 500 nm.Embodiment 28. The method of Embodiment 27, wherein the plurality of nucleic acid particles in the pharmaceutical composition have an average particle size of about 50 nm to about 250 nm.Embodiment 29. The method of any one of Embodiments 1-20, wherein the plurality of nucleic acid particles in the pharmaceutical composition have an average particle size of about 100 nm.Page 171 of 20712885786vlAttorney Docket No. 2013237-1455Embodiment 30. The method of any one of Embodiments 1-29, wherein the method further comprises purifying the pharmaceutical composition.Embodiment 31. The method of Embodiment 30, wherein the purification comprises dialysis.Embodiment 32. The method of Embodiment 31, wherein the pharmaceutical composition is dialyzed against a buffer having a pH of from about 5 to about 8.Embodiment 33. The method of Embodiment 32, wherein the pharmaceutical composition is dialyzed against a buffer having a pH of about 6.Embodiment 34. The method of Embodiment 30, wherein the purification comprises tangential flow filtration (TFF).Embodiment 35. The method of any one of Embodiments 1-34, wherein the oligosaccharide comprises one or more cationic moieties bonded to a trehalose, a sucrose, or a gluco-n- oligosaccharide moiety, where n is 2-6.Embodiment 36. The method of any one of Embodiments 1-34, wherein the oligosaccharide is a compound represented by formula I:or a pharmaceutically acceptable salt thereof, wherein:Page 172 of 20712885786vlAttorney Docket No. 2013237-1455 each of R1and R2are independently selected, at each instance, from H, Ra, and -C(O)-Ra, wherein at least one instance of R1or R2is not H; each Rais independently selected from C1-C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein each Rais optionally substituted with one or more Rb; each Rbis independently selected from halogen, -N3, -Rc, -ORC, -SRC, -NHRC, -C(O)-RC, - OC(O)RC, -NHC(O)RC, -C(O)NHRC, and -NHC(O)NHRC; each Rcis independently selected from optionally substituted C1-C20 aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S;X1and X2are each independently selected from -S-, -S-S-, and -NH-;Y1and Y2are each independently an optionally substituted Ci-30 aliphatic group wherein one or more carbons are optionally and independently replaced by -Cy-, -NRY-, - NRYC(O)-, -C(O)NRY-, -NRYC(O)O-, -OC(O)NRY-, -NRYC(O)NRY-, - NRYC(S)NRY-, -C(S)NRY-, -C(O)NRYSO2-, -SO2NRYC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each RYis independently H or optionally substituted Ci-Ce aliphatic; each Cy is independently an optionally substituted C3-C14 cycloaliphatic, optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, and optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S;Z1and Z2are each independently a cationic or ionizable group selected from optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S, -N+(M)3,each M is independently -Co-Ce aliphatic-Rzor -Co-Ce aliphatic-N+(Rz)3;Page 173 of 20712885786vlAttorney Docket No. 2013237-1455 each Rzis independently selected from H, optionally substituted Ci-Ce aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; or two or more Rzcan come together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; and p is an integer selected from 1, 2, 3, 4, or 5.Embodiment 37. The method of any one of Embodiments 1-36, wherein the oligosaccharide is selected from Table 1.Embodiment 38. The method of any one of Embodiments 1-37, wherein the organic solution further comprises one or more additional lipids.Embodiment 39. The method of Embodiment 38, wherein the one or more additional lipids are selected from a helper lipid, a steroid, and a polymer conjugated lipid.Embodiment 40. The method of Embodiment 39, wherein the sterol is selected from P- sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol.Embodiment 41. The method of Embodiments 39 or 40, wherein the helper lipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl -phosphatidylcholine (POPC), 1,2- di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn- glycero-3-phosphocholine (Cl 6 Lyso PC), dioleoylphosphatidylethanolamine (DOPE),Page 174 of 20712885786vlAttorney Docket No. 2013237-1455 distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and combinations thereof.Embodiment 42. The method of any one of Embodiments 39-41, wherein the polymer- conjugated lipid is or comprises a polysorbate, a poloxamer, and / or a compound comprising an amphiphilic moiety selected from polyalkylene glycols (e.g., polyethylene glycol), poly(2-oxazoline), poly(2-oxazine), polysarcosine, polyvinylpyrrolidone, and poly[N-(2-hydroxypropyl)methacrylamide, wherein the amphiphilic moiety is bonded to one or more C12-C20 aliphatic groups.Embodiment 43. The method of any one of Embodiments 1-42, wherein the nucleic acid isRNA.Embodiment 44. The method of Embodiment 43, wherein the RNA is mRNA.Embodiment 45. The method of Embodiment 44, wherein the mRNA is modRNA, saRNA, taRNA, or uRNA.Embodiment 46. The method of any one of Embodiments 1-42, wherein the nucleic acid is DNA.Embodiment 47. The method of any one of Embodiments 1-46, wherein the plurality of nucleic acid particles are stable (e.g., do not substantially change in size or PDI) after one or more freeze-thaw cycles.Embodiment 48. A drug production system for producing a pharmaceutical composition by a continuous flow manufacturing process, the drug production system comprising a first composition containment unit comprising a nucleic acid solution and a second composition containment unit comprising an organic solution, wherein the first composition containment unit and the second composition containment unit are connected via a mixer unit configured to receive the nucleic acid solution and the organic solution, and wherein the organic solution comprises an organic solvent and an oligosaccharide comprising a plurality of cationic moieties.Embodiment 49. The drug production system of Embodiment 48, wherein the first composition containment unit and the second composition containment unit are connectedPage 175 of 20712885786vlAttorney Docket No. 2013237-1455 via a mixer unit configured to receive the nucleic acid solution and the organic solution at a total flow rate (TFR) of about 25 ml / min to about 360 ml / min.Embodiment 50. The drug production system of Embodiments 48 or 49, further comprising one or more precursor containment units, configured to deliver a nucleic acid and / or a buffer to the first composition containment unit.Embodiment 51. The drug production system of Embodiment 50, wherein the buffer has a pH of about 4 to about 7.Embodiment 52. The drug production system of any one of Embodiments 48-51 , wherein the drug production system further comprises a purification module configured to receive and purify the pharmaceutical composition from the mixer unit.Embodiment 53. The drug production system of Embodiment 52, wherein the purification module dialyzes the pharmaceutical composition.Embodiment 54. The drug production system of Embodiment 53, wherein the pharmaceutical composition is dialyzed in the purification module against a buffer having a pH of from about 5 to about 8.Embodiment 55. The drug production system of Embodiment 53 , wherein the pharmaceutical composition is dialyzed in the purification module against a buffer having a pH at about physiological pH.Embodiment 56. The drug production system of Embodiment 54 or 55, wherein the purification module is configured for tangential flow filtration (TFF).Embodiment 57. The drug production system of any one of Embodiments 48-56, configured to perform the method of preparing a pharmaceutical composition in any one of Embodiments 1-47.EXAMPLES

[0264] The following examples are intended to illustrate certain embodiments of the subject matter described in the present disclosure. These examples are not intended to be limiting.

[0265] The following abbreviations are used herein: cone. = concentrationCPLX = nucleic acid particlesCQA = clinical quality attributePage 176 of 20712885786vlAttorney Docket No. 2013237-1455FT = freeze-thaw h or hr = hour or hoursMES = 2-(N-morpholino)ethanesulfonic acidMWCO = molecular weight cut-off paOs = polycationic amphiphilic oligosaccharide(s)PDI = polydispersity indexPES = polyethersulfoneRT = room temperatureTFF = tangential flow filtrationTFR = total flow rateTween 40, 60, 80, etc. = polysorbate 40, 60, 80, etc.Materials

[0266] The following table provides a summary of materials usedDrug Substance

[0267] modRNA-Luciferase used for the manufacturing of nucleic acid particles was prepared by known methods. The RNA was delivered at a cone, of 4.01 mg / ml and was buffered in milliQ water at neutral pH. The final mRNA was stored at -20 °C and thawed at 4 °C prior to usage.Page 177 of 20712885786vlAttorney Docket No. 2013237-1455MethodsVisual Appearance

[0268] The appearance of the drug product was determined by visual inspection. The drug product is an off-white suspension.Size and size distribution determination

[0269] Particle sizes were determined by dynamic light scattering (DLS) using DynaPro Plate Reader II (Wyatt, Dernbach, Germany). Measurements were conducted in a 96-well plate with nucleic acid particle samples diluted to 0.005 mg / mL (RNA concentration) in 10 mM MES buffer, in respective pH and in presence or absence of cryoprotectant and measured in triplicates at 23 °C. A run of 10 acquisitions were used as settings for the measurement. Average size and size distribution expressed as polydispersity index (PDI) were obtained using Dynamics software 7.8.1.3 (Wyatt Technology, Santa Barbara, CA, USA). pH determination

[0270] Samples pH values were measured using a pH meter. The pH meter was calibrated within the expected pH range prior to measurement. The electrode of the pH meter was dipped into a 100 pL sample volume in a 500 pL Eppendorf tube; to allow the membrane of the electrode to be in contact with the suspension.Osmolality

[0271] Osmolality was determined using an osmometer (Osmomat 3000, Gonotec, USA), and a pre-measurement calibration is performed with reference standards covering the expected range (used standards are as follows: 100, 290, and 500 mOsmol / kg). A sample of 50 pL nucleic acid particles in a 500 pL Eppendorf tube was used to determine the osmolality value.Free RNA and RNA content determination

[0272] The Agarose gel electrophoresis technique was performed to determine free RNA percentage in nucleic acid particles formulations. The agarose gel ( 1 %) was prepared by dissolving 1 g Agarose in 100 mL of lx Tris-acetate-EDTA buffer (ROTIPHORESE®50x TAE Buffer, CarlPage 178 of 20712885786vlAttorney Docket No. 2013237-1455Roth, Karlsruhe, Germany). In addition, 1 mL of 5% sodium hypochlorite (NaOCl) and 10 pL of GelRed® Nucleic acid Stain (Biotium, Hayward, CA, USA) were added to the mixture and heated in the microwave for 1 min (3 cycles to ensure complete dissolution). Using appropriate casting tray and combs, the agarose solution was poured at room temperature and the gel was allowed to solidify for approximately 20-25 min. Samples were prepared at a concentration of 0.1 mg / ml either directly (only nucleic acid particles to determine free RNA) or diluted 1:1 with release solution (5% v / v Triton X-100 and 0.72 mmol heparin solution in water) and incubated at 40 °C for 7 min (to determine total RNA) before adding the loading dye (6X DNA Gel Loading Dye, Thermo Fisher Scientific, Waltham, MA, USA). Free modRNA-luc was prepared in different concentrations between 0.00625 - 0.1 mg / ml and used as a calibration curve to determine the RNA content in the nucleic acid particle samples. An electrophoresis chamber containing lx TAE running buffer (pH 7.4) was used to run the gel at 80 V for 40 min, and GEL Doc EZ System was used to take gel images (Bio-Rad Laboratories GmbH, Postfach, Germany).RNA integrity

[0273] To assess the quality and the integrity of the RNA encapsulated into the nucleic acid particles formulations, a capillary electrophoresis technique was conducted using the Agilent 3367 Fragment Analyzer (FA) system (Agilent Technologies Inc., Germany). High sensitivity RNA kit (Agilent Technologies Inc., Germany) was used for this purpose. Briefly, 5 pL sample volume was mixed with 25 p L lysing buffer (Triton + heparin), the suspension was then incubated at 30 °C for 20 min under shaking (600 rpm). A volume of 2 pL of the suspension was then diluted 1:10 with Diluent marker of the RNA kit in a 96-well plate and covered with an auto-adhesive lid. The samples were subjected to a denaturation process for 2 min at 70 °C in a thermal cycler after a gentle shaking of the plate and then placed directly into the FA for analysis.Frozen stability of the test material

[0274] The frozen stability studies on the nucleic acid particle samples were conducted at -80 °C for a period of 3 days by freeze-thaw cycles. At each time point, the number of requisite vials were thawed or brought to room temperature and subjected to analytical testing.Page 179 of 20712885786vlAttorney Docket No. 2013237-1455In-vitro cell viability and transfection efficiency

[0275] Luciferase assays are a reliable and sensitive method to detect the expression of luciferase in live cells. In this assay, firefly luciferase catalyzes the ATP and Mg2+-dependent mono-oxygenation of beetle luciferin. This results in emission of light in the range of 550 to 620 nm. For cell transfections, HepG2, C2C12 and RAW 264.7 cells were seeded at respective cell number of 20,000 cells / well, 5,000 cells / well and 25,000 cells / well in a Nunc white flat-bottom96-well plate (Merck KGaA, Darmstadt, Germany) and centrifuged at 500 x g for 5 min. After 18- 24 h, the medium was replaced by fresh medium at 90 pl / well DPBS and only medium were used as negative and blank control, respectively. Formulation samples were tested at mRNA assay concentration between 25 to 100 ng per well. Samples and controls were added at 10 pl / well. After 24 h, luciferase expression was determined by Bright-Glo™ Luciferase assay (Promega, Madison, WI, USA) according to manufacturer’s protocol. Briefly, the reagent was added to the cells in medium in 1 : 1 (v / v) ratio, followed by an incubation of 5 min in the dark to allow for complete cell lysis. Viability was measured by CellTiter-Glo® (Promega, Madison, WI, USA) according to manufacturer’s protocol. Briefly, the reagent was added to the cells in medium in 1:1 (v / v) ratio, followed by an incubation of 10 min in shaker followed by 20 min to allow for stabilization the signal. Alternatively, luciferase and viability were determined by ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega GmbH, Madison, WI, USA) according to manual instructions. Briefly, 20 pl of 5X CellTiter-Fluor™ Reagent was added to the wells, mix by orbital shaking (300-500rpm for ~30 sec). After incubation for 30 min at 37 °C, the fluorescence was measured with excitation wavelength at 400 nm and emission at 505 nm (viability). Then, ONE- Glo™ Reagent was added at 100 pl / well. After incubation for three min, the bioluminescence was measured (luciferase expression). Bioluminescence signals (photons per second [p / s])) and fluorescence were measured using a microplate luminescence reader Infinite M200 (Tecan, Mannedorf, Switzerland). Relative luminescence was calculated by subtracting the signal of DPBS control from the sample control. Relative viability was calculated by: 100Page 180 of 20712885786vlAttorney Docket No. 2013237-1455Example 1 — Preparing Nucleic Acid Particles by a Hand Mixing Process

[0276] The present example provides hand mixing process for preparing nucleic acid particles described herein. Example nucleic acid particles were prepared by adding an RNA phase on top of the polycationic amphiphilic oligosaccharide (paOs) phase, in a 9: 1 ratio, respectively. The RNA phase consisted of modRNA-luc in water. The organic phase consisted of 25mM JLF99 in DMSO and lOOmM Tween 40 in DMSO mixed in a molar ratio of 1:0.5 respectively. For the manufacturing of nucleic acid particles, the RNA phase was preconditioned with 60 mM MES, 30% D-glucose (w / v), pH 6.1 (referred hereafter as 6x MBG buffer) at a concentration of 0.222 mg / ml. The raw nucleic acid particles obtained by mixing RNA and the organic phases had the manufacturing concentration of 0.2 mg / ml. The raw colloids were diluted with 10 mM MES, 5% glucose (w / v) pH 6.1 (referred hereafter as lx MBG buffer) to a final RNA concentration of 0.1 mg / ml. The nucleic acid particles obtained with hand mixing protocol has shown good frozen stability at -80 °C up to 12 months, in terms of both size and RNA integrity (see FIGs. 34A-34C)) The schematic representation of the hand mixing manufacturing process is depicted in FIG. 2A. The nucleic acid particles were analyzed to determine the size and PDI and confirm they were within acceptance criteria of <100 nm and <0.3 respectively as shown in FIG. 2B.Example 2 — Transfer of Hand-Mixing Process to Fluid Path Process

[0277] Before evaluation of the certain process parameters, the first step in the process development of nucleic acid particles involved transfer of hand mixing method to the fluid path process. Nucleic acid particles were manufactured in a semi-automated process by mixing RNA and organic phases in 3:1 ratios respectively. Y type mixing element with lengths 5 cm for RNA and organic phase each and 20 cm for the mixing channel was tested. The tubing used as a mixing element had a diameter of 1 mm. The total flow rate (TFR) of 50 ml / min was selected for the process. The RNA phase was preconditioned with 6x MBG buffer, pH 6.1. The organic phase consisted of 25 mM paOs and 100 mM Tween 40 solutions in DMSO and were mixed in the molar ratios 1 :0.5 respectively. The raw nucleic acid particles obtained by mixing RNA and the organic phases using fluid path process had the manufacturing cone, of 0.2 mg / ml. The raw colloids were diluted with lx MBG pH 6.1 buffer to a final RNA cone, of 0.1 mg / ml. The schematics of the fluid path manufacturing process is shown in FIG. 3. The nucleic acid particles obtained with the fluidPage 181 of 20712885786vlAttorney Docket No. 2013237-1455 path process were analyzed to determine their size and PDI and compared with the hand mixing process as shown in FIG. 2B.

[0278] In the next step, DMSO was replaced with ethanol as the organic solvent and the fluid path process was carried out keeping the other process parameters intact as mentioned earlier in this section. In this process, the organic phase consisted of 20 mM paOS and 100 mM Tween 40 in ethanol as organic solvent and were mixed in molar ratios 1:0.5 respectively. The raw colloids were diluted with lx MBG buffer, pH 6.1 buffer to a final RNA cone, of 0.1 mg / ml. The schematic representation of the fluid path manufacturing process is depicted in FIG. 3.The nucleic acid particles obtained with the fluid path process were analyzed to determine their size and PDI, and compared with the hand mixing process as shown in FIG. 2B. It was observed that the transfer of hand mixing process to fluid path using DMSO as the organic phase gives the comparable particle sizes, however, higher PDI was obtained.Example 3 — Effect of Mixing Geometry and Flow Rate on Colloidal Stability

[0279] The present example analyzes mixing geometries and the effect of total flow rates (TFR) on the nucleic acid particles. The present example provides two different mixing elements Y and T type that were tested, both with lengths 5 cm for RNA and organic phase each and 20 cm for the mixing channel. The tubings (Y and T) used as a mixing element has a diameter of 1 mm. Unless specifically mentioned, the mixing ratios were fixed to 3: 1 for RNA and organic phases respectively and N / P ratio was fixed to 6 in all the semi-automated fluid path process investigations.

[0280] To investigate the optimal mixing geometry and TFR, nucleic acid particles were manufactured using two different mixing geometries Y and T type with variable TFR from 25-200 ml / min. For the fluid path manufacturing of nucleic acid particles, RNA phase was preconditioned with 6x MBG buffer pH 6.1. The organic phase consisted of 20 mM JLF99 and 100 mM Tween 40 solutions in ethanol and were mixed in the molar ratios 1:0.5 respectively. The raw nucleic acid particles were obtained by mixing RNA and the organic phase using fluid path with a RNA manufacturing concentration of 0.2 mg / ml. The schematic representation of the fluid path manufacturing process is depicted in FIG. 4. Certain physiological parameters like size and PDI were investigated for the nucleic acid particles (raw colloids) at different TFR (25-200 ml / min) and using two different mixing geometries. In FIG. 5A, it was observed that the particles sizePage 182 of 20712885786vlAttorney Docket No. 2013237-1455 decreases as the TFR increases in case of Y mixer and the particles shows size <100 nm between TFR 50-200 ml / min. In case of T mixer, FIG. 5B, no considerable effect of TFR was observed and the particles shows acceptable size <100 nm at TFR 25-150 ml / min.

[0281] The purification of nucleic acid particles by removing the organic solvent was performed using dialysis method. Dialysis cassettes with 3 ml capacity and MWCO 10,000 were used. The raw nucleic acid particles were dialyzed against lx MBG buffer, pH 6.1 overnight at 4 °C to ensure complete removal of organic phase and obtain the final drug product. Certain physicochemical parameters like size, PDI, pH and osmolality of the nucleic acid particles were tested.

[0282] In FIGs. 6A-6D, it is shown that particle sizes obtained using both Y and T mixers at all the flow rates tested were found to be < 100 nm after removal of the organic solvent by dialysis. In the case of Y mixer (FIG. 6A), a bell trend was observed for nucleic acid particles sizes when the flow rate was varied in the range 25-200 mL / min, with biggest sizes obtained at 50 mL / min after dialysis. In comparison to raw colloids (FIG. 5A), a decrease in size of the nucleic acid particles was observed in Y mixer at TFR 25 ml / min after dialysis, whereas the sizes were comparable at other TFR tested 50-200 ml / min. In case of T mixer, it was observed that the TFR does not play a significant role on the particle size at all TFR tested both before (FIG. 5B) and after dialysis (FIG. 6C) of the nucleic acid particles. The PDI of the nucleic acid particles after dialysis were higher than the acceptance value in both Y and T mixers and at all TFR tested. The osmolality and the pH of nucleic acid particles obtained after dialysis were in the range for all tested conditions, confirming the optimization of dialysis process (FIGs. 6 B and D).Example 4 — TFR Variation and Mixing Geometry Evaluation

[0283] For the present example, et seq. , unless specified, for the fluid path manufacturing of provided nucleic acid particles, the RNA phase was preconditioned with 6x MBG buffer pH 6.1 and the organic phase consisted of 20 mM JLF99 in ethanol.

[0284] For efficient mixing of the aqueous and organic phases, more understanding in mixing geometries and TFR was required without Tween 40. Two different mixing elements (T and Y, see more details in Example 3) and at different TFR between 50-200 ml / min were investigated. The raw nucleic acid particles obtained by mixing RNA and the organic phase using fluid path have the RNA manufacturing cone, of 0.2 mg / ml. The raw nucleic acid particles were dialyzedPage 183 of 20712885786vlAttorney Docket No. 2013237-1455 against lx MBG buffer, pH 6.1 overnight at 4 °C. Certain physicochemical parameters such as particle size and PDI were investigated to understand the effect of two different types of mixing geometries and TFR variation on colloidal stability of the particles. The schematic representation of the fluid path manufacturing process is shown in FIG. 7.

[0285] As shown in FIGs. 8 A and 8B, particle sizes obtained using both Y and T mixers at all the flow rates tested were found to be >100nm with PDI <0.3. No significant effect of TFR variation was observed in case of Y mixer, in contradiction to previous observation (FIG. 6A). A slight increase in particle size was observed with T mixer when the TFR was increased from 100 to 200 ml / min (FIG. 8B). T mixer and a flow rate of 100 ml / min were selected for further optimization of the process to obtain nucleic acid particles in the desired size range.Example 5 —Manufacturing Concentration Variation and Removal of Glucose

[0286] The present example analyzes the effect of varying manufacturing concentration and viscosity during the mixing process. To see the effect of mentioned parameters on size of the particles, a T mixer with diameter 1 mm and TFR 100 mL / min was fixed, and RNA manufacturing concentration was varied in the range 0.05 - 0.3 mg / ml. Furthermore, RNA phase was prepared in 6x MES buffer, pH 6.1 (with no glucose). The raw nucleic acid particles were dialyzed against lx MES buffer, pH 6.1 overnight at 4 °C. The effect of manufacturing concentration was assessed on particle size and PDI. The schematic representation of the fluid path manufacturing process is shown in FIG. 9.

[0287] As illustrated in the data in FIG. 10, RNA manufacturing concentration impacts the size of nucleic acid particles when manufacturing process was carried out via fluid path. A trend of increasing particle size was observed with increasing manufacturing concentration with size >100 nm at higher manufacturing cone, of 0.15-0.3 mg / ml.Example 6 — Further Analysis of TFR

[0288] Based on the data presented in FIG. 10, RNA manufacturing concentration of 0.1 mg / ml was selected for further optimization of manufacturing process as the nucleic acid particles obtained were close to the desired size range of <100 nm. To further investigate the optimal flow rate at the selected manufacturing cone., nucleic acid particles were prepared with varying TFR from 25-200 ml / min. The raw nucleic acid particles were dialyzed against lx MES buffer, pH 6.1Page 184 of 20712885786vlAttorney Docket No. 2013237-1455 overnight at 4 °C. The schematic representation of the fluid path manufacturing process is shown in FIG. 11.

[0289] In FIG. 12, it is shown that at RNA manufacturing cone, of 0.1 mg / ml, the particles with desired size <100 nm and PDI <0.3 can be obtained using T mixer at TFR 100 ml / min. In subsequent examples, the mentioned process parameters are fixed and other parameters under study were investigated to obtain colloidal stability after purification of raw nucleic acid particles using dialysis method.Example 7 —Analysis of Storage Matrix pH on Colloidal Stability

[0290] For the nucleic acid particle manufacturing, an electrostatic interaction between the negatively charged RNA and positively charged JLF99 is required. The pKa of JLF99 is 6.7 and thus fluid path manufacturing in MES buffer, pH 6.1 results in electrostatic interaction between the RNA and JLF99 to form stable polyplexes. The dialysis buffer selected (lx mM MES buffer, pH 6.1) additionally confirms that nucleic acid particles can be purified from the organic solvent without compromising nanoparticles colloidal stability. However, pH drifts can result in aggregation of the nucleic acid particles as a consequence of change in the protonation state. The present example analyzes the pH of the MES dialysis buffer (fixed concentration of lx) to define the pH tolerances in which nucleic acid particles show acceptable colloidal stability after purification by dialysis.

[0291] As described in the previous example and illustrated in FIG. 12, nucleic acid particles were manufactured using T mixer at TFR 100 ml / min at manufacturing concentration of 0.1 mg / ml as the particle size and PDI obtained were within the acceptable range (<100nm and <0.3 respectively). The raw colloids were aliquoted and dialyzed against lx MES buffer, at variable pH between 5-6.1 overnight at 4 °C. The schematic representation of the fluid path manufacturing process is depicted in FIG. 13. Certain physicochemical parameters like size, PDI, pH and osmolality were investigated.

[0292] FIG. 14 A illustrates that nucleic acid particles dialyzed against lx MES buffer and pH between 5-5.75 show similar particle size of < lOOnm and PDI <0.3, whereas nucleic acid particles dialyzed against buffer pH 6.1 shows bigger particle >100 nm. The particles sizes obtained after dialysis in lx MES buffer, pH 6.1 are different than previous efforts, see, e.g., FIG. 12. The pH of the dialyzed samples was measured and showed no significant variation from the buffers used forPage 185 of 20712885786vlAttorney Docket No. 2013237-1455 dialysis and osmolality values indicated complete removal of the organic solvent from the nucleic acid particles after dialysis (FIG. 14B).Example 8 —Analysis of Surfactant and Frozen Colloidal Stability

[0293] The data obtained in Example 7 and illustrated in FIG. 14A illustrates that nucleic acid particles can be manufactured in absence of Tween 40 while maintaining the CQAs within acceptance criteria after dialysis against lx MES pH < 5.75. However, as described above, Tween 40 imparts certain biological activity to the nucleic acid particles. In the present example, the frozen colloidal stability of nucleic acid particles was tested and compared for samples dialyzed against lx MES buffer at different pH and in presence and absence of Tween 40 as the stealth moiety before assessing in-vitro biological activity. Sucrose (10% w / v), commonly used for cryopreservation in pharmaceutical compositions, was selected as the cryoprotectant for freezing the manufactured nucleic acid particles.

[0294] The frozen colloidal stability of the formulations was tested by stressing the nucleic acid particles through freeze-thaw experiments. The nucleic acid particles were subjected to three freeze-thaw cycles at -80 °C with a holding time of 8 h before the next freezing event. For each thawing cycle, one vial was taken out, kept at RT for 30 min for thawing process and evaluated for particle size and PDI. The rest of the sample was frozen again at -80 °C for the next day evaluation of QCs.Frozen colloidal stability in absence of Tween 40

[0295] Nucleic acid particles manufactured in the prior example were diluted with storage matrix consisting of lx MES buffer, at respective pH and 10% w / v sucrose to a final manufacturing cone, of 0.025 mg / ml and were subjected to three freeze thaw cycles at -80 °C. Certain physicochemical parameters like size and PDI of the nucleic acid particles were tested in 3 FT cycles and compared with the FT0 data.

[0296] In FIGs. 15A-C, it is shown that nucleic acid particles dialyzed against lx MES buffer pH 5, 5.25 and 5.5 do not show any deviation in both size and PDI after 3 FT cycles at -80 °C. Nucleic acid particles dialyzed against lx MES buffer pH 5.75 (FIG. 15D) provides a 2-fold increase in particle size after the first FT cycle whereas nucleic acid particles dialyzed against lxPage 186 of 20712885786vlAttorney Docket No. 2013237-1455MES buffer pH 6.1 shows aggregation with particles resulting in sizes >200nm after the first FT cycle (FIG. 15E).Frozen colloidal stability in the presence of Tween 40

[0297] Nucleic acid particles manufactured in the previous example and dialyzed against lx MES buffer at different pH were spiked with Tween 40 at molar ratios 0.5 relative to JLF99. The samples were diluted with storage matrix consisting of lx MES buffer, at respective pH and 10% w / v sucrose to a final manufacturing cone, of 0.025 mg / ml and were subjected to three freeze thaw cycles at -80 °C. Certain physicochemical parameters like size and PDI of the nucleic acid particles were tested in 3 FT cycles and compared with the FT0 data.

[0298] The data presented in FIGs. 16A-D illustrate that nucleic acid particles dialyzed against lx MES buffer pH 5, 5,25, 5.5 and 5.75 do not show any deviation in both size (<100 nm) and PDI (<0.3) after 3 FT cycles at -80 °C in presence of Tween 40. Without wishing to be bound by theory, it is hypothesized that this result suggested that Tween 40 at 0.5 mol ratio has a stabilizing effect on nucleic acid particles when frozen at -80 °C for repeated cycles at pH 5.75 (as illustrated in FIG. 15D, frozen stability is impacted without Tween40 at the same pH). Nucleic acid particles dialyzed against lx MES buffer pH 6.1 shows ~1.5 times increase in particle size after first FT cycle (FIG. 16E).Example 9 —Analysis of Tween concentration

[0299] Nucleic acid particles were manufactured using T mixer at TFR 100 ml / min at manufacturing cone, of 0.1 mg / ml. The raw colloids were dialyzed against lx MES buffer, pH 5.25 for 4 h at R.T. The dialyzed nucleic acid particles were aliquoted and spiked with Tween 40 in different molar ratios (0-5) relative to JEF99. The samples were diluted with storage matrix consisting of lx MES buffer, pH 5.25 and 10% w / v sucrose to a final manufacturing cone, of 0.025 mg / ml and were subjected to three freeze thaw cycles at -80 °C. Certain physicochemical parameters like size and PDI of the nucleic acid particles were tested. As shown in FIGs. 17A-D, the nucleic acid particles in presence of different cone, of Tween 40 were comparable in size and polydispersity after 3FT cycles at -80 °C and all samples tested were within the acceptable criteria of size and PDI.Page 187 of 20712885786vlAttorney Docket No. 2013237-1455In-vitro cell viability and transfection efficiency

[0300] The present example investigates the role of Tween 40 on biological activity. Nucleic acid particles samples were manufactured as provided in the previous example, dialyzed against lx MES buffer, pH 5.25 and spiked with different molar ratios of Tween 40 (0-0.5) relative to JLF 99. The samples were diluted with storage matrix consisting of lx MES buffer, pH 5.25 and 10% w / v sucrose to a final manufacturing cone, of 0.020 mg / ml. In-vitro cell viability and transfection efficiency were performed on three different cell lines C2C12, RAW and HepG2 and in different doses between 12.5-100 ng. The in-vitro biological activity of the nucleic acid particles obtained via fluid path containing different ratios of Tween 40 was compared with the hand mixing nucleic acid particles samples. From FIGs. 18A-18C, it was observed that all the samples containing Tween 40 in different ratios obtained via fluid path and hand mixing process shows > 80% cell viability at doses 12.5, 25 and 50 ng in all three cell lines tested. Nucleic acid particles obtained via fluid path in absence of Tween 40 shows dose dependent toxicity to the cells (more prominent in RAW cell lines). The transfection efficiency of the nucleic acid particles samples obtained via fluid path in both absence and presence of Tween 40 in different ratios shows higher cell transfection efficiency in all three cell lines and all doses tested as compared to the hand mixing nucleic acid particles (FIGs. 19A-19C).Example 10 — Purification Methods

[0301] The present example investigates the use of a tangential flow filtration (TFF) process for the removal of organic phase and up-concentration of the nucleic acid particles manufactured by fluid path. The nucleic acid particles were manufactured using the process as described in the previous section. The schematics of manufacturing and TFF purification process are shown in FIG. 20. The obtained intermediate nucleic acid particles were purified by TFF, employing the relevant purification steps as described in Table 10-1. After TFF process, the nucleic acid particles were transferred into a sterile hood and diluted to the appropriate concentrations using IxMES buffer, pH 5.25 and 10% w / v sucrose cryoprotectant solution, followed by filtration using 0.22 pm PES filter capsules (Table 10-1). Certain physicochemical parameters like size, PDI, pH and osmolality of the nucleic acid particles were evaluated. In FIGs. 21 A-B, it was observed that the CQAs of the nucleic acid particles were within the acceptable criteria after TFF process.Page 188 of 20712885786vlAttorney Docket No. 2013237-1455Table 10-1Tangential flow filtration process description for nucleic acid particlesFrozen Colloidal Stability after TFF

[0302] Nucleic acid particles can be manufactured in absence of Tween 40 and the nucleic acid particles have CQAs within the acceptance criteria after TFF process. The present example evaluates the effect of TFF on the particles frozen colloidal stability and to investigate the role of Tween 40 on nucleic acid particles assembly, stability and biological activity, further investigations were carried out by the addition of different amounts of Tween 40 to nucleic acid particles after TFF process. Nucleic acid particles obtained after TFF process were aliquoted and spiked with Tween 40 at molar ratios between 0-0.5 relative to JLF99. The samples were diluted with storage matrix consisting of lx MES buffer, pH 5.25 and 10% w / v sucrose to a final manufacturing cone, of 0.15 mg / ml and were subjected to three freeze thaw cycles at -80 °C. Certain physicochemical parameters like size and PDI of the nucleic acid particles were tested in 3 FT cycles and compared with the FT0 data.

[0303] As shown in FIG. 22, the nucleic acid particles in presence of different cone, of Tween 40 were comparable in size and polydispersity after three freeze-thaw cycles at -80 °C. All samples tested were within the acceptable criteria of size and PDI. The study indicated that TFF process was successfully employed without compromising the CQAs of the particles.In-vitro Cell Viability and Transfection Efficiency After TFF

[0304] In the current studies, investigations were carried out to understand the effect of TFF process on the biological activity of the nucleic acid particles in absence and presence of different amounts of Tween 40. Nucleic acid particles obtained after TFF process were aliquoted and spiked with Tween 40 at molar ratios between 0-0.5 relative to JLF99. The samples were diluted withPage 189 of 20712885786vlAttorney Docket No. 2013237-1455 storage matrix of lx MES buffer, pH 5.25 and 10% w / v sucrose to a final manufacturing cone, of 0.025 mg / ml.

[0305] The in-vitro biological activity of the nucleic acid particles obtained via fluid path containing different ratios of Tween 40 was compared with the hand mixing nucleic acid particles samples (benchmark). In-vitro cell viability and transfection efficiency were performed on three different cell lines C2C12, RAW and HepG2 and in different doses 12.5-100 ng. FIG 23 illustrates that the in-vitro cell viability was comparable to the previous studies (see, e.g., FIG 18) wherein nucleic acid particles samples obtained via fluid path in absence of Tween 40 shows dose dependent cell toxicity. All the nucleic acid particles samples in presence of different amounts of Tween 40 including hand mixing samples shows >80% cell viability in three cell lines and at all the doses tested. The transfection efficiency of nucleic acid particles obtained after TFF process and in presence of different amounts of Twee 40 (FIG. 24) were comparable to the nucleic acid particles obtained after hand mixing method. Similar to the previous observation (see, e.g., FIG. 19), the nucleic acid particles obtained by fluid path in absence of Tween 40 shows higher transfection efficiency as compared to samples in presence of Tween 40 (including hand mixing nucleic acid particles) in all three cell lines and at all doses tested. The data showed that TFF process can be employed for the removal of organic phase without compromising the biological activity of the nucleic acid particles.Example 11 — Nucleic acid particles ( CPLXs) manufacturing using JLF218 as alternative paOsManufacturing of CPLXs

[0306] For CPLXs manufacturing, modRNA-Luc phase was preconditioned with 6x MES buffer pH 6.1. The organic phase consisted of 20 mM JLF218 and 100 mM Tween 40 solutions in ethanol and were mixed in the molar ratios 1 :0.5 respectively. The raw CPLXs were obtained by mixing modRNA and the organic phase by fluid path using a T mixer (1mm diameter) at N / P ratio 6 and RNA manufacturing concentration of 0.1 mg / ml. The organic solvent in the raw colloids was removed by dialysis against 10 mM MES, pH 6.1 overnight at 4 °C using dialysis cassettes of MWCO 10,000. The final drug product consisted of RNA cone. <0.05 mg / ml in 10 mM MES, pH 6.1 and 10% sucrose as the final storage matrix.Page 190 of 20712885786vlAttorney Docket No. 2013237-1455Characterization of formulations

[0307] Particle size of CPLX was measured on particles after dialysis using dynamic light scattering using a DynaPro plate reader (Wyatt, Dernbach, Germany). From the measurements, size (Z-average) and polydispersity indices (PDI) were calculated from the cumulant analysis using Dynamics 7.8.1.3 software. For measurements, samples were diluted 1: 10 in 10 mM MES pH 6.1 and analysis was performed in triplicates. Successful cargo incorporation was performed and verified using Agarose gel electrophoresis.Freeze Thaw studies

[0308] Freeze thaw studies were conducted by cycling the formulations from -80 °C (24h) to 25 °C (30 min) at least three times. The particle size and polydispersity index of the formulations were measured for freeze-thaw samples. The formulations between thaw and freeze cycles were mixed by gentle inversions before the next freezing cycle.

[0309] Size and PDI analysis showed that CPLXs with JLF 218 can be obtained with size < 100 nm and PDI < 0.3 as observed in FIG. 25. Successful cargo incorporation was verified via Agarose gel electrophoresis wherein no free RNA band was observed (FIG. 26). FIG. 27 shows the results from freezing of CPLX at -80 °C and storage for at least 3 freeze / thaw cycles. As shown in the figure, no significant changes in size and PDI could be observed at freezing temperatures tested (-80 °C).Example 12 — Nucleic acid particles (CPLXs) manufacturing using JLF99 and DNA as an alternative cargo (dialysis in MES buffer, pH 6.1)Manufacturing of CPLXs

[0310] For CPLXs manufacturing, DNA phase was preconditioned with 6x MES buffer pH 6.1. The organic phase consisted of 20 mM JLF99 and 100 mM Tween 40 solutions in ethanol and were mixed in the molar ratios 1 :0.5 respectively. The raw CPLXs were obtained by mixing DNA and the organic phase by fluid path using a T mixer (1mm diameter) at N / P ratio 6 and DNA manufacturing concentration of 0.1 mg / ml. The organic solvent in the raw colloids was removed by dialysis against 10 mM MES, pH 6.1 overnight at 4 °C using dialysis cassettes of MWCOPage 191 of 20712885786vlAttorney Docket No. 2013237-145510,000. The final drug product consisted of DNA cone. <0.05 mg / ml in 10 mM MES, pH 6.1 and 10% sucrose as the final storage matrix.Characterization of formulations

[0311] Particle size of CPLX was measured on particles after dialysis using dynamic light scattering using a DynaPro plate reader (Wyatt, Dernbach, Germany). From the measurements, size (Z-average) and polydispersity indices (PDI) were calculated from the cumulant analysis using Dynamics 7.8.1.3 software. For measurements, samples were diluted 1: 10 in 10 mM MES pH 6.1 and analysis was performed in triplicates. Successful cargo incorporation was performed and verified using Agarose gel electrophoresis.Freeze Thaw studies

[0312] Freeze thaw studies were conducted by cycling the formulations from -80 °C (24h) to 25 °C (30 min) at least three times. The particle size and polydispersity index of the formulations were measured for freeze-thaw samples. The formulations between thaw and freeze cycles were mixed by gentle inversions before the next freezing cycle.

[0313] Size and PDI analysis showed that CPEXs with JEF 99 can be obtained with size < 200 nm and PDI < 0.3 as observed in FIG. 28. Successful cargo incorporation was verified via Agarose gel electrophoresis wherein no free DNA band was observed (FIG. 29). FIG. 30 shows the results from freezing of CPEX at -80 °C and storage for at least 3 freeze / thaw cycles. As shown in the figure, no significant changes in size and PDI could be observed at freezing temperatures tested (- 80 °C).Example 13 — Nucleic acid particles (CPLXs) manufacturing using JLF99 and DNA as an alternative cargo (dialysis in MES buffer, pH 5.25)Manufacturing of CPLXs

[0314] For CPLXs manufacturing, DNA phase was preconditioned with 6x MES buffer pH 6.1. The organic phase consisted of 20 mM JLF99 and 100 mM Tween 40 solutions in ethanol and were mixed in the molar ratios 1 :0.5 respectively. The raw CPLXs were obtained by mixing DNA and the organic phase by fluid path using a T mixer (1mm diameter) at N / P ratio 6 and DNA manufacturing concentration of 0.1 mg / ml. The organic solvent in the raw colloids was removedPage 192 of 20712885786vlAttorney Docket No. 2013237-1455 by dialysis against 10 mM MES, pH 5.25 overnight at 4 °C using dialysis cassettes of MWCO 10,000. The final drug product consisted of DNA cone. <0.05 mg / ml in 10 mM MES, pH 5.25 and 10% sucrose as the final storage matrix.Characterization of formulations

[0315] Particle size of CPLX was measured on particles after dialysis using dynamic light scattering using a DynaPro plate reader (Wyatt, Dernbach, Germany). From the measurements, size (Z-average) and polydispersity indices (PDI) were calculated from the cumulant analysis using Dynamics 7.8.1.3 software. For measurements, samples were diluted 1: 10 in 10 mM MES pH 5.25 and analysis was performed in triplicates. Successful cargo incorporation was performed and verified using Agarose gel electrophoresis.Freeze Thaw studies

[0316] Freeze thaw studies were conducted by cycling the formulations from -80 °C (24h) to 25 °C (30 min) at least three times. The particle size and polydispersity index of the formulations were measured for freeze-thaw samples. The formulations between thaw and freeze cycles were mixed by gentle inversions before the next freezing cycle.

[0317] Size and PDI analysis showed that CPLXs with JLF 99 can be obtained with size < 100 nm and PDI < 0.3 as observed in FIG. 31. Successful cargo incorporation was verified via Agarose gel electrophoresis wherein no free DNA band was observed (FIG. 32). FIG. 33 shows the results from freezing of CPEX at -80 °C and storage for at least 3 freeze / thaw cycles. As shown in the figure, no significant changes in size and PDI could be observed at freezing temperatures tested (- 80 °C).

[0318] The embodiments of the disclosure described above are intended to be merely exemplary, numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.Example 14 — Nucleic acid particles (CPLXs) manufacturing using JLF218 and up- concentration with final matrix in both acidic and physiological pH conditionsPage 193 of 20712885786vlAttorney Docket No. 2013237-1455Manufacturing of CPLXs

[0319] For CPLXs manufacturing, modRNA-Luc phase was preconditioned with 6x MES buffer pH 6.1. The organic phase consisted of 20 mM JLF218 and 100 mM Tween 40 solutions in ethanol and were mixed in the molar ratios 1 :0.5 or 1 :0.25 or 1 :0.125 respectively. The raw CPLXs were obtained by mixing modRNA and the organic phase by fluid path using a T mixer (1mm diameter) at N / P ratio 6 and RNA manufacturing concentration of 0.1 mg / ml. Three different dialysis buffers, lOmM MES pH 5.25, lOmM HEPES pH 7.4, and IX PBS pH 7.4, were used overnight at 4 °C using dialysis cassettes of MWCO 10,000. The final drug product consisted of RNA cone. <0.05 mg / ml in lOmM MES pH 5.25, lOmM HEPES pH 7.4 and IX PBS pH 7.4 with 10% sucrose as the final storage matrix.Characterization of formulations

[0320] Particle size of CPLX was measured after dialysis using dynamic light scattering in a DynaPro plate reader (Wyatt, Dernbach, Germany). From the measurements, size (Z-average) and polydispersity indices (PDI) were calculated from the cumulant analysis using Dynamics 7.8.1.3 software. For measurements, samples were diluted to 1:10 in respective buffers (lOmM MES pH 5.25, lOmM HEPES pH 7.4 and IX PBS pH 7.4) and analysis was performed in triplicates. Successful cargo incorporation was performed and verified using Agarose gel electrophoresis.Up-concentration of nanoparticles

[0321] Up-concentration of the particles after dialysis with different buffers as final matrix were carried out using Eppendorf 5910 Ri. First, 100K Amicon filters (50 ml tubes) were preconditioned using respective buffers, followed by centrifugation under 2000 ref, 5 min at 4 ° C. Particles with respective buffers (5 ml, RNA cone. < 0.5 mg / ml) were then up -concentrated using preconditioned Amicon filters using similar protocol as provided herein. The centrifugation steps were repeated 2 to 3 times to obtain ~ 1 ml as the final volume in the supernatant.Freeze Thaw Studies

[0322] Freeze thaw studies were conducted by cycling the up-concentrated formulations from -80 °C (24 h) to 25 °C (30 min) at least three times. The particle size and polydispersity index of the formulations were measured for freeze-thaw samples. The formulations between thaw and freeze cycles were mixed by gentle inversions before the next freezing cycle.In-vitro cell viability and transfection efficiency in HepG2, C2C12 and RAW 264.7 cell linesPage 194 of 20712885786vlAttorney Docket No. 2013237-1455

[0323] Luciferase assays are a reliable and sensitive method to detect the expression of luciferase in live cells. In this assay, firefly luciferase catalyzes the mono -oxygenation of beetle luciferin via ATP and Mg2+-dependent. This results in emission of light in the range of 550 to 620 nm. For cell transfections, HepG2, C2C12 and RAW 264.7 cells were seeded at respective cell number of 20,000 cells / well, 5,000 cells / well and 25,000 cells / well in Nunc white flat-bottom 96- well plate (Merck KGaA, Darmstadt, Germany) and centrifuged at 500 x g for 5 min. After 18-24 h, the medium was replaced by fresh medium at 90 pl / well DPBS and only medium were used as negative and blank control, respectively. Formulation samples were tested at mRNA assay concentration between 12.5 to 50 ng per well. Samples and controls were added at 10 pl / well. After 24 h, luciferase expression was determined by Bright-Glo™ Luciferase assay (Promega, Madison, WI, USA) according to manufacturer’s protocol.

[0324] Briefly, the reagent was added to the cells in medium in 1: 1 (v / v) ratio, followed by an incubation of 5 min in the dark to allow for complete cell lysis. Viability was measured by CellTiter-Glo® (Promega, Madison, WI, USA) according to manufacturer’s protocol. Briefly, the reagent was added to the cells in medium in 1:1 (v / v) ratio, followed by an incubation of 10 min in shaker followed by 20 min to allow for stabilization the signal. Alternatively, luciferase and viability were determined by ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega GmbH, Madison, WI, USA) according to manual instructions. Briefly, 20 pl of 5X CellTiter-Fluor™ Reagent was added to the wells, mixed by orbital shaking (300-500 rpm for ~30 sec). After incubation for 30 min at 37 °C, the fluorescence was measured with excitation wavelength at 400 nm and emission at 505 nm (viability). Then, ONE-Glo™ Reagent was added at 100 pl / well. After incubation for three min, the bioluminescence was measured (luciferase expression). Bioluminescence signals (photons per second [p / s]) and fluorescence were measured using a microplate luminescence reader Infinite M200 (Tecan, Mannedorf, Switzerland). Relative luminescence was calculated by subtracting the signal of DPBS control from the sample control.Relative viability was calculated by:RLUsampleViability % = x 100 RLU cells only.Results

[0325] Size and PDI analysis showed that CPLXs with JLF 218 can be obtained with size <100 nm and PDI < 0.3 as observed in FIGs. 35A-35B with different amounts of Tween 40 in thePage 195 of 20712885786vlAttorney Docket No. 2013237-1455 upstream (0.5 and 0.25 molar ratio) and upon dialysis in different buffer conditions (lOmM MES pH 5.25, lOmM HEPES pH 7.4 and IX PBS pH 7.4). In presence of Tween 40 in molar ratio 0.125 in upstream, particles show sizes > 100 nm upon dialysis in neutral buffer conditions (lOmM HEPES pH 7.4 and IX PBS pH 7.4) with PDI >0.3 as shown in FIG. 35C.

[0326] Successful cargo incorporation was verified via Agarose gel electrophoresis: small amount of free RNA was observed when Tween 40 was present in upstream at molar ratio 0.5 whereas no free RNA was observed when Tween was present in upstream in molar ratios 0.25 and 0.125 under both acidic and neutral pH as the final matrix (FIG. 36).

[0327] Particles were up-concentrated by centrifugation method to a maximum final concentration of 1 mg / ml (concentration > 1 mg / ml was not tested). Up-concentration was carried out on dialyzed samples (FIG. 37A) in different buffer conditions (acidic and physiological). All the up-concentrated samples were prepared with Tween 40 in upstream. From FIG. 37B, it was observed that in presence of 0.5 molar ratio of Tween 40 in upstream, CPLXs can be up- concentrated successfully up to 0.7 mg / ml with particle sizes <100 nm and PDI <0.3 under all buffer conditions tested (acidic and physiological).

[0328] In presence of 0.25 and 0.125 molar ratio of Tween 40 in upstream, particles can be up-concentrated to a cone. Of ~1 mg / ml and ~0.5 mg / ml (size <100 nm) respectively under acidic pH conditions (lOmM MES pH 5.25) (FIG. 37B). On the contrary, when up-concentration was carried out in neutral buffer conditions (lOmM HEPES pH 7.4 and IX PBS pH 7.4), particles show aggregation with size between 100-150 nm. The results indicate that higher amount of Tween 40 in upstream has a positive influence on the colloidal stability of particles both after dialysis and upon up concentration under physiological conditions. However, a trend of higher free RNA in AGE was observed with increasing amount of Tween 40 in upstream..

[0329] FIGs. 38A-38B show representative data of up-concentrate CPLXs formulations subjected to three freeze-thaw cycles at -80 °C when stored in different storage matrices. The frozen stability of the up concentrated particles could be achieved at higher concentrations (-0.5- 0.7 mg / ml) under both acidic and physiological conditions, as no significant changes in size and PDI could be observed at freezing temperatures tested (-80 °C) when Tween is present.

[0330] The role of different amounts of Tween 40 in upstream (0.125, 0.25 and 0.5 molar ratio with respect to JLF 218) and the effect of final storage matrix (lOmM MES pH 5.25, lOmM HEPES pH 7.4 and IX PBS pH 7.4) on biological activity of the CPLX particles was furtherPage 196 of 20712885786vlAttorney Docket No. 2013237-1455 investigated and compared with lipid nanoparticle benchmark (LNP BM). From FIGs. 39A-39C it was observed that all samples show > 80% cell viability at doses 12.5, 25 and 50 ng irrespective of Tween40 amount or the final storage matrix in all three cell lines tested. CPLXs obtained via fluid path in presence of different amounts of Tween 40 in upstream show cell transfection efficiency either lower or comparable to LNP BM in all cell line tested at lower doses (FIGs. 40A- 40C). Furthermore, it was observed that CPLXs with lower amount of Tween 40 in upstream shows higher transfection efficiency under all pH conditions tested (more prominent in RAW cell line, FIG. 40C). The effect of storage matrix pH on transfection efficiency can be observed evidently in FIGs. 40A and 40C, wherein, a trend of higher transfection efficiency was observed with CPLXs in IX PBS pH 7.4 as the storage matrix (irrespective of Tween 40 amounts in upstream).

[0331] The embodiments of the disclosure described above are intended to be merely exemplary, numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.Page 197 of 20712885786vl

Claims

1. Attorney Docket No. 2013237-1455CLAIMS1. A method of preparing a pharmaceutical composition comprising a plurality of nucleic acid particles, the method comprising contacting a nucleic acid solution with an organic solution via a mixer configured to receive the nucleic acid solution and the organic solution, wherein: the organic solution comprises an organic solvent and an oligosaccharide comprising one or more cationic moieties.

2. The method of claim 1, wherein a N / P ratio of the nucleic acid solution and the organic solution is from about 2 to about 12.

3. The method of claims 1 or 2, wherein a total flow rate (TFR) of the nucleic acid solution and the organic solution into the mixer is about 25 ml / min to about 360 ml / min.

4. The method of claim 3, wherein the total flow rate of nucleic acid solution and the organic solution into the mixer is about 25 ml / min to about 250 ml / min.

5. The method of any one of claims 1-4, wherein the organic solution further comprises a surfactant.

6. The method of claim 5, wherein the surfactant is or comprises a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

7. The method of claims 5 or 6, wherein a molar ratio of the oligosaccharide to the surfactant is from about 1:0.5 to about 1:0.01.

8. The method of any one of claims 5-7, wherein a concentration of the surfactant in the organic solution is about 50 to about 200 mM.

9. The method of claim 8, wherein a concentration of the surfactant in the organic solution is about 100 mM.Page 198 of 20712885786vlAttorney Docket No. 2013237-145510. The method of any one of claims 1-9, wherein a concentration of the oligosaccharide in the organic solution is from about 10 to about 50 mM.

11. The method of claim 10, wherein a concentration of the oligosaccharide in the organic solution is from about 20 mM to about 25 mM.

12. The method of any one of claims 1-11, wherein the TFR is about 25 ml / min to about 200 ml / min.

13. The method of any one of claims 1-12, wherein the TFR is about 100 ml / min.

14. The method of any one of claims 1-13, wherein the mixer is a T-shaped mixer.

15. The method of any one of claims 1-13, wherein the mixer is a Y-shaped mixer.

16. The method of any one of claims 1-15, wherein a diameter of the mixer is about 1 mm.

17. The method of any one of claims 1-16, wherein a pH of the nucleic acid solution is from about 4 to about 7.

18. The method of claim 17, wherein a pH of the nucleic acid solution is about 6.

19. The method of any one of claims 1-18, wherein the organic solvent is a polar organic solvent.

20. The method of claim 19, wherein the organic solvent is selected from methanol, ethanol, isopropanol, and DMSO.

21. The method of claim 20, wherein the organic solvent is ethanol or DMSO.

22. The method of claim 21, wherein the organic solvent is ethanol.Page 199 of 20712885786vlAttorney Docket No. 2013237-145523. The method of any one of claims 1-22, wherein the pharmaceutical composition has a nucleic acid concentration from about 0.025 to about 1.0 mg / ml.

24. The method of claim 23, wherein the pharmaceutical composition has a nucleic acid concentration of about 0.1 mg / ml.

25. The method of claim 23, wherein the pharmaceutical composition has a nucleic acid concentration of about 0.9 mg / ml.

26. The method of any one of claims 1-25, wherein the plurality of nucleic acid particles in the pharmaceutical composition have a PDI of less than 0.4.

27. The method of any one of claims 1-26, wherein the plurality of nucleic acid particles in the pharmaceutical composition have an average particle size of about 20 nm to about 500 nm.

28. The method of claim 27, wherein the plurality of nucleic acid particles in the pharmaceutical composition have an average particle size of about 50 nm to about 250 nm.

29. The method of any one of claims 1-20, wherein the plurality of nucleic acid particles in the pharmaceutical composition have an average particle size of about 100 nm.

30. The method of any one of claims 1-29, wherein the method further comprises purifying the pharmaceutical composition.

31. The method of claim 30, wherein the purification comprises dialysis.

32. The method of claim 31, wherein the pharmaceutical composition is dialyzed against a buffer having a pH of from about 5 to about 8.Page 200 of 20712885786vlAttorney Docket No. 2013237-145533. The method of claim 32, wherein the pharmaceutical composition is dialyzed against a buffer having a pH of about 6.

34. The method of claim 30, wherein the purification comprises tangential flow filtration (TFF).

35. The method of any one of claims 1-34, wherein the oligosaccharide comprises one or more cationic moieties bonded to a trehalose, a sucrose, or a gluco-n-oligosaccharide moiety, where n is 2-6.

36. The method of any one of claims 1-34, wherein the oligosaccharide is a compound represented by formula I:or a pharmaceutically acceptable salt thereof, wherein:each of R1and R2are independently selected, at each instance, from H, Ra, and -C(O)-Ra, wherein at least one instance of R1or R2is not H;Page 201 of 20712885786vlAttorney Docket No. 2013237-1455 each Rais independently selected from C1-C20 aliphatic, C3-C20 cycloaliphatic, C5-C6 aryl, 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein each Rais optionally substituted with one or more Rb; each Rbis independently selected from halogen, -N3, -Rc, -ORC, -SRC, -NHRC, -C(O)-RC, - OC(O)RC, -NHC(O)RC, -C(O)NHRC, and -NHC(O)NHRC; each Rcis independently selected from optionally substituted C1-C20 aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl, optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S;X1and X2are each independently selected from -S-, -S-S-, and -NH-;Y1and Y2are each independently an optionally substituted Ci-30 aliphatic group wherein one or more carbons are optionally and independently replaced by -Cy-, -NRY-, - NRYC(O)-, -C(O)NRY-, -NRYC(O)O-, -OC(O)NRY-, -NRYC(O)NRY-, - NRYC(S)NRY-, -C(S)NRY-, -C(O)NRYSO2-, -SO2NRYC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each RYis independently H or optionally substituted Ci-Ce aliphatic; each Cy is independently an optionally substituted C3-C14 cycloaliphatic, optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, and optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S;Z1and Z2are each independently a cationic or ionizable group selected from optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S, -N+(M)3,each M is independently -Co-Ce aliphatic-Rzor -Co-Ce aliphatic-N+(Rz)3; each Rzis independently selected from H, optionally substituted Ci-Ce aliphatic, optionally substituted C3-C20 cycloaliphatic, optionally substituted C5-C6 aryl,Page 202 of 20712885786vlAttorney Docket No. 2013237-1455 optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; or two or more Rzcan come together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 12-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S; and p is an integer selected from 1, 2, 3, 4, or 5.

37. The method of any one of claims 1-36, wherein the oligosaccharide is selected from Table 1.

38. The method of any one of claims 1-37, wherein the organic solution further comprises one or more additional lipids.

39. The method of claim 38, wherein the one or more additional lipids are selected from a helper lipid, a steroid, and a polymer conjugated lipid.

40. The method of claim 39, wherein the sterol is selected from P-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol.

41. The method of claims 39 or 40, wherein the helper lipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl -phosphatidylcholine (POPC), 1,2-Page 203 of 20712885786vlAttorney Docket No. 2013237-1455 di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn- glycero-3-phosphocholine (Cl 6 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and combinations thereof.

42. The method of any one of claims 39-41, wherein the polymer-conjugated lipid is or comprises a polysorbate, a poloxamer, and / or a compound comprising an amphiphilic moiety selected from polyalkylene glycols (e.g., polyethylene glycol), poly(2-oxazoline), poly(2-oxazine), polysarcosine, polyvinylpyrrolidone, and poly[N-(2- hydroxypropyl)methacrylamide, wherein the amphiphilic moiety is bonded to one or more C12-C20 aliphatic groups.

43. The method of any one of claims 1-42, wherein the nucleic acid is RNA.

44. The method of claim 43, wherein the RNA is mRNA.

45. The method of claim 44, wherein the mRNA is modRNA, saRNA, taRNA, or uRNA.

46. The method of any one of claims 1 -42, wherein the nucleic acid is DNA.

47. The method of any one of claims 1-46, wherein the plurality of nucleic acid particles are stable (e.g., do not substantially change in size or PDI) after one or more freeze-thaw cycles.

48. A drug production system for producing a pharmaceutical composition by a continuous flow manufacturing process, the drug production system comprising a first composition containment unit comprising a nucleic acid solution and a second composition containment unit comprising an organic solution, wherein the first composition containment unit andPage 204 of 20712885786vlAttorney Docket No. 2013237-1455 the second composition containment unit are connected via a mixer unit configured to receive the nucleic acid solution and the organic solution, and wherein the organic solution comprises an organic solvent and an oligosaccharide comprising a plurality of cationic moieties.

49. The drug production system of claim 48, wherein the first composition containment unit and the second composition containment unit are connected via a mixer unit configured to receive the nucleic acid solution and the organic solution at a total flow rate (TFR) of about 25 ml / min to about 360 ml / min.

50. The drug production system of claims 48 or 49, further comprising one or more precursor containment units, configured to deliver a nucleic acid and / or a buffer to the first composition containment unit.

51. The drug production system of claim 50, wherein the buffer has a pH of about 4 to about7.

52. The drug production system of any one of claims 48-51, wherein the drug production system further comprises a purification module configured to receive and purify the pharmaceutical composition from the mixer unit.

53. The drug production system of claim 52, wherein the purification module dialyzes the pharmaceutical composition.

54. The drug production system of claim 53, wherein the pharmaceutical composition is dialyzed in the purification module against a buffer having a pH of from about 5 to about8.

55. The drug production system of claim 53, wherein the pharmaceutical composition is dialyzed in the purification module against a buffer having a pH at about physiological pH.Page 205 of 20712885786vlAttorney Docket No. 2013237-145556. The drug production system of claim 54 or 55, wherein the purification module is configured for tangential flow filtration (TFF).

57. The drug production system of any one of claims 48-56, configured to perform the method of preparing a pharmaceutical composition in any one of claims 1-47.Page 206 of 20712885786vl

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