Method for forming polycomposite

Abstract

The present disclosure relates to methods for forming polycomplexes for use as safe and non-toxic nucleic acid transfection agents in gene therapy applications.

Description

[0001] This application is a divisional application of an invention patent application with an application date of December 30, 2019, application number 201980079914.2 (international application number PCT / US2019 / 068954), and name “Method for forming a polycomplex”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 786,050, filed on December 28, 2018, the contents of which are hereby incorporated by reference in their entirety. Background Art

[0004] Gene therapy is a rapidly growing field of nanomedicine used to improve health conditions and correct genetic disorders. [1] Currently, most gene delivery schemes utilize viral vectors as gene carriers. However, the problems associated with viral vectors remain unresolved, including the risk of eliciting immunogenic responses and transgene insertional mutagenesis, limitations associated with large-scale production and low "cargo capacity" of genetic material, and unpredictable vector migration. [2,3]

[0005] Nonviral vectors have many advantages over viral systems, such as their minimal immunogenicity, non-tumorigenicity, cost-effective manufacturing, high payload of nucleic acids, and potential for local gene expression. Since 2010, the number of clinical trials using nonviral gene vectors for gene therapy has increased significantly. Among at least 40 nanoparticle-based gene therapies, plasmid DNA and small interfering RNA (siRNA) have been formulated for gene correction, therapeutic protein expression, and antigen vaccination, of which 27 clinical trials have investigated 12 major liposomal systems and 13 clinical trials have investigated 7 polymer-based systems. [3] The readily available cationic polymer polyethyleneimine (PEI) has shown promise in polymer-based gene therapy clinical trials. However, PEI is non-degradable and has been severely hampered by safety concerns. [4] Therefore, great efforts have been made to improve the gene transfection efficiency and safety of polymeric gene carriers to bring polymer-based gene therapy closer to clinical application. Several factors such as polymer size, molecular weight, degree of polymer branching, charge density, as well as the composition of the formulation medium and the ratio of polymer to nucleic acid components can affect the transfection efficiency and biological activity of polyplexes. [5]

[0006] To be commercially useful, reproducible and scalable methods with tightly controlled physical parameters are needed to assemble uniform and stable polyplex complexes. In some embodiments, the present disclosure provides improved methods for preparing polyplexes, including methods that are scalable for commercial-scale production. Summary of the invention

[0007] In some embodiments, the present disclosure provides a method for preparing one or more polyplexes, the method comprising:

[0008] (a) Provide:

[0009] i. a polymer in a first liquid stream;

[0010] ii. a nucleic acid component in a second liquid stream;

[0011] (b) contacting the polymer in the first liquid stream with the nucleic acid component in the second liquid stream to form a polyplex having a size and charge suitable for therapeutic administration; and

[0012] (c) separating the polyplex to provide a stable polyplex.

[0013] In some embodiments, the method further comprises evaluating and harvesting one or more polycomplexes. In some embodiments, the method of the present invention is a continuous process, whereby each polycomplex formed is separated from other polycomplexes formed in the method. In some embodiments, after contacting the first liquid stream and the second liquid stream, each polycomplex is separated from other polycomplexes during the stabilization phase.

[0014] In some embodiments, the method further comprises filtering, washing, freezing and / or lyophilizing the stable polyplex. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Exemplary methods of the present disclosure for making polyplexes are described. DETAILED DESCRIPTION

[0016] As used above, and throughout this disclosure, unless otherwise indicated, the following terms should be understood to have the following meanings. In the absence of a term, conventional terms known to those skilled in the art prevail.

[0017] As used herein, the terms "including," "containing," and "comprising" are used in their open, non-limiting sense.

[0018] The articles "a / an" are used in this disclosure to refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0019] Unless stated otherwise, the term "and / or" means "and" or "or" in this disclosure.

[0020] The term "about" immediately before a numerical value refers to a range (e.g., plus or minus 10% of the value). For example, "about 50" can mean 45 to 55, "about 25,000" can mean 22,500 to 27,500, etc., unless the context of the present disclosure otherwise indicates or is inconsistent with such an interpretation. For example, in a numerical list such as "about 49, about 50, about 55, ...", "about 50" refers to a range extending to less than half of the interval between the previous value and the subsequent value, for example, greater than 49.5 to less than 52.5. In addition, the phrase "less than about" a certain value or "greater than about" a certain value should be understood according to the definition of the term "about" provided herein. Similarly, the term "about" refers to all values ​​in the series, or the endpoints of the range, respectively, when it is in front of a series of numerical values ​​or a series of values ​​(e.g., "about 10, 20, 30" or "about 10 to 30"). Unless otherwise specified, the concentration given as a percentage refers to the mass ratio.

[0021] Unless otherwise indicated, the term "disorder" is used in this disclosure to mean, and is used interchangeably with, the terms disease, illness, or condition.

[0022] The term "pharmaceutically acceptable" or "pharmaceutically acceptable" means that a substance is non-biological or undesirable and can be administered to an individual without causing any substantial unacceptable biological effect or interacting in a deleterious manner with any component of a composition in which it is contained.

[0023] All documents referenced herein are incorporated by reference in their entirety for all purposes.

[0024] As used herein, "alkyl" refers to a straight or branched saturated chain with 1 to 40 carbon atoms. Representative saturated alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl and the like, and longer alkyls such as heptyl and octyl and the like. Alkyl can be unsubstituted or substituted. Alkyl groups containing three or more carbon atoms may be straight chain or branched. As used herein, "lower alkyl" refers to an alkyl group having 1 to 10 carbon atoms.

[0025] As used herein, "alkenyl" includes unbranched or branched hydrocarbon chains containing 2 to 40 carbon atoms. "Alkenyl" contains at least one double bond. The double bond of an alkenyl group may be unconjugated or conjugated with another unsaturated group. Examples of alkenyl groups include, but are not limited to, ethylenyl, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl, etc. Alkenyl groups may be unsubstituted or substituted. As defined herein, alkenyl groups may also be branched or straight-chain.

[0026] As used herein, "alkynyl" includes unbranched or branched unsaturated hydrocarbon chains containing 2 to 40 carbon atoms. "Alkynyl" contains at least one triple bond. The triple bond of an alkynyl group may be unconjugated or conjugated with another unsaturated group. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, 4-butyl-2-hexynyl, etc. Alkynyl groups may be unsubstituted or substituted.

[0027] It should also be noted that any carbon and heteroatoms described herein having unsatisfied valences are assumed to have a sufficient number of hydrogen atoms to satisfy the valences.

[0028] The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine.

[0029] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl, and the like.

[0030] Unless specifically defined otherwise, the term "aryl" refers to a cyclic aromatic hydrocarbon with 1 to 3 aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl or naphthyl. In the case of containing two aromatic rings (bicyclic, etc.), the aromatic rings of the aryl can be combined (such as biphenyl) or fused (such as naphthyl) at a single point. The aryl can be optionally substituted by one or more substituents (for example, 1 to 5 substituents) at any attachment point. The substituent itself can be optionally substituted. In addition, when containing two fused rings, the aryl defined herein can have an unsaturated or partially saturated ring fused to a completely saturated ring. The exemplary ring systems of these aryls include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenalenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthalene, tetrahydrobenzoannulenyl, etc.

[0031] Unless specifically defined otherwise, "heteroaryl" refers to a monovalent monocyclic or polycyclic aromatic radical of 5 to 18 ring atoms, or a polycyclic aromatic radical containing one or more ring heteroatoms selected from N, O or S, the remaining ring atoms being C. Heteroaryl as defined herein also refers to a bicyclic heteroaromatic radical, wherein the heteroatoms are selected from N, O or S. Aromatic radicals are optionally substituted independently by one or more substituents as described herein. The substituents themselves may be optionally substituted. Examples include, but are not limited to, benzothiophene, furanyl, thienyl, pyrrolyl, pyridinyl, pyrazolyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, imidazo[1,2-b]pyrazolyl, furano[2 ,3-c]pyridyl, imidazo[1,2-a]pyridyl, indazolyl, pyrrolo[2,3-c]pyridyl, pyrrolo[3,2-c]pyridyl, pyrazolo[3,4-c]pyridyl, benzimidazolyl, thieno[3,2-c]pyridyl, thieno[2,3-c]pyridyl, thieno[2,3-b]pyridyl, benzothiazolyl, indolyl, indolyl, indole linonyl, dihydrobenzothiophenyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzooxanyl, quinolinyl, isoquinolinyl, 1,6-naphthyridinyl, benzo[de]isoquinolinyl, pyrido[4,3-b][1,6]naphthyridinyl, thieno[2,3-b]pyrazinyl, quinazolinyl, tetrazolo[ 1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, isoindolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b]pyridinyl, pyrrolo[1,2-a]pyrimidinyl, tetrahydropyrrolo[1,2-a]pyrimidinyl, 3,4-dihydro-2H-1λ 2-pyrrolo[2,1-b]pyrimidine, dibenzo[b,d]thiophene, pyridin-2-one, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl, 1H-pyrido[3,4-b][1,4]thiazinyl, benzoxazolyl, benzisoxazolyl, furo[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl, furo[3,2-b]pyridine, [1,2,4]triazolo[1,5-a]pyridinyl, benzo[1,2,3]triazolyl, imidazo[1,2-a]pyrimidinyl, [1,2, 4]triazolo[4,3-b]pyridazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole, 1,3-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl, thiazolo[5,4-d]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2,3-b]pyrrolyl, 3H-indolyl and derivatives thereof. In addition, when containing two fused rings, the heteroaryl group defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring.

[0032] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated monocyclic, fused or spirocyclic polycyclic carbocyclic ring having 3 to 18 carbon atoms per ring. The cycloalkyl ring or carbocyclic ring may be optionally substituted with one or more substituents (e.g., 1 to 5 substituents) at any point of attachment. The substituent itself may be optionally substituted. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, norbornyl, bicyclo [2.2.2] octyl, bicyclo [2.2.2] octenyl, decahydronaphthalene, octahydro-1H-indenyl, cyclopentenyl, cyclohexenyl, cyclohexa-1,4-dienyl, cyclohexa-1,3-dienyl, 1,2,3,4-tetrahydronaphthalene, octahydropentalenyl, 3a,4,5,6 ,7,7a-hexahydro-1H-indenyl, 1,2,3,3a-tetrahydropentalenyl, bicyclo[3.1.0]hexyl, bicyclo[2.1.0]pentyl, spiro[3.3]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]hept-2-enyl, bicyclo[2.2.2]octyl, 6-methylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl and derivatives thereof.

[0033] As used herein, the term "cycloalkenyl" refers to a partially saturated monocyclic, fused or spirocyclic polycyclic carbocyclic ring having 3 to 18 carbon atoms per ring and containing at least one double bond. The cycloalkenyl ring may be optionally substituted with one or more substituents (e.g., 1 to 5 substituents) at any point of attachment. The substituents themselves may be optionally substituted.

[0034] As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially unsaturated and non-aromatic monocyclic or fused or spirocyclic polycyclic ring structure of 4 to 18 atoms containing carbon and heteroatoms selected from oxygen, nitrogen or sulfur, and wherein the ring carbons or heteroatoms share undelocalized π electrons (aromaticity). The heterocycloalkyl or heterocyclyl ring structure may be substituted with one or more substituents. The substituents themselves may be optionally substituted.Examples of heterocycloalkyl or heterocyclyl rings include, but are not limited to, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, isotropanyl, dihydrothiophene-2(3H)-yl, tetrahydrothiophene 1,1-dioxide, 2,5-dihydro-1H-pyrrolyl, imidazolidin-2- ... ketone, pyrrolidin-2-one, dihydrofuran-2(3H)-one, 1,3-dioxolane-2-one, isothiazolidine 1,1-dioxide, 4,5-dihydro-1H-imidazolyl, 4,5-dihydrooxazole, oxiranyl, pyrazolidinyl, 4H-1,4-thiazinyl, thiomorpholinyl, 1,2,3,4-tetrahydropyridinyl, 1,2,3,4-tetrahydropyrazinyl, 1,3-oxazinan-2-one, tetrahydro-2H-thiopyran 1,1-dioxide, 7-oxabicyclo[2.2.1]heptyl, 1,2-thiazoline 1,1-dioxide, octahydro-2H-quinolizinyl, 1,3-diazabicyclo[2.2.2]octanyl, 2 ,3-dihydrobenzo[b][1,4]dioxin, 3-azabicyclo[3.2.1]octanyl, 8-azaspiro[4.5]decane, 8-oxa-3-azabicyclo[3.2.1]octanyl, 2-azabicyclo[2.2.1]heptane, 2,8-diazaspiro[5.5]undecane, 2-azaspiro[5.5]undecane, 3-azaspiro[5.5]undecane, decahydroisoquinolyl, 1-oxa-8-azaspiro[4.5]decane, 8-azabicyclo[3.2.1]octanyl, 1,4'-bipiperidinyl, azaalkyl, 8-oxa-3-azabicyclo[3.2.1]octanyl, 3,4-dihydro -2H-benzo[b][1,4]oxazinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyridinyl, 1,4-diazaalkyl, phenoxathiinyl, benzo[d][1,3]dioxolanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzo[b][1,4]dioxinyl, 4-(piperidin-4-yl)morpholinyl, 3-azaspiro[5.5]undecyl, decahydroquinolinyl, piperazin-2-one, 1-(pyrrolidin-2-ylmethyl)pyrrolidinyl, 1,3'-bipyrrolidinyl, and 6,7,8,9-tetrahydro-1H,5H-pyrazolo[1,2-a][1,2]diazepinyl.

[0035] Unless otherwise indicated, numerical ranges used herein are intended to include sequential integers. For example, a range expressed as "0 to 5" would include 0, 1, 2, 3, 4, and 5.

[0036] As used herein, the term "substituted" means that the specified group or moiety bears one or more suitable substituents, wherein the substituents may be attached to the specified group or moiety at one or more positions. For example, a cycloalkyl-substituted aryl may indicate that the cycloalkyl is attached to one atom of the aryl by a bond or by being fused to the aryl and sharing two or more common atoms.

[0037] As used herein, the term "unsubstituted" means that the designated group bears no substituents.

[0038] The term "optionally substituted" is understood to mean that a given chemical moiety (e.g., an alkyl group) can (but is not required to) be bonded to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group can be a fully saturated alkyl chain (i.e., a pure hydrocarbon). Alternatively, the same optionally substituted alkyl group can have a substituent other than hydrogen. For example, the alkyl group can be bonded to a halogen atom, a hydroxyl group, or any other substituent described herein at any point along the chain. Thus, the term "optionally substituted" means that a given chemical moiety has the potential to contain other functional groups, but does not necessarily have any other functional groups. If not otherwise specified, suitable substituents used in the optional substitution of the group include, but are not limited to, oxo, -halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl ... 1- C6 haloalkoxy, -OC1-C6 alkenyl, -OC1-C6 alkynyl, -C1-C6 alkenyl, -C1-C6 alkynyl, -OH, CN (cyano), -CH2CN, -OP(O)(OH)2, -C(O)OH, -OC(O)C1-C6 alkyl, -C(O)C1-C6 alkyl, -C(O)-C0-C6 alkylene-cycloalkyl, -C(O)-C0-C6 alkylene-heterocycloalkyl, -C(O)-C0-C6 alkylene-aryl, -C(O)-C0-C6 alkylene-heteroaryl, -OC(O)OC1-C6 alkyl, NH2, NH(C1-C6 alkyl -C(O)NH, -C(O)NH(C1-C6 alkyl), -C(O)N(C1-C6 alkyl)2, -C(O)NHcycloalkyl, -C(O)N(C1-C6 alkyl)cycloalkyl, -C(O)NHheterocycloalkyl, -C(O)N(C1-C6 alkyl)heterocycloalkyl, -C(O)NHaryl, -C(O)N(C1-C6 alkyl)aryl, -C(O)NHheteroaryl, -C(O)N(C1-C6 alkyl)heteroaryl, -S(O)2-C1-C6 alkyl, -S(O)2-C1-C6 haloalkyl, -S(O)2- Cycloalkyl, -S(O) 2- Heterocycloalkyl, -S(O) 2- Aryl, -S(O) 2- heteroaryl-C0-C6 alkylene-S(O)2NH2, -S(O)2NHC1-C6 alkyl, -S(O)2N(C1-C6 alkyl)2, -S(O)2NH cycloalkyl, -S(O)2NH heterocycloalkyl, -S(O)2NH aryl, -S(O)2NH heteroaryl, -NHS(O)2C1-C6 alkyl, -N(C1-C6 alkyl)S(O)2(C1-C6 alkyl), -NHS(O)2 aryl, -N(C1-C6 alkyl)S(O)2 aryl, -NHS(O)2 heteroaryl, -N(C1-C6 alkyl)S(O)2heteroaryl, -NHS(O)2cycloalkyl, -N(C1-C6 alkyl)S(O)2cycloalkyl, -NHS(O)2heterocycloalkyl, -N(C1-C6 alkyl)S(O)2heterocycloalkyl, -N(C1-C6 alkyl)S(O)2aryl, -C0-C6 alkylene-aryl, -C0-C6 alkylene-heteroaryl, -C0-C6 alkylene-cycloalkyl, -C0-C6 alkylene-heterocycloalkyl, -O-aryl, -NH-aryl and N(C1-C6 alkyl)aryl. The substituents themselves may be optionally substituted. When a multifunctional moiety is shown, the point of attachment to the core is represented by a line, for example, (cycloalkyloxy)alkyl- means that the alkyl group is the point of attachment to the core and the cycloalkyl group is attached to the alkyl group through the oxy group. "Optionally substituted" also refers to "substituted" or "unsubstituted", which have the above meanings.

[0039] As used herein, the term "linker" or "linking moiety" refers to a group that connects two groups and has a main chain between 0 and 100 atoms. The linking group or linkage can be a covalent bond connecting two groups or a chain between 1 and 100 atoms in length (i.e., a backbone of 0 atoms), for example, about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100 carbon atoms in length, wherein the linking group can be linear, branched, cyclic or a single atom. In some cases, one or more carbon atoms of the linking group backbone can be optionally substituted by sulfur, nitrogen or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated. The linker may include one or more substituent groups, such as alkyl, aryl or alkenyl. The linker may include, but is not limited to, oligo(ethylene glycol), ether, thioether, tertiary amine, alkyl, which may be straight or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl) and the like. The linker backbone may include cyclic groups, such as aryl, heterocycle or cycloalkyl, wherein 2 or more atoms of the cyclic group are included in the backbone, such as 2, 3 or 4 atoms. The linker may be cleavable or non-cleavable.

[0040] Unless otherwise stated, the term molecular weight refers to the weight average molecular weight (M W ).

[0041] The term "heteroalkylene" refers to a group in which one or more carbon atoms are replaced by sulfur, oxygen, or NR d A substituted divalent alkylene group, wherein R d The heteroalkylene group may be linear, branched, cyclic, or a combination thereof.

[0042] The term "heteroalkenylene" refers to a divalent straight or branched chain hydrocarbon group having at least one carbon-carbon double bond and one or more heteroatoms (eg, N, S or O) in its backbone.

[0043] The term "heteroalkynylene" refers to a divalent straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond and one or more heteroatoms (eg, N, S or O) in its backbone.

[0044] As used herein, the term "polyplex" refers to a complex between a nucleic acid and a polymer. The nucleic acid is bonded, encapsulated or linked to the polymer through non-covalent forces such as electrostatic interactions.

[0045] The term "plasmid" refers to an extra chromosomal component, which usually carries genes that are not part of the central metabolism of the cell, and is usually in the form of a circular double-stranded DNA molecule. Such a component can be an autonomously replicating sequence of single-stranded or double-stranded DNA or RNA derived from any source, a genome integration sequence, a phage or a nucleotide sequence (linear, circular or supercoiled), in which many nucleotide sequences have been combined or recombined into a unique structure that can introduce the promoter fragment and DNA sequence of the selected gene product and the appropriate 3' non-translated sequence into the cell. As used herein, the term "plasmid" refers to a construct consisting of genetic material (i.e., nucleic acid). Typically, a plasmid contains a functional origin of replication in a bacterial host cell (e.g., Escherichia coli), and a selection marker for detecting a bacterial host cell containing the plasmid.

[0046] The term "nanoplasmid" refers to a circular DNA sequence with reduced bacterial sequences that provides a smaller plasmid with a desired gene insert. For example, nanoplasmids produced by an antibiotic-free RNA-OUT selection system and methods for making the same are described in U.S. Pat. No. 9,109,012, which is hereby incorporated by reference in its entirety and patented by Nature Technology.

[0047] The term "nucleic acid" refers to a biological polymer of nucleotide bases and may include, but is not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), microRNA (miRNA), and peptide nucleic acid (PNA).

[0048] The term "minicircle" refers to a small, minimal-sized circular DNA derived from a parental plasmid by removal of bacterial replication sequences through intramolecular recombination.

[0049] The term "gene editing system" refers to a system that is able to alter a target nucleic acid through one of many DNA repair pathways.

[0050] The term "evaluating" as used in this disclosure refers to evaluating one or more properties of a polyplex to determine whether the polyplex meets specifications, including but not limited to size, shape, zeta potential, form (e.g., non-agglomerated form), or refractive index.

[0051] method

[0052] The present disclosure provides methods for making polyplexes, wherein the methods include mixing a polymer and a nucleic acid component (eg, DNA) to provide isolated stable polyplexes.

[0053] The conventional process for preparing polyplexes is to prepare for mixing a solution of a suitable polymer with a solution of a suitable DNA (or RNA). The resulting mixture usually forms a complex between the DNA (or RNA) and the polymer through electrostatic interactions. The resulting polyplexes exist in various sizes and include agglomerated polyplexes that are not suitable for absorption into target cells. The methods described herein provide for controllably forming such polyplexes by contacting a selected volume and concentration of DNA and polymer solutions under controlled conditions, so that the resulting polyplexes have a relatively narrow and well-defined distribution of polyplex properties (e.g., size, charge, etc.). In addition, since the polyplexes are formed in sequence, automated quality control methods can be used to discard polyplexes whose properties exceed defined manufacturing specifications, and / or select and separate polyplexes that meet defined manufacturing specifications. In this way, the resulting polyplexes have more uniform properties and improved therapeutic effects.

[0054] In some embodiments, the present disclosure provides a method for preparing one or more polyplexes, the method comprising:

[0055] (a) Provide:

[0056] i. a polymer in a first liquid stream;

[0057] ii. a nucleic acid component in a second liquid stream;

[0058] (b) contacting the polymer in the first liquid stream with the nucleic acid component in the second liquid stream to form a polyplex having a size and charge suitable for therapeutic administration (e.g., transdermal administration); and

[0059] (c) separating the polyplex to provide a stable polyplex.

[0060] In some embodiments, the first liquid stream and the second liquid stream are solutions.

[0061] In some embodiments, the present disclosure provides a continuous method for preparing a polyplex.

[0062] In some embodiments of the disclosed methods, the polymer in the first liquid stream is contacted with the nucleic acid component in the second liquid stream in an amount that provides a specific ratio of nucleic acid component to polymer, as described herein.

[0063] In some embodiments, the method includes flowing the first liquid stream and the second liquid stream through a flow regulating device at a rate that provides a polyplex having a size and charge suitable for transdermal administration or injection.

[0064] In some embodiments, the method includes flowing the first liquid stream and the second liquid stream through a flow regulating device at a microliter rate.

[0065] In some embodiments, the first liquid stream containing the polymer and the second liquid stream containing the nucleic acid each independently flow through the flow regulating device at a rate of about 1 μL / min to about 1000 μL / min, including about 1.0 μL / min, about 2.0 μL / min, about 3.0 μL / min, about 4.0 μL / min, about 5.0 μL / min, about 6.0 μL / min, about 7.0 μL / min, about 8.0 μL / min, about 9.0 μL / min, about 10 μL / min, about 15 μL / min, about 20 μL / min, about 25 μL / min, about 30 μL / min, about 35 μL / min, about 40 μL / min, about 45 μL / min, about 50 μL / min, about 55 μL / min, about 60 μL / min, about 65 μL / min / min, about 70 μL / min, about 75 μL / min, about 80 μL / min, about 85 μL / min, about 90 μL / min, about 95 μL / min, about 100 μL / min, about 150 μL / min, about 200 μL / min, about 250 μL / min, about 300 μL / min, about 350 μL / min, about 400 μL / min, about 450 μL / min, about 500 μL / min, about 550 μL / min, about 600 μL / min, about 650 μL / min, about 700 μL / min, about 750 μL / min, about 800 μL / min, about 850 μL / min, about 900 μL / min, about 950 μL / min and about 1000 μL / min, including all ranges therebetween.

[0066] In some embodiments, the first liquid stream containing the polymer and the second liquid stream containing the nucleic acid each independently flow through the flow regulating device at the following rates: about 1 μL / min, about 2.0 μL / min, about 3.0 μL / min, about 4.0 μL / min, about 5.0 μL / min, about 6.0 μL / min, about 7.0 μL / min, about 8.0 μL / min, about 9.0 μL / min, about 10 μL / min, about 15 μL / min, about 20 μL / min, about 25 μL / min, about 30 μL / min, about 35 μL / min, about 40 μL / min, about 45 μL / min, about 50 μL / min, about 55 μL / min, about 60 μL / min, about 65 μL / min, about 70 μL / min, about 75 μL / min, about 76 μL / min, about 77 μL / min, about 78 μL / min, about 79 μL / min, about 80 μL / min, about 81 μL / min, about 82 μL / min, about 83 μL / min, about 84 μL / min, about 86 μL / min, about 88 μL / min, about 89 μL / min, about 90 μL / min, about 91 μL / min, about 92 μL / min, about 94 μL / min, about 95 μL / min, about 96 μL / min, about 98 μL / min, about 99 μL / min, about 100 μL / min 0 μL / min, about 75 μL / min, about 80 μL / min, about 85 μL / min, about 90 μL / min, about 95 μL / min, about 100 μL / min, about 150 μL / min, about 200 μL / min, about 250 μL / min, about 300 μL / min, about 350 μL / min, about 400 μL / min, about 450 μL / min, about 500 μL / min, about 550 μL / min, about 600 μL / min, about 650 μL / min, about 700 μL / min, about 750 μL / min, about 800 μL / min, about 850 μL / min, about 900 μL / min, about 950 μL / min or about 1000 μL / min.

[0067] In some embodiments, the method is carried out at a pressure of about 500 mbar to about 2 bar, including about 500 mbar, about 550 mbar, about 600 mbar, about 650 mbar, about 700 mbar, about 750 mbar, about 800 mbar, about 850 mbar, about 900 mbar, about 950 mbar, about 1 bar, about 1.1 bar, about 1.2 bar, about 1.3 bar, about 1.4, about 1.5 bar, about 1.6 bar, about 1.7 bar, about 1.8 bar, about 1.9 bar, and about 2 bar, including all ranges therebetween. In some embodiments, the method is carried out at a pressure of about 500 mbar, about 550 mbar, about 600 mbar, about 650 mbar, about 700 mbar, about 750 mbar, about 800 mbar, about 850 mbar, about 900 mbar, about 950 mbar, about 1 bar, about 1.1 bar, about 1.2 bar, about 1.3 bar, about 1.4, about 1.5 bar, about 1.6 bar, about 1.7 bar, about 1.8 bar, about 1.9 bar, or about 2 bar. In some embodiments, the method is carried out at a pressure of about 1 bar to about 2 bar. All pressures described herein are absolute pressures.

[0068] In some embodiments, the first and second liquid streams conveying the polymer and nucleic acid components are enclosed in a channel having a width of about 1 mm to about 12 mm, including about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, and about 12 mm, including all ranges therebetween. In some embodiments, the width of the channel is about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, or about 12 mm.

[0069] In some embodiments, the first and second liquid streams conveying polymers and nucleic acid components are enclosed in a channel having a height of about 1 mm to about 12 mm, the height comprising about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm and about 12 mm, including all ranges therebetween. In some embodiments, the height of the channel is about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm or about 12 mm. The cross section of the channel can be approximately rectangular, elliptical or circular.

[0070] In some embodiments, the flow regulating device is selected from the group consisting of a positive displacement pump, a syringe driven pump, a pressure driven pump, and a gravity feed pump or system.

[0071] In some embodiments, the contacting step uses a nozzle, a microfluidic mixing device, a contact tube, a liquid bridge, a vertical mixer, a rotating double tube, or an atomizer.

[0072] The following embodiments provide non-limiting examples of methods of contacting a first liquid stream comprising a polymer and a second liquid stream comprising nucleic acids.

[0073] Nozzle. In some embodiments, a nozzle is used in the methods of the present disclosure. In some embodiments, one nozzle injects a controlled amount of a nucleic acid component into an oil / liquid channel at a specified time and position determined by a sensor (e.g., a camera) through a control system, and a second nozzle injects a first liquid stream containing a polymer into a second liquid stream containing nucleic acids to provide a polyplex. The precision of multiple injections under precisely controlled time, speed, volume, density, and position parameters provides a controlled formation of a polyplex with relatively uniform and controlled properties. In some embodiments, two nozzles (one for the second liquid stream containing nucleic acids and one for the first liquid stream containing polymers) are oppositely configured to inject two opposing liquid streams into a mixing chamber at high speed (i.e., impact jet mixing).

[0074] Microfluidics. In some embodiments, microfluidics are used in the methods of the present disclosure. In some embodiments, two microfluidic streams (e.g., a first liquid stream containing a polymer and a second liquid stream containing nucleic acids) are combined to form a polyplex. In some embodiments, a pulse of liquid containing a controlled amount of a nucleic acid component and a polymer (e.g., HPAE) travels along each microfluidic stream to discharge the correct amount of raw material (e.g., nucleic acid component or HPAE) from each microfluidic stream into a mixing region. Mixing is instantaneous and precise.

[0075] Touch tube. In some embodiments, a touch tube is used in the methods of the present disclosure. In some embodiments, a first liquid stream containing a polymer and a second liquid stream containing nucleic acids converge together and mix in a contact tube after being discharged from respective nozzles or microfluidic streams. In some embodiments, the tubes face each other, separated by a small air gap. In some embodiments, multiple contact tubes discharge or discharge the first liquid stream containing a polymer and the second liquid stream containing nucleic acids into a carrier stream individually or collectively.

[0076] Liquid bridging: In some embodiments, liquid bridging is used in the methods of the present disclosure. In some embodiments, a first liquid stream containing a polymer and a second liquid stream containing nucleic acids are delivered to a vertically oriented platform through substantially parallel tubes, where the liquid streams meet and complex. The resulting polyplexes can then slide from the vertical platform into a liquid channel as described herein, which delivers the formed polyplexes to, for example, a container for harvesting.

[0077] Vertical mixer: In some embodiments, vertical mixing is used in the methods of the present disclosure. In some embodiments, a first liquid stream containing a polymer and a second liquid stream containing nucleic acids are fed into two concentrically oriented tubes, one tube inside the other. After discharge, the contents of the inner tube (e.g., nucleic acid components or polymers) are mixed with the contents of the concentrically oriented outer tube (e.g., nucleic acid components or polymers) to form polyplexes. Once polyplexes are formed, the density changes, and the formed polyplexes move outward and flow into a liquid channel, as described herein, which transports the formed polyplexes to, for example, a container for harvesting.

[0078] Rotating vertical mixer: In some embodiments, rotating vertical mixing is used in the methods of the present disclosure. In some embodiments, a first liquid stream containing a polymer and a second liquid stream containing nucleic acids are fed into a tube in a tube case, and centrifugal force causes the two materials to move out of the center at different rates, thereby mixing. Once polyplexes are formed, the density changes, whereby the formed polyplexes move outward and flow into a liquid channel, as described herein, which transports the formed polyplexes to, for example, a container for harvesting.

[0079] In some embodiments, the contacting step uses a nozzle. In some embodiments, the nozzle is directed to provide a pulsed flow contact between the first liquid stream and the second liquid stream, the contact being 90° to the direction of travel of each individual material. The angle of the nozzle relative to the liquid delivery channel can be varied from 0° to 90°.

[0080] In some embodiments, as described herein, the contacting step occurs in an air gap in a liquid channel or in a carrier oil. In some embodiments, in the absence of external mixing (e.g., mechanical mixing), polycomplexes are formed immediately. In some embodiments, after the contacting step, external mixing is required to form polycomplexes. In some embodiments, external mixing is included in blowing air streams through the mixture after the contacting step. In some embodiments, air streams assist the movement of newly formed polycomplexes during the stable phase.

[0081] In some embodiments, the volumes of the first liquid stream containing polymers and the second liquid stream containing nucleic acids in the contacting step are in the microliter range (e.g., about 1 μL to about 1000 μL). In some embodiments, the volumes of the first liquid stream containing polymers and the second liquid stream containing nucleic acids in the contacting step are less than about 5 μL.

[0082] In some embodiments, the volume of the first liquid stream and the second liquid stream is each independently about 1 μL to about 1000 μL, including about 1 μL, about 5 μL, about 10 μL, about 15 μL, about 20 μL, about 25 μL, about 30 μL, about 35 μL, about 40 μL, about 45 μL, about 50 μL, about 55 μL, about 60 μL, about 65 μL, about 70 μL, about 75 μL, about 80 μL, about 85 μL, about 90 μL, about 95 μL, about 100 μL, about 105 μL, about 110 μL, about 115 μL, about 120 μL, about 125 μL, about 130 μL, about 135 μL, about 140 μL, about 145 μL, about 150 μL, about 155 μL, about 160 μL, about 165μL, about 170μL, about 175μL, about 180μL, about 185μL, about 190μL, about 195μL, about 200μL, about 205μL, about 210μL, about 215μL, about 220μL, about 225μL, about 230μL, about 235μL, about 240μL, about 245μL, about 25 0 μL, about 255 μL, about 260 μL, about 265 μL, about 270 μL, about 275 μL, about 280 μL, about 285 μL, about 290 μL, about 295 μL, about 300 μL, about 305 μL, about 310 μL, about 315 μL, about 320 μL, about 325 μL, about 330 μL, about 335 μL, about 340 μL 0μL, about 345μL, about 350μL, about 355μL, about 360μL, about 365μL, about 370μL, about 375μL, about 380μL, about 385μL, about 390μL, about 395μL, about 400μL, about 405μL, about 410μL, about 415μL, about 420μL, about 425μL, about 4 30μL, about 435μL, about 440μL, about 445μL, about 450μL, about 455μL, about 460μL, about 465μL, about 470μL, about 475μL, about 480μL, about 485μL, about 490μL, about 495μL, about 500μL, about 505μL, about 510μL, about 515μL, about 520μL, about 525μL, about 530μL, about 535μL, about 540μL, about 545μL, about 550μL, about 555μL, about 560μL, about 565μL, about 570μL, about 575μL, about 580μL, about 585μL, about 590μL, about 595μL, about 600μL, about 605μL, about 610 μL, about 615 μL, about 620 μL, about 625 μL, about 630 μL, about 635 μL, about 640 μL, about 645 μL, about 650 μL, about 655 μL, about 660 μL, about 665 μL, about 670 μL, about 675 μL, about 680 μL, about 685 μL, about 690 μL, about 695 μL,about 700 μL, about 705 μL, about 710 μL, about 715 μL, about 720 μL, about 725 μL, about 730 μL, about 735 μL, about 740 μL, about 745 μL, about 750 μL, about 755 μL, about 760 μL, about 765 μL, about 770 μL, about 775 μL, about 780 μL, about 785 μL, about 790 μL, about 795 μL, about 800 μL, about 810 μL, about 815 μL, about 820 μL, about 825 μL, about 830 μL, about 835 μL, about 840 μL, about 845 μL, about 850 μL, about 855 μL, about 860 μL, about 865 μL, about 870 μL, about 875 μL, about 880 μL, about 885 μL, about 890 μL, about 895 μL, about 900 μL, about 905 μL, about 910 μL, about 915 μL, about 920 μL, about 925 μL, about 930 μL, about 935 μL, about 940 μL, about 945 μL, about 950 μL, about 955 μL, about 960 μL, about 965 μL, about 970 μL, about 975 μL, about 980 μL, about 985 μL, about 990 μL, about 995 μL, and about 1000 μL, including all ranges therebetween.

[0083] In some embodiments, the volumes of the first liquid stream and the second liquid stream are each independently about 1 μL, about 5 μL, about 10 μL, about 15 μL, about 20 μL, about 25 μL, about 30 μL, about 35 μL, about 40 μL, about 45 μL, about 50 μL, about 55 μL, about 60 μL, about 65 μL, about 70 μL, about 75 μL, about 80 μL, about 85 μL, about 90 μL, about 95 μL, about 100 μL, about 105 μL, about 110 μL, about 115 μL, about 120 μL, about 125 μL, about 130 μL, about 135 μL, about 140 μL, about 145 μL, about 150 μL, about 155 μL, about 160 μL, about 165 μL, about 170 μL, about μL, about 175 μL, about 180 μL, about 185 μL, about 190 μL, about 195 μL, about 200 μL, about 205 μL, about 210 μL, about 215 μL, about 220 μL, about 225 μL, about 230 μL, about 235 μL, about 240 μL, about 245 μL, about 250 μL, about 255 μL, about 260 μL, about 265 μL, about 270 μL, about 275 μL, about 280 μL, about 285 μL, about 290 μL, about 295 μL, about 300 μL, about 305 μL, about 310 μL, about 315 μL, about 320 μL, about 325 μL, about 330 μL, about 335 μL, about 340 μL, about 345 μL, about 350 μL μL, about 355 μL, about 360 μL, about 365 μL, about 370 μL, about 375 μL, about 380 μL, about 385 μL, about 390 μL, about 395 μL, about 400 μL, about 405 μL, about 410 μL, about 415 μL, about 420 μL, about 425 μL, about 430 μL, about 435 μL, about 44 0μL, about 445μL, about 450μL, about 455μL, about 460μL, about 465μL, about 470μL, about 475μL, about 480μL, about 485μL, about 490μL, about 495μL, about 500μL, about 505μL, about 510μL, about 515μL, about 520μL, about 525μL, about 5 30μL, about 535μL, about 540μL, about 545μL, about 550μL, about 555μL, about 560μL, about 565μL, about 570μL, about 575μL, about 580μL, about 585μL, about 590μL, about 595μL, about 600μL, about 605μL, about 610μL, about 615μL, about 620μL, about 625μL, about 630μL, about 635μL, about 640μL, about 645μL, about 650μL, about 655μL, about 660μL, about 665μL, about 670μL, about 675μL, about 680μL, about 685μL, about 690μL, about 695μL, about 700μL, about 705μL,about 710 μL, about 715 μL, about 720 μL, about 725 μL, about 730 μL, about 735 μL, about 740 μL, about 745 μL, about 750 μL, about 755 μL, about 760 μL, about 765 μL, about 770 μL, about 775 μL, about 780 μL, about 785 μL, about 790 μL, about 795 μL, about 800 μL, about 810 μL, about 815 μL, about 820 μL, about 825 μL, about 830 μL, about 835 μL, about 840 μL, about 845 μL, about 850 μL, about 855 μL, About 860 μL, about 865 μL, about 870 μL, about 875 μL, about 880 μL, about 885 μL, about 890 μL, about 895 μL, about 900 μL, about 905 μL, about 910 μL, about 915 μL, about 920 μL, about 925 μL, about 930 μL, about 935 μL, about 940 μL, about 945 μL, about 950 μL, about 955 μL, about 960 μL, about 965 μL, about 970 μL, about 975 μL, about 980 μL, about 985 μL, about 990 μL, about 995 μL, or about 1000 μL.

[0084] In some embodiments, the volume of the first liquid stream and the second liquid stream are each independently a volume of about 1 μL to about 300 μL. In some embodiments, the volume of the first liquid stream and the second liquid stream are each independently about 10 μL.

[0085] In some embodiments, the volume of the first liquid stream and the second liquid stream (e.g., each pulse of the first liquid stream and the second liquid stream from the nozzle) is each independently about 0.1 μL, about 0.5 μL, about 1 μL, about 2 μL, about 3 μL, about 4 μL, about 5 μL, about 6 μL, about 7 μL, about 8 μL, about 9 μL, about 10 μL, about 11 μL, about 12 μL, about 13 μL, about 14 μL, about 15 μL, about 16 μL, about 17 μL, about 18 μL, about 19 μL, about 20 μL, about 21 μL, about 22 μL, about 23 μL, about 24 μL, about 25 μL, about 26 μL, about 27 μL, about 28 μL, about 29 μL, about 30 μL, about 31 μL, about 32 μL, about 33 μL, about 34 μL, about 35 μL, about 36 μL, about 37 μL, about 38 μL, about 39 μL, about 40 μL, about 41 μL, about 42 μL, about 43 μL, about 44 μL 2μL, about 33μL, about 34μL, about 35μL, about 36μL, about 37μL, about 38μL, about 39μL, about 40μL, about 41μL, about 42μL, about 43μL, about 44μL, about 45μL, about 46μL, about 47μL, about 48μL, about 49μL, about 50μL, about 51μL, about 52μL, about 53μL, about 54μL, about 55μL, about 56μL, about 57μL, about 58μL, about 59μL, about 60μL, about 61μL, about 62μL, about 63μL, about 64μL, about 65μL, about 67μL, about 68μL, about 69μL, about 70μL, about 71μL, about 72μL, about 73μL, about 74μL, About 75μL, about 76μL, about 77μL, about 78μL, about 79μL, about 80μL, about 81μL, about 82μL, about 83μL, about 84μL, about 85μL, about 86μL, about 87μL, about 88μL, about 89μL, about 90μL, about 91μL, about 92μL, about 93μL, about 94μL, about 95μL , about 96μL, about 97μL, about 98μL, about 99μL, about 100μL, about 101μL, about 102μL, about 103μL, about 104μL, about 105μL, about 106μL, about 107μL, about 108μL, about 109μL, about 110μL, about 111μL, about 112μL, about 113μL, about 114μL 14μL, about 115μL, about 116μL, about 117μL, about 118μL, about 119μL, about 120μL, about 121μL, about 122μL, about 123μL, about 124μL, about 125μL, about 126μL, about 127μL, about 128μL, about 129μL, about 130μL, about 131μL, about 132μL, about 133μL, about 134μL, about 135μL, about 136μL, about 137μL, about 138μL, about 139μL, about 140μL, about 141μL, about 142μL, about 143μL, about 144μL, about 145μL, about 146μL, about 147μL, about 148μL, about 149μL,About 150 μL, about 151 μL, about 152 μL, about 153 μL, about 154 μL, about 155 μL, about 156 μL, about 157 μL, about 158 ​​μL, about 159 μL, about 160 μL, about 161 μL, about 162 μL, about 163 μL, about 164 μL, about 165 μL, about 166 μL, about 167 μL L, about 168μL, about 169μL, about 170μL, about 171μL, about 172μL, about 173μL, about 174μL, about 175μL, about 176μL, about 177μL, about 178μL, about 179μL, about 180μL, about 181μL, about 182μL, about 183μL, about 184μL, about 185 μL, about 186μL, about 187μL, about 188μL, about 189μL, about 190μL, about 191μL, about 192μL, about 193μL, about 194μL, about 195μL, about 196μL, about 197μL, about 198μL, about 199μL, about 200, about 201μL, about 202μL, about 203μ L, about 204 μL, about 205 μL, about 206 μL, about 207 μL, about 208 μL, about 209 μL, about 210 μL, about 211 μL, about 212 μL, about 213 μL, about 214 μL, about 215 μL, about 216 μL, about 217 μL, about 218 μL, about 219 μL, about 220 μL, about 221 μL, about 222 μL, about 223 μL, about 224 μL, about 225 μL, about 226 μL, about 227 μL, about 228 μL, about 229 μL, about 230 μL, about 231 μL, about 232 μL, about 233 μL, about 234 μL, about 235 μL, about 236 μL, about 237 μL, about 238 μL, about 239 μL 9μL, about 240μL, about 241μL, about 242μL, about 243μL, about 244μL, about 245μL, about 246μL, about 247μL, about 248μL, about 249μL, about 250μL, about 251μL, about 252μL, about 253μL, about 254μL, about 255μL, about 256μL, about 2 57μL, about 258μL, about 259μL, about 260μL, about 261μL, about 262μL, about 263μL, about 264μL, about 265μL, about 266μL, about 267μL, about 268μL, about 269μL, about 270μL, about 271μL, about 272μL, about 273μL, about 274μL, about 275μL, about 276μL, about 277μL, about 278μL, about 279μL, about 280μL, about 281μL, about 282μL, about 283μL, about 284μL, about 285μL, about 286μL, about 287μL, about 288μL, about 289μL, about 290μL, about 291μL, about 292μL,about 293 μL, about 294 μL, about 295 μL, about 296 μL, about 297 μL, about 298 μL, about 299 μL, 300 μL, about 305 μL, about 310 μL, about 315 μL, about 320 μL, about 325 μL, about 330 μL, about 335 μL, about 340 μL, about 345 μL, about 350 μL, About 355 μL, about 360 μL, about 365 μL, about 370 μL, about 375 μL, about 380 μL, about 385 μL, about 390 μL, about 395 μL, about 400 μL, about 405 μL, about 410 μL, about 415 μL, about 420 μL, about 425 μL, about 430 μL, about 435 μL, about 440 μL , about 445μL, about 450μL, about 455μL, about 460μL, about 465μL, about 470μL, about 475μL, about 480μL, about 485μL, about 490μL, about 495μL, about 500μL, about 505μL, about 510μL, about 515μL, about 520μL, about 525μL, about 530μ L, about 535 μL, about 540 μL, about 545 μL, about 550 μL, about 555 μL, about 560 μL, about 565 μL, about 570 μL, about 575 μL, about 580 μL, about 585 μL, about 590 μL, about 595 μL, about 600 μL, about 605 μL, about 610 μL, about 615 μL, about 620 μL, about 625μL, about 630μL, about 635μL, about 640μL, about 645μL, about 650μL, about 655μL, about 660μL, about 665μL, about 670μL, about 675μL, about 680μL, about 685μL, about 690μL, about 695μL, about 700μL, about 705μL, about 71 0μL, about 715μL, about 720μL, about 725μL, about 730μL, about 735μL, about 740μL, about 745μL, about 750μL, about 755μL, about 760μL, about 765μL, about 770μL, about 775μL, about 780μL, about 785μL, about 790μL, about 795μL, about 8 00μL, about 805μL, about 810μL, about 815μL, about 820μL, about 825μL, about 830μL, about 835μL, about 840μL, about 845μL, about 850μL, about 855μL, about 860μL, about 865μL, about 870μL, about 875μL, about 880μL, about 885μL, about 890μL, about 895μL, about 900μL, about 905μL, about 910μL, about 915μL, about 920μL, about 925μL, about 930μL, about 935μL, about 940μL, about 945μL, about 950μL, about 955μL, about 960μL, about 965μL, about 970μL, about 975μL,About 980 μL, about 985 μL, about 990 μL, about 995 μL, or about 1000 μL.

[0086] In some embodiments, the first liquid stream and the second liquid stream are contacted (or mixed) at about room temperature (e.g., at about 20°C to 22°C). In some embodiments, the first liquid stream and the liquid second stream are contacted (or mixed) at the following temperatures: about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C , about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C or about 60°C. In some embodiments, the first liquid stream and the second liquid stream are contacted (or mixed) at 25°C + / - 5°C. In some embodiments, the first liquid stream and the second liquid stream are contacted (or mixed) at 20°C + / - 10°C.

[0087] In some embodiments, separating comprises transporting a polyplex (eg, when the polyplex is formed after mixing the polymer and nucleic acid components) in a fluid channel for a residence time sufficient for the polyplex to stabilize.

[0088] In some embodiments, the residence time is from about 1 second (sec) to about 20 minutes (min), including about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 5.5 minutes, about 6 minutes, about 6.5 minutes, about 7 minutes, about 7.5 minutes, about 8 minutes, about 8.5 minutes, about 9 minutes, about 9.5 minutes, about 10 minutes, about 10.5 minutes, about 11 minutes, about 11.5 minutes, about 12 minutes, about 12.5 minutes, about 13 minutes, about 13.5 minutes, about 14 minutes, about 14.5 minutes, about 15.0 minutes, about 15.5 minutes, about 16 minutes, about 16.5 minutes, about 17 minutes, about 17.5 minutes, about 18 minutes, about 18.5 minutes, about 19 minutes, about 19.5 minutes and about 20 minutes, including all ranges therebetween.

[0089] In some embodiments, the residence time is about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 5.5 minutes, about 6 minutes, about 6.5 minutes, about 7 minutes, about 7.5 minutes, about 8 minutes , about 8.5 minutes, about 9 minutes, about 9.5 minutes, about 10 minutes, about 10.5 minutes, about 11 minutes, about 11.5 minutes, about 12 minutes, about 12.5 minutes, about 13 minutes, about 13.5 minutes, about 14 minutes, about 14.5 minutes, about 15.0 minutes, about 15.5 minutes, about 16 minutes, about 16.5 minutes, about 17 minutes, about 17.5 minutes, about 18 minutes, about 18.5 minutes, about 19 minutes, about 19.5 minutes or about 20 minutes.

[0090] In some embodiments, the residence time is no less than 1 second and no more than 10 minutes. In some embodiments, the residence time is about 10 minutes.

[0091] Different polyplex products (ie, different combinations of polymer and nucleic acid components) may require different sedimentation or formation times. Sedimentation and formation times can be altered by increasing or decreasing the residence time in the liquid channel.

[0092] In some embodiments, the methods disclosed herein prevent the newly formed polyplexes from agglomerating, coalescing, condensing, grouping or sticking together. For example, by contacting the first and second liquid streams in a pulsed manner, the resulting polyplexes can be spatially separated from each other during formation and / or prior to separation.

[0093] In some embodiments, the liquid channel is slightly negatively charged. In some embodiments, the liquid channel comprises an aqueous phase surrounded by a carrier fluid. In some embodiments, the density of the carrier fluid is about 1,300 kg / m 3 Up to 2,000kg / m 3 The density of the aqueous phase is about 900 kg / m 3 To about 1200kg / m 3 In some embodiments, the density of the carrier fluid is about 1,300 kg / m 3 , about 1,400kg / m 3 , about 1,500kg / m 3 , about 1,600kg / m 3 , about 1,700kg / m 3 , about 1,800kg / m 3 , about 1,900kg / m 3 or about 2,000kg / m 3 , including all ranges between any values ​​herein. In some embodiments, the density of the aqueous phase is about 900 kg / m 3 , about 1,000kg / m 3 , about 1,100kg / m 3 or about 1,200kg / m 3 , including all ranges between any values ​​herein. In some embodiments, the aqueous phase comprises a sodium acetate buffer.

[0094] In some embodiments, the carrier fluid is oil (as described herein). In some embodiments, the oil channel is about 5 mm to 100 mm wide. Without wishing to be bound by theory, the slightly negative charge of the oil / liquid channel electrostatically attracts the positively charged polyplexes, thereby orderly transporting the polyplexes through the liquid channel and enhancing the stability of the system.

[0095] In some embodiments, the oil is selected from the group consisting of: (a) Fluorinert FC-40 (fluorinated carbon oil), (b) silicone oil, (c) mineral oil, (d) perfluoroamine oil, (e) phenylmethylpolysiloxane or (f) phenylmethylpolysiloxane-based oil and additives. In some embodiments, the additive has a hydrophile-lipophile balance number in the range of 2 to 8. In some embodiments, the additive is a polysorbate additive.

[0096] In some embodiments, the polysorbate additive is SPAN 80 (sorbitan oleate), SPAN 65 (sorbitan tristearate), or Tween 20 (polyethylene glycol sorbitan monolaurate).

[0097] In some embodiments, the concentration of the additive in the aqueous phase is between about 0.001% and about 10% (wt / wt %).

[0098] In some embodiments, the additive is added to a final concentration of 10 mg / mL to 50 mg / mL in the aqueous phase. On the one hand, the polyplexes are spatially separated or isolated in the liquid channel. In some embodiments, the space between the isolated polyplexes is controlled by the speed of the liquid in the channel. That is, because each polyplex is formed by contacting a portion of the DNA solution and a portion of the polymer solution at a discrete rate before being introduced into the liquid channel, the greater the speed of the liquid in the liquid channel, the smaller the spatial separation between the polyplexes.

[0099] In some embodiments, the speed at which the liquid moves in the channel is about 0.1 m / s to about 1 m / s, including about 0.1 m / s, about 0.11 m / s, about 0.12 m / s, about 0.13 m / s, about 0.14 m / s, about 0.15 m / s, about 0.16 m / s, about 0.17 m / s, about 0.18 m / s, about 0.19 m / s, about 0.2 m / s, about 0.21 m / s, about 0.22 m / s, about 0.23 m / s, about 0.24 m / s, about 0.25 m / s, about 0.26 m / s, about 0.27 m / s, about 0.28 m / s, about 0.29 m / s, about 0.30 m / s, about 0.31 m / s, about 0.32 m / s, about 0.33 m / s, about 0.34 m / s, about 0.35 m / s, about 0.36 m / s, about 0.37 m / s, about 0.38 m / s, about 0.39 m / s, about 0.40 m / s, about 0.41 m / s, about 0.42 m / s, about 0.43 m / s, about 0.44 m / s, about 0.45 m / s, about 0.46 m / s, about 0.47 m / s, about 0.48 m / s, about 0.49 m / s, about 0.50 m / s, about 0.51 m / s, about 0.5 9m / s, about 0.30m / s, about 0.31m / s, about 0.32m / s, about 0.33m / s, about 0.34m / s, about 0.35m / s, about 0.36m / s, about 0.37m / s, about 0.38m / s, about 0.39m / s, about 0.4m / s, about 0.41m / s, about 0.42m / s, about 0.43m / s, about 0.44m / s, about 0.45m / s, about 0.46m / s, about 0.47m / s, about 0.48m / s, about 0.49m / s, about 0.5m / s, about 0.51m / s, about 0.52m / s, about 0.53m / s, about 0.54m / s, about 0.55m / s, about 0.56m / s, about 0.57m / s, about 0.57m / s, about 0.59m / s, about 0.6m / s, about 0.61m / s, about 0.62m / s, about 0.63m / s, about 0.64m / s, about 0.65m / s, about 0.66m / s, about 0.67m / s, about 0.68m / s, about 0.69m / s, about 0.7m / s, about 0.71m / s, about 0.72m / s, about 0.73m / s, about 0.74m / s, about 0.75m / s, about 0.76m / s, about 0.77 m / s, about 0.78 m / s, about 0.79 m / s, about 0.8 m / s, about 0.81 m / s, about 0.82 m / s, about 0.83 m / s, about 0.84 m / s, about 0.85 m / s, about 0.86 m / s, about 0.87 m / s, about 0.88 m / s, about 0.9 m / s, about 0.91 m / s, about 0.92 m / s, about 0.93 m / s, about 0.94 m / s, about 0.95 m / s, about 0.96 m / s, about 0.97 m / s, about 0.98 m / s and about 1.0 m / s, including all ranges therebetween.

[0100] In some embodiments, the speed at which the liquid moves in the channel is about 0.10 m / s, about 0.11 m / s, about 0.12 m / s, about 0.13 m / s, about 0.14 m / s, about 0.15 m / s, about 0.16 m / s, about 0.17 m / s, about 0.18 m / s, about 0.19 m / s, about 0.20 m / s, about 0.21 m / s, about 0.22 m / s, about 0.23 m / s, about 0.24 m / s, about 0.25 m / s, about 0.26 m / s, about 0.27 m / s, about 0.28 m / s, about 0.29 m / s, about 0.30 m / s, about 0.31 m / s, about 0.32 m / s, about 0.33 m / s, about 0.34 m / s, about 0.36 m / s, about 0.37 m / s, about 0.38 m / s, about 0.39 m / s, about 0.40 m / s, about 0.41 m / s, about 0.42 m / s, about 0.43 m / s, about 0.44 m / s, about 0.45 m / s, about 0.46 m / s, about 0.47 m / s, about 0.48 m / s, about 0.49 m / s, about 0.50 m / s, about 0.51 m / s, about 0.52 m / s, about 0.53 m / s, about 0.54 m / s, about 0.55 m / s, about 0.56 m / s, about 0.57 m / s, about 0.58 m / s, about 0.59 m / s 1m / s, about 0.32m / s, about 0.33m / s, about 0.34m / s, about 0.35m / s, about 0.36m / s, about 0.37m / s, about 0.38m / s, about 0.39m / s, about 0.40m / s, about 0.41m / s, about 0.42m / s, about 0.43m / s, about 0.44m / s, about 0.45m / s, about 0.46m / s, about 0.47m / s, about 0.48m / s, about 0.49m / s, about 0.50m / s, about 0.51m / s, about 0.52m / s, about 0.53m / s, about 0.54m / s, about 0. .55m / s, about 0.56m / s, about 0.57m / s, about 0.58m / s, about 0.59m / s, about 0.60m / s, about 0.61m / s, about 0.62m / s, about 0.63m / s, about 0.64m / s, about 0.65m / s, about 0.66m / s, about 0.67m / s, about 0.68m / s, about 0.69m / s, about 0.70m / s, about 0.71m / s, about 0.71m / s, about 0.72m / s, about 0.73m / s, about 0.74m / s, about 0.75m / s, about 0.76m / s, about 0.77m / s, About 0.78m / s, about 0.78m / s, about 0.79m / s, about 0.80m / s, about 0.81m / s, about 0.82m / s, about 0.83m / s, about 0.84m / s, about 0.85m / s, about 0.86m / s, about 0.87m / s, about 0.88m / s, about 0.89m / s, about 0.90m / s, about 0.91m / s, about 0.92m / s, about 0.93m / s, about 0.94m / s, about 0.95m / s, about 0.96m / s, about 0.97m / s, about 0.98m / s, about 0.99m / s or about 1.0m / s.

[0101] In some embodiments, the fluid stream in which the polyplex is formed moves under laminar flow conditions. In some embodiments, the fluid stream moves under partially laminar flow conditions.

[0102] In some embodiments, the method further comprises evaluating and harvesting the one or more polyplexes.

[0103] In some embodiments, when the polycomplex moves in the liquid channel to determine whether the polycomplex meets the specifications, the evaluation of the polycomplex occurs (e.g., by an online QC test). For example, in some embodiments, the region of interest in the liquid channel and the polycomplex therein are monitored by a high-speed camera that measures the optical properties (e.g., refractive index) of the polycomplex. Polycomplexes that fail to meet the specifications will be separated and waste will result. In some embodiments, polycomplexes that do not meet the specifications are separated by applying air pressure to the region of interest. Polycomplexes that meet the specifications are harvested.

[0104] In some embodiments, the evaluation includes measuring the refractive index of the polyplexes in the liquid channel; and optionally removing polyplexes whose refractive index does not meet the specification. The refractive index of the polyplexes is related to the desired properties of properly formed polyplexes (e.g., the desired polyplex particle size).

[0105] In some embodiments, when the polyplex particle is fully formed, the polyplex particle and the oil / liquid channel into a region where the negative charge is removed from the oil / liquid, allowing the particle to be easily separated from the oil / liquid and harvested.

[0106] In some embodiments, harvesting the stable polyplex comprises:

[0107] a) collecting the polyplex in a reservoir; or

[0108] b) collecting aliquots of a defined amount of the liquid polyplex and freezing said aliquots for therapeutic use; or

[0109] c) collecting the polyplex in a container for freezing or freeze-drying the polyplex to prepare the final therapeutic product.

[0110] In some embodiments, the method further comprises filtering, washing, freezing and / or lyophilizing the stable polyplex.

[0111] In some embodiments, the method further comprises first filtering and washing the polyplex.

[0112] In some embodiments, the method comprises freezing and / or lyophilizing the polyplex.

[0113] In some embodiments, the lyophilized polyplex can be packaged and securely stored.

[0114] In some embodiments, the polymer is positively charged and the nucleic acid component is negatively charged.

[0115] In some embodiments, the polymer is first dissolved in an organic solvent at a concentration of no more than 300 μg / μL and then suspended in a sodium acetate solution. In some embodiments, the sodium acetate solution is 25 mM and pH 5.2.

[0116] In some embodiments, the concentration of the polymer in the organic solvent is about 1 μg / μL to about 300 μg / μL, including about 1 μg / μL, about 5 μg / μL, about 10 μg / μL, about 15 μg / μL, about 20 μg / μL, about 25 μg / μL, about 30 μg / μL, about 35 μg / μL, about 40 μg / μL, about 45 μg / μL, about 50 μg / μL, about 55 μg / μL, about 60 μg / μL, about 65 μg / μL, about 70μg / μL, about 75μg / μL, about 80μg / μL, about 85μg / μL, about 90μg / μL, about 95μg / μL, about 100μg / μL, about 105μg / μL, About 110 μg / μL, about 115 μg / μL, about 120 μg / μL, about 125 μg / μL, about 130 μg / μL, about 135 μg / μL, about 140 μg / μL, about 145 μg / μL, about 150μg / μL, about 155μg / μL, about 160μg / μL, about 165μg / μL, about 170μg / μL, about 175μg / μL, about 180μg / μL, about 185μg / μL, about 190μg / μL, about 195μg / μL, about 200μg / μL, about 205μg / μL, about 210μg / μL, about 215μg / μL, about 220μg / μL, about 225μg / μL, about 2 30μg / μL, about 235μg / μL, about 240μg / μL, about 245μg / μL, about 250μg / μL, about 255μg / μL, about 260μg / μL, about 265μg / μL, about 270μg / μL, about 275μg / μL, about 280μg / μL, about 285μg / μL, about 290μg / μL, about 295μg / μL and about 300μg / μL, including all ranges therebetween.

[0117] In some embodiments, the concentration of the polymer in the organic solvent is about 1 μg / μL, about 2 μg / μL, about 3 μg / μL, about 4 μg / μL, about 5 μg / μL, about 6 μg / μL, about 7 μg / μL, about 8 μg / μL, about 9 μg / μL, about 10 μg / μL, about 11 μg / μL, about 12 μg / μL, about 13 μg / μL, about 14 μg / μL, about 15 μg / μL, about 16 μg / μL, about 17 μg / μL, about 18 μg / μL, about 19 μg / μL, about 20 μg / μL, about 21 μg / μL, about 22 μg / μL, about 23 μg / μL, about 24 μg / μL, about 25 μg / μL, about 26 μg / μL, about 27μg / μL, about 28μg / μL, about 29μg / μL, about 30μg / μL, about 31μg / μL, about 32μg / μL, about 33μg / μL, About 34μg / μL, about 35μg / μL, about 36μg / μL, about 37μg / μL, about 38μg / μL, about 39μg / μL, about 40μg / μL , about 41μg / μL, about 42μg / μL, about 43μg / μL, about 44μg / μL, about 45μg / μL, about 46μg / μL, about 47μg / μ L, about 48μg / μL, about 49μg / μL, about 50μg / μL, about 51μg / μL, about 52μg / μL, about 53μg / μL, about 54μg / μ L, about 55μg / μL, about 56μg / μL, about 57μg / μL, about 58μg / μL, about 59μg / μL, about 60μg / μL, about 61μg / μL, about 62μg / μL, about 63μg / μL, about 64μg / μL, about 65μg / μL, about 67μg / μL, about 68μg / μL, about 69μg / μL, about 70μg / μL, about 71μg / μL, about 72μg / μL, about 73μg / μL, about 74μg / μL, about 75μg / μL, about 76μ g / μL, about 77μg / μL, about 78μg / μL, about 79μg / μL, about 80μg / μL, about 81μg / μL, about 82μg / μL, about 83μ g / μL, about 84μg / μL, about 85μg / μL, about 86μg / μL, about 87μg / μL, about 88μg / μL, about 89μg / μL, about 90 μg / μL, about 91 μg / μL, about 92 μg / μL, about 93 μg / μL, about 94 μg / μL, about 95 μg / μL, about 96 μg / μL, about 9 7μg / μL, about 98μg / μL, about 99μg / μL, about 100μg / μL, about 101μg / μL, about 102μg / μL, about 103μg / μL, about 104μg / μL, about 105μg / μL, about 106μg / μL, about 107μg / μL, about 108μg / μL, about 109μg / μL,Approximately 110 μg / μL, approximately 111 μg / μL, approximately 112 μg / μL, approximately 113 μg / μL, approximately 114 μg / μL, approximately 115 μg / μL, approximately 116 μg / μL, approximately 117 μg / μL, approximately 118 μg / μL, approximately 119 μg / μL, approximately 120 μg / μL, approximately 121 μg / μL, approximately 122 μg / μL, approximately 123 μg / μL, approximately 124 μg / μL, approximately 125 μg / μL, approximately 126 μg / μL, approximately 127 μg / μL, approximately 128 μg / μL, approximately 129 μg / μL, approximately 130 μg / μL, approximately 131 μg / μL, approximately 132 μg / μL, approximately 133 μg / μL, approximately 134 μg / μL, approximately 135 μg / μL, approximately 136 μg / μL, approximately 137 μg / μL, approximately 138 μg / μL, approximately 139 μg / μL, approximately 140 μg / μL, approximately 141 μg / μL, approximately 142 μg / μL, approximately 143 μg / μL, approximately 144 μg / μL, approximately 145 μg / μL, approximately 146 μg / μL, approximately 147 μg / μL, approximately 148 μg / μL, approximately 149 μg / μL, approximately 150 μg / μL, approximately 151 μg / μL, approximately 152 μg / μL, approximately 153 μg / μL, approximately 154 μg / μL, approximately 155 μg / μL, approximately 156 μg / μL, approximately 157 μg / μL, approximately 158 μg / μL, approximately 159 μg / μL, approximately 160 μg / μL, approximately 161 μg / μL, approximately 162 μg / μL, approximately 163 μg / μL, approximately 164 μg / μL, approximately 165 μg / μL, approximately 166 μg / μL, approximately 167 μg / μL, approximately 168 μg / μL, approximately 169 μg / μL, approximately 170 μg / μL, approximately 171 μg / μL, approximately 172 μg / μL, approximately 173 μg / μL, approximately 174 μg / μL, approximately 175 μg / μL, approximately 176 μg / μL, approximately 177 μg / μL, approximately 178 μg / μL, approximately 179 μg / μL, approximately 180 μg / μL, approximately 181 μg / μL, approximately 182 μg / μL, approximately 183 μg / μL, approximately 184 μg / μL, approximately 185 μg / μL, approximately 186 μg / μL, approximately 187 μg / μL, approximately 188 μg / μL, approximately 189 μg / μL, approximately 190 μg / μL, approximately 191 μg / μL, approximately 192 μg / μL, approximately 193 μg / μL, approximately 194 μg / μL, approximately 195 μg / μL, approximately 196 μg / μL, approximately 197 μg / μL, approximately 198 μg / μL, approximately 199 μg / μL, approximately 200 μg / μL, approximately 201 μg / μL, approximately 202 μg / μL, approximately 203 μg / μL, approximately 204 μg / μL, approximately 205 μg / μL, approximately 206 μg / μL, approximately 207 μg / μL, approximately 208 μg / μL, approximately 209 μg / μLAbout 210μg / μL, about 211μg / μL, about 212μg / μL, about 213μg / μL, about 214μg / μL, about 215μg / μL, about 216μg / μL, about 217μg / μL, about 218μg / μL, about 219μg / μL, about 220μg / μL, about 221μg / μL, about 222μg / μL, about 223μg / μL, about 224μg / μL, about 225μg / μL, about 226μg / μL, about 227μg / μL, about 228μg / μL, about 229μg / μL, about 230μg / μL, about 231μg / μL, about 232μg / μL, about 233μg / μL, about 234μg / μL, about 235μg / μL, about 236μg / μL, about 237μg / μL, about 238μg / μL, about 239μg / μL, about 240μg / μL, about 241μg / μL, about 242μg / μL, about 243μg / μL, about 244μg / μL, about 245μg / μL, about 246μg / μL, about 247μg / μL, about 248μg / μL, about 249μg / μL, about 250μg / μL, about 251μg / μL, about 252μg / μL, about 253μg / μL, about 254μg / μL, about 255μ g / μL, about 256μg / μL, about 257μg / μL, about 258μg / μL, about 259μg / μL, about 260μg / μL, about 261μg / μL, about 262μg / μL, about 263μg / μL, about 264μg / μL, about 265μg / μL, about 266μg / μL, about 267μg / μL, about 268μg / μL, about 269μg / μL, about 270μg / μL, about 271μg / μL, about 272μg / μL, about 273μg / μL, about 274μg / μL, about 275μg / μL, about 276μg / μL, about 277μg / μL, about 27 8μg / μL, about 279μg / μL, about 280μg / μL, about 281μg / μL, about 282μg / μL, about 283μg / μL, about 284μg / μL, about 285μg / μL, about 286μg / μL, about 287μg / μL, about 288μg / μL, about 289μg / μL, about 290μg / μL, about 291μg / μL, about 292μg / μL, about 293μg / μL, about 294μg / μL, about 295μg / μL, about 296μg / μL, about 297μg / μL, about 298μg / μL, about 299μg / μL or about 300μg / μL.,

[0118] In some embodiments, the organic solvent used to initially dissolve the polymer is an organic solvent that is miscible with water. In some embodiments, the organic solvent is dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, tetrahydrofuran, methanol, ethanol, or isopropanol. In some embodiments, the organic solvent is DMSO, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), etc.; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.; or ethers such as tetrahydrofuran (THF). In some embodiments, the organic solvent is DMSO.

[0119] In some embodiments, the nucleic acid component (e.g., DNA) is first dissolved in a solvent at a concentration of no more than 4 mg / mL and then suspended in a sodium acetate solution. In some embodiments, the sodium acetate solution is 25 mM and pH 5.2.

[0120] In some embodiments, the nucleic acid component (e.g., DNA) is dissolved to provide a solution having a concentration of no more than about 4 mg / mL, including no more than about 3.5 mg / mL, no more than about 3.0 mg / mL, no more than about 2.5 mg / mL, no more than about 2.0 mg / mL, no more than about 1.5 mg / mL, no more than about 1.0 mg / mL, no more than about 0.5 mg / mL, no more than about 0.1 mg / mL, no more than about 0.05 mg / mL, and no more than about 0.01 mg / mL; including all concentrations in between.

[0121] In some embodiments, the nucleic acid component (e.g., DNA) is dissolved to provide a solution having a concentration of about 0.01 mg / mL to 4 mg / mL, including about 0.01 mg / mL, about 0.02 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.16 mg / mL, about 0.17 mg / mL, about 0.18 mg / mL, about 0.19 mg / mL, about 0.20 mg / mL, about 0.25 mg / mL, about 0.30 mg / mL, about 0.35 mg / mL, about 0.40 mg / mL, about 0.45 mg / mL, about 0.50 mg / mL, about 0.55 mg / mL, about 0.60 mg / mL, about 0.65 mg / mL, about 0.70 mg / mL, about 0.75 mg / mL, about 0.80 mg / mL, about 0.85 mg / mL, about 0.90 mg / mL, about 0.95 mg / mL, about 1.0 mg / mL, about 1.05 mg / mL, about 1.10 mg / mL, about 1.15 mg / mL, about 1.20 mg / mL, about 1.25 mg / mL, about 1.30 mg / mL, about 1.35 mg / mL, about 1.40 mg / mL mL, about 1.45 mg / mL, about 1.50 mg / mL, about 1.55 mg / mL, about 1.60 mg / mL, about 1.65 mg / mL, about 1.70 mg / mL, about 1.75 mg / mL, about 1.80 mg / mL, about 1.85 mg / mL, about 1.90 mg / mL, about 1.95 mg / mL, about 2.0 mg / mL, about 2.10 mg / mL, about 2.15 mg / mL, about 2.20 mg / mL, about 2.25 mg / mL, about 2.30 mg / mL, about 2.35 mg / mL, about 2.40 mg / mL, about 2.45 mg / mL, about 2.50 mg / mL, about 2.55 mg / mL, about 2.60 .0 mg / mL, about 2.65 mg / mL, about 2.70 mg / mL, about 2.75 mg / mL, about 2.80 mg / mL, about 2.85 mg / mL, about 2.90 mg / mL, about 2.95 mg / mL, about 3.0 mg / mL, about 3.10 mg / mL, about 3.15 mg / mL, about 3.20 mg / mL, about 3.25 mg / mL, about 3.30 mg / mL, about 3.35 mg / mL, about 3.40 mg / mL, about 3.45 mg / mL, about 3.50 mg / mL, about 3.55 mg / mL, about 3.60 mg / mL, about 3.65 mg / mL, about 3.70 mg / mL, about 3.75 mg / mL, about 3.80 mg / mL, about 3.85 mg / mL, about 3.90 mg / mL, about 3.95 mg / mL, and about 4.0 mg / mL, including all values ​​therebetween. In some embodiments, the polymer and nucleic acid components (e.g., DNA) are suspended in a liquid containing a sugar. For example, trehalose, sucrose, glucose, and mannitol. In some embodiments, the sugar is sucrose. .

[0122] In some embodiments, the methods of the present disclosure employ any polymer capable of forming a polyplex, including polyethyleneimine, linear poly(amino esters) (LPAE) [e.g., those described in Mol Ther. 2005, 11(3), 426-434; and Methods Mol Biol. 2009; 480, 53–63, the contents of which are hereby incorporated by reference in their entireties], chitosan, poly(lactic-co-glycolic acid) polymers, polylactic acid polymers, and highly branched poly(amino esters), such as highly branched poly(b-amino esters) [e.g., those described in Biomacromolecules 2016, 17, 3640-3647; Biomacromolecules 2015, 16, 2609-2617; ACS Appl. Mater. Interfaces 2016, 8, 50, 34218-34226; J. Control Release, 2016, 244(Pt B), 336-346; Drug Discov. Today. 2013 November; 18(21-22), 1090-1098; Sci. Adv. 2016; 2:e1600102; WO 2014 / 053654; US 2015 / 0266986; and polymers described in WO 2016 / 020474, the contents of which are hereby incorporated by reference in their entirety]. In some embodiments, the polymer is a highly branched poly(amino ester) (HPAE), such as described below.

[0123] In one aspect, the methods described herein provide a polyplex comprising a nucleic acid component and a polymer comprising:

[0124] (a)

[0125] (b)

[0126] (c)

[0127]

[0128] Each J is –O–

[0129] "Z" is the connecting part;

[0130] Z is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched carbon chain of 1 to 30 atoms containing heteroatoms, a carbocyclic ring containing 3 to 30 carbon atoms, or a heterocyclic ring containing 3 to 30 carbon atoms; or Z is Where x is from 1 to 1000;

[0131] wherein Z is unsubstituted or substituted by at least one of the following: halogen, hydroxy, amino, sulfonyl, sulfonamido, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C6 alkyl, carboxyl, cyano, nitro, nitroso, —OC(O)NR'R', —N(R')C(O)NR'R', —N(R')C(O)O—C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 Aryl; wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl;

[0132] A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched carbon chain of 2 to 30 atoms containing heteroatoms, a carbocyclic ring containing 3 to 30 carbon atoms, or a heterocyclic ring containing 3 to 30 atoms;

[0133] wherein A is optionally substituted with one or more halogen, hydroxy, amino, sulfonyl, sulfonamido, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C6 alkyl, carboxyl, cyano, nitro, nitroso, —OC(O)NR'R', —N(R')C(O)NR'R', —N(R')C(O)O—C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 Aryl; wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl;

[0134] G is –C–;

[0135] Each Q is H;

[0136] Each E1 is heteroalkylene;

[0137] R1 and R2 are each independently C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl or heteroaryl; wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P and O; wherein C1-C 40 Alkyl, C2-C 40 alkenyl, C4-C8 cycloalkenyl, C2-C 40 wherein R1 and R2 are each independently unsubstituted or substituted with at least one of halogen, hydroxy, amino, sulfonyl, sulfonamido, thiol, C1-C6 alkyl ... 1- C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C6 alkyl, carboxyl, cyano, nitro, nitroso, —OC(O)NR'R', —N(R')C(O)NR'R', —N(R')C(O)O—C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 ) aryl; wherein each R 'is independently selected from the group consisting of hydrogen and C1-C6 alkyl; and

[0138] Each n is at least 1

[0139] In some embodiments, Z is a linear or branched carbon chain of 1 to 30 carbon atoms, or a linear or branched carbon chain of 1 to 30 atoms containing heteroatoms; wherein Z is unsubstituted or substituted with at least one of halogen, hydroxy, amino, sulfonyl, sulfonamido, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C6 alkyl, carboxyl, cyano, nitro, nitroso, —OC(O)NR'R', —N(R')C(O)NR'R', —N(R')C(O)O—C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C6 heteroaryl. 10 Aryl; wherein each R 'is independently selected from the group consisting of hydrogen and C1-C6 alkyl; or Z is Where x is from 1 to 1000.

[0140] In some embodiments, Z" is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched carbon chain of 1 to 30 atoms containing heteroatoms, a carbocyclic ring containing 3 to 30 carbon atoms, or a heterocyclic ring containing 3 to 30 atoms; wherein Z is unsubstituted or substituted with at least one of the following: halogen, hydroxyl, amino, sulfonyl, sulfonamido, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C6 alkyl, carboxyl, cyano, nitro, nitroso, —OC(O)NR'R', —N(R')C(O)NR'R', —N(R')C(O)O—C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 Aryl; wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl

[0141] In some embodiments, Z" is a linear carbon chain of 1 to 30 carbon atoms. For example, Z" can be an alkylene group, including but not limited to C1-C 24 Alkylene, C1-C 20 Alkylene, C1-C 16 Alkylene, C1-C 12 Alkylene, C1-C 8个 Alkylene, C1-C6 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, C1 alkylene. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butenyl, propynylene, n-butenyl, etc.

[0142] In some embodiments, Z" is a linear or branched carbon chain of 1 to 30 carbon atoms or a linear or branched carbon chain of 1 to 30 atoms containing heteroatoms.

[0143] In some embodiments, Z is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched carbon chain of 1 to 30 atoms containing heteroatoms, a carbocyclic ring containing 3 to 30 carbon atoms, or a heterocyclic ring containing 3 to 30 atoms; wherein Z is unsubstituted or substituted with at least one of halogen, hydroxy, amino, sulfonyl, sulfonamido, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 sulfide, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C6 alkyl, carboxyl, cyano, nitro, nitroso, —OC(O)NR'R', —N(R')C(O)NR'R', —N(R')C(O)O—C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl and C6-C 10Aryl; wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl.

[0144] In some embodiments, Z is a linear or branched carbon chain of 1 to 30 carbon atoms or a linear or branched carbon chain containing heteroatoms of 1 to 30 atoms. In some embodiments, Z is a linear carbon chain of 1 to 30 carbon atoms. For example, Z can be an alkylene group, including but not limited to C1-C 24 Alkylene, C1-C 20 Alkylene, C1-C 16 Alkylene, C1-C 12 Alkylene, C1-C 8个 Alkylene, C1-C6 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, C1 alkylene. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butenyl, propynylene, n-butenyl, etc. In some embodiments, Z is a branched carbon chain of 1 to 30 carbon atoms. In some embodiments, Z is a linear or branched carbon chain of 1 to 30 atoms containing heteroatoms. For example, Z can be a linear or branched carbon chain in which one or more carbon atoms are substituted with heteroatoms including, but not limited to, O, N, S, or P. In some embodiments, Z is a linear or branched carbon chain of 1 to 10 carbon atoms. In some embodiments, Z is wherein x is 1 to 1000. In some embodiments, Z is

[0145] In some embodiments, R1 and R2 are independently C1-C 20 For example, R1 and R2 can be C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 or C 20Alkyl, for example methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl or n-eicosyl. In some embodiments, R1 and R2 are independently unsubstituted. In some embodiments, R1 and R2 are independently substituted. In some embodiments, R1 and R2 are independently selected from the group consisting of:

[0146]

[0147] as well as In some embodiments, R1 is And R2 is

[0148]

[0149] In some embodiments, R1 is And R2 is

[0150]

[0151] In some embodiments, the polymer comprises:

[0152] (a)

[0153] (b)

[0154] (c) In some embodiments, the polymer comprises:

[0155] (a)

[0156] (b)

[0157] (c) In some embodiments, the polymer comprises:

[0158] (a)

[0159] (c) In some embodiments, the polymer comprises:

[0160] (a)

[0161] (c) in

[0162] J is O, Z is Where x is from 1 to 1000.

[0163] In some embodiments, the polymer comprises:

[0164] (b)

[0165] In some embodiments, the polymer comprises:

[0166] (a)

[0167] (b)

[0168] (c) in

[0169] R1 is and

[0170] R2 is selected from

[0171] In some embodiments, the polyplex comprises a nucleic acid component and a polymer comprising:

[0172] (a)

[0173] (b)

[0174] (c) in

[0175] J is O and Z is Where x is from 1 to 1000;

[0176] R1 is and

[0177] R2 is

[0178] In some embodiments, the polymers of the present disclosure have an alpha parameter defined by the Mark-Houwink equation of less than about 0.5. For example, the polymers of the present disclosure have an alpha parameter defined by the Mark-Houwink equation in the range of about 0.01 to about 0.49. For example, the polymers of the present disclosure have an alpha parameter defined by the Mark-Houwink equation in the following ranges: about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.50, about 0.51, about 0.52, about 0.53, about 0.54, about 0.55, about 0.56, about 0.57, about 0 23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43 about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about to about 0.49, including all ranges therebetween. In some embodiments, the polymers of the present disclosure have an alpha parameter defined by the Mark-Houwink equation of about 0.2 to about 0.5.

[0179] In some embodiments, the polymers of the present disclosure have the following alpha parameters defined by the Mark-Houwink equation: about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.50, about 0.51, about 0.52 .22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43 about 0.44, about 0.45, about 0.46, about 0.47, about 0.48 or about 0.49.

[0180] In some embodiments, the polymer has an alpha parameter defined by the Mark-Houwink equation of less than about 0.5. In some embodiments, the polymer has an alpha parameter defined by the Mark-Houwink equation of about 0.2 to about 0.5.

[0181] The term "polydispersity index" (PDI) refers to a measure of the molecular weight distribution in a given polymer sample. The polydispersity index is calculated by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn). As used herein, the term "weight average molecular weight" generally refers to a molecular weight measurement that depends on the contribution of polymer molecules according to their size. As used herein, the term "number average molecular weight" generally refers to a molecular weight measurement calculated by dividing the total weight of all polymer molecules in a sample by the total number of polymer molecules in the sample. These terms are well known to those of ordinary skill in the art.

[0182] In some embodiments, the polymers of the present disclosure have a PDI of about 1.01 to about 8.0. For example, the PDI can be in the range of about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 to about 8.0, including all ranges therebetween.

[0183] In some embodiments, the polymers of the present disclosure have a PDI of about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about In some embodiments, the polymers of the present disclosure have a PDI of about 2.5.

[0184] In some embodiments, the polymers of the present disclosure have a PDI of about 1.01 to about 8.0. In some embodiments, the polymers of the present disclosure have a PDI of about 2.5.

[0185] In some embodiments, the polymers of the present disclosure have an M of at least 3 kDa. W In some embodiments, the polymers of the present disclosure have an M of about 3 kDa to about 200 kDa. W Therefore, the polymers of the present disclosure have an M in the range W: about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kDa, about 65 kDa, about 66 kDa, about 67 kDa, about 68 kDa, about 69 kDa, about 70 kDa, about 71 kDa, about 72 kDa, about 73 kDa, about 74 kDa kDa, about 9kDa, about 90kDa, about 91kDa, about 92kDa, about 93kDa, about 94kDa, about 95kDa, about 96kDa, about 97kDa, about 98kDa, about 99kDa, about 100kDa, about 101kDa, about 102kDa, about 103kDa, about 104kDa, about 105kDa, about 106kDa, about 107kDa, about 108kDa, about 109kDa, about 110kDa, about 111kDa, about 112kDa, about 113kDa, about 114kDa, about 115kDa, about 116kDa, about 117kDa, about 118kDa, about 119kDakDa, about 101 kDa, about 102 kDa, about 103 kDa, about 104 kDa, about 105 kDa, about 106 kDa, about 107 kDa, about 108 kDa, about 109 kDa, about 110 kDa, about 111 kDa, about 112 kDa, about 113 kDa, about 114 kDa, about 115 kDa, about 116 kDa, about 117 kDa, about 118 kDa, about 119 kDa, about 120 kDa, about 121 kDa, about 122 kDa, about 12 kDa, about 124 kDa, about 125 kDa, about 126 kDa, about 127 kDa, about 128 kDa, about 129 kDa, about 130 kDa, about 131 kDa, about 132 kDa, about 133 kDa, about 134 kDa, about 135 kDa, about 136 kDa, about 137 kDa, about 138 kDa, about 139 kDa, about 140 kDa, about 141 kDa, about 142 kDa, about 143 kDa, about 144 kDa, about 145 kDa a, about 146 kDa, about 147 kDa, about 148 kDa, about 149 kDa, about 150 kDa, about 151 kDa, about 152 kDa, about 153 kDa, about 154 kDa, about 155 kDa, about 156 kDa, about 157 kDa, about 158 ​​kDa, about 159 kDa, about 160 kDa, about 161 kDa, about 162 kDa, about 163 kDa, about 164 kDa, about 165 kDa, about 166 kDa, about 167 kDa kDa, about 190 kDa, about 191 kDa, about 192 kDa, about 193 kDa, about 194 kDa, about 195 kDa, about 196 kDa, about 197 kDa, about 198 kDa, about 199 kDa to about 200 kDa, including all ranges therebetween. In some embodiments, the polymer has an M between about 5 kDa and 50 kDa. W In some embodiments, the polymer has an M between about 10 kDa and 50 kDa. W .

[0186] In some embodiments, the polymers of the present disclosure have the following M W: about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa , about 39kDa, about 40kDa, about 41kDa, about 42kDa, about 43kDa, about 44kDa, about 45kDa, about 46kDa, about 47kDa, about 48kDa, about 49kDa, about 50kDa, about 51kDa, about 52kDa, about 53kDa, about 54kDa, about 55kDa, about 56kDa, about 57kDa, about 58kDa, about 59kDa, about 60kDa, about 61kDa, about 62kDa, about 63kDa, about 64kDa, about 65kDa, about 66kDa, about 67kDa, about 68kDa, about 69kDa, about 70kDa, about 71kDa, about 72kDa, about 73kDa, about 74kDa, about 75kDa, about 76kDa, about 77kDa, about 78kDa, about 79kDa, about 80kDa, about 81kDa, about 82kDa, about 83kDa, about 84kDa, about 85kDa, about 86kDa, about 87kDa, about 88kDa, about 89kDa, about 90kDa, about 91kDa, about 92kDa, about 93kDa, about 94kDa, about 95kDa, about 96kDa, about 97kDa, about 98kDa, about 99kDa, about 100kDa, about 101kDa, about 102kDa, about 103kDa, about 104kDa, about 105kDa, about 106kDa, about 107kDa kDa, about 110 kDa, about 111 kDa, about 112 kDa, about 113 kDa, about 114 kDa, about 115 kDa, about 116 kDa, about 117 kDa, about 118 kDa, about 119 kDa, about 120 kDa, about 121 kDa, about 122 kDa, about 123 kDa, about 124 kDa, about 125 kDa, about 126 kDa, about 127 kDa, about 128 kDa, about 129 kDa, about 130 kDa, about 131 kDa, about 132 kDa, about 133 kDa, about 134 kDa, about 135 kDa, about 136 kDa, about 137 kDa, about 138 kDa,kDa, about 139 kDa, about 140 kDa, about 141 kDa, about 142 kDa, about 143 kDa, about 144 kDa, about 145 kDa, about 146 kDa, about 147 kDa, about 148 kDa, about 149 kDa, about 150 kDa, about 151 kDa, about 152 kDa, about 153 kDa, about 154 kDa, about 155 kDa, about 156 kDa, about 157 kDa, about 158 ​​kDa, about 159 kDa, about 160 kDa, about 161 kDa, about 162 kDa, about 163 kDa, about 164 kDa kDa, about 165kDa, about 166kDa, about 167kDa, about 168kDa, about 169kDa, about 170kDa, about 171kDa, about 172kDa, about 173kDa, about 174kDa, about 175kDa, about 176kDa, about 177kDa, about 178kDa, about 179kDa, about 180kDa, about 181kDa, about 182kDa, about 183kDa, about 184kDa, about 185kDa, about 186kDa, about 187kDa, about 188kDa, about 189kDa, about 190kDa In some embodiments, the polymer has an M between about 5 kDa and 50 kDa, W In some embodiments, the polymer has an M between about 10 kDa and 50 kDa. W In some embodiments, the polymer has an M of about 10 kDa. W In some embodiments, the polymer has an M of about 20 kDa. W In some embodiments, the polymer has an M of about 30 kDa. W .

[0187] In some embodiments, the polymer has an M of about 40 kDa. W .

[0188] In some embodiments, the polymers of the present disclosure have an M of at least 3 kDa. W In some embodiments, the polymers of the present disclosure have an M between about 5 kDa and 50 kDa. W In some embodiments, the polymers of the present disclosure have an M of about 10 kDa. W .

[0189] In some embodiments, the polymer and nucleic acid components are mixed in a ratio of about 0.1:1 to about 200:1 (w / w). For example, the polymer and nucleic acid components are mixed in a ratio within the following ranges: about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1 about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1 1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, about 67: 1, about 68:1, about 69:1, about 70:1, about 71:1, about 72:1, about 73:1, about 74:1, about 75:1, about 76:1, about 77:1, about 78:1, about 79:1, about 80:1, about 81:1, about 82:1, about 83:1, about 84:1, about 85:1, about 86:1, about 87:1, about 88:1, about 89:1, about 90:1, about 91:1, about 92:1, about 93:1, about 94:1, about 95:1, about 96:1, about 97:1, about 98:1, about 99:1, about 100:1 about 101:1, about 102:1, about 103:1, about 104:1, about 105:1, about 106: 1, about 107:1, about 108:1, about 109:1, about 110:1, about 111:1, about 112:1, about 113:1, about 114:1, about 115:1, about 116:1, about 117:1, about 118:1, about 119:1, about 120:1, about 121:1, about 122:1, about 123:1, about 124:1, about 125:1, about 126:1, about 127:1, about 128:1, about 129:1, about 130:1, about 131:1, about 132:1, about 133:1, about 134:1, about 135:1, about 136:1, about 137:1, about 138:1, about 139:1, about 140:1,about 141:1, about 142:1, about 143:1, about 144:1, about 145:1, about 146:1, about 147:1, about 148:1, about 149:1, about 150:1, about 151:1, about 152:1, about 153:1, about 154:1, about 155:1, about 156:1, about 157:1, about 158:1, about 159:1, about 160:1, about 161:1, about 162:1, about 163:1, about 164:1, about 165:1, about 166:1, about 167:1, about 168:1, about 169:1, about 170:1, about 171:1 , about 172:1, about 173:1, about 174:1, about 175:1, about 176:1, about 177:1, about 178:1, about 179:1, about 180:1, about 181:1, about 182:1, about 183:1, about 184:1, about 185:1, about 186:1, about 187:1, about 188:1, about 189:1, about 190:1, about 191:1, about 192:1, about 193:1, about 194:1, about 195:1, about 196:1, about 197:1, about 198:1, about 199:1 to about 200:1, including all ranges therebetween. In some embodiments, the polymer and nucleic acid components are mixed in a ratio of about 20:1 to about 80:1 (w / w).

[0190] In some embodiments, the polymer and nucleic acid components are mixed in a ratio of about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1 about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1 1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50 :1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, about 67:1, about 68:1, about 69:1, about 70:1, about 71:1 1:1, about 72:1, about 73:1, about 74:1, about 75:1, about 76:1, about 77:1, about 78:1, about 79:1, about 80:1, about 81:1, about 82:1, about 83:1, about 84:1, about 85:1, about 86:1, about 87:1, about 88:1, about 89:1, about 90:1, about 91:1, about 92:1, about 93:1, about 94:1, about 95:1, about 96:1, about 97:1, about 98:1, about 99:1, about 100:1, about 101:1, about 102:1, about 103:1, about 104:1, about 105:1, about 106:1, about 107:1, about 108:1, about 109:1, about 110:1, about 111:1 11:1, about 112:1, about 113:1, about 114:1, about 115:1, about 116:1, about 117:1, about 118:1, about 119:1, about 120:1, about 121:1, about 122:1, about 123:1, about 124:1, about 125:1, about 126:1, about 127:1, about 128:1, about 129:1, about 130:1, about 131:1, about 132:1, about 133:1, about 134:1, about 135:1, about 136:1, about 137:1, about 138:1, about 139:1, about 140:1, about 141:1, about 142:1, about 143:1, about 144:1, about 145:1, about 146:1,about 147:1, about 148:1, about 149:1, about 150:1, about 151:1, about 152:1, about 153:1, about 154:1, about 155:1, about 156:1, about 157:1, about 158:1, about 159:1, about 160:1, about 161:1, about 162:1, about 163:1, about 164:1, about 165:1, about 166:1, about 167:1, about 168:1, about 169:1, about 170:1, about 171:1, about 172:1, about 173:1, In some embodiments, the polymer and nucleic acid components are mixed at a ratio of about 30:1 (w / w).

[0191] In some other embodiments, the polymer has an M of about 3 kDa to about 200 kDa. W In some other embodiments, the polymer has an M of about 5 kDa to about 50 kDa. W In some other embodiments, the polymer has an M between about 10 kDa and 50 kDa. W In some other embodiments, the polymer has an M of about 5 kDa to about 15 kDa. W In some other embodiments, the polymer has an M of about 10 kDa. W In some other embodiments, the polymer has an M of about 20 kDa. W In some other embodiments, the polymer has an M of about 30 kDa. W In some other embodiments, the polymer has an M of about 40 kDa. W In some other embodiments, the polymer has an alpha parameter defined by the Mark-Houwink equation of less than about 0.5. In some other embodiments, the polymer has an alpha parameter defined by the Mark-Houwink equation in the range of about 0.3 to about 0.5. In some other embodiments, the polymer has a PDI of about 1.0 to about 8.0. In some other embodiments, the polymer has a PDI of about 2.5.

[0192] In one aspect, the polymer is HPAE.

[0193] In some embodiments, the polymer and nucleic acid components are present in the polyplex in a ratio of about 0.1:1 to about 200:1 (w / w). For example, the polymer and nucleic acid components are present in the polyplex at a ratio in the following ranges: about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1 about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1 6:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, about 67:1, about 68:1, about 69:1, about 70:1, about 71:1, about 72:1, about 73:1, about 74:1, about 75:1, about 76:1, about 77:1, about 78:1, about 79:1, about 80:1, about 81:1, about 82:1, about 83:1, about 84:1, about 85:1, about 86:1, about 87:1, about 88:1, about 89:1, about 90:1, about 91:1, about 92:1, about 93:1, about 94:1, about 95:1, about 96:1, about 97:1, about 98:1, about 99:1, about 100:1 about 101:1, about 102:1, about 103:1, about 104:1, about 105:1, about 106:1, about 107:1, about 108:1, about 109:1, about 110:1, about 111:1, about 112:1, about 113:1, about 114:1, about 115:1, about 116:1, about 117:1, about 118:1, about 119:1, about 120:1, about 121:1, about 122:1, about 123:1, about 124:1, about 125:1, about 126:1, about 127:1, about 128:1, about 129:1, about 130:1, about 131:1, about 132:1, about 133:1, about 134:1, about 135:1, about 136:1, about 137:1, about 138:1,about 139:1, about 140:1, about 141:1, about 142:1, about 143:1, about 144:1, about 145:1, about 146:1, about 147:1, about 148:1, about 149:1, about 150:1, about 151:1, about 152:1, about 153:1, about 154:1, about 155:1, about 156:1, about 157:1, about 158:1, about 159:1, about 160:1, about 161:1, about 162:1, about 163:1, about 164:1, about 165:1, about 166:1, about 167:1, about 168:1, about 169:1, about 170:1 , about 171:1, about 172:1, about 173:1, about 174:1, about 175:1, about 176:1, about 177:1, about 178:1, about 179:1, about 180:1, about 181:1, about 182:1, about 183:1, about 184:1, about 185:1, about 186:1, about 187:1, about 188:1, about 189:1, about 190:1, about 191:1, about 192:1, about 193:1, about 194:1, about 195:1, about 196:1, about 197:1, about 198:1, about 199:1 to about 200:1, including all ranges therebetween. In some embodiments, the polymer and nucleic acid components are present in the polyplex in a ratio of about 20:1 to about 80:1 (w / w).

[0194] For example, the polymer and nucleic acid components are present in the polyplex at a ratio of about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1 about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49: 1. about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, about 67:1, about 68:1, about 69:1, about 70:1 :1, about 71:1, about 72:1, about 73:1, about 74:1, about 75:1, about 76:1, about 77:1, about 78:1, about 79:1, about 80:1, about 81:1, about 82:1, about 83:1, about 84:1, about 85:1, about 86:1, about 87:1, about 88:1, about 89:1, about 90:1, about 91:1, about 92:1, about 93:1, about 94:1, about 95:1, about 96:1, about 97:1, about 98:1, about 99:1, about 100:1, about 101:1, about 102:1, about 103:1, about 104:1, about 105:1, about 106:1, about 107:1, about 108:1, about 109:1, about 11 0:1, about 111:1, about 112:1, about 113:1, about 114:1, about 115:1, about 116:1, about 117:1, about 118:1, about 119:1, about 120:1, about 121:1, about 122:1, about 123:1, about 124:1, about 125:1, about 126:1, about 127:1, about 128:1, about 129:1, about 130:1, about 131:1, about 132:1, about 133:1, about 134:1, about 135:1, about 136:1, about 137:1, about 138:1, about 139:1, about 140:1, about 141:1, about 142:1, about 143:1, about 144:1, about 145:1,about 146:1, about 147:1, about 148:1, about 149:1, about 150:1, about 151:1, about 152:1, about 153:1, about 154:1, about 155:1, about 156:1, about 157:1, about 158:1, about 159:1, about 160:1, about 161:1, about 162:1, about 163:1, about 164:1, about 165:1, about 166:1, about 167:1, about 168:1, about 169:1, about 170:1, about 171:1, about 172:1, about In some embodiments, the polymer and nucleic acid components are present in the polyplex at a ratio of about 30:1 (w / w).

[0195] In some embodiments, the particle size of the stable polyplex is less than 2 μm. In some embodiments, the particle size of the stable polyplex is less than about 300 nm. In some embodiments, the particle size of the stable polyplex is about 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, about 75 nm, about 76 nm, about 77 nm, about 78 nm, about 79 nm, about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm , about 86nm, about 87nm, about 88nm, about 89nm, about 90nm, about 91nm, about 92nm, about 93nm, about 94nm, about 95nm, about 96nm, about 97nm, about 98nm, about 99nm, about 100nm, about 101nm, about 102nm, about 103nm, about 104nm, about 105nm, about 106nm, about 107nm, about 108nm, about 109nm, about 110nm, about 111nm, about 112nm, about 113nm, about 114nm, about 115nm, about 116nm, about 117nm, about 118nm, about 119nm, about 120nm, about 121nm nm, about 122nm, about 123nm, about 124nm, about 125nm, about 126nm, about 127nm, about 128nm, about 129nm, about 130nm, about 131nm, about 132nm, about 133nm, about 134nm, about 135nm, about 136nm, about 137nm, about 138nm, about 139nm, about 140nm, about 141nm, about 142nm, about 143nm, about 144nm, about 145nm, about 146nm, about 147nm, about 148nm, about 149nm, about 150nm, about 151nm, about 152nm, about 153nm, about 154nm, about 155nm about 170nm, about 171nm, about 172nm, about 173nm, about 174nm, about 175nm, about 176nm, about 177nm, about 178nm, about 179nm, about 180nm, about 181nm, about 182nm, about 183nm, about 184nm, about 185nm, about 186nm, about 187nm, about 188nm,about 189nm, about 190nm, about 191nm, about 192nm, about 193nm, about 194nm, about 195nm, about 196nm, about 197nm, about 198nm, about 199nm, about 200nm, about 201nm, about 202nm, about 203nm, about 204nm, about 205nm, about 206nm, about 207nm, about 208nm, about 209nm, about 210nm, about 211nm, about 212nm, about 213nm, about 214nm, about 215nm, about 216nm, about 217nm, about 218nm, about 219nm, about 220nm, about 221nm, about 222nm, about 223nm, about 224nm, about 225nm, about 226nm, about 227nm, about 228nm, about 229nm, about 230nm, about 231nm, about 232nm, about 233nm, about 234nm, about 235nm, about 236nm, about 237nm, about 238nm, about 239nm, about 240nm, about 241nm, about 242nm, about 243nm, about 244nm, about 245nm, about 246nm, about 247nm, about 248nm, about 249nm, about 250nm, about 251nm, about 252nm, about 253nm, about 254nm, about 255nm, about 256nm, about 257nm, about 258nm, about 259nm, about 260nm, about 261nm, about 262nm, about 263nm, about 264nm, about 265nm, about 266nm, about 267nm, about 268nm, about 269nm, about 270nm, about 271nm, about 272nm, About 273nm, about 274nm, about 275nm, about 276nm, about 277nm, about 278nm, about 279nm, about 280nm, about 281nm, about 282nm, about 283nm, about 284nm, about 285nm, about 286nm, about 287nm, about 288nm, about 289nm, about 290nm, about 291nm, about 292nm, about 293nm, about 294nm, about 295nm, about 296nm, about 297nm, about 298nm, about 299nm or about 300nm. In some embodiments, the stable polyplex of the present disclosure has a particle size of about 60nm to about 250nm. In some embodiments, the stable polyplex of the present disclosure has a particle size of about 175nm to about 250nm.

[0196] In some embodiments, the stable polyplex is slightly positively charged. In some embodiments, slightly positively charged is a zeta potential of about 10 mV and 40 mV.

[0197] In some embodiments, the stable polyplexes of the present disclosure have a zeta potential of about 0 mV to about 100 mV, including about 0 mV, about 1 mV, about 2 mV, about 3 mV, about 4 mV, about 5 mV, about 6 mV, about 7 mV, about 8 mV, about 9 mV, about 10 mV, about 11 mV, about 12 mV, about 13 mV, about 14 mV, about 15 mV, about 16 mV, about 17 mV, about 18 mV, about 19 mV, about 20 mV, about 21 mV, about 22 mV, about 23 mV, about 24 mV, about 25 mV, about 26 mV, about 27 mV, about 28 mV, about 29 mV, about 30 mV, about 31 mV, about 32 mV, about 33 mV, about 34 mV, about 35 mV, about 36 mV, about 37 mV, about 38 mV, about 39 mV, about 40 mV, about 41 mV, about 42 mV, about 43 mV, about 44 mV 2mV, about 23mV, about 24mV, about 25mV, about 26mV, about 27mV, about 28mV, about 29mV, about 30mV, about 31mV, about 32mV, about 33mV, about 34mV, about 35mV, about 36mV, about 37mV, about 38mV, about 39mV, about 40mV, about 41mV, about 42mV, about 43mV, about 44mV, about 45mV, about 46mV, about 47mV, about 48mV, about 49mV V, about 50mV, about 51mV, about 52mV, about 53mV, about 54mV, about 55mV, about 56mV, about 57mV, about 58mV, about 59mV, about 60mV, about 61mV, about 62mV, about 63mV, about 64mV, about 65mV, about 66mV, about 67mV, about 68mV, about 69mV, about 70mV, about 71mV, about 72mV, about 73mV, about 74mV, about 75mV, about 76mV, In some embodiments, the zeta potential of the stable polyplex is about 30 mV to about 34 mV.

[0198] In some embodiments, the stable polyplexes of the present disclosure have a zeta potential of 0 mV, about 1 mV, about 2 mV, about 3 mV, about 4 mV, about 5 mV, about 6 mV, about 7 mV, about 8 mV, about 9 mV, about 10 mV, about 11 mV, about 12 mV, about 13 mV, about 14 mV, about 15 mV, about 16 mV, about 17 mV, about 18 mV, about 19 mV, about 20 mV, about 21 mV, about 22 mV, about 2 3mV, about 24mV, about 25mV, about 26mV, about 27mV, about 28mV, about 29mV, about 30mV, about 31mV, about 32mV, about 33mV, about 34mV, about 35mV, about 36mV, about 37mV, about 38mV, about 39mV, about 40mV, about 41mV, about 42mV, about 43mV, about 44mV, about 45mV, about 46mV, about 47mV, about 48mV, about 49mV mV, about 50mV, about 51mV, about 52mV, about 53mV, about 54mV, about 55mV, about 56mV, about 57mV, about 58mV, about 59mV, about 60mV, about 61mV, about 62mV, about 63mV, about 64mV, about 65mV, about 66mV, about 67mV, about 68mV, about 69mV, about 70mV, about 71mV, about 72mV, about 73mV, about 74mV, about 75mV V, about 76mV, about 77mV, about 78mV, about 78mV, about 79mV, about 80mV, about 81mV, about 82mV, about 83mV, about 84mV, about 85mV, about 86mV, about 87mV, about 88mV, about 89mV, about 90mV, about 91mV, about 92mV, about 93mV, about 94mV, about 95mV, about 96mV, about 97mV, about 98mV, about 99 or about 100mV.

[0199] In some embodiments, the stable polyplex is spherical.

[0200] In some embodiments, the nucleic acid component of the polyplex is a plasmid, a nanoplasmid, a nucleic acid, a minicircle, or a gene editing system. In some embodiments, the nucleic acid component of the polyplex is a plasmid. In some embodiments, the nucleic acid component of the polyplex is a nanoplasmid. In some embodiments, the nanoplasmid comprises a eukaryotic transgene and a bacterial backbone less than 0.5 kb in size. In some embodiments, the plasmid or nanoplasmid is a plasmid without an antibiotic resistance marker or a nanoplasmid without an antibiotic resistance marker. In some embodiments, the plasmid or nanoplasmid comprises a sucrose selection marker or a nonsense suppressor marker.

[0201] In some embodiments, the nucleic acid component of the polyplex is a gene editing system. In some embodiments, the gene editing system is (i) a clustered, regularly interspaced palindromic repeat (CRISPR)-associated (Cas) system; (ii) a transcription activator-like effector nuclease (TALEN) system; or (iii) a zinc finger nuclease (ZFN) system.

[0202] In some embodiments, the nucleic acid component is an RNAi-inducing molecule. The RNAi-inducing molecule can be selected from the group consisting of siRNA, dsRNA, shRNA and microRNA.

[0203] In some embodiments, the nucleic acid component comprises a tissue-specific promoter.

[0204] In some embodiments, the nucleic acid component comprises a gene associated with a hereditary disease or condition. Hereditary diseases or conditions may be caused by mutations in one or more genes, which result in low protein expression levels, lack of protein expression, or dysfunction. In some embodiments, the gene is selected from the group consisting of COL7A1, LAMB3, ADA, SERPINA1, CFTR, HTT, NF1, PHA, HBS, FERMT1, KRT14, DSP, SPINK5, and FLG.

[0205] In some embodiments, the gene is COL7A1 and the inherited disease or disorder is a form of epidermolysis bullosa. Epidermolysis bullosa includes dystrophic epidermolysis bullosa (autosomal recessive), dystrophic epidermolysis bullosa (localized variant), prurigo epidermolysis bullosa, epidermolysis bullosa (pretibial), simplex epidermolysis bullosa (Dowling-Meara type), simplex epidermolysis bullosa (Koebner type), simplex epidermolysis bullosa (recessive 1), simplex epidermolysis bullosa (Weber-Cockayne type), epidermolysis bullosa (lethal acantholysis). In some embodiments, the genetic disorder or genetic disease is adenosine deaminase (ADA) deficiency, alpha-1 antitrypsin deficiency, cystic fibrosis, Huntington's disease, neurofibromatosis type 1, phenylketonuria, sickle cell disease, sporadic inclusion body myositis, Duchenne muscular dystrophy, Kindler syndrome, epidermolysis bullosa conjunctiva, dermatosis pigmentosa reticularis, Naegeli-Franceschetti-Jadassohn syndrome, Netherton syndrome, ichthyosis vulgaris, atopic dermatitis, Usher syndrome, Ehlers-Danlos syndrome, homozygous familial hypercholesterolemia (HoFH), or Crohn's disease.

[0206] In some embodiments, the sequence of a gene is optimized for maximum protein expression when the polyplex is delivered to a cell.

[0207] Pharmaceutical composition

[0208] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of one or more polycomplexes described herein, and a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of glucose, sucrose, trehalose, lactose, mannitol, sorbitol, aerosil (colloidal silicon dioxide), maltose, poly (vinyl pyrrolidone), fructose, dextran, glycerol, poly (vinyl alcohol), glycine, hydroxypropyl-β-cyclodextrin and gelatin. In some embodiments, the cryoprotectant is selected from the group consisting of trehalose, sucrose, glucose and mannitol. In some embodiments, the cryoprotectant is sucrose.

[0209] In some embodiments, the pharmaceutical composition is a lyophile. In some embodiments, the lyophile comprises an effective amount of one or more polyplexes described herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of trehalose, sucrose, glucose, and mannitol. In some embodiments, the cryoprotectant is sucrose.

[0210] In some embodiments, the method comprises combining one or more polycomplexes described herein with a suitable solvent. In some embodiments, the suitable solvent is selected from the group consisting of water, dimethyl sulfoxide, and mixtures thereof. In some embodiments, the suitable solvent comprises water.

[0211] In some embodiments, the method comprises:

[0212] (a) combining one or more polycomplexes described herein with a suitable solvent;

[0213] (b) adding one or more pharmaceutically acceptable excipients and

[0214] (c) lyophilizing the mixture of step (b) to provide a lyophilic colloid.

[0215] In some embodiments, the one or more pharmaceutically acceptable excipients of step (b) comprise a cryoprotectant. In some embodiments, the concentration of the cryoprotectant is from about 1% to about 20%, including about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18% and about 19%, including all ranges therebetween, based on the weight of the mixture of step (b). In some embodiments, the concentration of the cryoprotectant is about 1% about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or about 20% by weight of the mixture of step (b). In some embodiments, the concentration of the cryoprotectant is about 1% by weight of the mixture of step (b). In some embodiments, the concentration of the cryoprotectant is about 3% by weight of the mixture of step (b). In some embodiments, the concentration of the cryoprotectant is about 5% by weight of the mixture of step (b).

[0216] Examples

[0217] The following examples are provided to illustrate the present disclosure and should not be construed as limiting the present disclosure. In these examples, unless otherwise indicated, all parts and percentages are by weight. The abbreviations in the examples are indicated below.

[0218] Example 1.

[0219] Synthesis of an exemplary hyperbranched PAE (HPAE) polymer: 0.28 g of trimethylolpropane triacrylate (TMPTA), 0.29 g of bisphenol ethoxylate diacrylate (BE), and 0.18 g of 4-amino-1-butanol (S4) were dissolved in 7.5 ml of DMSO and reacted at 90°C. Once the molecular weight was in the range of 5000 Da to 7000 Da, 0.288 g of 3-morpholinopropylamine (MPA) dissolved in 2.88 mL of DMSO was added to cap the acrylate-terminated base polymer at room temperature for 24 hours. The polymer product was precipitated into diethyl ether three times, dried under vacuum for 24 hours, and then stored at -20°C for subsequent studies.

[0220] Preparation of polyplexes: HPAE polymer was first dissolved in DMSO to a concentration of about 100 μg / μL. The required amount of HPAE polymer stock solution and 4 mg / mL nanoplasmid COL7A1 solution were diluted to the required volume with sodium acetate buffer (0.025 M, pH = 5.2) according to a 30:1 (w / w) HPAE polymer / DNA weight ratio. Then 10 μL of HPAE solution and 10 μL of DNA solution were dispensed into a mixing chamber through separate nozzles, so that the two solutions contacted and mixed in the mixing chamber. The newly formed polyplex droplets flowed into a slightly negatively charged liquid channel containing silicone oil / sodium acetate buffer. The polyplex was transported through the liquid channel at a speed of 0.1 m / s to 1 m / s, and the residence time was about 10 minutes. The polyplexes with a size and charge suitable for transdermal administration were frozen and lyophilized to be resuspended for therapeutic use.

[0221] Implementation plan:

[0222] 1. A method for preparing one or more polycomplexes, the method comprising:

[0223] (a) Provide:

[0224] i. a polymer in a first liquid stream;

[0225] ii. a nucleic acid component in a second liquid stream;

[0226] (b) contacting the polymer in the first liquid stream with the nucleic acid component in the second liquid stream to form a polyplex having a size and charge suitable for therapeutic administration; and

[0227] (c) separating the polyplex to provide a stable polyplex.

[0228] 2. The method of embodiment 1, wherein the separation comprises transporting the polyplex in a liquid channel for a residence time sufficient to stabilize the polyplex.

[0229] 3. The method of any of the preceding embodiments, further comprising evaluating and harvesting one or more polyplexes.

[0230] 4. The method of any one of the preceding embodiments, wherein the first liquid stream and the second liquid stream are solutions.

[0231] 5. The method of any of the preceding embodiments, further comprising flowing the first liquid stream and the second liquid stream through a flow regulating device at a rate that provides a polycomplex having a size and charge suitable for transdermal administration.

[0232] 6. The method of embodiment 5, wherein the flow regulating device is selected from the group consisting of a positive displacement pump, a syringe driven pump, a pressure driven pump, and a gravity feed pump.

[0233] 7. The method of any of the preceding embodiments, wherein the contacting step uses a nozzle, a microfluidic mixing device, a touch tube, a liquid bridge, a vertical mixer, a rotating double tube, or an atomizer.

[0234] 8. The method of any one of the preceding embodiments, wherein the contacting step occurs in an air gap or in a carrier fluid within the liquid channel.

[0235] 9. The method of any one of the preceding embodiments, wherein the stable polycomplex is slightly positively charged.

[0236] 10. The method of any one of embodiments 2 to 9, wherein the liquid channel is slightly negatively charged.

[0237] 11. The method of embodiment 10, wherein the liquid channel comprises an aqueous phase surrounded by a carrier fluid.

[0238] 12. The method of embodiment 11, wherein the density of the carrier fluid is between 1,300 and 2,000 kg / m 3 The density of the aqueous phase is between 900 and 1200 kg / m 3 value in the range of .

[0239] 13. A method as described in embodiment 12, wherein the aqueous phase comprises a sodium acetate buffer.

[0240] 14. The method of embodiment 11, wherein the carrier fluid is oil.

[0241] 15. A method as described in embodiment 14, wherein the oil is selected from the group consisting of: (a) Fluorinert FC-40 (fluorocarbon oil), (b) silicone oil, (c) mineral oil, (d) perfluoroamine oil, (e) phenylmethylpolysiloxane and (f) phenylmethylpolysiloxane-based oils and additives.

[0242] 16. The method of embodiment 15, wherein the additive has a hydrophile-lipophile balance number in the range of 2 to 8.

[0243] 17. The method of embodiment 15, wherein the additive is a polysorbate additive.

[0244] 18. The method of embodiment 17, wherein the polysorbate additive is SPAN 80, SPAN 65 or Tween 20.

[0245] 19. The method of embodiment 15, wherein the concentration of the additive in the aqueous phase is between about 0.001% and about 10% (wt / wt%).

[0246] 20. The method according to any one of the preceding embodiments, wherein the separating comprises isolating the polycomplex in the liquid channel.

[0247] 21. The method of embodiment 20, wherein the spaces between the isolated polyplexes are controlled by the velocity of the liquid within the channel.

[0248] 22. The method of any one of embodiments 2 to 21, wherein the residence time is from about 1 second to about 20 minutes.

[0249] 23. The method of embodiment 22, wherein the residence time is about 10 minutes.

[0250] 24. The method of any one of embodiments 3 to 23, wherein the evaluating comprises:

[0251] measuring a refractive index of the polycomplex in the liquid channel; and

[0252] Polycomposites having a refractive index that is out of specification are optionally removed.

[0253] 25. The method of any one of the preceding embodiments, further comprising filtering, washing, freezing and / or lyophilizing the stable polyplex.

[0254] 26. The method of any one of the preceding embodiments, wherein the polymer is positively charged and the nucleic acid component is negatively charged.

[0255] 27. The method of any one of the preceding embodiments, wherein the polymer and the nucleic acid component are mixed in a ratio of about 0.1:1 to about 200:1 (w / w).

[0256] 28. The method of embodiment 27, wherein the polymer and the nucleic acid component are mixed in a ratio of about 20:1 to about 80:1 (w / w).

[0257] 29. The method of embodiment 27, wherein the polymer and the nucleic acid component are mixed at a ratio of about 30:1 (w / w).

[0258] 30. The method of any one of the preceding embodiments, wherein the polymer is HPAE.

[0259] 31. The method of any one of the preceding embodiments, wherein the polymer and the nucleic acid component are present in the polyplex in a ratio of about 0.1:1 to about 200:1 (w / w).

[0260] 32. The method of embodiment 31, wherein the polymer and nucleic acid components are present in the polyplex in a ratio of about 20:1 to about 80:1 (w / w).

[0261] 33. The method of embodiment 31, wherein the polymer and nucleic acid components are present in the polyplex in a ratio of about 30:1 (w / w).

[0262] 34. The method of any one of the preceding embodiments, wherein the particle size of the stable polyplex is less than about 2 μm.

[0263] 35. The method of embodiment 34, wherein the particle size of the stable polycomplex is about 60 nm to about 250 nm.

[0264] 36. The method of embodiment 34, wherein the particle size of the stable polyplex is about 175 nm to about 250 nm.

[0265] 37. The method of any one of the preceding embodiments, wherein the zeta potential of the stable polycomplex is from about 0 mV to about 100 mV.

[0266] 38. A method as described in embodiment 37, wherein the zeta potential of the stable polycomplex is about 30 mV to about 34 mV.

[0267] 39. The method according to any of the preceding embodiments, wherein the stable polycomplex is spherical.

[0268] 40. The method of any one of the preceding embodiments, wherein the polymer has an M of about 10 kDa. W .

[0269] 41. The method of any of the preceding embodiments, wherein the nucleic acid component is a plasmid, a nanoplasmid, a nucleic acid, a minicircle, or a gene editing system.

[0270] 42. The method of embodiment 41, wherein the nucleic acid component comprises a gene associated with a genetic disease or disorder.

[0271] 43. The method of embodiment 42, wherein the genetic disease or condition is caused by a mutation in one or more genes, which results in low protein expression, absence or dysfunction.

[0272] 44. The method of embodiment 43, wherein the gene is selected from the group consisting of COL7A1, LAMB3, ADA, SERPINA1, CFTR, HTT, NF1, PHA, HBS, FERMT1, KRT14, DSP, SPINK5 and FLG.

[0273] 45. The method of embodiment 44, wherein the gene is COL7A1 and the genetic disease or disorder is in the form of epidermolysis bullosa.

[0274] 46. ​​The method of embodiment 45, wherein the sequence of the gene is optimized to achieve maximum protein expression when the polyplex is delivered to a cell.

[0275] 47. The method of any of the preceding embodiments, wherein the polymer has an alpha parameter defined according to the Mark-Houwink equation of less than about 0.5.

[0276] 48. The method of any of the preceding embodiments, wherein the polymer has an alpha parameter defined according to the Mark-Houwink equation of about 0.2 to about 0.5.

[0277] 49. The method of any of the preceding embodiments, wherein the polymer has a PDI of about 1.01 to about 8.0.

[0278] 50. The method of any one of the preceding embodiments, wherein the polymer has a PDI of about 2.5.

[0279] 51. The method of any one of the preceding embodiments, wherein the polymer has an M of at least 3 kDa. W .

[0280] 52. The method of any one of the preceding embodiments, wherein the polymer has an M between about 5 kDa and 50 kDa. W .

[0281] 53. The method of any one of the preceding embodiments, wherein the polymer has an M of about 10 kDa. W .

[0282] 54. The method of any one of the preceding embodiments, wherein the polymer comprises:

[0283] (a)

[0284] (b)

[0285] (c) in

[0286] J is O and Z is Where x is from 1 to 1000;

[0287] R1 is and

[0288] R2 is

[0289] References

[0290] [1] L. Naldini. Gene therapy returns to centrestage. Nature 2015.

[0291] [2] H. Yin, RL Kanasty, AA Eltoukhy, AJ Vegas, JR Dorkin, DG Anderson. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 2014.

[0292] [3] M. Foldvari, DW Chen, N. Nafissi, D. Calderon, L. Narsineni, A. Rafiee, J. Control. Release 2016.

[0293] [4] H. Lv, S. Zhang, B. Wang, S. Cui, J. Yan, Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control. Release 2006.

[0294] [5]C.Tros de Ilarduya, Y.Sun, N. Gene delivery by lipoplexes and polyplexes. Eur. J. Pharm. Sci. 2010, pp. 159-170.

Claims

1. A method for preparing one or more polycomplexes, the method comprising: (a) Provide: i. a polymer in a first liquid stream; ii. a nucleic acid component in a second liquid stream; (b) contacting the polymer in the first liquid stream with the nucleic acid component in the second liquid stream to form a polyplex having a size and charge suitable for therapeutic administration; and (c) separating the polyplex to provide a stable polyplex.

2. The method of claim 1, wherein the separating comprises transporting the polyplex in a liquid channel for a residence time sufficient to stabilize the polyplex.

3. The method of any of the preceding claims, further comprising evaluating and harvesting one or more polyplexes.

4. A method as claimed in any one of the preceding claims, wherein the first liquid stream and the second liquid stream are solutions.

5. The method of any of the preceding claims, further comprising flowing the first and second liquid streams through a flow regulating device at a rate that provides a polycomplex having a size and charge suitable for transdermal administration.

6. The method of claim 5, wherein the flow regulating device is selected from the group consisting of a positive displacement pump, a syringe driven pump, a pressure driven pump, and a gravity feed pump.

7. The method of any of the preceding claims, wherein the contacting step uses a nozzle, a microfluidic mixing device, a touch tube, a liquid bridge, a vertical mixer, a rotating double tube, or an atomizer.

8. A method as claimed in any one of the preceding claims, wherein the contacting step occurs in an air gap or in a carrier fluid within the liquid channel.

9. The method of any one of the preceding claims, wherein the stable polycomplex is slightly positively charged.

10. The method of any one of claims 2 to 9, wherein the liquid channel is slightly negatively charged.

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