Methods and compositions for continuous production of nucleic acids

Abstract

Described are methods, systems, and compositions for continuous production of RNA molecules in a continuous-flow in vitro transcription reaction with immobilized non- consumables. Also described are methods and systems for continuous production of RNA drug substance and RNA drug product from raw materials within an integrated, end-to-end, continuous-flow manufacturing skid.

Description

[0001] PC073007A - 1 - METHODS AND COMPOSITIONS FOR CONTINUOUS PRODUCTION OF NUCLEIC ACIDS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. 63 / 692,166, filed September 8, 2024, and U.S. Provisional Patent Application Serial 5 No.63 / 582,905, filed September 15, 2023, the disclosures of which are hereby incorporated by reference in their entirety. FIELD The present invention relates to synthesis of nucleic acid molecules for therapeutic and prophylactic purposes, in particular continuous synthesis of RNA molecules via in vitro 10 transcription (IVT) and purification of RNA drug substance and drug product formulation. BACKGROUND While state-of-the-art batch and fed-batch IVT processes have proven commercially successful for RNA therapeutic and prophylactic manufacture, there remains an urgent need in the art for 15 further improved and intensified RNA manufacturing processes in terms of speed, throughput, quality, footprint, cost, and ability to meet a broad range of dose demand. Such improvements will accelerate the development of RNA therapeutics and prophylactics for effective treatment and prevention of a wider variety of diseases for more patients. 20 SUMMARY OF THE INVENTION The present invention relates to 1) improved and intensified processes for the synthesis of nucleic acid molecules, in particular, continuous-flow in vitro transcription (CF-IVT) for the synthesis of RNA via continuous flow of reactants through reactors containing stationary non-consumables (e.g., enzymes, DNA, or both), 2) continuous purification of RNA from the CF-IVT product to 25 obtain RNA drug substance, 3) continuous encapsulation of the drug substance RNA within lipid nanoparticles (LNPs) and consequent processing into RNA-loaded LNP drug product, and 4) integration of the above modules into an end-to-end skid for continuous RNA therapeutic manufacturing from raw materials to drug product. The resulting integrated, modular, and continuous manufacturing process for on-demand manufacturing of RNA therapeutics from DNA 30 to drug product is termed “mSKID.” In some embodiments, disclosed herein are methods for continuous production of an RNA molecule, wherein the methods comprise: (a) incubating a circular double-stranded DNA (dsDNA) template with a restriction endonuclease to obtain a linearized dsDNA fragment; (b) incubating the linearized dsDNA fragment with an aminoallyl-dNTP and a DNA polymerase for a time period between about three and twenty-four hours at a temperature between about 30 and 45 °C to obtain a labeled composition; (c) purifying the labeled composition to obtain a labeled dsDNA fragment; (d) immobilizing the labeled dsDNA fragment within a substrate to obtain an immobilized DNA reactor; (e) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain a continuous-flow in vitro transcription (CF-IVT) product comprising the RNA molecule, thereby continuously producing the RNA molecule. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule, wherein the methods comprise: (a) incubating a circular double-stranded DNA (dsDNA) template with a restriction endonuclease to obtain a linearized dsDNA fragment; (b) incubating the linearized dsDNA fragment with an aminoallyl-dNTP and a DNA polymerase for a time period between about three and twenty-four hours at a temperature between about 30 and 45 °C to obtain a labeled composition; (c) purifying the labeled composition to obtain a labeled dsDNA fragment; (d) immobilizing the labeled dsDNA fragment within a substrate to obtain an immobilized DNA reactor; (e) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain a continuous-flow in vitro transcription (CF-IVT) product comprising the RNA molecule: (f) incubating the CF-IVT product with an additive to obtain a treated CF-IVT product; (g) contacting the treated CF-IVT product with a diluent to obtain a diluted treated CF-IVT product; and (h) contacting the diluted treated CF-IVT product with a continuous in-line purification device to obtain a drug substance. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule, wherein the methods comprise: (a) incubating a circular double-stranded DNA (dsDNA) template with a restriction endonuclease to obtain a linearized dsDNA fragment; (b) incubating the linearized dsDNA fragment with an aminoallyl-dNTP and a DNA polymerase for a time period between about three and twenty-four hours at a temperature between about 30 and 45 °C to obtain a labeled composition; (c) purifying the labeled composition to obtain a labeled dsDNA fragment; (d) immobilizing the labeled dsDNA fragment within a substrate to obtain an immobilized DNA reactor; (e) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain a continuous-flow in vitro transcription (CF-IVT) product comprising the RNA molecule: (f) incubating the CF-IVT product with an additive to obtain a treated CF-IVT product; (g) contacting the treated CF-IVT product with a diluent to obtain a diluted treated CF-IVT product; (h) contacting the diluted treated CF-IVT product with a continuous in-line purification device to obtain a drug substance; (i) contacting the drug substance with an aqueous solution to obtain an aqueous phase, wherein contacting the drug substance with the aqueous solution adjusts a concentration and a pH of the drug substance; (j) mixing the aqueous phase with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (k) contacting the RNA-loaded LNP dispersion with a diluent to obtain a quenched LNP dispersion; (l) contacting the quenched LNP dispersion with a diluent to obtain a diluted quenched LNP dispersion; (m) contacting the diluted quenched LNP dispersion with a continuous in-line purification device to obtain a re-concentrated LNP dispersion; (n) contacting the re-concentrated LNP dispersion with a diluent to obtain a diluted LNP dispersion; (o) contacting the diluted LNP dispersion with a continuous in-line purification device to obtain a purified LNP dispersion; (p) filtering the purified LNP dispersion through an in-line filtration device to obtain a filtered purified LNP dispersion; (q) contacting the filtered purified LNP dispersion with a diluent to obtain a diluted, filtered purified LNP dispersion; and (r) filtering the diluted, filtered purified LNP dispersion through an in-line filtration device thereby, continuously producing an RNA drug product. In some embodiments, the aminoallyl-dNTP comprises aminoallyl-dUTP. In some embodiments, the additive is selected from the group consisting of proteinase K, ethylenediaminetetraacetic acid (EDTA), and DNaseI. In some embodiments, a yield of the CF-IVT product is measured in-line via a pH sensor. In some embodiments, late migrating species of the CF-IVT product are reduced by up to 60% compared to an IVT product obtained by non-continuous production. In some embodiments, the circular dsDNA template comprises a synthetic dsDNA template. In some embodiments, the substrate comprises an N- hydroxysuccinimide-activated resin-packed column or a carbonylimidazole-activated resin- packed column. In some embodiments, the in vitro transcription reaction system comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a residence time of between 100 minutes and 2 minutes at a temperature between about 20 and 45 °C. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a time period between 10 minutes and 1,200 minutes. In some embodiments, following the step of flowing the in vitro transcription reaction system across the immobilized DNA reactor, the immobilized DNA reactor remains functional for at least 14, 20, 42, or 64 days. In some embodiments, the continuous in-line purification device is selected from the group consisting of an oligo(dT) column and a single-pass tangential flow filtration (SPTFF) device. In some embodiments, the oligo(dT) column comprises a resin-packed column or a monolith column. In some embodiments, the aqueous solution comprises an acidifying solution. In some embodiments, the diluent is selected from the group consisting of a HEPES-buffered diluent, a Tris-buffered diluent, and water. In some embodiments, the acidifying solution comprises a low pH citrate solution. In some embodiments, contacting the drug substance with the aqueous solution comprises passing the purified RNA molecule through a device selected from the group consisting of a continuous in-line purification device, an in-line mixing device, an in-line UV absorbance sensor, and a surge vessel. In some embodiments, the lipid is selected from the group consisting of a cationic lipid, a phospholipid, a sterol, and a polymer-conjugated lipid. In some embodiments, contacting the RNA-loaded LNP dispersion with a diluent comprises passing the RNA-loaded LNP dispersion through an in-line mixing device. In some embodiments, the in-line filtration device is selected from the group consisting of a bioburden reduction filtration device and a sterile filtration device. In some embodiments, the steps of contacting the treated CF-IVT product with a diluent and contacting the diluted treated CF-IVT product with a continuous in-line purification device are repeated one or more times. In some embodiments, the steps of contacting the re-concentrated LNP dispersion with a diluent and contacting the diluted LNP dispersion with a continuous in-line purification device are repeated one or more times. In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, the DNA polymerase comprises phi29 DNA polymerase or a Klenow fragment DNA polymerase. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule comprising: (a) obtaining a composition comprising: a. a circular double-stranded DNA (dsDNA) template, b. a primer, c. a deoxyribonucleotide triphosphate (dNTP), d. an aminoallyl-dNTP, and e. a DNA polymerase; (b) incubating the composition for a time period between about three and twenty-four hours at a temperature between about 30 and 45 °C to obtain an incubated composition; (c) contacting the incubated composition with a restriction endonuclease to obtain a digested composition; (d) purifying the digested composition to obtain a labeled linearized dsDNA fragment; (e) immobilizing the labeled linearized dsDNA fragment within a substrate to obtain an immobilized DNA reactor; and (f) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain the RNA molecule, thereby continuously producing the RNA molecule. In some embodiments, the deoxyribonucleotide triphosphate comprises dATP, dCTP, dGTP, and dTTP, and the aminoallyl-dNTP comprises aminoallyl-dUTP. In some embodiments, the ratio of aminoallyl-dUTP to dTTP is 1:100. In some embodiments, the DNA polymerase is a phi29 DNA polymerase. In some embodiments, contacting the incubated composition with a restriction endonuclease occurs for a time period between about ten and twenty-four hours at a temperature between about 30 and 45 °C. In some embodiments, purifying the digested composition to obtain a labeled linearized dsDNA fragment comprises isopropanol precipitation. In some embodiments, the substrate comprises an N-hydroxysuccinimide-activated resin-packed column or a carbonylimidazole-activated resin-packed column. In some embodiments, the in vitro transcription reaction system comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNAse inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a residence time of between 100 minutes and 2 minutes at a temperature between about 20 and 45 °C. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a time period between 10 minutes and 1,200 minutes. In some embodiments, following the step of flowing the in vitro transcription reaction system across the immobilized DNA reactor, the immobilized DNA reactor remains functional for at least 14, 20, 42, or 64 days. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule comprising: (a) obtaining a composition comprising: a. a circular double-stranded DNA (dsDNA) template, b. a primer, c. a deoxyribonucleotide triphosphate (dNTP), d. an aminoallyl-dNTP, and e. a DNA polymerase; (b) incubating the composition for a time period between about three and twenty-four hours at a temperature between about 30 and 45 °C to obtain an incubated composition; (c) contacting the incubated composition with a restriction endonuclease to obtain a digested composition; (d) purifying the digested composition to obtain a labeled linearized dsDNA fragment; (e) immobilizing the labeled linearized dsDNA fragment within a substrate to obtain an immobilized DNA reactor; (f) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain the RNA molecule; (g) contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule; (h) adjusting the purified RNA molecule with an aqueous solution to obtain an aqueous phase; (i) mixing the aqueous phase with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (j) contacting the RNA-loaded LNP dispersion with a diluent to obtain a diluted RNA- loaded LNP dispersion; and (k) filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device thereby continuously producing an RNA drug product. In some embodiments, the continuous in-line purification device is an oligo(dT) column. In some embodiments, the oligo(dT) column is a resin-packed column or a monolith column. In some embodiments, the continuous in-line purification device is a single-pass tangential flow filtration (SPTFF) device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device further comprises contacting the RNA molecule with a diluent. In some embodiments, the aqueous solution is an acidifying solution. In some embodiments, the diluent is selected from the group consisting of a HEPES-buffered diluent, a Tris-buffered diluent, and water. In some embodiments, the acidifying solution is a low pH citrate solution. In some embodiments, adjusting the purified RNA molecule with an aqueous solution comprises passing the purified RNA molecule through a device selected from the group consisting of a continuous in-line purification device, an in-line mixing device, an in-line UV absorbance sensor, and a surge vessel. In some embodiments, the lipid is selected from the group consisting of a cationic lipid, a phospholipid, a sterol, and a polymer-conjugated lipid. In some embodiments, mixing the RNA-loaded LNP dispersion with a diluent comprises passing the RNA-loaded LNP dispersion through an in-line mixing device. In some embodiments, the continuous in-line filtration device is selected from the group consisting of a single-pass tangential flow filtration (SPTFF) device, a bioburden reduction filtration device, and a sterile filtration device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule is repeated one or more times. In some embodiments, filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device is repeated one or more times. In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, the DNA polymerase comprises phi29 DNA polymerase or a Klenow fragment DNA polymerase. In some embodiments, disclosed herein are methods for continuous production of an RNA drug product, wherein the methods comprise: (a) immobilizing an RNA polymerase within a substrate to obtain an immobilized polymerase reactor; (b) incubating the immobilized polymerase reactor at a temperature between 20 °C and 45 °C; (c) flowing an in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor to obtain an RNA molecule; (d) contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule; (e) adjusting the purified RNA molecule with an aqueous solution to obtain an aqueous phase; (f) mixing the aqueous phase with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (g) contacting the RNA-loaded LNP dispersion with a diluent to obtain a diluted RNA-loaded LNP dispersion; and (h) filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device; thereby continuously producing the RNA drug product. In some embodiments, the substrate is selected from the group consisting of a N-hydroxysuccinimide-activated resin- packed column, carbonylimidazole-activated resin-packed column, and a carbonylimidazole- functionalized monolithic column. In some embodiments, immobilizing the RNA polymerase comprises flowing a solution comprising the RNA polymerase across the substrate at a residence time of at least 20 minutes followed by a longer residence time of at least 200 minutes. In some embodiments, flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor comprises a time period between 10 minutes and 36 h. In some embodiments, following the step of flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor, the immobilized polymerase reactor remains functional for at least 14, 20, 42, or 64 days. In some embodiments, flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor comprises a residence time of between 1000 minutes and 2 minutes at a temperature between about 35 and 45 °C. In some embodiments, the in vitro transcription reaction system comprising a DNA template further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNase inhibitor, and a pyrophosphatase. In some embodiments, the continuous in-line purification device is an oligo(dT) column. In some embodiments, the oligo(dT) column is a resin-packed column or a monolith column. In some embodiments, the continuous in-line purification device is a single- pass tangential flow filtration (SPTFF) device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device further comprises contacting the RNA molecule with a diluent. In some embodiments, the aqueous solution is an acidifying solution. In some embodiments, the diluent is selected from the group consisting of a HEPES-buffered diluent, a Tris-buffered diluent, and water. In some embodiments, the acidifying solution is a low pH citrate solution. In some embodiments, adjusting the purified RNA molecule with an aqueous solution comprises passing the purified RNA molecule through a device selected from the group consisting of a continuous in-line purification device, an in-line mixing device, an in- line UV absorbance sensor, and a surge vessel. In some embodiments, the lipid is selected from the group consisting of a cationic lipid, a phospholipid, a sterol, and a polymer-conjugated lipid. In some embodiments, mixing the RNA-loaded LNP dispersion with a diluent comprises passing the RNA-loaded LNP dispersion through an in-line mixing device. In some embodiments, the continuous in-line filtration device is selected from the group consisting of a single-pass tangential flow filtration (SPTFF) device, a bioburden reduction filtration device, and a sterile filtration device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule is repeated one or more times. In some embodiments, filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device is repeated one or more times. In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, the DNA polymerase comprises phi29 DNA polymerase or a Klenow fragment DNA polymerase. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule, wherein the methods comprise: (a) obtaining a composition comprising: a. a linear double-stranded DNA (dsDNA) template comprising a 5’ overhang, b. a DNA polymerase, and c. an aminoallyl-dNTP; (b) incubating the composition for a time period between about twelve and twenty-four hours at a temperature between about 20 °C and 40 °C to obtain a labeled dsDNA template; (c) immobilizing the labeled dsDNA template within a substrate to obtain an immobilized DNA reactor; and (d) flowing an in vitro transcription reaction system across the immobilized DNA reactor, thereby continuously producing the RNA molecule. In some embodiments, the 5’ overhang comprises a 5’ adenine overhang. In some embodiments, the DNA polymerase is selected from the group consisting of a Klenow fragment DNA polymerase and a Phi29 DNA polymerase. In some embodiments, the aminoallyl-dNTP comprises aminoallyl-dUTP. In some embodiments, the 5’ adenine overhang is upstream of a T7 promoter site on the linear dsDNA template and the linear dsDNA template further comprises a 5’ thymidine overhang downstream of the T7 promoter site. In some embodiments, the substrate comprises an N-hydroxysuccinimide-activated resin-packed column or a carbonylimidazole-activated resin-packed column. In some embodiments, the substrate comprises a carbonylimidazole-functionalized monolithic column. In some embodiments, the in vitro transcription reaction system comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me- pUTP, a magnesium ion, an RNAse inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a residence time of between 100 minutes and 2 minutes at a temperature between about 20 and 45 °C. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a time period between 2 minutes and 60 hours. In some embodiments, following the step of flowing the in vitro transcription reaction system across the immobilized DNA reactor, the immobilized DNA reactor remains functional for at least 14, 20, 42, or 64 days. In some embodiments, the RNA polymerase is at a concentration between about 5,000 U / mL and about 30,000 U / mL. In some embodiments, immobilizing the labeled dsDNA template within a substrate comprises a binding density of at least 0.013 mg of dsDNA per mL of substrate volume. In some embodiments, following the step of flowing the in vitro transcription reaction system across the immobilized DNA reactor, the immobilized DNA reactor produces at least 7 mg / mL of the RNA molecule after at least 40 days. In some embodiments, following the step of flowing the in vitro transcription reaction system across the immobilized DNA reactor, the RNA molecule is contacted with a chelating agent and / or a protease. In some embodiments, the chelating agent comprises ethylenediaminetetraacetic acid (EDTA) and the protease comprises proteinase K. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule, wherein the methods comprise: (a) obtaining a composition comprising: a. a linear double-stranded DNA (dsDNA) template comprising a 5’ overhang, b. a DNA polymerase, and c. an aminoallyl-dNTP; (b) incubating the composition for a time period between about twelve and twenty-four hours at a temperature between about 20 °C and 40 °C to obtain a labeled dsDNA template; (c) immobilizing the labeled dsDNA template within a substrate to obtain an immobilized DNA reactor; (d) flowing an in vitro transcription reaction system across the immobilized DNA reactor; (e) contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule; (f) adjusting the purified RNA molecule with an aqueous solution to obtain an aqueous phase; (g) mixing the aqueous phase with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (h) mixing the RNA-loaded LNP dispersion with a diluent to obtain a diluted RNA-loaded LNP dispersion; and (i) filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device thereby continuously producing an RNA drug product. In some embodiments, the continuous in-line purification device is an oligo(dT) column. In some embodiments, the oligo(dT) column is a resin-packed column or a monolith column. In some embodiments, the continuous in-line purification device is a single-pass tangential flow filtration (SPTFF) device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device further comprises contacting the RNA molecule with a diluent. In some embodiments, the aqueous solution is an acidifying solution. In some embodiments, the diluent is selected from the group consisting of a HEPES-buffered diluent, a Tris-buffered diluent, and water. In some embodiments, the acidifying solution is a low pH citrate solution. In some embodiments, adjusting the purified RNA molecule with an aqueous solution comprises passing the purified RNA molecule through a device selected from the group consisting of a continuous in-line purification device, an in-line mixing device, an in-line UV absorbance sensor, and a surge vessel. In some embodiments, the lipid is selected from the group consisting of a cationic lipid, a phospholipid, a sterol, and a polymer-conjugated lipid. In some embodiments, mixing the RNA-loaded LNP dispersion with a diluent comprises passing the RNA-loaded LNP dispersion through an in-line mixing device. In some embodiments, the continuous in-line filtration device is selected from the group consisting of a single-pass tangential flow filtration (SPTFF) device, a bioburden reduction filtration device, and a sterile filtration device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule is repeated one or more times. In some embodiments, filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device is repeated one or more times. In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, the DNA polymerase comprises phi29 DNA polymerase or a Klenow fragment DNA polymerase. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule comprising: (a) transforming a bacterial cell with a vector encoding an RNA polymerase comprising a histidine tag; (b) incubating the bacterial cell at a temperature between 34 °C and 40 °C for at least 12 hours; (c) lysing the bacterial cell to obtain a solution comprising the RNA polymerase; (d) flowing the solution comprising the RNA polymerase across a nickel-nitrilotriacetic acid (Ni-NTA)-functionalized resin-packed column to obtain an immobilized polymerase reactor; (e) incubating the immobilized polymerase reactor at a temperature between 20 °C and 40 °C; and (f) flowing an in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor to obtain an RNA molecule, thereby continuously producing the RNA molecule. In some embodiments, In some embodiments, the bacterial cell comprises E. coli. In some embodiments, the in vitro transcription reaction system comprising a DNA template further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNase inhibitor, and a pyrophosphatase. In some embodiments, flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor comprises a residence time of between 1000 minutes and 2 minutes at a temperature between about 20 and 45 °C. In some embodiments, flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor comprises a time period between 10 minutes and 120 minutes. In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, the DNA polymerase comprises phi29 DNA polymerase or a Klenow fragment DNA polymerase. In some embodiments, disclosed herein are methods for continuous production of an RNA drug product, wherein the methods comprise: (a) incubating a circular double-stranded DNA (dsDNA) template with a restriction endonuclease to obtain a linearized dsDNA fragment; (b) incubating the linearized dsDNA fragment with an aminoallyl-dNTP and a DNA polymerase for a time period between about three and twenty-four hours at a temperature between about 30 and 45 °C to obtain a labeled composition; (c) purifying the labeled composition to obtain a labeled dsDNA fragment; (d) immobilizing the labeled dsDNA fragment within a substrate to obtain an immobilized DNA reactor; (e) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain a continuous-flow in vitro transcription (CF-IVT) product; (f) incubating the CF-IVT product with an additive to obtain a treated CF-IVT product; (g) contacting the treated CF-IVT product with a diluent to obtain a diluted treated CF-IVT product; (h) contacting the diluted treated CF-IVT product with a continuous in-line purification device to obtain a drug substance; (i) contacting the drug substance with an aqueous solution to obtain an aqueous phase, wherein contacting the drug substance with the aqueous solution adjusts a concentration and a pH of the drug substance; (j) mixing the aqueous phase with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (k) contacting the RNA-loaded LNP dispersion with a diluent to obtain a quenched LNP dispersion; (l) contacting the quenched LNP dispersion with a diluent to obtain a diluted quenched LNP dispersion; (m) contacting the diluted quenched LNP dispersion with a continuous in-line purification device to obtain a re-concentrated LNP dispersion; (n) contacting the re-concentrated LNP dispersion with a diluent to obtain a diluted LNP dispersion; (o) contacting the diluted LNP dispersion with a continuous in-line purification device to obtain a purified LNP dispersion; (p) filtering the purified LNP dispersion through an in-line filtration device to obtain a filtered purified LNP dispersion; (q) contacting the filtered purified LNP dispersion with a diluent to obtain a diluted, filtered purified LNP dispersion; and (r) filtering the diluted, filtered purified LNP dispersion through an in-line filtration device, thereby continuously producing the RNA drug product. In some embodiments, the aminoallyl-dNTP comprises aminoallyl-dUTP. In some embodiments, the additive is selected from the group consisting of proteinase K and ethylenediaminetetraacetic acid (EDTA). In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, the DNA polymerase comprises phi29 DNA polymerase or a Klenow fragment DNA polymerase. In some embodiments, contacting the drug substance with an aqueous solution further comprises an in-line UV absorbance sensor. In some embodiments, disclosed herein are continuous-flow RNA drug substance manufacturing systems, wherein the systems comprise: (a) an immobilized DNA reactor comprising a reactor inlet and a reactor outlet, wherein the immobilized DNA reactor is configured such that an in vitro transcription reaction system is passed into the reactor inlet and an RNA molecule exits the DNA reactor through the reactor outlet; (b) a first in-line mixing device in fluid communication with the reactor outlet, wherein the first in-line mixing device is configured such that a chelating agent contacts the RNA molecule; (c) a hold-up volume in fluid communication with the first in-line mixing device, wherein the hold-up volume comprises a loop of tubing, a loop of piping, or a vessel and wherein the hold-up volume is configured such that the RNA molecule has a desired residence time; (d) a second in-line mixing device in fluid communication with the hold-up volume, wherein the second in-line mixing device is configured such that a single-pass tangential flow filtration (SPTFF) load buffer contacts the RNA molecule; and (e) a single-pass tangential flow filtration (SPTFF) device comprising a membrane inlet and a membrane outlet, wherein the membrane inlet is in fluid communication with the second in-line mixing device, and a drug substance exits the SPTFF device through the membrane outlet. In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, disclosed herein are continuous-flow RNA drug substance manufacturing systems, wherein the systems comprise: (a) an immobilized DNA reactor comprising a reactor inlet and a reactor outlet, wherein the immobilized DNA reactor is configured such that an in vitro transcription reaction system is passed into the reactor inlet and an RNA molecule exits the DNA reactor through the reactor outlet; (b) a first in-line mixing device in fluid communication with the reactor outlet, wherein the first in-line mixing device is configured such that a protease contacts the RNA molecule; (c) a second in-line mixing device in fluid communication with the reactor outlet, wherein the second in-line mixing device is configured such that a chelating agent contacts the RNA molecule; (d) a hold-up volume in fluid communication with the second in-line mixing device, wherein the hold-up volume comprises a loop of tubing, a loop of piping, or a vessel and wherein the hold-up volume is configured such that the RNA molecule has a desired residence time; (e) a third in-line mixing device in fluid communication with the hold-up volume, wherein the third in-line mixing device is configured such that a single-pass tangential flow filtration (SPTFF) load buffer contacts the RNA molecule; and (f) a single-pass tangential flow filtration (SPTFF) device comprising a membrane inlet and a membrane outlet, wherein the membrane inlet is in fluid communication with the third in-line mixing device, and a drug substance exits the SPTFF device through the membrane outlet. In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, disclosed herein are continuous-flow RNA drug product manufacturing systems, wherein the systems comprise: (a) an immobilized DNA reactor comprising a reactor inlet and a reactor outlet, wherein the immobilized DNA reactor is configured such that an in vitro transcription reaction system is passed into the reactor inlet and an RNA molecule exits the DNA reactor through the reactor outlet; (b) a first in-line mixing device in fluid communication with the reactor outlet, wherein the first in-line mixing device is configured such that a protease contacts the RNA molecule; (c) a second in-line mixing device in fluid communication with the reactor outlet, wherein the second in-line mixing device is configured such that a chelating agent contacts the RNA molecule; (d) a hold-up volume in fluid communication with the second in-line mixing device, wherein the hold-up volume comprises a loop of tubing, a loop of piping, or a vessel and wherein the hold-up volume is configured such that the RNA molecule has a desired residence time; (e) a third in-line mixing device in fluid communication with the hold-up volume, wherein the third in-line mixing device is configured such that a single-pass tangential flow filtration (SPTFF) load buffer contacts the RNA molecule; (f) a single-pass tangential flow filtration (SPTFF) device comprising a membrane inlet and a membrane outlet, wherein the membrane inlet is in fluid communication with the third in-line mixing device, and a drug substance exits the SPTFF device through the membrane outlet; (g) a pump in fluid communication with the membrane outlet for contacting the drug substance with an aqueous solution via an in-line mixing device to yield an aqueous phase; (h) an in-line dispersion formation mixer in fluid communication with the aqueous phase for concurrent mixing of the aqueous phase and an organic phase to produce an RNA-loaded lipid nanoparticle (LNP) dispersion; (i) a pump in fluid communication with the RNA-loaded LNP dispersion to deliver a diluent to the RNA-loaded LNP dispersion and produce a quenched LNP dispersion; (j) an in-line mixing device in fluid communication with the quenched LNP dispersion and a diluent to dilute the quenched LNP dispersion and produce a diluted quenched LNP dispersion; (k) a single-pass tangential flow filtration (SPTFF) device in fluid communication with the diluted quenched LNP dispersion to produce a re-concentrated LNP dispersion; (l) a pump in fluid communication with the re-concentrated LNP dispersion to deliver a diluent to the re-concentrated LNP dispersion and produce a diluted LNP dispersion; (m) a SPTFF device in fluid communication with the diluted LNP dispersion to produce a purified LNP dispersion; (n) a pump in fluid communication with the purified LNP dispersion to transfer the purified LNP dispersion through a filtration device to produce an RNA drug product. In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the RNA molecule is a vaccine RNA molecule. In some embodiments, the RNA molecule is a therapeutic RNA molecule. In some embodiments, contacting the drug substance with an aqueous solution further comprises an in-line UV absorbance sensor. In some embodiments, the methods and systems disclosed herein comprise immobilized DNA reactors, wherein the immobilized DNA reactors comprise in-line sensors selected from the group consisting of an in-line pH sensor, an in-line UV absorbance sensor, an in-line conductivity sensor, and an in-line pressure sensor. In some embodiments, the methods and systems disclosed herein comprise single-pass tangential flow filtration (SPTFF) devices, wherein the SPTFF devices comprise in-line sensors selected from the group consisting of a feed pressure sensor, a permeate pressure sensor, and a retentate pressure sensor. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule comprising: (a) obtaining a composition comprising: a circular double-stranded DNA (dsDNA) template, a primer, a deoxyribonucleotide triphosphate (dNTP), an aminoallyl-dNTP, and a DNA polymerase; (b) incubating the composition for a time period between about three and twenty-four hours at a temperature between about 30 and 45 °C to obtain an incubated composition; (c) contacting the incubated composition with a restriction endonuclease to obtain a digested composition; (d) purifying the digested composition to obtain a linearized dsDNA fragment; (e) immobilizing the linearized dsDNA fragment within a substrate to obtain an immobilized DNA reactor; and (f) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain the RNA molecule, thereby continuously producing the RNA molecule. In some embodiments, the deoxyribonucleotide triphosphate comprises dATP, dCTP, dGTP, and dTTP, and the aminoallyl-dNTP comprises aminoallyl-dUTP. In some embodiments, the ratio of aminoallyl-dUTP to dTTP is 1:100. In some embodiments, the DNA polymerase is a phi29 DNA polymerase. In some embodiments, contacting the incubated composition with a restriction endonuclease occurs for a time period between about ten and twenty-four hours at a temperature between about 30 and 45 °C. In some embodiments, purifying the digested composition to obtain a linearized dsDNA fragment comprises isopropanol precipitation. In some embodiments, the substrate comprises an N-hydroxysuccinimide-activated resin-packed column. In some embodiments, the in vitro transcription reaction system comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNAse inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a flow rate of between 0.01 column volumes (CV) per minute and 0.1 CV per minute at a temperature between about 20 and 40 °C. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a time period between 10 minutes and 1,200 minutes. In some embodiments, following the step of flowing the in vitro transcription reaction system across the immobilized DNA reactor, the immobilized DNA reactor remains functional for at least 14, 20, 42, or 64 days. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule comprising: (a) obtaining a composition comprising: a circular double-stranded DNA (dsDNA) template, a primer, a deoxyribonucleotide triphosphate (dNTP), an aminoallyl-dNTP, and a DNA polymerase; (b) incubating the composition for a time period between about three and twenty- four hours at a temperature between about 30 and 45 °C to obtain an incubated composition; (c) contacting the incubated composition with a restriction endonuclease to obtain a digested composition; (d) purifying the digested composition to obtain a linearized dsDNA fragment; (e) immobilizing the linearized dsDNA fragment within a substrate to obtain an immobilized DNA reactor; (f) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain the RNA molecule; (g) contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule; (h) adjusting the purified RNA molecule with an aqueous solution to obtain an adjusted RNA solution; (i) mixing the adjusted RNA solution with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (j) mixing the RNA-loaded LNP dispersion with a diluent to obtain a diluted RNA-loaded LNP dispersion; and (k) filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device. In some embodiments, the continuous in-line purification device is an oligo(dT) column. In some embodiments, the oligo(dT) column is a resin-packed column or a monolith column. In some embodiments, the continuous in-line purification device is a single-pass tangential flow filtration (SPTFF) device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device further comprises contacting the RNA molecule with an acidifying solution. In some embodiments, the aqueous solution is an acidifying solution. In some embodiments, the diluent is an acidifying solution. In some embodiments, the acidifying solution is a low pH citrate solution. In some embodiments, adjusting the purified RNA molecule with an aqueous solution comprises passing the purified RNA molecule through a device selected from the group consisting of a continuous in-line filtration device, an in-line mixer, and a surge vessel. In some embodiments, the lipid is selected from the group consisting of a cationic lipid, a phospholipid, a sterol, and a polymer-conjugated lipid. In some embodiments, mixing the RNA-loaded LNP dispersion with a diluent comprises passing the RNA-loaded LNP dispersion through an in-line mixer. In some embodiments, the continuous in-line filtration device is selected from the group consisting of a single-pass tangential flow filtration (SPTFF) device, a bioburden reduction filtration device, and a sterile filtration device. In some embodiments, disclosed herein are methods for continuous production of an RNA drug product comprising: (a) immobilizing a polymerase within a substrate to obtain an immobilized polymerase reactor; (b) incubating the immobilized polymerase reactor at a temperature between 20 °C and 40 °C; (c) flowing an in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor to obtain an RNA molecule; (d) contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule; (e) adjusting the purified RNA molecule with an aqueous solution to obtain an adjusted RNA solution; (f) contacting the adjusted RNA solution with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (g) mixing the RNA-loaded LNP dispersion with a diluent to obtain a diluted RNA-loaded LNP dispersion; and (h) filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device; thereby continuously producing the RNA drug product. In some embodiments, the substrate is selected from the group consisting of a N-hydroxysuccinimide-activated resin-packed column and a carbonylimidazole- functionalized monolithic column. In some embodiments, immobilizing the polymerase comprises flowing a solution comprising the polymerase across the substrate at a fast flow rate of at least 0.05 CV / min followed by a slow flow rate of at least 0.005 CV / min. In some embodiments, flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor comprises a time period between 10 minutes and 36 h. In some embodiments, following the step of flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor, the immobilized polymerase reactor remains functional for at least 14, 20, 42, or 64 days. In some embodiments, flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor comprises a flow rate of between 0.001 CV per minute and 0.1 CV per minute at a temperature between about 20 and 40 °C. In some embodiments, the in vitro transcription reaction system comprising a DNA template further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNAse inhibitor, and a pyrophosphatase. In some embodiments, the continuous in-line purification device is an oligo(dT) column. In some embodiments, the oligo(dT) column is a resin-packed column or a monolith column. In some embodiments, the continuous in-line purification device is a single-pass tangential flow filtration (SPTFF) device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device further comprises contacting the RNA molecule with an acidifying solution. In some embodiments, the aqueous solution is an acidifying solution. In some embodiments, the diluent is an acidifying solution. In some embodiments, the acidifying solution is a low pH citrate solution. In some embodiments, adjusting the purified RNA molecule with an aqueous solution comprises passing the purified RNA molecule through a device selected from the group consisting of a continuous in-line filtration device, an in-line mixer, and a surge vessel. In some embodiments, the lipid is selected from the group consisting of a cationic lipid, a phospholipid, a sterol, and a polymer-conjugated lipid. In some embodiments, mixing the RNA-loaded LNP dispersion with a diluent comprises passing the RNA-loaded LNP dispersion through an in-line mixer. In some embodiments, the continuous in-line filtration device is selected from the group consisting of a single-pass tangential flow filtration (SPTFF) device, a bioburden reduction filtration device, and a sterile filtration device. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule comprising: (a) obtaining a composition comprising: a linear double-stranded DNA (dsDNA) template comprising a 5’ overhang, a DNA polymerase, and an aminoallyl-dNTP; (b) incubating the composition for a time period between about twelve and twenty-four hours at a temperature between about 20 °C and 40 °C to obtain a labeled dsDNA template; (c) immobilizing the labeled dsDNA template within a substrate to obtain an immobilized DNA reactor; and (d) flowing an in vitro transcription reaction system across the immobilized DNA reactor, thereby continuously producing the RNA molecule. In some embodiments, the 5’ overhang comprises a 5’ adenine overhang. In some embodiments, the DNA polymerase is selected from the group consisting of a Klenow fragment DNA polymerase and a Phi29 DNA polymerase. In some embodiments, the aminoallyl-dNTP comprises aminoallyl-dUTP. In some embodiments, the 5’ adenine overhang is upstream of a T7 promoter site on the linear dsDNA template and the linear dsDNA template further comprises a 5’ thymidine overhang downstream of the T7 promoter site. In some embodiments, the substrate comprises an N-hydroxysuccinimide-activated resin-packed column. In some embodiments, the substrate comprises a carbonylimidazole-functionalized monolithic column. In some embodiments, the in vitro transcription reaction system comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a flow rate of between 0.01 CV per minute and 0.5 CV per minute at a temperature between about 20 and 40 °C. In some embodiments, flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a time period between 2 minutes and 60 hours. In some embodiments, following the step of flowing the in vitro transcription reaction system across the immobilized DNA reactor, the immobilized DNA reactor remains functional for at least 14, 20, 42, or 64 days. In some embodiments, the RNA polymerase is at a concentration between about 5,000 U / mL and about 30,000 U / mL. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule comprising: (a) obtaining a composition comprising: a linear double-stranded DNA (dsDNA) template comprising a 5’ overhang, a DNA polymerase, and an aminoallyl-dNTP; (b) incubating the composition for a time period between about twelve and twenty-four hours at a temperature between about 20 °C and 40 °C to obtain a labeled dsDNA template; (c) immobilizing the labeled dsDNA template within a substrate to obtain an immobilized DNA reactor; (d) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain the RNA molecule; (e) contacting the RNA molecule with a continuous in-line purification device to obtain a purified RNA molecule; (f) adjusting the purified RNA molecule with an aqueous solution to obtain an adjusted RNA solution; (g) mixing the adjusted RNA solution with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (h) mixing the RNA-loaded LNP dispersion with a diluent to obtain a diluted RNA-loaded LNP dispersion; and (i) filtering the diluted RNA-loaded LNP dispersion via a continuous in-line filtration device. In some embodiments, the continuous in-line purification device is an oligo(dT) column. In some embodiments, the oligo(dT) column is a resin-packed column or a monolith column. In some embodiments, the continuous in-line purification device is a single-pass tangential flow filtration (SPTFF) device. In some embodiments, contacting the RNA molecule with a continuous in-line purification device further comprises contacting the RNA molecule with an acidifying solution. In some embodiments, the aqueous solution is an acidifying solution. In some embodiments, the diluent is an acidifying solution. In some embodiments, the acidifying solution is a low pH citrate buffer. In some embodiments, adjusting the purified RNA molecule with an aqueous solution comprises passing the purified RNA molecule through a device selected from the group consisting of a continuous in-line filtration device, an in-line mixer, and a surge vessel. In some embodiments, the lipid is selected from the group consisting of a cationic lipid, a phospholipid, a sterol, and a polymer-conjugated lipid. In some embodiments, mixing the RNA-loaded LNP dispersion with a diluent comprises passing the RNA-loaded LNP dispersion through an in-line mixer. In some embodiments, the continuous in-line filtration device is selected from the group consisting of a single-pass tangential flow filtration (SPTFF) device, a bioburden reduction filtration device, and a sterile filtration device. In some embodiments, disclosed herein are methods for continuous production of an RNA molecule comprising: (a) transforming a bacterial cell with a vector encoding a polymerase comprising a histidine tag; (b) incubating the bacterial cell at a temperature between 34 °C and 40 °C for at least 12 hours; (c) lysing the bacterial cell to obtain a solution comprising the polymerase; (d) flowing the solution comprising the polymerase across a nickel-nitrilotriacetic acid (Ni-NTA)-functionalized resin-packed column to obtain an immobilized polymerase reactor; (e) incubating the immobilized polymerase reactor at a temperature between 20 °C and 40 °C; and (f) flowing an in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor to obtain an RNA molecule, thereby continuously producing the RNA molecule. In some embodiments, the bacterial cell comprises E. coli. In some embodiments, the in vitro transcription reaction system comprising a DNA template further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNAse inhibitor, and a pyrophosphatase. In some embodiments, flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor comprises a flow rate of between 0.05 CV per minute and 0.1 CV per minute at a temperature between about 20 and 40 °C. In some embodiments, flowing the in vitro transcription reaction system comprising a DNA template across the immobilized polymerase reactor comprises a time period between 10 minutes and 120 minutes. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 depicts a Coomassie-stained SDS PAGE gel showing harvest from 18 hours TB culture of E. coli BL21 expressing histidine-tagged T7 RNA polymerase (T7 RNAP). FIG.2 depicts a Coomassie-stained SDS PAGE gel showing purified histidine-tagged T7 RNA polymerase after elution from Ni-NTA-functionalized resin-packed columns. FIG. 3 depicts reaction kinetics for continuous-flow IVT from packed-bed reactors containing covalently-immobilized T7 RNAP. mRNA concentration was obtained from the same reactor on different days as indicated on the right side. FIG.4 depicts the effect of reactor age on mRNA percent integrity for continuous-flow IVT from packed-bed reactors containing covalently-immobilized T7 RNAP. mRNA percent integrity is shown for both batch IVT and continuous-flow IVT. Days on the x-axis represent the day on which continuous-flow IVT product was obtained. FIG.5 depicts mRNA productivity from a packed-bed reactor containing unlabeled linear DNA operated at 0.05 CV / min flow rate (20 minute residence time) over the course of 12 days of operation. FIG.6 depicts mRNA productivity from a packed-bed reactor containing unlabeled linear DNA operated at 0.1 CV / min flow rate (10 min residence time) over the course of 12 days of operation. FIG. 7 depicts mRNA yield vs sample time for continuous-flow IVT from packed-bed reactors containing covalently-immobilized DNA that was randomly amine-tagged via RCA. FIG.8 depicts flow rate effects on productivity and product quality for continuous-flow IVT from packed-bed reactors containing covalently immobilized DNA that was randomly amine-tagged via RCA. FIG.9 depicts residual NTP measurements for continuous-flow IVT from a packed-bed reactor containing covalently immobilized DNA that was randomly amine tagged via RCA. FIG.10 depicts residual DNA measurements for continuous-flow IVT from a packed-bed reactor containing covalently immobilized DNA that was randomly amine tagged via RCA. FIG. 11 depicts a schematic for Klenow polymerase-mediated site-specific fill-in of 5’-AAA overhangs with aminoallyl-dUTP upstream of the T7 promoter on a BspQI digested mRNA therapeutic DNA template. FIG. 12 depicts mRNA productivity, 5’-capping, and percent integrity measurements for continuous-flow IVT from a packed-bed reactor containing covalently immobilized DNA that was site-specifically amine tagged via Klenow polymerase. FIG.13 depicts double-stranded RNA and residual DNA measurements for continuous-flow IVT from a packed-bed reactor containing covalently immobilized DNA that was site-specifically amine tagged via Klenow polymerase. FIG.14 depicts residual NTP measurements for continuous-flow IVT from a packed-bed reactor containing covalently immobilized DNA that was site-specifically amine tagged via Klenow polymerase. FIG.15 depicts mRNA productivity from a packed-bed reactor containing covalently immobilized DNA that was site-specifically amine tagged via Klenow polymerase at 0.05 CV / min flow rate (20 min residence time) over the course of 12 days of operation. FIG.16 depicts mRNA productivity from a packed-bed reactor containing covalently immobilized DNA that was site-specifically amine tagged via Klenow polymerase at 0.1 CV / min flow rate (10 min residence time) over the course of 12 days of operation. FIG.17 depicts mRNA productivity from a packed-bed reactor containing covalently immobilized DNA that was site-specifically amine tagged via Klenow polymerase at 0.1 CV / min flow rate (10 min residence time) at standard, half, and double T7 RNAP concentrations (13,636 U / mL, 6,818 U / mL, and 27,272 U / mL, respectively). FIG.18 depicts the breakthrough percent, relative to the load concentration, plotted vs. the resin load for oligo(dT) monolith. The dynamic binding capacity, breakthrough at 10% is 2.6 mg / mL of resin. FIG.19 depicts particle size and size distribution for samples of RNA-loaded LNP dispersions for batch-based (2 mL / min, 5 mL / min, and 8 mL / min) and continuous-flow (12 mL / min) IVT. FIG.20 depicts reactor productivity, mRNA integrity, and residual DNA (resDNA), from a 10mL packed-bed reactor containing covalently immobilized DNA (CF-IVT reactor). Reactor productivity was measured over the course of CF-IVT operation at 0.04 CV / min (25 min residence time) on Day 0, 1, 14, 26, 28, and 42 of reactor life. FIG.21 depicts stagnating conversion and retentate RNA concentration observed with increasing TMP for SPTFF of CF-IVT “neat” product with no treatments. FIG.22 depicts stagnating conversion and retentate RNA concentration observed with increasing TMP for SPTFF of CF-IVT product after ProK only treatment. FIG.23 depicts increased conversion and retentate RNA concentration with increasing TMP for SPTFF of CF-IVT product after EDTA-only treatment. FIG.24 depicts increased conversion and retentate RNA concentration with increasing TMP for SPTFF of CF-IVT product after both ProK and EDTA treatment. FIG.25 depicts a schematic of integrated CF-IVT and SPTFF with in-line ProK treatment. IVT reaction system is flowed through a 10mL reactor at 0.04 CV / min (25 min residence time), producing an RNA yield of approximately 8 mg / mL. CF-IVT product is then mixed in-line through a static mixing tee (IDEX) with flow of 30 U / mL ProK (THERMO FISHER) stock solution in 1x TE buffer pH 8.0 (THERMO FISHER) to yield a final ProK concentration of 5 U / mL. The mix then proceeds through two 150 µL static in-line mixers (RESTEK) into a 15 mL hold-up volume loop of 1.0 mm i.d. PEEK tubing (CYTIVA) incubated at 42 °C to provide an average residence time of approximately 30 min. The ProK treated CF-IVT product is then diluted in-line through a static mixing tee (IDEX) with SPTFF load buffer (10 mM HEPES, 0.1 mM EDTA, pH 7.0) to provide a total of approximately 20-fold dilution of the CF-IVT product prior to passing through a 50 cm2, 300 kDa MWCO regenerated cellulose TFF membrane (SARTORIUS) to re-concentrate the purified RNA in the SPTFF retentate. FIG.26 depicts retentate RNA concentration and permeate conversion for integrated CF-IVT and SPTFF with in-line ProK treatment and pressure excursion up to a target TMP of 8 psi. FIG.27 depicts retentate RNA concentration and permeate conversion for integrated CF-IVT and SPTFF with in-line ProK treatment and pressure excursion up to a target TMP of 30 psi. FIG.28 depicts a schematic of integrated CF-IVT and SPTFF with in-line EDTA treatment. IVT reaction system is flowed through a 10mL reactor at 0.04 CV / min (25 min residence time), producing an RNA yield of approximately 8 mg / mL. CF-IVT product is then mixed in-line through a static mixing tee (IDEX) with flow of 500 mM EDTA pH 8 stock solution (INVITROGEN) to yield a final EDTA concentration of approximately 75 mM. The mix then proceeds through two 150 µL static in-line mixers (RESTEK) into a 15 mL hold-up volume loop of 1.0 mm i.d. PEEK tubing (CYTIVA) incubated at 42 °C to provide an average residence time of approximately 30 min. The EDTA treated CF-IVT product is then diluted in-line through a static mixing tee (IDEX) with SPTFF load buffer (10 mM HEPES, 0.1 mM EDTA, pH 7.0) to provide a total of approximately 20-fold dilution of the CF-IVT product prior to passing through a 50 cm2, 300 kDa MWCO regenerated cellulose TFF membrane (SARTORIUS) to re-concentrate the purified RNA in the SPTFF retentate. FIG.29 depicts retentate RNA concentration and permeate conversion for integrated CF-IVT and SPTFF with in-line EDTA treatment and pressure excursion up to a target TMP of 8 psi. FIG. 30 depicts a schematic of integrated CF-IVT and SPTFF with in-line ProK and EDTA treatment. IVT reaction system is flowed through a 10mL reactor at 0.04 CV / min (25 min residence time), producing an RNA yield of approximately 8 mg / mL. CF-IVT product is then mixed in-line through a static mixing tee (IDEX) with flow of 25 U / mL ProK (THERMO FISHER) stock solution in 1x TE buffer pH 8.0 (THERMO FISHER) to yield a final ProK concentration of approximately 0.5 U / mL. This mix is then mixed in-line through another static mixing tee (IDEX) with flow of 500 mM EDTA pH 8 stock solution (INVITROGEN) to yield a final EDTA concentration of approximately 75 mM. The mix then proceeds through two 150 µL static in-line mixers (RESTEK) into a 15 mL hold-up volume loop of 1.0 mm i.d. PEEK tubing (CYTIVA) incubated at 42 °C to provide an average residence time of approximately 30 min. The ProK and EDTA treated CF-IVT product is then diluted in-line through a static mixing tee (IDEX) with SPTFF load buffer (10 mM HEPES, 0.1 mM EDTA, pH 7.0) to provide a total of approximately 20-fold dilution of the CF-IVT product prior to passing through a 50 cm2, 300 kDa MWCO regenerated cellulose TFF membrane (SARTORIUS) to re-concentrate the purified RNA in the SPTFF retentate. FIG.31 depicts retentate RNA concentration and permeate conversion for integrated CF-IVT and SPTFF with in-line ProK and EDTA treatment with pressure excursion up to a target TMP of 8 psi. FIG. 32 depicts schematically an integrated, end-to-end, continuous-flow RNA drug product manufacturing skid . “2x” and dashed brackets indicate 2 sequential repetitions each of the DS- SPTFF and DP-SPTFF operations within the brackets. DS indicates drug substance. “APV” indicates an aqueous phase vessel. “AP” indicates aqueous phase. “OP” indicates organic phase. “CCV” indicates a continuous cure vessel. “DP” indicates drug product. “BPS” indicates a booster pump supply. “PLV” indicates a purified LNP vessel. “DPV” indicates a final drug product vessel. FIG.33 depicts schematically an exemplary integrated, end-to-end, continuous-flow RNA drug product manufacturing skid. “nx” and dashed brackets indicate (n) number of serial repetitions each of the DS-SPTFF and DP-SPTFF operations within the brackets to reach a desired reduction of impurities in the RNA drug substance or RNA-loaded LNP dispersion, respectively. “DS” indicates drug substance. “APV” indicates an aqueous phase vessel. “AP” indicates aqueous phase. “OP” indicates organic phase. “CCV” indicates a continuous cure vessel. “DP” indicates drug product. “BPS” indicates a booster pump supply. “PLV” indicates a purified LNP vessel. “DPV” indicates a final drug product vessel. FIG.34 depicts pH and conductivity of the CF-IVT product exiting the CF-IVT reactor as a function of time of day during the course of continuous operation of the end-to-end, integrated RNA drug product manufacturing skid. FIG. 35 depicts total transmembrane pressure (TMP) of the two DS-SPTFF membranes and aqueous phase (AP) RNA concentration as a function of time of day during the course of continuous operation of the end-to-end, integrated RNA drug product manufacturing skid as a function of time. The dashed line indicates target aqueous phase RNA concentration. FIG.36 depicts transmembrane pressures (TMP) for each of the three DP-SPTFF membranes as a function of time of day during the course of continuous operation of the end-to-end, integrated RNA drug product manufacturing skid. FIG.37 depicts the relationship between CF-IVT product pH and total volume flowed through the CF-IVT reactor at several different flow rates indicated in column volumes (CV) per minute. FIG.38 depicts the correlation between ΔpH and CF-IVT product RNA concentration with linear fit equation. FIG. 39 depicts SPTFF conversion and retentate RNA concentration as a function of transmembrane pressure (TMP) during RNA purification from EDTA-treated CF-IVT product in 10 mM HEPES buffer using a 1000 kDa MWCO membrane. FIG. 40 depicts SPTFF conversion and retentate RNA concentration as a function of transmembrane pressure (TMP) during RNA purification from EDTA-treated CF-IVT product in 10 mM Tris, 250 mM KCl buffer using a 1000 kDa MWCO membrane. DETAILED DESCRIPTION RNA therapeutics and vaccines have tremendous potential to treat and prevent diseases. To achieve that potential, high-quality RNA for vaccine or therapeutic purposes is needed. In vitro transcription (IVT) can be carried out to synthesize RNA from a DNA template using a variety of natural or engineered RNA polymerases including SP6, T7 or T3 RNA polymerases. Traditionally, the RNA molecules are made using batch in vitro transcription where the reaction components are combined and then incubated for a few hours. Another approach to produce RNA molecules is by continuous-flow in vitro transcription (CF-IVT). CF-IVT can be achieved by contacting a constantly or near constantly flowing solution of reactants (in vitro transcription reaction system) with stationary non-consumables / catalysts (e.g., RNA polymerase, DNA template, or combination of both). The non-consumables can be made stationary through covalent immobilization, adsorption, entrapment, or co-polymerization, on or within a substrate (e.g., reactors, columns, membranes, microfluidic channels, resins, particles, and gels). Additionally, CF-IVT can be followed by continuous purification of the in vitro transcribed RNA via chromatographic or filtration methods (e.g., oligo(dT), hydroxyapatite, phenyl boronate, ion exchange, hydrophobic interaction, tangential flow filtration, single-pass tangential flow filtration) to obtain RNA drug substance. Components of the IVT reaction system (e.g., NTPs, 5’ cap analog, RNA polymerase, template DNA), having been separated from the in vitro transcribed RNA molecules through the above-mentioned continuous purification methods, can then be recovered and returned to the CF-IVT reaction if desired. Continuous purification of the RNA to obtain RNA drug substance can then be followed by continuous encapsulation of the drug substance RNA within an encapsulating agent (e.g., lipid nanoparticles [LNPs]) by concurrent mixing of the RNA drug substance with a solution of encapsulating agent through a mixing device (e.g., co-axial, impinging jet, or microfluidic bifurcating mixing devices) to form an RNA-loaded LNP dispersion. The RNA-loaded LNP dispersion can then be continuously adjusted, purified, and processed via in-line mixing and filtration (e.g., single-pass tangential flow filtration, bioburden reduction filtration, and sterile filtration) to form the RNA drug product. The entire process of CF-IVT, RNA purification, and encapsulation of the drug substance RNA within LNPs to form RNA drug product can be unified into an integrated, end-to-end (E2E) skid for continuous- flow conversion of raw material reactants into RNA drug product with overall greatly improved productivity, speed, throughput, quality, footprint, cost, and ability to meet a broad range of dose demand. The resulting integrated, modular, and continuous manufacturing process for on- demand manufacturing of RNA therapeutics from DNA to drug product is termed “mSKID.” Disclosed herein are continuous-flow methods for the production, purification, and formulation of RNA molecules (e.g., mRNA, including modified mRNA molecules, self-amplifying RNA (saRNA) molecules, guide RNA (gRNA) molecules, and / or circular-RNA (circRNA) molecules) that are useful for producing clinical grade RNA, such as mRNA, of high purity and potency, consistently, reproducibly, and in compliance with current good manufacturing practices (cGMP). The methods use a DNA template in a CF-IVT reaction with immobilized non-consumables (e.g., DNA template, enzyme, or both) to continuously generate the RNA molecule and can be applied to a wide variety of constructs with varying 5’ UTRS, coding sequence lengths, 3’ UTRs, and 3’ ends. In some aspects, the in vitro transcribed RNA molecule is continuously purified from the CF-IVT product to form RNA drug substance via chromatographic methods (e.g., through the use of an adsorbent resin or monolith such as oligo(dT), hydroxyapatite, phenyl boronate, ion exchange, or hydrophobic interaction) or filtration methods (e.g., through tangential flow filtration or single-pass tangential flow filtration). In some aspects, components of the IVT reaction system (e.g., NTPs, 5’ cap analog, RNA polymerase, template DNA), having been separated from the in vitro transcribed RNA molecules through the above-mentioned continuous purification methods, are then recovered and returned to the CF-IVT reaction. In some aspects, the purified RNA is subsequently continuously encapsulated within an encapsulating agent (e.g., LNPs) by continuous concurrent mixing of the RNA drug substance with a solution of encapsulating agent through a mixing device (e.g., co-axial, impinging jet, or microfluidic bifurcating mixing devices) to form an RNA-loaded LNP dispersion. The RNA-loaded LNP dispersion can then be continuously adjusted, purified, and processed via in-line mixing and filtration (e.g., single-pass tangential flow filtration, bioburden reduction filtration, and sterile filtration) to form the RNA drug product. In some aspects, the entire process of CF-IVT, RNA purification, and encapsulation of the drug substance RNA within LNPs to form RNA drug product is unified into an integrated, end- to-end (E2E) skid for continuous-flow conversion of raw material reactants into RNA drug product with overall greatly improved productivity, speed, throughput, quality, footprint, cost, and ability to meet a broad range of dose demand. The resulting integrated, modular, and continuous manufacturing process for on-demand manufacturing of RNA therapeutics from DNA to drug product is termed “mSKID.” Also disclosed herein are methods for various enzyme catalyzed reactions for the tagging of linear template DNA with reactive chemistries to facilitate subsequent immobilization, adsorption, entrapment, or co-polymerization, on or within a substrate (e.g., reactors, columns, membranes, microfluidic channels, resins, particles, and gels) for use in continuous-flow IVT reactions for production of RNA molecules, e.g., mRNA, including modified mRNA molecules, unmodified mRNA molecules, and / or saRNA, that are useful for producing clinical-grade RNA, such as mRNA, of high purity and potency, consistently, reproducibly, and in compliance with current good manufacturing practices (cGMP). Also disclosed herein are methods for various “one pot” reactions for the production of linear template DNA molecules labeled with reactive chemistries to allow subsequent immobilization, adsorption, entrapment, or co-polymerization, on or within substrate (e.g., reactors, columns, membranes, microfluidic channels, resins, particles, and gels) for use in continuous-flow IVT reactions for production of RNA molecules, e.g., mRNA, including modified mRNA molecules, unmodified mRNA molecules, and / or saRNA, that are useful for producing clinical-grade RNA, such as mRNA, of high purity and potency, consistently, reproducibly, and in compliance with current good manufacturing practices (cGMP). One or more steps of the methods (e.g., amplification, linearization, and / or labeling) are performed in the same reaction vessel without intermediate nucleic acid purification processes between the step(s). In some aspects, all steps of the method (e.g., amplification, linearization, and / or labeling) are performed in the same reaction vessel without intermediate nucleic acid purification processes between the steps. Certain Definitions Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the inherent variation or standard deviation of error for the measurement or quantitation method being employed to determine the value. For example, in some aspects, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the measurement or quantitation. The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or. The phrase “essentially all” is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some instances, essentially all means equal to any one of, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100 % of members of the group have that property. The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Throughout this specification, unless the context requires otherwise, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. It is contemplated that aspects described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of” or “consisting essentially of.” Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure. The words “consisting of” (and any form of consisting of, such as “consist of” and “consists of”) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” “a further embodiment,” “some embodiments”, “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” “a further aspect,” “some aspects” or combinations thereof means that a particular feature, structure or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects. The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. The terms “improve,” “promote,” or “increase” or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule. As used herein, the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and / or determined substantially simultaneously and / or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and / or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment. The term “DNA,” as used herein, means a nucleic acid molecule that includes deoxyribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or thymine (T)). For example, DNA can contain all, or a majority of, deoxyribonucleotide residues. As used herein, the term “deoxyribonucleotide” means a nucleotide lacking a hydroxyl group at the 2′ position of a β-D-ribofuranosyl group. Without any limitation, DNA can encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA. The term “RNA,” as used herein, means a nucleic acid molecule that includes ribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or uracil (U) or N-1-methylpseudouridine). For example, RNA can contain all, or a majority of, ribonucleotide residues. As used herein, the term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranosyl group. In one aspect, RNA can be messenger RNA (mRNA) that relates to an RNA transcript which encodes a peptide or protein. As known to those of skill in the art, mRNA generally contains a 5′ untranslated region (5′-UTR), a polypeptide coding region, and a 3′ untranslated region (3′-UTR). Without any limitation, RNA can encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, circular RNA, self-amplifying RNA (saRNA), guide RNA (gRNA), and modified RNA (modRNA). The terms “continuous” or “continuous-flow,” as used herein, are meant to describe any operation or process that generates a desired product without pauses or interruptions between batches and wherein the equipment of the operation or process (e.g., reactors, columns, filters, mixers) are integrated within an assembly line or skid, with reactants or materials constantly or near constantly passing through the operation or process to constantly or near constantly generate a desired product. The term “substrate," as used herein, means any form of device, material, matrix, or surface within which DNA, enzyme, or both may be covalently immobilized, adsorbed, entrapped, or co- polymerized. Examples of substrate included, but are not limited to reactors, columns, membranes, microfluidic channels, resins, particles, and gels. The term “in vitro transcription reaction system” (IVT reaction system) or “continuous-flow in vitro transcription reaction system” (IVT reaction system), are used interchangeably herein to refer to any mixture of reactants, buffers, salts, or other chemical components enabling or facilitating transcription of DNA into RNA. The terms label, labeling, labeled, tag, tagging, or tagged, are used interchangeably herein to refer to the intentional addition or presence of certain reactive chemistries or functional groups (e.g., carboxyls, sulfhydryls, aldehydes, hydroxyls, azides, epoxides, or biotin) into or within the structure of certain non-consumable components of an in vitro transcription reaction system (e.g., enzymes, DNA, or both), in order to facilitate covalent immobilization, adsorption, entrapment, or co-polymerization of the non-consumables on or within a substrate. The term “continuous-flow in vitro transcription product” or “CF-IVT product,” as used herein, means the mixture of in vitro transcription reaction system and in vitro transcribed RNA exiting the continuous-flow in vitro transcription reactor following continuous-flow in vitro transcription. The terms “RNA drug substance,” or “drug substance,” as used herein, mean a purified RNA that is solubilized in any form of aqueous solution appropriate to permit subsequent encapsulation of the RNA within encapsulating agents as described below. The term “aqueous phase,” as used herein, means the RNA drug substance that has been further adjusted to a desired RNA concentration and pH to facilitate subsequent encapsulation of the RNA within encapsulating agents as described below. The term “RNA drug substance” and “aqueous phase” are in some cases herein used interchangeably. The term “drug substance RNA,” as used herein, means the RNA component of the RNA drug substance. The terms “RNA drug product,” “RNA-loaded LNP drug product,” “drug product,” or “DP,” are used interchangeably herein to refer to a purified RNA that has been encapsulated in any form of encapsulating agents (e.g., lipid nanoparticles) described herein and forms a colloidal dispersion (e.g., RNA-loaded LNP dispersion) and where the resulting colloidal dispersion has been adjusted and purified to stabilize the encapsulated RNA. As contemplated herein, without any limitations, RNA can be used as a therapeutic modality to treat and / or prevent a number of conditions in mammals, including humans. Methods contemplated comprise administration of the RNA described herein to a mammal, such as a human. For example, in one aspect, such methods of use for RNA include an antigen-coding RNA vaccine to induce robust neutralizing antibodies and accompanying / concomitant T-cell response to achieve protective immunization with preferably minimal vaccine doses. The RNA administered is preferably in vitro transcribed RNA. An “isolated RNA” is defined as an RNA molecule that can be recombinant or has been isolated from total genomic nucleic acid. A “modified RNA” or “modRNA” refers to an RNA molecule, e.g., an mRNA molecule, having at least one addition, deletion, substitution, and / or alteration of one or more nucleotides as compared to naturally occurring RNA. Such alterations can refer to the addition of non-nucleotide material to internal RNA nucleotides, or to the 5′ and / or 3′ end(s) of RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as an alteration to the base of the nucleotide. For example, a modified nucleotide can replace one or more uridine and / or cytidine nucleotides. For example, these replacements can occur for every instance of uridine and / or cytidine in the RNA sequence, or can occur for only select uridine and / or cytidine nucleotides. Such alterations to the standard nucleotides in RNA can include non- standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide can be replaced with 1-methylpseudouridine in an RNA sequence. Other such altered nucleotides are known to those of skill in the art. Such altered RNAs are considered analogs of naturally-occurring RNA. In some aspects, the RNA is produced by continuous-flow in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides. In some aspects, the RNA can be replicon RNA (replicon), in particular self-replicating RNA, or self-amplifying RNA (saRNA). In some aspects, the RNA can be guide RNA (gRNA) or circular RNA. As used herein, a “protein,” “polypeptide,” or “peptide” refers to a molecule comprising at least two amino acid residues. As used herein, the term “wild-type” or “native” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, produced by solid-phase peptide synthesis (SPPS), or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antigen or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule. The term “isolated” can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (e.g., when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (e.g., when chemically synthesized). Moreover, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel. Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and / or is not typically in the functional state and / or that is altered or removed from the natural state through human intervention. For example, a DNA naturally present in a living animal is not “isolated,” but a synthetic DNA, or a DNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the nucleic acid has been delivered. All patents, published patent applications, other publications, and databases referred to herein are incorporated by reference in their entirety with respect to the related technology. DNA Template In some aspects, the method for producing an RNA molecule, e.g., mRNA, includes providing a sample that includes a linear DNA template (in solution or stationary). The DNA template includes a sequence coding for a gene of interest that encodes, e.g., a peptide or polypeptide of interest. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the sequence coding for a gene of interest. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. For example, in some preferred aspects, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence operably linked to the RdRp gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. In some aspects, a DNA template lacks a plasmid backbone. In some aspects, the linear DNA template is a linearized plasmid DNA used as the template for continuous-flow in vitro transcription (in solution or stationary). In some aspects, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH10B cells, are transfected with the plasmid DNA template. The transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some aspects, the linear DNA template is synthesized in a cell-free environment. In some aspects, the linear DNA template is synthesized by rolling circle amplification (RCA). In some aspects, RCA includes an amplification target circle (ATC) that forms a template on which new DNA is made, thereby extending the initial sequence as a continuous sequence of repeated sequences complementary to the circle but generating only about several thousand copies per hour. In some aspects, the linear DNA template is provided by exponential RCA, including hyperbranched RCA (also termed ramification amplification). In other aspects, the linear DNA template is provided by a cell-free process for synthesizing DNA that includes contacting a DNA template with at least one polymerase in the presence of nucleotides to form a reaction mixture, wherein the DNA template is amplified by strand displacement replication, and wherein further nucleotides are supplied to the reaction mixture continuously or at intervals during the process. In some aspects the nucleotides can include an aminoallyl-dNTP (e.g. aminoallyl-dUTP) so that nucleophilic primary amines are inserted into the amplified DNA to enable subsequent covalent immobilization of the resulting linear DNA template. In some aspects, the nucleotides can include other functional groups (including, but not limited to amines, carboxyls, sulfhydryls, aldehydes, hydroxyls, azides, epoxides, or biotin) within their structure to facilitate covalent immobilization, adsorption, entrapment, or co-polymerization of the resulting DNA template on or within a substrate In some aspects, the DNA template also includes an RNA polymerase promoter sequence, e.g., a T7 promoter, located 5′ to and operably linked to the gene of interest. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. In some preferred aspects, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence, located 5′ to and operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like. For example, a gene of interest operably linked to an RNA polymerase promoter allows transcription of the gene of interest. Any RNA polymerase or variants thereof may be used in the methods described here. The RNA polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and / or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids. As used herein, “gene of interest” refers to a polynucleotide which encodes a polypeptide or protein of interest. Depending on the context, the gene of interest refers to a deoxyribonucleic acid, e.g., a gene of interest in a DNA template which may be transcribed to an RNA molecule, or a ribonucleic acid, e.g., a gene of interest in an RNA molecule which may be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. As described in more detail below, a polypeptide of interest includes but is not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, etc. Flanking Regions: Untranslated Regions (UTRs) In some aspects, the methods for continuous production of an RNA molecule disclosed herein include (a) providing a sample having a linear DNA template, the DNA template includes an RNA polymerase promoter sequence operably linked to a sequence coding for a gene of interest and a 5′ untranslated region (UTR) and / or a 3′ UTR. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. For example, in some aspects, the methods for continuous production of an RNA molecule disclosed herein include (a) providing a sample having a linear DNA template, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence, located 5′ to and operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest and a 5′ untranslated region (UTR) and / or a 3′ UTR. The DNA template and RNA molecule may include UTRs. Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon, whereas the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory features of a UTR may be incorporated into the polynucleotides, primary constructs and / or mRNA of the present invention to enhance the stability of the molecule. The specific features may also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organ sites. Natural 5′ UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’.5′ UTR also have been known to form secondary structures which are involved in elongation factor binding. By engineering the features typically found in abundantly expressed genes of specific target organs, one may enhance the stability and protein production of the polynucleotides, primary constructs. For example, use of 5′ UTRs from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), and for endothelial cells (Tie-1, CD36), among others. Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR) UTRs. For example, introns or portions of introns sequences may be incorporated into the flanking regions of the polynucleotides, primary constructs or mRNA described here. Incorporation of intronic sequences may increase protein production as well as mRNA levels. Cap-dependent translation involves recruitment of the pre-initiation complex (PIC) to the 5′ end of an mRNA followed by scanning to find an AUG initiation codon in an optimum sequence context. AUG recognition promotes scanning cessation, release of most initiation factors, and recruitment of the large ribosomal subunit to initiate elongation. Efficient recognition of an initiation codon depends on its surrounding sequence. In some aspects, the sequence CRCCaugG (R = purine, A or G) may provide optimal context for AUG recognition in eukaryotes. 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU-rich elements (AREs) may be separated into three classes: Class I AREs include several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD include class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules including this type of AREs include GM-CSF and TNF-alpha. Class III ARES are less well defined. These U rich regions do not include an AUUUA motif c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules may lead to HuR binding and thus, stabilization of the message in vivo. Introduction, removal or modification of 3′ UTR AREs may be used to modulate the stability of polynucleotides, and primary constructs. When engineering specific polynucleotides, and / or primary constructs, one or more copies of an ARE may be introduced to make polynucleotides, and / or primary constructs less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs may be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments may be conducted in relevant cell lines, using polynucleotides, and / or primary constructs and protein production may be assayed at various time points post-transfection. For example, cells may be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection. Poly(A) tail In some aspects, the methods for continuous production of an RNA molecule disclosed herein include providing a sample having a DNA template, the DNA template having an RNA polymerase promoter sequence operably linked to a sequence coding for a gene of interest and a poly(A) tail sequence of 20-100 nucleotides. The poly(A) tail can prevent degradation of the RNA molecule in a cell. Accordingly, in some aspects, the plasmid DNA template includes a sequence coding for a poly(A) tail located 3′ to the gene of interest. As used herein, “poly(A) tail” refers to a chain of adenine nucleotides. In some aspects, the poly(A) tail includes 5-300 adenine nucleotides in length, e.g., at least, at most, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 adenine nucleotides in length, or any range or value derivable therein. In preferred aspects, the DNA template includes a poly(A) tail that includes about 40 adenines. In preferred aspects, the DNA template includes a poly(A) tail that includes about 80 adenines. In some aspects, the poly(A) tail is encoded in the DNA template. In other aspects, the poly(A) tail is added to the RNA molecule by enzymatic treatment with a Poly(A) polymerase. In some aspects, the RNA molecule does not include a poly(A) tail. In some aspects, immediately downstream of the poly(A) tail coding sequence on the plasmid DNA template is a recognition site for a restriction endonuclease to linearize the plasmid. Linearization of the plasmid can mitigate transcriptional readthrough. In some aspects, following linearization, the plasmid DNA template is filtered into an appropriate solvent, e.g., water, HEPES, and EDTA. In a preferred aspect, the solvent includes 10 mM HEPES, 0.1 mM EDTA, and the like. Filtration occurs via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration. The linear DNA template may be purified before use as a template for continuous-flow in vitro transcription. For example, the linear DNA template may be purified chromatographically or by ethanol or isopropanol precipitation. In vitro Transcription In vitro transcription (IVT) and continuous-flow IVT refer to a procedure that allows for DNA- directed synthesis of RNA molecules of any sequence, ranging in size from short oligonucleotides to several kilobases. In some aspects, in vitro transcription involves engineering of a DNA template to include a bacteriophage promoter sequence (e.g., from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In some aspects, the resulting RNA molecules are subsequently modified (e.g., by capping, splicing, the addition of a poly(A) tail, etc.). In some aspects, the methods described herein for continuous production of an RNA molecule, e.g., mRNA, includes contacting a DNA template with an in vitro transcription reaction system. In some aspects, the IVT reaction system includes an RNA polymerase and ribonucleotides, which may be natural and / or modified ribonucleotides. In some aspects, the IVT reaction system includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and an RNA polymerase. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified, such as, for example, N-1-methylpseudouridine triphosphate) NTPs. In some aspects, the methods described herein for continuous production of an RNA molecule, e.g., mRNA, includes contacting a stationary DNA template with an IVT reaction system. In some aspects, the IVT reaction system includes an RNA polymerase and ribonucleotides, which may be natural and / or modified ribonucleotides. In some aspects, the IVT reaction system includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and an RNA polymerase. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified, such as, for example, N-1-methylpseudouridine triphosphate) NTPs. In some aspects, the methods described herein for continuous production of an RNA molecule, e.g., mRNA, includes contacting a stationary RNA polymerase with an IVT reaction system. In some aspects, the IVT reaction system includes a DNA template and ribonucleotides, which may be natural and / or modified ribonucleotides. In some aspects, the IVT reaction system includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a DNA template. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified, such as, for example, N-1-methylpseudouridine triphosphate) NTPs. In some aspects, the methods described herein for continuous production of an RNA molecule, e.g., mRNA, includes contacting a stationary RNA polymerase and stationary DNA template with an IVT reaction system. In some aspects, the IVT reaction system includes ribonucleotides, which may be natural and / or modified ribonucleotides. In some aspects, the IVT reaction system includes a transcription buffer, nucleotide triphosphates (NTPs), and an RNase inhibitor. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified, such as, for example, N-1-methylpseudouridine triphosphate) NTPs. RNA Polymerase In some aspects, the RNA polymerase used to generate the mRNA transcript may also be referred to as a “DNA-dependent RNA polymerase” which transcribes DNA into RNA molecules. Exemplary RNA polymerases include bacteriophage T7, T3, Syn5, and SP6 RNA polymerases, or variants thereof (including thermostable / thermophilic variants), which may be used to transcribe the mRNA, self-amplifying RNA, or guide RNA from a DNA template. RNA polymerases represent the primary machinery that drives transcription. RNA polymerases have been isolated and purified sufficiently that they are useful for producing RNA in vitro. In some aspects, the RNA polymerase is a T7 RNA polymerase, which refers to a monomeric T7 bacteriophage-encoded DNA directed RNA polymerase that catalyzes the formation of RNA in the 5′ to 3′ direction. The wild-type T7 RNA polymerase includes 883 amino acids. It is homologous to T3 RNA polymerase and somewhat homologous to SP6 RNA polymerase. In some aspects, the RNA polymerase includes an engineered T7 RNA polymerase variant, such as a variant that allows for selective incorporation of a 5’ cap analog (e.g. m7G(5′)ppp(5′)m7G, m7G(5’)ppp(5’)(2’OMeA)pG, or m7G(5')ppp(5')(2'OMeA)pU cap analogs) over GTP at the initiation of in vitro transcription. For example, in some aspects, the RNA polymerase has been modified to preferentially accept a cap (also referred to as an RNA cap, an RNA 7- methylguanosine cap or an RNA m7G cap) or cap analog (e.g., the “Anti Reverse Cap Analog” (3′-O-Me-m7G(5′)ppp(5′)G; “ARCA”), or a methylated cap analog with one or more nucleotides at the transcription initiation site (e.g., m7G(5′)ppp(5′)N, wherein N is any nucleotide) to begin transcription during transcription initiation. The 5′ cap is an altered nucleotide on the 5′ end of some eukaryotic primary transcripts such as precursor messenger RNA. The typical cap structure includes a 7-methylguanosine (m7G) linked to the first nucleotide of the transcript via a 5′-5′ triphosphate bridge. Cap analogs may include, for example, one, two or more methyl (or other substitution) groups at specific positions. Exemplary 5’ cap analogs include, but are not limited to, m7G(5′)ppp(5′)m7G, 3′-O-Me-m7G(5′)ppp(5′)G, m7G(5′)ppp(5′)G, G(5′)ppp(5′)G, m7G(5′)ppp(5′)A, G(5′)ppp(5′)A, m7G(5’)ppp(5’)(2’OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, or m7(3’OMeG)(5’)ppp(5’)m6(2’OMeA)pG. Cap molecules or 5’ cap analogs may be added either upfront in the continuous-flow IVT reaction or after the synthesis of mRNA, by enzymatic capping. Cap molecules added upfront in the continuous-flow IVT reaction can make the mRNA production more straightforward. Ribonucleotides In the methods described herein, the IVT reaction system includes nucleotides (for example, non- modified ribonucleotide triphosphates or modified ribonucleotide triphosphates). The nucleotides may be selected from any one of natural nucleotides, e.g., A, G, C, and U ribonucleotides; modified nucleotides (such as, for example, N-1-methylpseudouridine triphosphate); or a combination thereof. In some aspects, the ribonucleotides are Tris buffered, such as a 100 mM aqueous solution of ribonucleotide titrated to pH 7.3-7.5 with Tris base. In some aspects, the ribonucleotides are in sodium salt. Modified nucleobases which may be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules generated by the IVT reaction system include, for example, m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-0-methyluridine), mlA (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O- methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6- glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2- methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2- methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-0-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (l,2'-0-dimethylinosine); m3C (3-methylcytidine); Cm (2T- 0-methylcytidine); s2C (2- thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm (5,2-0-dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); mlG (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-0- methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-0- dimethylguanosine); m22Gm (N2,N2,2'-0-trimethylguanosine); Gr(p) (2'-0- ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl- queuosine); manQ (mannosyl-queuosine); preQo (7-cyano- 7-deazaguanosine); preQi (7- aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-0- dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2'- O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5- hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5- (carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5- methoxycarbonylmethyl- 2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2- selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0- methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1 aminomethyl-2-L-Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-0-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-0-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,T-0-dimethyladenosine); rn62Am (N6,N6,0-2- trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-0- methylcytidine); mlGm (l,2'-0-dimethylguanosine); m'Am (1,2-0- dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7- substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5- aminouracil, 5-(Ci-C6)-alkyluracil, 5-methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)- alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5- hydroxycytosine, 5-(Ci-C6 )- alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2- C6)-alkynylcytosine, 5- chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2- C6)alkynylguanine, 7-deaza- 8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8- oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8- azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-0-methyl-U. Additional exemplary modified nucleotides include any one of N-1-methylpseudouridine ; pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine. In some aspects, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. In some aspects, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5′ cap that may include, for example, 7-methylguanosine. In other aspects, the RNA may include a 5′ cap comprising a 7′- methylguanosine, and the first 1, 2 or 35′ ribonucleotides may be methylated at the 2′ position of the ribose. Exemplary In Vitro Transcription Reaction Systems In some aspects, the in vitro transcription reaction system includes the following: an RNA polymerase, e.g., a T7 RNA polymerase, DNA template; nucleotide triphosphates (NTPs); magnesium; and a buffer such as, e.g., HEPES or Tris (or both HEPES and Tris). In some aspects, the in vitro transcription reaction system does not include RNA polymerase. In some aspects, the in vitro transcription reaction system does not include DNA template. In some aspects, the in vitro transcription reaction system includes neither RNA polymerase nor DNA template. In some aspects, the RNA polymerase is stationary. In some aspects, the DNA template is stationary. In some aspects, both RNA polymerase and DNA template are stationary. In some aspects, the in vitro transcription reaction system is flowing across a substrate containing stationary RNA polymerase and / or DNA template with a residence time within the substrate of between 1 second and 3 hours at a temperature between about 20 °C and 50 °C. In some aspects, the total time that the in vitro transcription reaction system is flowing across a substrate, i.e., the total amount of time the continuous-flow in vitro transcription reaction proceeds, is between 1 second and 365 days. In some aspects, the total amount of time the continuous-flow in vitro transcription reaction proceeds is more than 365 days. In some aspects, the in vitro transcription reaction system includes the RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 1000-44000 U / mL, e.g., at least, at most, or about 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000, 20500, 21000, 21500, 22000, 22500, 23000, 23500, 24000, 24500, 25000, 25500, 26000, 26500, 27000, 27500, 28000, 28500, 29000, 29500, 30000, 30500, 31000, 31500, 32000, 32500, 33000, 33500, 34000, 34500, 35000, 35500, 36000, 36500, 37000, 37500, 38000, 38500, 39000, 39500, 40000, 40500, 41000, 41500, 42000, 42500, 43000, 43500, or 44000 U / mL, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes a T7 RNA polymerase at either 8U / uL, 10U / uL, 13U / uL, or 15U / uL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 7000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 8000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 14000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 17000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 25000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 40000 U / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 nM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 40 nM. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 144 nM. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 5-24 nM DNA, e.g., at least, at most, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 36 to 144 nM DNA, e.g., at least, at most, or about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144 nM, or any range or valuable derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of, e.g., at least, at most, or about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.075, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.50 mg / mL, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.025 mg / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.05 mg / mL. In some aspects, in vitro transcription reaction system includes the DNA template at a final concentration of 0.075 mg / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.1 mg / mL. In some aspects, the in vitro transcription reaction system includes each nucleotide triphosphate (NTP) at a final concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription system includes each nucleotide triphosphate (NTP) at a starting concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM. In some aspects, the in vitro transcription reaction system includes the nucleotide triphosphates (NTPs) at a final concentration of about 8 mM each. In some aspects, the in vitro transcription reaction system includes the nucleotide triphosphate ATP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the nucleotide triphosphate CTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the nucleotide triphosphate GTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the UTP or nucleotide triphosphate modified UTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In certain aspects the concentration of NTPs in the reaction is 0.4mM, 0.8mM, 1mM, 1.25mM, 3.mM, 5mM, 6mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, or 10mM. In some aspects, the in vitro transcription reaction system includes a 5’ cap analog. Exemplary 5’ cap analogs include, but are not limited to, m7G(5′)ppp(5′)m7G, 3′-O-Me-m7G(5′)ppp(5′)G, m7G(5′)ppp(5′)G, G(5′)ppp(5′)G, m7G(5′)ppp(5′)A, G(5′)ppp(5′)A, m7G(5’)ppp(5’)(2’OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, or m7(3’OMeG)(5’)ppp(5’)m6(2’OMeA)pG. In some aspects, the in vitro transcription reaction system includes a 5’ cap analog at a final concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription system includes a 5’ cap analog at a starting concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM. In some aspects, the in vitro transcription reaction system includes a 5’ cap analog at a final concentration of about 4 mM. In certain aspects the concentration of 5’ cap analog in the reaction is 0.4mM, 0.8mM, 1mM, 1.25mM, 3.mM, 5mM, 6mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, or 10mM. In some aspects, the in vitro transcription reaction system includes magnesium ion, for example, as a magnesium salt, such as any one of magnesium chloride and magnesium acetate. In some aspects, the in vitro transcription reaction system includes the magnesium at a final concentration of, e.g., at least, at most, or about 12, 13, 14, 15, 16, 16.5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, or 220 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 30 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 40 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 16.5 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 33 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 36 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 50 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 110 mM. In some aspects, the Mg:NTP ratio can be maintained at a ratio of, e.g., at least, at most, or about, 0, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, or 2.2 mM Mg / mM NTP, or any range or value derivable therein. In some aspects, during the continuous flow of the in vitro transcription reaction system across a substrate, one or more reaction components are added during in vitro transcription by occasional bolus feeds, semi-continuous feeds, or continuous feeds. Bolus feeds can be delivered at intervals of, e.g., at least, at most, or about, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 minutes, or any range or value derivable therein. These components can include, but are not limited to, one or more NTPs and a cation such as magnesium. These components can be combined into a single feed, or they can be delivered separately in the form of multiple feeds. In some aspects, a continuous feed of at least 1 NTP can be delivered at flow rates of, e.g, at least, at most, or about 0, 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 mL / L / min. In some aspects, a continuous feed of a cation such as magnesium can be delivered at concentrations of, e.g., at least, at most, or about 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 mM / min, or any range or value derivable therein In some aspects, the in vitro transcription reaction system includes a buffer. Exemplary buffers for the in vitro transcription reaction system may include Tris and / or HEPES. In some aspects, the in vitro transcription reaction system includes the buffer at a pH of, e.g., at least, at most, or about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In some aspects, the buffer is Tris-HCl, pH 8.0. In some aspects, the in vitro transcription reaction system includes 40 mM Tris HCl, pH 8.0. Alternative buffers for the in vitro transcription reaction system include 40 mM Tris pH 7.5, 80 mM HEPES. In some aspects, the in vitro transcription reaction system does not include HEPES. In some aspects, the in vitro transcription reaction system includes HEPES and Tris. In some aspects, the buffer does not comprise dithiothreitol (DTT). In some aspects, an Rnase inhibitor is included in the in vitro transcription reaction system. The Rnase inhibitor may reduce Rnase-induced degradation during the transcription reaction. For example, murine Rnase inhibitor may be utilized at a final concentration of 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 U / mL. In some aspects, the in vitro transcription reaction system comprises Rnase inhibitor at a final concentration of 100 U / mL. In some aspects, the in vitro transcription reaction system comprises Rnase inhibitor at a final concentration of 1000 U / mL. In some aspects, a pyrophosphatase is included in the in vitro transcription reaction system. The pyrophosphatase may cleave the inorganic pyrophosphate generated following each nucleotide incorporation into two units of inorganic phosphate, which may reduce the likelihood of magnesium co-precipitating with pyrophosphate to form magnesium pyrophosphate. Pyrophosphatase in certain aspects may be diluted in pyrophosphatase buffer and present in the reaction at concentrations of 0.01mU / uL, 0.02mU / uL, 0.05mU / uL, 0.08mU / uL, 0.1mU / uL, 0.2mU / uL, 0.8mU / uL, or 2mU / uL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 0.25 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 0.5 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 1 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 2 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 3 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 6 U / mL. In some aspects, the in vitro transcription reaction system described herein includes an inorganic pyrophosphatase at a final concentration between 0.25 to 100 U / mL, e.g., at least, at most, or about 0.25, 0.50, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 U / mL, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes a polyamine. Exemplary polyamines include spermine, putrescene, and spermidine. In some aspects 1 mM spermidine is included. In some aspects 2.0 mM spermidine is included. In some aspects 2.15 mM spermidine is included. In some aspects, the in vitro transcription reaction system lacks a polyamine. In some aspects, the in vitro transcription reaction system lacks spermidine. In some aspects, the IVT reaction system includes a reducing reagent, such as, for example, DTT (dithiothreitol), e.g., at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mM, or any range or value derivable therein. In some aspects, the reducing agent is selected from the group consisting of dithiothreitol (DTT), dithioerythritol (DTE), Tris(2- carboxyethyl)phosphine (TCEP) and beta-mercaptoethanol. In some aspects, the IVT reaction system includes 1 mM DTT. In some aspects, the IVT reaction system includes 5 mM DTT. In some aspects, the IVT reaction system includes 10 mM DTT. In some aspects, the IVT reaction system includes 20 mM DTT. In some aspects, the IVT reaction system lacks a reducing agent. In some aspects, the IVT reaction system does not contain DTT. In some aspects, the IVT reaction system does not contain added DTT beyond the protective amount of DTT present in the RNA polymerase storage solution. In some aspects, the continuous-flow in vitro transcription reaction proceeds, for example, at about 37°C for about 12 days, e.g., at least, at most, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, or any range or value derivable therein. In some aspects, the continuous-flow in vitro transcription reaction proceeds, for example, at about 37°C for about 365 days, e.g., at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, or 365 days or any range or value derivable therein. In some aspects, the continuous-flow in vitro transcription reaction proceeds, for example, at about 37°C for any number of days less than 0.1 days. In some aspects, the continuous-flow in vitro transcription reaction proceeds, for example, at about 37°C for any number of days greater than 365 days. In some aspects, the continuous-flow in vitro transcription reaction proceeds, for example, at less than 50°C for less than 365 days, such as for example, at least, at most, or about 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41°C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, or about 20°C, for at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, or 365 days or any range or value derivable therein. As disclosed herein, a continuous-flow in vitro transcription reaction proceeding at about 37°C for about 365 days results in high yields of RNA. In some aspects, RNA yield of the continuous-flow IVT reaction may be at least 0.1 mg RNA / mL to about 20 mg RNA / mL of flowed IVT reaction system (i.e., the total volume of IVT reaction system flowed over a substrate during the course of the CF-IVT reaction). For example, in some aspects, the RNA yield may be at least, at most, or about 0.1 mg RNA / mL, 0.2 mg RNA / mL, 0.3 mg RNA / mL, 0.4 mg RNA / mL, 0.5 mg RNA / mL, 0.6 mg RNA / mL, 0.7 mg RNA / mL, 0.8 mg RNA / mL, 0.9 mg RNA / mL, 1.0 mg RNA / mL, 2 mg RNA / mL, 3 mg RNA / mL, 4 mg RNA / mL, preferably at least 5 mg RNA / mL, 6 mg RNA / mL, 7 mg RNA / mL, 8 mg RNA / mL, 9 mg RNA / mL, 10 mg RNA / mL, 11 mg RNA / mL, 12 mg RNA / mL, 13 mg RNA / mL, 14 mg RNA / mL, 15 mg RNA / mL, 16 mg RNA / mL, 17 mg RNA / mL, 18 mg RNA / mL, 19 mg RNA / mL, or 20 mg RNA / mL of flowed IVT reaction system, or any range or value derivable therein. In some aspects, RNA yield of the continuous-flow IVT reaction may be any value less than 0.1 mg RNA / mL of flowed IVT reaction system. In some aspects, RNA yield of the continuous-flow IVT reaction may be any value greater than 20 mg RNA / mL of flowed IVT reaction system. In some aspects, the yield of the continuous-flow in vitro transcription reaction producing RNA molecules having at least 90% of the intended full length transcript may be at least, at most, or about 0.1 mg RNA / mL, 0.2 mg RNA / mL, 0.3 mg RNA / mL, 0.4 mg RNA / mL, 0.5 mg RNA / mL, 0.6 mg RNA / mL, 0.7 mg RNA / mL, 0.8 mg RNA / mL, 0.9 mg RNA / mL, 1.0 mg RNA / mL, 2 mg RNA / mL, 3 mg RNA / mL, 4 mg RNA / mL, preferably at least 5 mg RNA / mL, 6 mg RNA / mL, 7 mg RNA / mL, 8 mg RNA / mL, 9 mg RNA / mL, 10 mg RNA / mL, 11 mg RNA / mL, 12 mg RNA / mL, 13 mg RNA / mL, 14 mg RNA / mL, 15 mg RNA / mL, 16 mg RNA / mL, 17 mg RNA / mL, 18 mg RNA / mL, 19 mg RNA / mL, or 20 mg RNA / mL of flowed IVT reaction system. In some preferred aspects, the yield of the continuous-flow in vitro transcription reaction producing RNA molecules having at least 90% of the intended full length transcript is at least 9 mg RNA / mL of flowed IVT reaction system. As disclosed herein, a continuous-flow IVT reaction proceeding at about 37°C for about 365 days results in high productivity of RNA. Productivity in the case of continuous-flow in vitro transcription refers to the amount of RNA produced per volume of reactor per unit time. In some aspects, productivity of the continuous-flow IVT reaction may be at least 0.1 mg / mL / h to about 1000 mg / mL / h, where volume refers to the reactor volume. For example, in some aspects, the productivity of the continuous-flow IVT reaction may be at least, at most, or about 0.1 mg / mL / h, 0.2 mg / mL / h, 0.3 mg / mL / h, 0.4 mg / mL / h, 0.5 mg / mL / h, 0.6 mg / mL / h, 0.7 mg / mL / h, 0.8 mg / mL / h, 0.9 mg / mL / h, 1.0 mg / mL / h, 2 mg / mL / h, 3 mg / mL / h, 4 mg / mL / h, 5 mg / mL / h, 6 mg / mL / h, 7 mg / mL / h, 8 mg / mL / h, 9 mg / mL / h, 10 mg / mL / h, 11 mg / mL / h, 12 mg / mL / h, 13 mg / mL / h, 14 mg / mL / h, 15 mg / mL / h, 16 mg / mL / h, 17 mg / mL / h, 18 mg / mL / h, 19 mg / mL / h, 20 mg / mL / h, 25 mg / mL / h, 30 mg / mL / h, 35 mg / mL / h, 40 mg / mL / h, 45 mg / mL / h, 50 mg / mL / h, 55 mg / mL / h, 60 mg / mL / h, 65 mg / mL / h, 70 mg / mL / h, 75 mg / mL / h, 80 mg / mL / h, 85 mg / mL / h, 90 mg / mL / h, 95 mg / mL / h, 100 mg / mL / h, 150 mg / mL / h, 200 mg / mL / h, 250 mg / mL / h, 300 mg / mL / h, 350 mg / mL / h, 400 mg / mL / h, 450 mg / mL / h, 500 mg / mL / h, 600 mg / mL / h, 700 mg / mL / h, 800 mg / mL / h, 900 mg / mL / h, or 1000 mg / mL / h, or any range or value derivable therein, where volume refers to the reactor volume. In some aspects, the productivity of the continuous-flow IVT reaction may be any value less than 0.1 mg / mL / h, where volume refers to the reactor volume. In some aspects, the productivity of the continuous-flow IVT reaction may be any value greater than 1000mg / mL / h, where volume refers to the reactor volume. In some preferred aspects, the productivity of the continuous-flow IVT reaction producing RNA having at least 90% of the intended full length transcript is at least 30 mg / mL / h, where volume refers to the reactor volume. In some aspects, following a continuous-flow IVT reaction using a DNA template and an RNA polymerase as described here, a first composition that includes an uncapped RNA molecule is produced. In some aspects, the RNA molecule includes the coding sequence for a gene of interest and a poly(A) tail. As used herein, the RNA molecule includes an mRNA. The RNA molecule may include modifications, such as, modified nucleotides. As used herein, an “RNA molecule” produced by continuous-flow in vitro transcription may be referred to as an “RNA transcript” or an “in vitro transcribed RNA.” An “RNA molecule,” “RNA transcript,” or “in vitro transcribed RNA,” may encompass any one of modified mRNA (“modRNA”), unmodified mRNA, self-amplifying RNA (saRNA), and guide RNA (gRNA). In some aspects, the methods of continuous-flow IVT production of an RNA molecule described herein produce a first composition having an uncapped RNA molecule. In some aspects, at most 30% of the RNA molecules in the first composition includes uncapped RNA molecules. In some aspects, the first composition includes at least, at most, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, uncapped RNA molecules. RNA Molecule The RNA molecule produced by the methods described herein may be a non-coding and / or a coding RNA. A non-coding RNA (ncRNA) molecule includes a functional RNA molecule that is not translated into a peptide or polypeptide. Non-coding RNA molecules may include highly abundant and functionally important RNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAs such as snoRNAs, microRNAs, siRNAs, snRNAs, guideRNAs, circularRNAs, exRNAs, and piRNAs and long ncRNAs. In a preferred aspect, the RNA molecule is an mRNA molecule that includes a modified nucleotide (herein referred to as a “modified RNA molecule” or “modified mRNA molecule” or “modRNA”). In some preferred aspects, the RNA molecule is a self-amplifying RNA molecule. Coding RNA includes a functional RNA molecule that may be translated into a peptide or polypeptide. In some aspects, the coding RNA molecule includes at least one open reading frame coding for at least one peptide or polypeptide. The coding RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs). The coding RNA molecule may be a messenger RNA (mRNA) molecule, viral RNA molecule or self- amplifying RNA molecule (saRNA, also referred to as a replicon). Preferably, the RNA molecule is an mRNA molecule. The RNA molecule may encode more than one protein, e.g., two, three, four, five, ten or more polypeptides. Alternatively, or in addition, one RNA molecule may also encode more than one antigen, e.g., a bicistronic, or tricistronic RNA molecules that encode different or identical antigens. The sequence of the RNA molecule may be codon optimized or de-optimized for expression in a desired host, such as a human cell. The sequence of the RNA molecule may be modified if desired, for example to increase the efficacy of expression or replication of the RNA, or to provide additional stability or resistance to degradation. For example, the RNA sequence may be modified with respect to its codon usage, for example, to increase translation efficacy and half-life of the RNA. In some aspects, the RNA molecules may include one or more structural and / or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some aspects, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. As used herein, a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in an RNA molecule without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide. In some aspects, the RNA molecule may include one or more modified nucleotides in addition to any 5′ cap structure. Naturally occurring nucleoside modifications are known in the art. In some aspects, the RNA molecules produced continuously by the continuous-flow in vitro transcription reactions described herein include from at least, at most, or about 20 to at least, at most, or about 100,000 nucleotides, or any range or value derivable therein (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000). In preferred aspects, the RNA molecule includes at least 100 nucleotides. For example, in some aspects, the RNA has a length between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides. In some aspects, the RNA has a length between about 1,600 nucleotides and 9,600 nucleotides. In preferred aspects, the RNA molecule that is the polynucleotide product of the continuous-flow in vitro transcription reaction described herein includes a gene of interest and a poly(A) tail. In some aspects, the RNA molecule further includes a 5′ UTR and a 3′ UTR. In some aspects, the modified mRNA molecule encodes a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptide(s) generated from the modified mRNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences. In preferred aspects, the modified mRNA molecule encodes a single polypeptide of interest. In some preferred aspects, the RNA molecule is a saRNA. “Self-amplifying RNA,” “self-replicating RNA,” and “replicon” refer to RNA with the ability to replicate itself. Self-amplifying RNA molecules may be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs and so the encoded gene of interest, e.g., a viral antigen, becomes a major polypeptide product of the cells. In some aspects, the self-amplifying RNA includes at least one or more genes selected from any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins. In some aspects, the self-amplifying RNA may also include 5′- and 3’-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES). In preferred aspects, the self-amplifying RNA molecule is not encapsulated in a virus-like particle. Self-amplifying RNA molecules described herein may be designed so that the self-amplifying RNA molecule cannot induce production of infectious viral particles. This may be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary to produce viral particles in the self-amplifying RNA. For example, when the self-amplifying RNA molecule is based on an alpha virus, such as Sindbis virus (SINV), Semliki Forest virus and Venezuelan equine encephalitis virus (VEEV), one or more genes encoding viral structural proteins, such as capsid and / or envelope glycoproteins, may be omitted. In some aspects, a self-amplifying RNA molecule described herein encodes (i) an RNA- dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a viral antigen. In some aspects, the polymerase may be an alphavirus replicase e.g., including alphavirus protein nsP4. In some aspects, the self-amplifying RNA molecules described herein may include one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5- methylcytidine, 5-methyluridine). The saRNA construct may encode at least one non-structural protein (nsP), disposed 5′ or 3′ of the sequence encoding at least one peptide or polypeptide of interest. Preferably, the sequence encoding at least one nsP is disposed 5′ of the sequences encoding the peptide or polypeptide of interest. Thus, preferably the sequence encoding at least one nsP is disposed at the 5′ end of the RNA construct. In some aspects, at least one non-structural protein encoded by the RNA construct may be the RNA polymerase nsP4. Preferably, the saRNA construct encodes nsP1, nsP2, nsP3 and nsP4. As is known in the art, nsP1 is the viral capping enzyme and membrane anchor of the replication complex (RC). nsP2 is an RNA helicase and the protease responsible for the ns polyprotein processing. nsP3 interacts with several host proteins and may modulate protein poly- and mono-ADP-ribosylation. nsP4 is the core viral RNA-dependent RNA polymerase. In some aspects, the polymerase may be an alphavirus replicase, e.g., comprising one or more of alphavirus proteins nsP1 nsP2, nsP3 and nsP4. Whereas natural alphavirus genomes encode structural virion proteins in addition to the non- structural replicase polypeptide, in some aspects, the self-amplifying RNA molecules do not encode alphavirus structural proteins. In some aspects, the self-amplifying RNA may lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA that includes virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike a wild-type alphavirus, the self-amplifying RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild- type viruses are absent from self-amplifying RNAs of the present disclosure and their place is taken by gene(s) encoding the protein of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins. In some aspects, the self-amplifying RNA molecule may have two open reading frames. The first (5′) open reading frame encodes a replicase; the second (3′) open reading frame encodes a polypeptide comprising an antigen of interest. In some aspects the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides. Optionally, self-amplifying RNA molecules described herein may also be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection. When delivered to a vertebrate cell, a self-amplifying RNA molecule may lead to the production of multiple daughter RNAs by transcription from itself (or from an antisense copy of itself). The self-amplifying RNA may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces transcripts from the delivered RNA, thereby producing multiple daughter RNAs. These RNA molecules are antisense relative to the delivered RNA and may be translated themselves to provide in situ expression of a gene product, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the gene product. In some aspects, the saRNA molecule is alphavirus-based. Alphaviruses include a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Exemplary viruses and virus subtypes within the alphavirus genus include Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. As such, the self-amplifying RNA described herein may incorporate an RNA replicase derived from any one of Semliki Forest virus (SFV), Sindbis virus (SINV), Venezuelan equine encephalitis virus (VEEV), Ross-River virus (RRV), or other viruses belonging to the alphavirus family. In some aspects, the self-amplifying RNA described herein may incorporate sequences derived from a mutant or wild- type virus sequence, e.g., the attenuated TC83 mutant of VEEV has been used in saRNAs. Alphavirus-based saRNAs are (+)-stranded saRNAs that may be translated after delivery to a cell, which leads to translation of a replicase (or replicase- transcriptase). The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic (-)-strand copies of the (+)-strand delivered RNA. These (-)-strand transcripts may themselves be transcribed to give further copies of the (+)-stranded parent RNA and also to give a subgenomic transcript which encodes the desired gene product. Translation of the subgenomic transcript thus leads to in situ expression of the desired gene product by the infected cell. Suitable alphavirus saRNAs may use a replicase from a Sindbis virus, a Semliki Forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, or mutant variants thereof. In some aspects, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, preferably, a positive-stranded RNA virus, and more preferably a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wild-type alphavirus sequences are well-known and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR- 66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR- 372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCC VR- 1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR- 70, ATCC VR- 1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some aspects, the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., mRNA). Typically, the self-amplifying RNA molecules described herein include at least about 4 kb. For example, the self-amplifying RNA may include at least, at most, or about 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, or 12 kb or more, or any range or value derivable therein. In certain examples, the self-amplifying RNA is about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb. In some aspects, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences. In some aspects, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. Preferably epitopes capable of eliciting either a helper T-cell response or a cytotoxic T-cell response or both. In some aspects, the RNA molecule has a 3′ poly(A) tail, that is, a stretch of consecutive adenosine residues, that may be attached to the 3′ end of the RNA. The poly(A) tail may increase the half-life of the RNA molecule. The RNA molecule may further include a poly(A) polymerase recognition sequence (e.g., AAUAAA) near its 3′ end. In some aspects, the 3′ poly(A) tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. Preferably, the RNA molecule includes at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some preferred aspects, the RNA molecule includes about 40 consecutive adenosine residues. In some aspects, the RNA molecule includes about 80 consecutive adenosine residues. Poly(A) tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyper-polyadenylation may signal for RNA degradation. Exemplary designs include a poly(A) tails of about 40 As, about 80 As. In some aspects, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly(A) tail sequence. Capping of RNA Molecule In some aspects, the methods of continuous production of an RNA molecule described herein further include capping of uncapped RNA molecules by contacting the uncapped RNA molecules with a capping reaction system, which includes any one of guanylyltransferase (e.g., vaccinia capping enzyme or faustovirus capping enzyme), s-adenosyl-L-methionine (SAM), guanosine triphosphate (GTP), and 2′-O-methyltransferase, and any combination thereof, to produce a capped RNA molecule. In some aspects, the 5′ end of the RNA is capped with a modified ribonucleotide with the structure m7G(5′)ppp(5′)N (cap 0 structure) or a derivative thereof, which may be incorporated during RNA synthesis (co-transcriptional capping) or may be performed enzymatically after RNA transcription (post-transcriptional capping). In some aspects, the 5′ end of the RNA molecule is capped with a modified ribonucleotide via an enzymatic reaction after RNA transcription. In some aspects, capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule. An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) or Faustovirus Capping Enzyme, that include mRNA triphosphatase, guanylyltransferase and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures). Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule. The 5′ cap of the RNA molecule may be further modified by a 2’- O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp[2’Ome]N), which may further increase translation efficacy. In some aspects, the RNA molecule may be enzymatically capped at the 5′ end using Vaccinia or Faustovirus guanylyltransferase, guanosine triphosphate and S-adenosyl-L-methionine to yield cap 0 structure. An inverted 7-methylguanosine cap is added via a 5′ to 5′ triphosphate bridge. Alternatively, use of a 2′-O-methyltransferase with Vaccinia or Faustovirus guanylyltransferase yields the cap 1 structure where in addition to the cap 0 structure, the 2′-OH group is methylated on the first transcribed nucleotide. S-adenosyl-L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent. In some aspects, Rnase inhibitor is not included in the enzymatic capping reaction. In another aspect, the enzymatic capping reaction step is performed under constant mixing. In another aspect, the RNA molecule is not co-transcriptionally capped. Non-limiting examples of 5′ cap structures are those which, among other things, have enhanced binding of cap binding polypeptides, increased half-life, reduced susceptibility to 5′ endonucleases and / or reduced 5′ de-capping, as compared to synthetic 5′ cap structures known in the art (or to a wild-type, natural or physiological 5′ cap structure). For example, recombinant Vaccinia Virus or Faustovirus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme may create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine includes an N7 methylation and the 5′- terminal nucleotide of the mRNA includes a 2′-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5′ cap analog structures known in the art. Cap structures include, but are not limited to, m7G(5′)ppp(5′)N (cap 0) and m7G(5′)ppp(5′)Nm (cap 1). Cap 0 is a N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage, typically referred to as m7G cap or m7Gppp or m7G(5′)ppp(5′)N. In the cell, the cap 0 structure is essential for efficient translation of the mRNA that carries the cap. An additional methylation on the 2′-O position of the initiating nucleotide generates Cap 1, sometimes referred to as m7GpppNm- or m7G(5′)ppp(5′)Nm, wherein Nm denotes any nucleotide with a 2′-O methylation. In some aspects, the 5′ terminal cap includes a cap analog. Exemplary 5’ cap analogs include, but are not limited to, m7G(5′)ppp(5′)m7G, 3′-O- Me-m7G(5′)ppp(5′)G, m7G(5′)ppp(5′)G, G(5′)ppp(5′)G, m7G(5′)ppp(5′)A, G(5′)ppp(5′)A, m7G(5’)ppp(5’)(2’OmeA)pG, m7G(5’)ppp(5’)(2’OmeA)pU, or m7(3’OMeG)(5’)ppp(5’)m6(2’OmeA)pG. In some aspects, a 5′ terminal cap may include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl- guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine. In some aspects, the capping region may include a single cap or a series of nucleotides forming the cap. In this aspect the capping region may be from 1 to 10, e.g., at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or any range or value therein, e.g., 2-9, 3-8, 4-7, 1-5, 5- 10, or at least 2, or 10 or fewer nucleotides in length. In some aspects, the cap is absent. In some aspects, the first and second operational regions may range from 3 to 40, e.g., at least, at most, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides, or any range or value derivable therein, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and / or Stop codon, one or more signal and / or restriction sequences. In some aspects, the self-amplifying RNA molecules described herein have a 5′ cap (e.g., a 7- methylguanosine). This cap may enhance in vivo translation of the RNA. In some aspects, the self-amplifying RNA may include (in addition to any 5′ cap structure) one or more nucleotides having a modified nucleobase. In some aspects, the RNA molecule includes only phosphodiester linkages between nucleosides. In some aspects, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methyl phosphonate linkages. In some aspects, the modified mRNA molecules described herein have a 5′ cap (e.g., a 7- methylguanosine). This cap may enhance in vivo translation of the RNA. In some aspects, the modified mRNA may include (in addition to any 5′ cap structure) one or more nucleotides having a modified nucleobase. In some aspects, the RNA molecule includes only phosphodiester linkages between nucleosides. In some aspects, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methyl phosphonate linkages. In one aspect, the capping reaction system includes enzymatic 5′ capping that is performed as follows. The final 1X buffer conditions includes the following: at least, at most, or about 50 mM Tris HCl, pH 8, 5 mM KCl, 1 mM MgCl2, 0.5 mM GTP, 0.2 mM S-adenosyl-methionine and 1 mM dithiothreitol. In some aspects, the final 1X buffer does not include dithiothreitol. In some aspects, the capping reaction occurs in the reactor in which the continuous-flow IVT reaction was performed. In some aspects, the capping reaction occurs in a separate reactor. To degrade residual DNA template from the continuous-flow IVT reaction, Dnase I can be added. In some aspects, Dnase I is added at a concentration between at least, at most, or about 1 U / μg of DNA to 10 U / μg of DNA, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 U / μg of DNA, or any range or value derivable therein. In addition to Dnase I, CaCl2can be added as a co-factor for Dnase I at a concentration between at least, at most, or about 0.1 mM to 4 mM, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mM, or any range or value derivable therein. In some aspects, pyrophosphatase is added into the capping reaction. Pyrophosphatase assists with degrading pyrophosphate, which is the inhibitory by-product that is generated by the continuous-flow IVT reaction or by the capping reaction. In some aspects, the capping reaction is conducted under 37 °C for 1 minute to 2 hours, e.g., at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 minutes, or any range or value derivable therein. In some aspects, the capping reaction is conducted at a temperature greater than 20°C and less than 50°C, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50°C. In some aspects, the step of capping the uncapped RNA molecules results in at least, at most, or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% capped RNA molecules of the total of RNA molecules (capped and uncapped), or any range or value derivable therein. Purity may be determined as described herein, e.g., via reverse phase HPLC or Fragment analyzer or Bioanalyzer chip-based electrophoresis and measured by, e.g., peak area of full-length RNA molecule relative to total peak. Purification The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” or “purified RNA molecule” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment and / or purification method. Through purification of RNA, the RNA can be solubilized in an aqueous solution appropriate to permit subsequent encapsulation of the RNA within encapsulating agents (e.g., LNPs) and the solution is thus called “RNA drug substance” or “aqueous phase.” In some aspects, the RNA molecules produced by the methods described herein may be contacted with Dnase I and CaCl2to enzymatically digest DNA template following the continuous- flow in vitro transcription (CF-IVT) reaction. In some aspects, the RNA molecules produced by the methods described herein may be contacted with EDTA to chelate any cationic metal species, including magnesium following the continuous-flow in vitro transcription (CF-IVT) reaction, and thereby facilitate subsequent RNA purification from the CF-IVT product. In some aspects, the RNA molecules produced by the methods described herein may be contacted with EDTA and proteinase K. The EDTA may quench any cationic metal species, including magnesium, and the proteinase K may digest proteins present in the CF-IVT product, reducing their size. In some aspects, the RNA molecules produced by the methods described herein may be contacted with Dnase I and CaCl2followed by EDTA and proteinase K. In some aspects, the methods described herein do not include contacting the RNA molecules produced by the methods described herein with Dnase I and CaCl2to enzymatically digest DNA template following the CF-IVT reaction. In some aspects, the methods described herein do not include contacting the RNA molecules produced by the methods described herein with EDTA and proteinase K to digest proteins present in the CF-IVT product. In some aspects, the methods described herein do not include contacting the RNA molecules produced by the methods described herein with Dnase I and CaCl2followed by EDTA and proteinase K. In some aspects, the methods for continuous production of RNA described herein may be followed by continuous purification steps, e.g., an affinity chromatography step, an ion exchange chromatography step, a hydrophobic interaction chromatography (HIC) step, a ceramic hydroxyapatite (CHA) chromatography step, a phenyl boronate chromatography step, and / or a filtration step (e.g., ultrafiltration, diafiltration, tangential flow ultrafiltration / diafiltration, or single- pass variants thereof). In some aspects, the methods for continuous production of RNA described herein comprise a continuous in-line filtration device, wherein the continuous in-line filtration device comprises one or more diluent solutions. In some aspects, the RNA molecule produced by the CF-IVT reaction described herein is purified by methods including chromatographic adsorption (e.g., hydrogen bonding, hydrophobic interaction, ion-exchange, diol bonding, or metal affinity) or filtration (e.g., ultrafiltration, diafiltration, tangential flow ultrafiltration / diafiltration, or single-pass variants thereof). In some aspects, components of the CF-IVT product (e.g., NTPs, 5’ cap analog, RNA polymerase, template DNA), having been separated from the in vitro transcribed RNA molecule through the above-mentioned continuous purification methods, can then be recovered and returned to the CF-IVT reaction. In some aspects, at least a portion of the CF-IVT product is adsorbed on a substrate such as a resin bead or monolith. The RNA may bind to the substrate while impurities flow past the substrate or the impurities may bind to the substrate while RNA flows past the substrate. The mode of interaction may be based on hydrogen bonding, hydrophobic interaction, ion-exchange, diol bonding, or metal affinity, or any combination of these. In some aspects, the RNA molecule binds to an affinity substrate while the DNA template flows through and is removed. In some aspects, the RNA molecule binds to an affinity substrate while RNA polymerase flows through and is removed. In some aspects, the chromatographic method involves poly(A) capture-based affinity purification, i.e., oligo(dT) purification. For example, a polythymidine ligand may be immobilized to a derivatized chromatography resin or monolithic column. The mechanism of purification may involve hybridization of the poly(A) tail of the RNA molecule to the oligonucleotide ligand, wherein the DNA template will not bind. In preferred aspects, the RNA molecules that do not include Poly(A) stretches (e.g., abortive transcripts and other truncates formed during CF-IVT) will not bind to the resin and will not form a duplex with the affinity ligand. Poly-adenylated RNA may subsequently be eluted from the resin utilizing a low ionic strength buffer or a competitive binding oligonucleotide solution. In some aspects, adsorbed RNA may be eluted using a solution that prepares the RNA solution for further purification, encapsulation in encapsulating agents (e.g. LNPs), or formulation operations. In some aspects, the adsorption of RNA may occur on packed resin bead columns or a monolith. The process may operate with 1 or more columns in parallel and 1 or more columns in series. The columns may be operated independently or in a multi-column format where the effluent from one column is fed to the inlet of another column. After the first column is loaded, the CF-IVT product continues loading to the next column while the first loaded column is further processed and eluted. In some aspects, 2 or more columns with different modes of adsorption are used in series. The RNA may bind to 1 or more of the columns and RNA polymerase or other CF-IVT product components may bind to 1 or more of the columns. In some cases, the RNA polymerase and / or other CF-IVT product components may be eluted from the chromatography column and recycled for repeated use in the CF-IVT step. In some aspects, the amount of RNA loaded on the column is controlled by in-line, at-line, or offline concentration measurements. In some aspects, the measurements are by HPLC, NMR, Raman spectroscopy, or UV spectroscopy. In some aspects, the CF-IVT product may be mixed with additional buffer solutions (e.g., through in-line mixing, filtration, or surge vessel) before loading on the chromatographic step. In some aspects, where the RNA is bound to the chromatographic step, 1 or more wash steps may be applied to remove additional impurities or exchange the RNA into a new buffer solution. In some aspects, at least a portion of the CF-IVT product is filtered. The CF-IVT product may be filtered via ultrafiltration and / or diafiltration and / or single-pass variants of these to remove at least some impurities from the CF-IVT product and / or to change buffer solution for at least a portion of CF-IVT product to produce a concentrated RNA solution as a retentate. In some aspects, filtering the CF-IVT product comprises filtering the CF-IVT product as liquid feed in a single pass mode through a single pass tangential flow filtration (SPTFF) system and recovering the retentate (containing the RNA molecule) and permeate separately without recirculation through the SPTFF system, thereby filtering the liquid feed. In some aspects, the SPTFF system comprises a plurality of filtration modules that are fluidly connected. Each of the filtration modules comprises a manifold segment that includes a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module. In some aspects, each manifold allows the option to dilute the liquid feed or retentate prior to entering the filtration module to allow for in-line dilution. The filtration modules are fluidly connected through the manifold segments to provide a serial flow path between filtration modules, by coupling of the first manifold in a manifold segment to the second manifold of a manifold segment in an adjacent module, such that the retentate of one module serves as the feed for the next module. In some instances, the manifold is leveraged to dilute the retentate of one module prior to serving as feed for the next module. The manifold segment in each module is also fluidly connected to a plurality of TFF cassettes that are stacked on one or both faces of the manifold segment. In addition, the SPTFF system can comprise a feed inlet on the first module in the system and a retentate outlet on the last module in the system. In some aspects, the SPTFF system consists of one filtration module. The filtration module comprises a feed inlet, a retentate outlet, a manifold segment that comprises a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module, wherein the flow path through the manifold segment is serial, and a plurality of TFF cassettes that are stacked on one or both faces of, and are fluidly connected to, the manifold segment, wherein the liquid flow path is parallel through the cassettes. In some embodiments, described herein are methods of filtering a liquid feed, comprising passing a liquid feed through a tangential flow filtration (TFF) system, recovering permeate and a portion of the retentate from the system separately without recirculation through the TFF system, and recirculating the remainder of the retentate through the TFF system at least once, thereby filtering the liquid feed. In some aspects, the TFF system comprises a plurality of filtration modules that are fluidly connected. Each of the filtration modules comprises a manifold segment that includes a first manifold for receiving and carrying the feed into the filtration module, and in some aspects, receiving and carrying dilution buffer into the feed line, a second manifold for receiving and carrying retentate out of the filtration module, and in some aspects, receiving and carrying dilution buffer into said retentate line prior to feeding into an additional filtration module, and a third manifold for receiving and carrying permeate through the filtration module. The filtration modules are fluidly connected through the manifold segments to provide a serial flow path between filtration modules, by coupling of the first manifold in a manifold segment to the second manifold of a manifold segment in an adjacent module, such that the retentate of one module serves as the feed for the next module. In some aspects, a manifold segment is coupled to said manifold segments, such that the retentate of one module is diluted in-line and serves as the feed for the next module. The manifold segment in each module is also fluidly connected to a plurality of TFF cassettes that are stacked on one or both faces of the manifold segment. In addition, the TFF system comprises a feed inlet on the first module in the system, a retentate outlet on the last module in the system, a recirculation loop (e.g., a pump) for recirculating retentate through all or part of the system, and at least one conduit for recirculating retentate. In some aspects, the TFF system consists of one filtration module. The filtration module comprises a feed inlet, a retentate outlet, a recirculation loop (e.g., a pump) for recirculating retentate through all or part of the system, at least one conduit for recirculating retentate, a manifold segment that comprises a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module, wherein the flow path through the manifold segment is serial, and a plurality of TFF cassettes that are stacked on one or both faces of, and are fluidly connected to, the manifold segment, wherein the liquid flow path is parallel through the cassettes. In some aspects, a fourth manifold for receiving and carrying dilution buffer to the second manifold is fluidly connected to the manifold segment to allow for in-line dilution. The processes described herein can be performed using SPTFF and TFF systems that include diverter plates, thereby reducing the length of the flow path required to achieve effective serial processing and providing other advantages, such as improved flow distribution and multiplication of system size with a compact design and minimal external piping. In some aspects, filtration may be used to prepare the purified RNA in a solution that is appropriate for subsequent encapsulation in encapsulating agents (e.g. LNPs) and formulation operations. In some aspects, the RNA polymerase and / or other CF-IVT product components may be recovered from the CF-IVT product filtration permeate and recycled for repeated use in the CF- IVT step. In some aspects, the amount of RNA passed through the filter is controlled by in-line, at-line, or offline concentration measurements. In some aspects, the measurements are by HPLC, NMR, Raman spectroscopy, or UV spectroscopy. In some aspects, the CF-IVT product may be mixed with additional buffer solutions (e.g., through in-line mixing or surge vessel) before passing through the subsequent filtration step. In some aspects, the RNA purified by the methods described above may be mixed with additional buffer solutions (e.g., through in-line mixing, filtration, or surge vessel) to prepare the solution for subsequent encapsulation in encapsulating agents (e.g. LNPs) and formulation. In some aspects, the concentration of RNA after mixing with additional buffer solutions is controlled by in-line, at-line, or offline concentration measurements. In some aspects, the measurements are made by HPLC, NMR, Raman spectroscopy, or UV spectroscopy. In some aspects, both “ultrafiltration” and “diafiltration” refer to a membrane filtration process. Ultrafiltration typically uses membranes having pore sizes of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 µm. In some aspects, ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size. For example, the MWCO may be at least, at most, exactly, or between (inclusive or exclusive) any two of 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa, and 10000 kDa. A skilled artisan will understand that filtration membranes may comprise different suitable materials, including, e.g., polymers, cellulose, ceramic, etc., depending upon the application. In some aspects, membrane filtration may be more desirable for continuous and / or large volume purification process. In some aspects, the SPTFF systems may comprise any of the membranes described herein. In some embodiments, the RNA molecule has a clinical grade purity. In some embodiments, the purity of the RNA molecule is between about 60% and about 100%. In some embodiments, the purity of the RNA molecule is between about 80% and 99%. In some embodiments, the purity of the RNA molecule is between about 90% and about 99%. In some embodiments, the purified mRNA has a clinical grade purity without further purification. In some embodiments, the clinical grade purity is achieved through a method including adsorption or filtration purification. In some embodiments, the clinical grade purity is achieved with purification methods such as high-performance liquid chromatography (HPLC) purification, ligand or binding based purification, and / or ion exchange chromatography. In some embodiments, the method of purifying the RNA molecule removes short or long abortive RNA species, double-stranded RNA (dsRNA), residual template DNA, residual RNA polymerase, residual solvent, and / or residual salt. In some embodiments, the short abortive transcript contaminants comprise less than 15 bases. In some embodiments, the short abortive transcript contaminants comprise about 8-12 bases. In some embodiments, the method of purifying the RNA molecule also removes RNase inhibitor and inorganic yeast pyrophosphatase. In some embodiments, the method of purifying the RNA molecule also removes EDTA, proteinase K, and / or DNase I. In some embodiments, the method of purifying the RNA molecule may include, but is not limited to, phenol / chloroform extraction and / or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride for nucleic acid clean-up, quality assurance and quality control. Additional, non-limiting examples of RNA purification procedures include AGENCOURT® beads (BECKMAN COULTER GENOMICS), poly-T beads, LNA oligo-T capture probes (EXIQON INC), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction chromatography-HPLC (HIC-HPLC), size exclusion chromatography, and silica-based affinity chromatography and polyacrylamide gel electrophoresis. RNA purification can also be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (PROMEGA) and In vitro Transcription Cleanup and Concentration Kit (NORGEN BIOTEK). In some aspects, 1, 2, 3, 4, 5, or more of the foregoing purification methods may be excluded. Preferably, purified RNA material is substantially free of one or more impurities or contaminants including the linear DNA template and / or reverse complement transcription products described herein and for instance is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% pure; more preferably, at least 98% pure, and more preferably still at least 99% pure. Characterization and Analysis of the RNA Molecule The RNA molecule produced by the methods described herein may be analyzed and characterized using various methods. Analysis may be performed before or after capping. Alternatively, analysis may be performed before or after purification via poly(A) capture-based affinity, TFF, SPTFF, or other chromatographic and filtration purification methods. Analysis may be performed before or after purification via LiCl precipitation. In another aspect, analysis may be performed before or after additional purification steps, e.g., anion exchange chromatography and the like. RNA transcript integrity may be determined using electrophoresis (e.g., using the fragment analyzer capillary or Bioanalyzer chip systems) or through a reverse phase HPLC method. In other aspects, RNA purity is analyzed using analytical reverse phase HPLC. Capping efficiency may be analyzed using, e.g., total nuclease digestion followed by LC-UV or LC-MS quantitation of the dinucleotide cap species vs. uncapped GTP species. The level of residual DNA template (resDNA) can be measured using quantitative polymerase chain reaction (qPCR). The concentration of residual NTPs and / or 5’ cap analogs can be measured using anion- exchange chromatography on an HPLC-UV system. Next-Generation Sequencing (also referred to as massively parallel sequencing) which refers to non-Sanger sequencing technologies enables the determination of nucleic acid order which can be used to confirm the sequence identity of the mRNA transcripts as well as determine the location and frequency of sequence variants within said transcripts. In vitro efficacy may be analyzed by, e.g., transfecting RNA molecules into a human cell line. Protein expression of the polypeptide of interest may be quantified using methods such as ELISA or flow cytometry. Immunogenicity may be analyzed by, e.g., transfecting RNA molecules into cell lines that indicate innate immune stimulation, e.g., PBMCs. Cytokine induction may be analyzed using, e.g., methods such as ELISA to quantify a cytokine, e.g., Interferon-α. Residual enzymes may be quantified by microfluidic electrophoresis assays, e.g., Revvity LabChip pico protein assay. Double-stranded RNA (dsRNA) can be measured using methods such as a dot blotting assay or ELISA with, for example, an anti-dsRNA antibody. The methods of continuous production of an RNA molecule described herein may produce an RNA molecule that is at least 30% full-length transcript, or at least, at most, or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% full-length transcript, or any range or value derivable therein. Purity may be determined as described herein, e.g., via reverse phase HPLC or Bioanalyzer chip-based electrophoresis and measured by, e.g., peak area of full-length RNA molecule relative to total peak area. Genes of Interest The DNA template and resulting RNA molecule of the present invention include a gene of interest. The gene of interest encodes a polypeptide of interest selected from, e.g., biologics, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties or those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. The sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GenBank. In some aspects, the RNA molecule includes a coding region for an antigen preferably derived from a pathogen associated with infectious disease which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean- Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DENV-1, DENV-2, DENV-3 and DENV-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli O157:H7, 0111 and 0104:H4, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (including Influenza such as avian influenza and human influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, Coronavirus (e.g., SARS-CoV-2), Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis. In some aspects, the RNA molecules of the present disclosure encode a viral polypeptide or fragment thereof, including naturally occurring or engineered variants thereof, for prophylaxis against a virus in humans. Entrapment of RNA in Nanoparticles and Processing to Drug Product In some aspects, the in vitro transcribed RNA molecules within the RNA drug substance of the present disclosure may be encapsulated to form colloidal dispersions (e.g. RNA-loaded LNP dispersion) comprising at least one encapsulating agent. In one aspect, the encapsulating agent comprises one or more lipids, a lipid nanoparticle (LNP), lipoplexes, one or more polymers, polymeric particles, polyplexes, monolithic delivery systems, or a combination thereof. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing elements may be excluded as an encapsulating agent. In one aspect, the encapsulating agent is a lipid, and produced is an RNA-loaded LNP dispersion. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and / or the cationic polymer combine together with the nucleic acid to form colloidally stable dispersions. A lipid may be a naturally occurring lipid or a synthetic lipid. However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. As encapsulating agent, a lipid is a substance that is insoluble or partially insoluble in water and extractable with an organic solvent. Compounds other than those specifically described herein are understood by one of skill in the art as lipids and are encompassed by the compositions and methods of the present disclosure. A lipid component and a non-lipid may be attached to one another, either covalently or non-covalently. In some aspects, LNPs may be designed to protect RNA molecules with unmodified and / or modified nitrogenous bases and various sizes (e.g., mRNA, modified mRNA [modRNA], saRNA, gRNA and / or circRNA) from extracellular RNases and / or may be engineered for systemic delivery of the RNA to target cells. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intravenously administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intramuscularly administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intradermally administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intranasally administered to a subject in need thereof. In one aspect, the RNA in the RNA drug substance is at a concentration of < 1 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.05 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 1 mg / mL. In another aspect, the RNA concentration is from or from about 0.05 mg / mL to about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least 10 mg / mL. In another aspect, the RNA is at a concentration of at least 50 mg / mL. In some aspects, the RNA is or is not at a concentration of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, or more. The present disclosure provides for an RNA drug substance and a lipid preparation mixture or compositions thereof comprising at least one RNA encoding, e.g., an antigen complexed with, encapsulated in, and / or formulated with one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipoplexes and / or nanoliposomes. In some aspects, the composition comprises a lipid nanoparticle. A lipid nanoparticle or LNP refers to particles of any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and / or in the presence of RNA. In some aspects, lipid nanoparticles are included in a formulation that may be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some aspects, the lipid nanoparticles of the present disclosure comprise a nucleic acid (e.g., mRNA). Such lipid nanoparticles typically comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids, polymer conjugated lipids, or combinations thereof. In some aspects, the LNPs comprise at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., a steroid), and / or at least one polymer conjugated lipid (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, 1, 2, 3, or more of the foregoing excipients may be excluded from the LNPs. In some aspects, the LNPs comprise 20-60 mol% cationic (e.g., ionizable) lipid(s). For example, the LNPs may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30- 40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise or do not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise 45 to 55 mole percent (mol%) cationic (e.g., ionizable) lipid(s). For example, LNPs may comprise or not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise 5-25 mol% neutral (e.g., non-cationic) lipid(s). For example, the LNPs may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs comprise 5 to 15 mol% neutral (e.g., non-cationic) lipid(s). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs comprise 25-55 mol% structural lipid(s) (e.g., a steroid). For example, the LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30- 55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs comprise 35 to 40 mol% structural lipid(s) (e.g., a steroid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 35, 36, 37, 38, 39, or 40 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs comprise 0.5-15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). For example, the lipid nanoparticles (LNPs) may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% polymer conjugated lipid(s) (e.g., a PEG- conjugated lipid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). In some aspects, the LNPs comprise 1 to 2 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)- conjugated lipid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 1, 1.5, or 2 mol% polymer conjugated lipid(s) (e.g., a PEG- conjugated lipid). In some aspects, the LNPs comprise 20-75 mol% cationic (e.g., ionizable) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%), 0.5-25 mol% neutral (e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2.25%, 4%, 5.75%, 7.5%, 9.25%, 11%, 12.75%, 14.5%, 16.25%, 18%, 19.75%, 21.5%, 23.25%, and 25%), 5-55 mol% structural lipid(s) (e.g., a sterol) e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%), and 0.5-20 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2%, 3.5%, 5%, 6.5%, 8%, 9.5%, 11%, 12.5%, 14%, 15.5%, 17%, 18.5%, and 20%). In some aspects, 1, 2, 3, or more of the lipids may be excluded from the LNPs. In some non-limiting aspects, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 60 / 7.5 / 31 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 57.5 / 7.5 / 31.5 / 3.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 57.2 / 7.1 / 34.3 / 1.4 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 40 / 15 / 40 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 50 / 10 / 35 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 40 / 10 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 35 / 15 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), or 52 / 13 / 30 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid). In some aspects, the active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), may be encapsulated in the lipid portion of the lipid nanoparticle and / or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response. The nucleic acid (e.g., mRNA) or a portion thereof may also be associated and complexed with the lipid nanoparticle. A lipid nanoparticle may comprise any lipid capable of forming a particle to which the nucleic acids are attached, and / or in which the one or more nucleic acids are encapsulated. In some aspects, provided RNA molecules (e.g., mRNA, modRNA, saRNA, gRNA and / or circRNA) may be formulated with LNPs. In some aspects, the lipid nanoparticles may or may not have a mean diameter of or of about 1 to 500 nm (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nm). In some aspects, the lipid nanoparticles have a mean diameter of or of from about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or at least, at most, exactly, or between (inclusive or exclusive) of 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. The term “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys.57, 1972, pp 4814-4820, ISO 13321). Here, “mean diameter,” “diameter,” or “size” for particles is used synonymously with the value of the Z-average. LNPs described herein may exhibit a polydispersity index less than or less than about 0.5, 0.4, 0.3, or 0.2 or less. By way of example, the LNPs may or may not exhibit a polydispersity index of at least, at most, exactly, or between (inclusive or exclusive) of 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5. The polydispersity index is, in some aspects, calculated based on dynamic light scattering measurements by the so-called cumulant analysis referred to in the definition of “average diameter.” Under certain prerequisites, it may be taken as a measure of the size distribution of an ensemble of nanoparticles. In some aspects, an LNP of the disclosure comprises or does not comprise a molar ratio of positively chargeable nitrogen of tertiary amine in the cationic lipid to negatively charged phosphates of mRNA backbone (known as N:P ratio) of or of from about 2:1 to about 30:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 6:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 3:1. In some aspects, an LNP of the disclosure comprises or does not comprise a wt / wt ratio of the cationic lipid component to the RNA of or of from about 5:1 to about 100:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, or 100:1. In some aspects, an LNP of the disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of or of about 20:1. In some aspects, an LNP of the disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of or of about 10:1. In certain aspects, nucleic acids (e.g., RNA molecules), when present in provided LNPs, are resistant in aqueous solution to degradation with a nuclease. In some aspects, LNPs are liver- targeting lipid nanoparticles. In some aspects, LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some aspects, cationic LNPs may comprise at least one cationic lipid, at least one polymer conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid). In certain aspects, the mixture of RNA and lipid preparations or compositions thereof may have at least, at most, exactly, between (inclusive or exclusive) of, or about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a particular lipid, lipid type, or non-lipid component such as lipid-like materials and / or cationic polymers and / or an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid or other material disclosed herein or as would be known to one of skill in the art. LNPs described herein can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety. For example, methods of preparing LNPs may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. The term “colloid” as used herein relates to a type of mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal dispersion. Sometimes the term “colloid” refers only to the particles in the mixture and not the entire dispersion. While methods for preparing a colloid containing an organic solvent are described herein, other methods having organic solvent-free characteristics may also be used according to the present disclosure. In some aspects, an RNA-loaded LNP dispersion may be produced by inline mixing of an RNA solution or adjusted RNA solution described herein (e.g., an RNA drug substance) and a lipid preparation described herein (comprising, e.g., at least one cationic lipid and optionally one or more other lipid components, in an organic solvent) under conditions such that a sudden change in solubility of lipid component(s) is triggered, which drives the lipids towards self-assembly in the form of LNPs. In some aspects, suitable buffering agents comprise tris, histidine, citrate, acetate, phosphate, and / or succinate. In some aspects, 1, 2, 3, or more of the foregoing buffering agents are excluded. The pH of a liquid formulation relates to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer may be at least half a pH scale less than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half a pH scale greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, properties of a cationic lipid are chosen such that nascent formation of particles occurs by association with an oppositely charged backbone of a nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which, for example, in some aspects, may result in greater encapsulation efficiency than is achieved in the absence of interactions between nucleic acids and at least one of the lipid components. In certain aspects, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes. Some aspects described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species, such as RNA species. In an LNP formulation, it is possible that each nucleic acid species is separately formulated as an individual LNP formulation. In that case, each individual LNP formulation will comprise one nucleic acid species. The individual LNP formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations). The resulting dispersion following the formation of LNPs can be filtered as liquid feed in a single pass mode through a single pass tangential flow filtration (SPTFF) system and recovering the retentate (containing the LNPs) and permeate from the system in separate containers without recirculation through the SPTFF system, thereby filtering the liquid feed. In some aspects, the SPTFF system comprises a plurality of filtration modules that are fluidly connected. Each of the filtration modules comprises a manifold segment that includes a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module. The filtration modules are fluidly connected through the manifold segments to provide a serial flow path between filtration modules, by coupling of the first manifold in a manifold segment to the second manifold of a manifold segment in an adjacent module, such that the retentate of one module serves as the feed for the next module. The manifold segment in each module is also fluidly connected to a plurality of TFF cassettes that are stacked on one or both faces of the manifold segment. In addition, the SPTFF system comprises a feed inlet on the first module in the system and a retentate outlet on the last module in the system. In another aspect, the SPTFF system consists of one filtration module. The filtration module comprises a feed inlet, a retentate outlet, a manifold segment that comprises a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module, wherein the flow path through the manifold segment is serial, and a plurality of TFF cassettes that are stacked on one or both faces of, and are fluidly connected to, the manifold segment, wherein the liquid flow path is parallel through the cassettes. In another embodiment, described herein are methods of filtering a liquid feed, comprising passing a liquid feed through a tangential flow filtration (TFF) system, recovering permeate and a portion of the retentate from the system in separate containers without recirculation through the TFF system, and recirculating the remainder of the retentate through the TFF system at least once, thereby filtering the liquid feed. In another aspect, the TFF system comprises a plurality of filtration modules that are fluidly connected. Each of the filtration modules comprises a manifold segment that includes a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module. The filtration modules are fluidly connected through the manifold segments to provide a serial flow path between filtration modules, by coupling of the first manifold in a manifold segment to the second manifold of a manifold segment in an adjacent module, such that the retentate of one module serves as the feed for the next module. The manifold segment in each module is also fluidly connected to a plurality of TFF cassettes that are stacked on one or both faces of the manifold segment. In addition, the TFF system comprises a feed inlet on the first module in the system, a retentate outlet on the last module in the system, a recirculation loop (e.g., a pump) for recirculating retentate through all or part of the system, and at least one conduit for recirculating retentate. In another aspect, the TFF system consists of one filtration module. The filtration module comprises a feed inlet, a retentate outlet, a recirculation loop (e.g., a pump) for recirculating retentate through all or part of the system, at least one conduit for recirculating retentate, a manifold segment that comprises a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module, wherein the flow path through the manifold segment is serial, and a plurality of TFF cassettes that are stacked on one or both faces of, and are fluidly connected to, the manifold segment, wherein the liquid flow path is parallel through the cassettes. The processes described herein can be performed using SPTFF and TFF systems that include diverter plates, thereby reducing the length of the flow path required to achieve effective serial processing and providing other advantages, such as improved flow distribution and multiplication of system size with a compact design and minimal external piping. The method of filtration of a liquid feed in a single pass mode through a single pass tangential flow filtration (SPTFF) system is disclosed in, e.g., U.S. Patent No.11,679,349 and PCT Pub. No. WO 2016 / 033546A1, the full disclosures of which are herein incorporated by reference in their entirety for all purposes. In some aspects, the described SPTFF systems can be used to purify and adjust the concentration of the RNA-loaded LNP dispersion. The resulting composition of the post-SPTFF dispersion can be continuously adjusted through inline bifurcating mixers, followed by passing through filters for bioburden reduction and sterilization to obtain RNA drug product. In some aspects, the processes described herein comprise a bioburden reduction filter that removes aggregates at least 0.2 micron or larger. In some aspects, the RNA drug product can be passed to a surge tank for transferring of the RNA drug product into containers / closure systems. In some aspects, a composition such as a pharmaceutical composition comprises more than one individual LNP formulation. Respective pharmaceutical compositions are referred to as mixed LNP formulations. Mixed LNP formulations according to the invention are obtainable by forming, separately, individual LNP formulations, as described above, followed by a step of mixing of the individual LNP formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing LNPs is obtainable. Individual LNP populations may be together in one container, comprising a mixed population of individual LNP formulations. The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye / colorant, and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included or exclude. A liquid pharmaceutical composition, whether they be solutions, suspensions, or other like forms, may include or exclude one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile. Alternatively, it is possible that different nucleic acid species are formulated together as a combined LNP formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of different RNA species together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. As opposed to a mixed LNP formulation, a combined LNP formulation will typically comprise LNPs that comprise more than one RNA species. In a combined LNP composition, different RNA species are typically present together in a single particle. CATIONIC POLYMERIC MATERIALS Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically condense the negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L- lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic materials useful in some aspects herein. In addition, some investigators have synthesized polymeric materials specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. In some aspects, such synthetic materials may be suitable for use as cationic materials herein. A “polymeric material,” as used herein, is given its ordinary meaning, e.g., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. In some aspects, such repeat units may all be identical; alternatively, in some cases, there may be more than one type of repeat unit present within the polymeric material. In some cases, a polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties may also be present in the polymeric material, for example targeting moieties such as those described herein. Those skilled in the art are aware that, when more than one type of repeat unit is present within a polymer (or polymeric moiety), then the polymer (or polymeric moiety) is said to be a “copolymer.” In some aspects, a polymer (or polymeric moiety) utilized in accordance with the present disclosure may be a copolymer. Repeat units forming the copolymer may be arranged in any fashion. For example, in some aspects, repeat units may be arranged in a random order; alternatively or additionally, in some aspects, repeat units may be arranged in an alternating order, or as a “block” copolymer, e.g., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks. In certain aspects, a polymeric material for use in accordance with the present disclosure is biocompatible. Biocompatible materials are those that typically do not result in significant cell death at moderate concentrations. In certain aspects, a biocompatible material is biodegradable, e.g., is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body. In certain aspects, a polymeric material may be or comprise protamine or polyalkylene imine, in particular protamine. As those skilled in the art are aware, the term “protamine” is often used to refer to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (e.g., fish). In particular, the term “protamine” is often used to refer to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin. In some aspects, the term “protamine” as used herein is refers to a protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and / or multimeric forms of said amino acid sequence or fragment thereof, as well as (synthesized) polypeptides that are artificial and designed for specific purposes and cannot be isolated from native or biological sources. In some aspects, a polyalkylene imine comprises polyethylenimine and / or polypropylenimine. In some aspects, the polyalkylene imine is polyethyleneimine (PEI). In some aspects, the polyalkylene imine is a linear polyalkylene imine, e.g., linear polyethyleneimine (PEI). Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those which are able to electrostatically bind nucleic acid. In some aspects, cationic polymeric materials contemplated for use herein include any cationic polymeric materials with which nucleic acid may be associated, e.g., by forming complexes with the nucleic acid and / or forming vesicles in which the nucleic acid is enclosed or encapsulated. In some aspects, particles described herein may comprise polymers other than cationic polymers, e.g., non-cationic polymeric materials and / or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials. LIPIDS & LIPID-LIKE MATERIALS The terms “lipid” and “lipid-like material” are used herein to refer to molecules that comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH. The term “lipid” refers to a group of organic compounds that are characterized by being insoluble or partially soluble in water but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids as well as sterol-containing metabolites such as cholesterol, and prenol lipids. Examples of fatty acids include, but are not limited to, fatty esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycosylglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramides phosphosphingolipids (e.g., sphingomyelins, phosphocholine), and glycosphingolipids (e.g., cerebrosides, gangliosides). Examples of sterol lipids include, but are not limited to, cholesterol and its derivatives and tocopherol and its derivatives. In some aspects, 1, 2, 3, 4, 5, or more of the lipids may be excluded from the LNPs of the present disclosure. The term “lipid-like material,” “lipid-like compound,” or “lipid-like molecule” relates to substances that structurally and / or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment, and includes surfactants or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules that comprise hydrophilic and hydrophobic moieties with different structural organization that may or may not be similar to that of lipids. In some aspects, the mixture of RNA and lipid preparations or compositions thereof may comprise cationic lipids, neutral lipids, cholesterol, and / or polymer (e.g., polyethylene glycol)-conjugated lipids which form lipid nanoparticles that encompass the RNA molecules. Therefore, in some aspects, the LNP may comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids or steroid analogs (e.g., cholesterol), polymer conjugated lipids (e.g., PEG-lipid), or combinations thereof. In some aspects, 1, 2, 3, or more of the foregoing excipients may be excluded from the LNPs of the present disclosure. In some aspects, the lipids are present in a composition in an amount that is effective to form a lipid nanoparticle and deliver a therapeutic agent, e.g., an RNA molecule, for treating a particular disease or condition of interest. In some aspects, the LNPs encompass, or encapsulate, the nucleic acid molecules. CATIONIC LIPIDS Cationic or cationically ionizable lipids or lipid-like materials refer to a lipid or lipid-like material capable of being positively charged and able to electrostatically bind nucleic acid. As used herein, a “cationic lipid” or “cationic lipid-like material” refers to a lipid or lipid-like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl, or more acyl chains, and the head group of the lipid typically carries the positive charge. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Cationic lipids may encapsulate negatively charged RNA. In some aspects, cationic lipids are ionizable such that they may exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. Without wishing to be bound by theory, this ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH. For purposes of the present disclosure, such “cationically ionizable” lipids or lipid-like materials are comprised by the term “cationic lipid” or “cationic lipid-like material” unless contradicted by the circumstances. In some aspects, a cationic lipid may comprise from or from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle. In some aspects, a cationic lipid may or may not be at least, at most, exactly, or between (inclusive or exclusive) of 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, 90 mol %, or 100 mol %, or any range or value derivable therein, of the total lipid present in the particle. Examples of cationic lipids include, but are not limited to: ((4-hydroxybutyl)azanediyl)bis(hexane- 6,1-diyl)bis(2-hexyldecanoate), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N,N- dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-( N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propanes, 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl- 1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc- tadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-l- (cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy- N,N-dimethylpropylamine (DLinDAP), 1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin- KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3- DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1- propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl- 2,3-bis(tetradecyloxy)-1-propanaminium bromide (bAE-DMRIE), N-(4-carboxybenzyl)-N,N- dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3b)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl- CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3- dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn- glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8′- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2- dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}- ethylamino)propionamide (lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2- [bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid 02-200); C 12-200; or heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl) amino) octanoate (SM-102). In some aspects, 1, 2, 3, 4, 5, or more of the foregoing cationic lipids may be excluded from the LNPs of the present disclosure. In some aspects, an ionizable cationic lipid of the disclosure comprises a compound of Formula (I): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: R1is a C5-30alkyl, C5-20alkenyl, -R*YR”, -YR”, or -R”M’R’; R2and R3are independently a H, C1-14alkyl, C2-14alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4is a C3-6carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, or unsubstituted C1-6alkyl, where Q is a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, - CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, - N(R)C(S)N(R)2, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, - C(O)N(R)OR, or -C(R)N(R)2C(O)OR, and / or each n is independently a 1, 2, 3, 4, or 5; each R5is independently a C1-3alkyl, C2-3alkenyl, or H; each R6is independently a C1-3alkyl, C2-3alkenyl, or H; M and M’ are independently a -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group; R7is a C1-3alkyl, C2-3alkenyl, or H; R8is a C3-6carbocycle or heterocycle; R9is a H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6alkenyl, C3-6carbocycle, or heterocycle; each R is a C1-3alkyl, C2-3alkenyl, or H; each R' is a C1-18 alkyl, C2-18alkenyl, -R*YR”, -YR”, or H; each R” is a C3-14alkyl or C3-14alkenyl; each R* is independently a C1-12alkyl or C2-12alkenyl; each Y is independently a C3-6carbocycle; each X is independently a F, Cl, Br, or I; and m is a 5, 6, 7, 8, 9, 10, 11, 12, or 13. In some aspects, a subset of compounds of Formula (I) includes those in which when R4is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, then (i) Q is not -N(R)2when n is 1, 2, 3, 4, or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2. In some aspects, another subset of compounds of Formula (I) includes those in which R1is a C5-30alkyl, C5-20alkenyl, -R*YR”, -YR”, or -R”M’R’; R2and R3are independently an H, C1-14alkyl, C2-14alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4is a C3-6carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, or unsubstituted C1-6alkyl, where Q is a C3-6carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms comprising N, O, or S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, - CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, - CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, - OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, - C(O)N(R)OR, or a 5- to 14-membered heterocycloalkyl having one or more heteroatoms comprising N, O, and S which is substituted with one or more substituents comprising oxo (=O), OH, amino, mono- or di- alkylamino, or C1-3alkyl, and / or each n is independently 1, 2, 3, 4, or 5; each R5is independently a C1-3alkyl, C2-3alkenyl, or H; each R6is independently a C1-3alkyl, C2-3alkenyl, or H; M and M' are independently a -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group; R7is a C1-3alkyl, C2-3alkenyl, or H; R8is a C3-6carbocycle or heterocycle; R9is a H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6alkenyl, C3-6carbocycle or heterocycle; each R is independently a C1-3alkyl, C2-3alkenyl, or H; each R’ is independently a C1-18alkyl, C2-18alkenyl, -R* YR”, -YR”, or H; each R” is independently a C3-14alkyl or C3-14alkenyl; each R* is independently a C1-12alkyl or C2-12alkenyl; each Y is independently a C3-6carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11, 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof. In some aspects, another subset of compounds of Formula (I) includes those in which: R1is a C5-30alkyl, C5-20alkenyl, -R*YR”, -YR”, or -R”M’R’; R2and R3are independently an H, C1-14alkyl, C2-14alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4is a C3-6carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, or unsubstituted C1-6alkyl, where Q is a C3-6carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms comprising N, O, or S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, - CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, - CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, - OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, or - C(=NR9)N(R)2, and / or each n is independently 1, 2, 3, 4, or 5; and / or when Q is a 5- to 14- membered heterocycle and (i) R4is -(CH2)nQ in which n is 1 or 2, or (ii) R4is -(CH2)nCHQR in which n is 1, or (iii) R4is -CHQR, and -CQ(R)2, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl; each R5is independently a C1-3alkyl, C2-3alkenyl, or H; each R6is independently a C1-3alkyl, C2-3alkenyl, or H; M and M' are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group; R7is a C1-3alkyl, C2-3alkenyl, or H; R8is C3-6carbocycle or heterocycle; R9is H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6alkenyl, C3-6carbocycle, or heterocycle; each R is independently a C1-3alkyl, C2-3alkenyl, or H; each R’ is independently a C1-18alkyl, C2-18alkenyl, -R*YR”, -YR”, or H; each R” is independently a C3-14alkyl or C3-14alkenyl; each R* is independently a C1-12alkyl or C2-12alkenyl; each Y is independently a C3-6carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11, 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof. In some aspects, another subset of compounds of Formula (I) includes those in which: R1is a C5-30alkyl, C5-20alkenyl, -R*YR”, -YR”, or -R”M’R’; R2and R3are independently an H, C1-14alkyl, C2-14alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4is a C3-6carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, or unsubstituted C1-6alkyl, where Q is a C3-6carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms comprising N, O, or S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, - CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, - CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, - OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, or - C(=NR9)N(R)2, and / or each n is independently 1, 2, 3, 4, or 5; each R5is independently a C1-3alkyl, C2-3alkenyl, or H; each R6is independently a C1-3alkyl, C2-3alkenyl, or H; M and M' are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group; R7is a C1-3alkyl, C2-3alkenyl, or H; R8is a C3-6carbocycle or heterocycle; R9is an H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6alkenyl, C3-6carbocycle, or heterocycle; each R is independently a C1-3alkyl, C2-3alkenyl, or H; each R' is independently a C1-18alkyl, C2-18alkenyl, -R*YR”, -YR”, or H; each R” is independently a C3-14alkyl or C3-14alkenyl; each R* is independently a C1-12alkyl or C2-12alkenyl; each Y is independently a C3-6carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11, 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof. In some aspects, another subset of compounds of Formula (I) includes those in which: R1is a C5-30alkyl, C5-20alkenyl, -R*YR”, -YR”, or -R”M’R’; R2and R3are independently an H, C2-14alkyl, C2-14alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2, and / or n is 3, 4, or 5; each R5is independently a C1-3alkyl, C2-3alkenyl, or H; each R6is independently a C1-3alkyl, C2-3alkenyl, or H; M and M’ are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group; R7is a C1-3alkyl, C2-3alkenyl, or H; each R is independently a C1-3alkyl, C2-3alkenyl, or H; each R’ is independently a C1-18alkyl, C2-18alkenyl, -R*YR”, -YR”, or H; each R” is independently a C3-14alkyl or C3-14alkenyl; each R* is independently a C1-12alkyl or C1-12alkenyl; each Y is independently a C3-6carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11, 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof. In some aspects, another subset of compounds of Formula (I) includes those in which: R1is a C5-30alkyl, C5-20alkenyl, -R*YR”, -YR”, or -R”M’R’; R2and R3are independently a C1-14alkyl, C2-14alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4is a -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, where Q is -N(R)2, and / or n is 1, 2, 3, 4, or 5; each R5is independently a C1-3alkyl, C2-3alkenyl, or H; each R6is independently a C1-3alkyl, C2-3alkenyl, or H; M and M' are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group; R7is a C1-3alkyl, C2-3alkenyl, or H; each R is independently a C1-3alkyl, C2-3alkenyl, or H; each R' is independently a C1-18alkyl, C2-18alkenyl, -R*YR”, -YR”, or H; each R” is independently a C3-14alkyl or C3-14alkenyl; each R* is independently a C1-12alkyl or C1-12alkenyl; each Y is independently a C3-6carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11, 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof. In some aspects, a subset of compounds of Formula (I) includes those of Formula (IA): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein I is 1, 2, 3, 4, or 5; m is 5, 6, 7, 8, or 9; M1is a bond or M’; R4is unsubstituted C1-3alkyl, or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, - N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently a -C(O)O-, -OC(O)-, -C(O)N(R’)-, -P(O)(OR’)O-, - S-S-, an aryl group, or a heteroaryl group; and R2and R3are independently a H, C1-14alkyl, or C2-14alkenyl. In some aspects, a subset of compounds of Formula (I) includes those of Formula (II): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein I is 1, 2, 3, 4, or 5; M1is a bond or M’; R4is unsubstituted C1-3alkyl, or -(CH2)nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR’)O-, - S-S-, an aryl group, or a heteroaryl group; and R2and R3are independently a H, C1-14alkyl, or C2-14alkenyl. In some aspects, a subset of compounds of Formula (I) includes those of Formula (Ila), (lIb), (lIc), or (lIe): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein R4is as described herein. In some aspects, a subset of compounds of Formula (I) includes those of Formula (IId): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R2through R6are as described herein. For example, each of R2and R3may be independently a C5-14alkyl or C5-14alkenyl. In some aspects, an ionizable cationic lipid of the disclosure comprises a compound having structure: In some aspects, an ionizable cationic lipid of the disclosure comprises a compound having structure: In some aspects, an ionizable cationic lipid of the disclosure comprises a compound having structure: or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, - NRaC(=O)-, -C(=O) =NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, - C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- oradirect bond; G1and G2are each independently unsubstituted C1-C12alkylene or C1- C12alkenylene; G is C1-C24alkylene, C1-C24alkenylene, C3-C8cycloalkylene, C3-C8cycloalkenylene; Rais H or C1-C12 alkyl; R1and R2are each independently C6-C24alkyl or C6-C24alkenyl; R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4; R4is C1-C12alkyl; R5is H or C1-C6alkyl; and x is 0, 1, or 2. In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R6is, at each occurrence, independently H, OH or C1-C24alkyl; and n is an integer ranging from 1 to 15. In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein y and z are each independently integers ranging from 1 to 12. In any of the foregoing aspects, one of L1or L2is -OCCO)-. For example, in some aspects, each of L1and L2are -O(C=O)-. In some aspects of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some aspects, each of L1and L2is - (C=O)O-. In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof. In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof. In some of the foregoing aspects, n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some aspects, n is 3, 4, 5, or 6. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6. In some of the foregoing aspects, y and z are each independently an integer ranging from 2 to 10. For example, in some aspects, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6. In some of the foregoing aspects, R6is H. In other of the foregoing embodiments, R6is C1-C24alkyl. In other aspects, R6is OH. In some embodiments, G is unsubstituted. In other aspects, G3is substituted. In various different aspects, G3is linear C1-C24alkylene or linear C1- C24alkenylene. In some other foregoing embodiments, R1or R2, or both, is C6-C24alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure: wherein: R7aand R7bare, at each occurrence, independently H or C1-C12alkyl; and a is an integer from 2 to 12, wherein R7a, R7b, and a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12. In some of the foregoing aspects, at least one occurrence of R7ais H. For example, in some aspects, R7ais H at each occurrence. In other different aspects of the foregoing, at least one occurrence of R7bis C1-C8alkyl. For example, in some embodiments, C1-C8alkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl. In different aspects, R1or R2, or both, has one of the following: In some of the foregoing aspects, R is OH, CN, -C(=O)OR4-OC(=O)R4or -NHC(=O)R4. In some aspects, R4is methyl or ethyl. It is understood that any aspect of the compounds set forth above, and any specific substituent and / or variable in the compounds set forth above, may be independently combined with other aspects and / or substituents and / or variables of compounds to form aspects of the inventions not specifically set forth above. In addition, in the event that a list of substituents and / or variables is listed for any particular substituent and / or variable in a particular embodiment and / or claim, it is understood that each individual substituent and / or variable may be deleted from the particular aspect and / or claim and that the remaining list of substituents and / or variables will be considered to be within the scope of the disclosure. It is understood that in the present description, combinations of substituents and / or variables of the depicted formulae are permissible only if such contributions result in stable compounds. In some embodiments, the cationic lipid is In some embodiments, the cationic lipid is In some aspects, the lipid nanoparticles comprise one or more cationic lipids. In one aspect, the lipid nanoparticles comprise (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) (ALC-0315), having the formula: Exemplary cationic lipids are disclosed in, e.g., U.S.10,166,298, the full disclosure of which is herein incorporated by reference in its entirety for all purposes. Representative cationic lipids include: No. Structure 1 30 31 32 33 34 35 36 In some aspects, 1, 2, 3, 4, 5, or more of the foregoing cationic lipids may be excluded from the LNPs of the present disclosure. In some aspects, the RNA-loaded LNPs comprise a cationic lipid, an RNA molecule as described herein, and one or more of neutral lipids, steroids, pegylated lipids, or combinations thereof. In one aspect, the cationic lipid is or is not present in the LNP in an amount such as at least, at most, exactly, between (inclusive or exclusive) of, or about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent (mol %). In some aspects, two or more cationic lipids are incorporated within the LNP. If more than one cationic lipid is incorporated within the LNP, the foregoing percentages apply to the combined cationic lipids. In some aspects, an ionizable cationic lipid of the disclosure comprises a compound of Formula (I) Formula (I) or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof, wherein m, n, o, and p are each independently 1 - 3; G1is C1-12alkylene or C2-12alkenylene; R1is -N(R2)R3, -OR4, CN, -N(R4)(heteroaryl), -O(CH2)qOH, -(OCH2CH2)rOH, -OC(=O)R5, -N(R4)C(=O)R5, -N(R4)S(O)2R5, -N(R4)C(=O)N(R2)R3, -OC(=O)N(R2)R3, -N(R4)C(=O)OR5, - N(R4)C(=S)N(R2)R3, -N(R4)C(=NR6)N(R2)R3, or R2and R3are each independently H, C1-6alkyl, C3-8cycloalkyl, or aryl; or R2and R3together with the nitrogen atom to which they are attached form a heterocyclic ring; R4is H, C1-6alkyl or C3-8cycloalkyl; R5is C1-6alkyl or C3-8cycloalkyl; R6is H, CN, NO2, C1-6alkyl, OR5, S(O)2R5, S(O)2N(R2)R3; q is 2 – 6; r is 1 – 6; X is N or CH; G2and G3are each independently C1-12alkylene or C2-12alkenylene; L1and L2are each independently -C(=O)OR7, -OC(=O)R7, -OC(=O)(CH2)rC(=O)OR7, - OC(=O)(CH2)rOC(=O)R7, -OC(=O)N(R4)R7, -N(R4)C(=O)OR7, -N(R4)C(=O)N(R4)R7, - OC(=O)OR7, or -S-SR7; and R7is C6-24alkyl, C6-24alkenyl, or C6-24alkynyl where each is optionally substituted by F, C1-6alkoxy, C3-8cycloalkyl, or C3-8cycloalkenyl. In some aspects, the compound of Formula (I) has the structure of Formula (Ia) Formula (Ia) or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof, wherein m, n, o, and p are each independently 1 or 2. In some aspects, the compound of Formula (I) has the structure of Formula (Ib) Formula (Ib) or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof, wherein m, n, o, and p are each independently 1 or 2. In some aspects, the compound of Formula (I) has the structure of Formula (Ic) Formula (Ic) or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof, wherein m and n are each independently 1 or 2; and o and p are each 1. In some aspects, the compound of Formula (I) has a structure wherein G1is C1-12alkylene; R2and R3are each independently H, C1-6alkyl, or C3-8cycloalkyl; or R2and R3together with the nitrogen atom to which they are attached form a heterocyclic ring; R4is H, C1-6alkyl or C3-8cycloalkyl; G2and G3are each independently C1-12alkylene; L1and L2are each -OC(=O)R7; and R7is C6-24alkyl, C6-24alkenyl, or C6-24alkynyl wherein each is optionally substituted by F, C1-6alkoxy, C3-8cycloalkyl, or C3-8cycloalkenyl. In some aspects, the compound of Formula (I) has a structure wherein R7has the following structure: In some aspects, the compound of Formula (I) has a structure wherein R1is OH. In some aspects, the compound of Formula (I) has a structure wherein R1is . In some aspects, the compound of Formula (I) has a structure wherein G1is C2-C5alkylene. In some aspects, the compound of Formula (I) has a structure wherein G1is C3-C5alkylene. In some aspects, the compound of Formula (I) is selected from the group consisting of: (3-(4-hydroxybutyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene) bis(2- heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl bis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diyl bis(2- heptylnonanoate); and 2-(3-(4-hydroxybutyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diyl bis(2- heptylnonanoate), or a pharmaceutically acceptable salt thereof. In some aspects, an ionizable cationic lipid of the disclosure comprises a compound of Formula (II) Formula (II) or a pharmaceutically acceptable salt, tautomer, N-oxide, or stereoisomer thereof, wherein G1, G2and G3are independently C1-12alkylene or C2-12alkenylene; L1and L2are independently -C(=O)O- or -OC(=O)-; R1is C6-24alkyl, C6-24alkenyl, or C6-24alkynyl where each is substituted by W-Y; R2is C6-24alkyl, C6-24alkenyl, or C6-24alkynyl where each is optionally substituted by W-Y; W is independently C1-6alkylene or a bond; Y is independently C3-12cycloalkyl, C5-12cycloalkenyl, or a 4-6 membered heterocycloalkyl containing one oxygen atom, with the proviso that when R2is C6-24alkyl, R1is C6-24alkyl substituted by W-Y, and W is a bond, then Y is not cyclopropyl; R3is -N(R4)R5, -OR6, -O(CH2)qOH, -(OCH2CH2)rOH, -N(R6)C(=O)R7, -N(R6)S(O)2R7, - N(R6)C(=O)N(R4)R5, -OC(=O)N(R4)R5, -N(R6)C(=O)OR7, -N(R6)C(=S)N(R4)R5, R4and R5are each independently H, C1-6alkyl, C3-8cycloalkyl, or aryl; or R4and R5together with the nitrogen atom to which they are attached form a heterocyclic ring; R6is H, C1-6alkyl or C3-8cycloalkyl; R7is C1-6alkyl or C3-8cycloalkyl; q is 2 – 6; r is 1 – 6. In some aspects, the compound of Formula (II) has the structure of Formula (IIa) Formula (IIa) or a pharmaceutically acceptable salt, tautomer, N-oxide, or stereoisomer thereof, wherein R8is independently C4-16alkyl. In some aspects, the compound of Formula II has a structure wherein G1, G2, and G3are independently C1-12alkylene; L1is independently -C(=O)O- or -OC(=O)-; L2is independently -C(=O)O- or -OC(=O)-; R1is C6-24alkyl substituted by W-Y; R2is C6-24alkyl optionally substituted by W-Y; W is independently -CH2- or -CH2CH2-; Y is independently C3-12cycloalkyl, C5-12cycloalkenyl, or a 4-6 membered heterocycloalkyl containing one oxygen atom; R4and R5are each independently H, C1-6alkyl, C3-8cycloalkyl, or aryl; or R4and R5together with the nitrogen atom to which they are attached form a heterocyclic ring; R6is H, C1-6alkyl or C3-8cycloalkyl. In some aspects, the compound of Formula (II) has a structure wherein Y has the following structure: In some aspects, the compound of Formula (II) has a structure wherein R3is -OH. In some aspects, the compound of Formula (II) has a structure wherein R3is . In some aspects, the compound of Formula (II) has a structure wherein G3is C2-5alkylene. In some aspects, the compound of Formula (II) has a structure wherein R1and R2have the following structure: In some aspects, the compound of Formula (II) is ((4-Hydroxybutyl)azanediyl)bis(hexane-6,1- diyl) bis(2-(cyclobutylmethyl)decanoate), or a pharmaceutically acceptable salt thereof. In some aspects of the disclosure, the LNP comprises a combination or mixture of any the lipids described above. POLYMER CONJUGATED LIPIDS In some aspects, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid (e.g., polyethylene glycol-lipid, PEG-lipid). In certain aspects, the LNP comprises an additional, stabilizing lipid that is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and mixtures thereof. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, PEG-DSG, PEG-DPG, and PEG-s-DMG (1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol). In one aspect, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one aspect, the polyethylene glycol-lipid is PEG-2000-DMG. In one aspect, the polyethylene glycol-lipid is PEG- c-DOMG. In other aspects, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4- O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-((O-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. PEG-lipids are disclosed in, e.g., U.S.9,737,619, the full disclosures of which is herein incorporated by reference in its entirety for all purposes. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing pegylated lipids may be excluded from the LNPs of the present disclosure. In some aspects, the composition comprises a pegylated lipid having the following structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: R8and R9are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some aspects, R8 and R9are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some aspects, w has a mean value ranging from 43 to 53. In other aspects, the average w is or is about 45. In other different embodiments, the average w is or is about 49. In some aspects, the lipid nanoparticles comprise a polymer conjugated lipid. In one aspect, the lipid nanoparticle comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC- 0159), having the formula: In some aspects, the lipid nanoparticles comprise a polymer conjugated lipid compound of Formula (III): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1and R2are each independently hydrogen or methyl; R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor; R5is hydrogen or halogen; m is an integer from 1 to 6; Y is a polymer having Formula (IV):

[0002] n is an integer from 1 to 1000; x is an integer from 2 to 6; and w is an integer from 2 to 4. In a preferred embodiment, m is 3, x is 2 and w is 2. In another embodiment, R5is -Br. In some aspects, the polymer conjugated lipid compound of Formula (III) is Formula (IIIa): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor; R5is hydrogen or halogen; and n is an integer from 1 to 1000. In some aspects, the polymer conjugated lipid compound of Formula (III) is Formula (IIIb): or a pharmaceutically acceptable salt or stereoisomer thereof. In some aspects, the polymer conjugated lipid compound of Formula (III) comprises a structure wherein (i) n ranges from 15 to 19, (ii) n ranges from 20 to 24, (iii) n ranges from 25 to 29, (iv) n ranges from 30 to 33, or (v) n ranges from 34 to 38. In some aspects, the polymer conjugated lipid compound of Formula (III) comprises a structure wherein the polymer conjugated lipid compound has a polydispersity index of about 1.0 to about 2.0. In some aspects, the polymer conjugated lipid compound of Formula (III) comprises a structure wherein the polymer conjugated lipid compound has a polydispersity index of between 1.0 to 1.4. In a preferred embodiment, the polydispersity index is 1.2. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between 100 Daltons and 100,000 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between 4,500 Daltons and 12,000 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between about 4,800 Daltons and about 5,300 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between about 8,300 Daltons and about 8,900 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between about 11,200 Daltons and about 11,800 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) is selected from the group consisting of: (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-8k]; and (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-11k], or a pharmaceutically acceptable salt or stereoisomer thereof. In various aspects, the molar ratio of the cationic lipid to the pegylated lipid ranges from or from about 100:1 to about 20:1, e.g., 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1, or any range or value derivable therein. In certain aspects, the PEG-lipid is or is not present in the LNP in an amount from or from about 1 to about 10 mole percent (mol %) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol %), relative to the total lipid content of the nanoparticle. In some aspects, the ratio of PEG in the lipid nanoparticle formulations may be increased or decreased and / or the carbon chain length of the PEG lipid may be modified to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulations. ADDITIONAL LIPIDS In certain aspects, the LNP comprises one or more additional lipids or lipid-like materials that stabilize particles during their formation. Suitable stabilizing or structural lipids include non- cationic lipids, e.g., neutral lipids and anionic lipids. Without being bound by any theory, optimizing the formulation of LNPs by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable / cationic lipid or lipid-like material may enhance particle stability and efficacy of nucleic acid delivery. As used herein, an “anionic lipid” refers to any lipid that is negatively charged at a selected pH. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. In some aspects, additional lipids comprise one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Representative neutral lipids include phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines, ceramides, sphingomyelins, dihydro-sphingomyelins, cephalins, and cerebrosides. Exemplary phospholipids include, for example, phosphatidylcholines, e.g., diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl- phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and 1- hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC); and phosphatidylethanolamines, e.g., diacylphosphatidylethanolamines, such as dioleoyl-phosphatidylethanolamine (DOPE), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dilauroyl- phosphatidylethanolamine (DLPE), distearoyl-phosphatidylethanolamine (DSPE), 1-phytanoyl- phosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1- stearoyl-2-oleoylphosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (transDOPE), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing neutral lipids may be excluded from the LNPs of the present disclosure. In one aspect, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), having the formula: In some aspects, the LNPs comprise a neutral lipid, and the neutral lipid comprises one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and / or SM. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing neutral lipids may be excluded from the LNPs of the present disclosure. In various aspects, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton: In certain aspects, the steroid or steroid analogue is cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing steroid or steroid analogues may be excluded from the LNPs of the present disclosure. In certain aspects, the steroid or steroid analogue is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing cholesterol derivaties may be excluded from the LNPs of the present disclosure. In one aspect, the cholesterol has the formula: Without being bound by any theory, the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some aspects, the molar ratio of the cationic lipid to the neutral lipid ranges from or from about 2:1 to about 8:1, or from or from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In some aspects, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and / or cholesterol), may comprise from or from about 0 mol % to about 90 mol %, from or from about 0 mol % to about 80 mol %, from or from about 0 mol % to about 70 mol %, from or from about 0 mol % to about 60 mol %, or from or from about 0 mol % to about 50 mol %, of the total lipid present in the particle. In some aspects, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and / or cholesterol), may or may not be at least, at most, exactly, or between (inclusive or exclusive) of 0 mol %, 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, or 90 mol % of the total lipid present in the particle. CHARACTERIZATION AND ANALYSIS OF NANOPARTICLES Lipid nanoparticles (LNPs) produced by the methods described herein may be analyzed and characterized using various methods. Analyses may be performed after LNP generation, and before, during, or after drug product (DP) SPTFF. LNP population particle size and polydispersity measurement may be performed using dynamic light scattering analysis (e.g., using Malvern Zetasizer, Unchained Labs Stunner). RNA concentration and encapsulation efficiency in the LNPs may be determined by fluorescence-based method (e.g., using RiboGreen assay). Alternatively, RNA concentration may be determined by UV / Vis spectrographic analysis (e.g., using Unchained Labs Stunner). RNA integrity may be determined by capillary gel electrophoresis method (e.g., using Agilent Technologies, FA Automated CGE system). Lipid content may be measured using, for example, a reversed-phase high-performance liquid chromatography charged-aerosol-detector (RP-HPLC-CAD) method. LNP morphology may be examined by cryogenic electron microscopy (e.g., using Thermo Fisher Talos Artica CryoTEM). In-vitro efficacy may be analyzed by, e.g., transfecting the LNPs into a human cell line. Protein expression of the polypeptide of interest may be quantified using methods such as ELISA or flow cytometry. Immunogenicity may be analyzed by, e.g., transfecting the LNPs into cell lines which indicate innate immune stimulation, e.g., PBMCs. Cytokine induction may be analyzed using, e.g., methods such as ELISA to quantify a cytokine, e.g., interferon-α. INTEGRATED, END-TO-END SKID FOR CONTINUOUS RNA DRUG PRODUCT MANUFACTURING FROM RAW MATERIALS TO RNA DRUG PRODUCT (MSKID) Described herein are end-to-end modular process skids for integrated and continuous manufacture of RNA drug product (referred to as “mSKIDs”). In some aspects, the mSKIDs described herein comprise the following components: (1) a pump for delivery of an in vitro transcription reaction system; (2) a CF-IVT reactor configured to receive the in vitro transcription reaction system and produce a CF-IVT product comprising an RNA molecule; (3) sensors configured to allow measurement of in-line pressure, flow rate, pH, conductivity, and UV absorbance situated before and after the CF-IVT reactor; (4) one or more pumps for delivery of one or more additives (e.g., EDTA, proteinase K, DNase I) into the CF-IVT product via one or more in-line mixing devices to obtain an additive-treated CF-IVT product; (5) a length of tubing or piping to provide a desired incubation time (e.g., around 30 min) for incubating the additive- treated CF-IVT product; (6) a pump for delivery of diluent into the additive-treated CF-IVT product via an in-line mixing device; (7) a single-pass tangential flow filtration device for RNA purification (DS-SPTFF) comprising feed, permeate, and retentate lines for re-concentrating the RNA molecule within the DS-SPTFF retentate and passing impurities to the DS-SPTFF permeate to obtain an RNA drug substance; (8) pressure control devices on all DS-SPTFF permeate and retentate lines to control transmembrane pressure (TMP) over each membrane; (9) sensors to measure in-line pressure, flow rate, pH, conductivity, and UV absorbance on all DS-SPTFF feed, permeate, and retentate lines; (10) a pump for delivery of an acidifying diluent into the RNA drug substance via an in-line mixing device to yield an aqueous phase (AP) at a desired concentration of purified RNA molecule; (11) sensors to measure in-line pressure, flow rate, pH, conductivity, and UV absorbance of the AP; (12) a vessel (herein referred to as an “aqueous phase vessel” [APV]) wherein the AP has a desired mean residence time (e.g., 1.5 min); (13) an in-line dispersion formation mixer (e.g., a coaxial mixer) for mixing of the AP and an organic phase (OP) at a desired AP:OP ratio (e.g., 3:1) to yield an RNA-loaded LNP dispersion; (14) a pump for transfer of the AP from the APV into the in-line dispersion formation mixer; (15) a pump for transfer of the OP into the in-line dispersion formation mixer; (16) a pump to deliver a quenching diluent to the RNA-loaded LNP dispersion via an in-line mixing device to obtain a quenched LNP dispersion; (17) sensors to measure in-line pressure and flow rate on the AP, OP, and quenching diluent lines; (18) sensors to measure in-line LNP size and polydispersity index (PDI) of the quenched LNP dispersion; (19) a vessel (herein referred to as a “continuous cure vessel” [CCV]) wherein the quenched LNP dispersion has a desired mean residence time (e.g., 1.5 min); (20) a pump to transfer the quenched LNP dispersion from the CCV into a post-CCV in-line mixing device; (21) a pump to deliver diluent to the quenched LNP dispersion via the post-CCV in-line mixing device to obtain a diluted quenched LNP dispersion; (22) an SPTFF device for LNP dispersion purification (DP-SPTFF) to receive the diluted quenched LNP dispersion and re- concentrate the diluted quenched LNP dispersion within the DP-SPTFF retentate and passage impurities to the DP-SPTFF permeate to obtain a re-concentrated LNP dispersion; (23) a vessel (herein referred to as a “booster pump supply” [BPS]) providing the re-concentrated LNP dispersion a desired mean residence time (e.g., 1 min); (24) a pump to transfer the re- concentrated LNP dispersion from the BPS into a post-BPS in-line mixing device, (25) a pump to deliver diluent to the re-concentrated LNP dispersion via the post-BPS in-line mixing device to obtain a diluted LNP dispersion; (26) a DP-SPTFF device to receive the diluted LNP dispersion and re-concentrate the diluted LNP dispersion within the DP-SPTFF retentate and passage impurities to the DP-SPTFF permeate to obtain a purified LNP dispersion; (27) pressure control devices on all DP-SPTFF feed, permeate, and retentate lines to control transmembrane pressure (TMP) over each membrane; (28) sensors to measure in-line pressure, flow rate, pH, and conductivity on all DP-SPTFF feed, permeate, and retentate lines; (29) sensors to measure in- line LNP size and polydispersity index (PDI) on all SPTFF retentate lines; (30) a vessel (herein referred to as a “purified LNP vessel” [PLV]) wherein the purified LNP dispersion has a desired mean residence time (e.g., 1.5 min); (31) a pump to transfer the purified LNP dispersion from the PLV through a filtration device to obtain an RNA drug product; (32) a post-filtration in-line mixing device to receive the RNA drug product; (33) a pump to deliver diluent into the RNA drug product within the post-filtration in-line mixing device to obtain a diluted RNA drug product; (34) a final filtration device through which to pass the diluted RNA drug product and thereby obtain a final RNA drug product; (35) a vessel (herein referred to as a final “drug product vessel” [DPV]) to receive the final RNA drug product; and (36) a pump to transfer the final RNA drug product into one of any desired container systems (e.g., vials or syringes) for storage and delivery of the final RNA drug product. In some aspects, the mSKIDs described herein comprise the foregoing components arranged sequentially. In some aspects, the mSKIDs described herein comprise one or more of the foregoing components arranged in fluid communication. In some embodiments, the mSKIDs described herein may exclude one or more foregoing components, for example, in some aspects, a vessel (e.g., APV, CCV, BPS, or PLV) and corresponding downstream pump may be removed and replaced with a length of tubing or piping. In some aspects, the steps of delivering diluent into the additive-treated CF-IVT product and re- concentrating the RNA molecule within the DS-SPTFF retentate are repeated sequentially to reach a desired purity level, a desired reduction of impurities, a desired pH, a desired ionic strength, and / or a desired RNA concentration in the resulting RNA drug substance within the DS- SPTFF retentate. In some aspects, the steps of providing the re-concentrated LNP dispersion a desired mean residence time, delivering diluent to the re-concentrated LNP dispersion, and re-concentrating the diluted LNP dispersion within the DP-SPTFF retentate are repeated sequentially to reach a desired purity level, a desired reduction of impurities, a desired pH, a desired ionic strength, and / or a desired RNA concentration in the resulting purified LNP dispersion within the DP-SPTFF retentate. In some embodiments, the mSKIDs described herein may be operated integrated and continuous up to any point in the process prior to the final drug product, for example, the mSKID may be operated integrated and continuous up to the point of the CF-IVT product, treated CF-IVT product, drug substance, aqueous phase, quenched LNP dispersion, or RNA drug product. In some embodiments, multiple mSKIDs may be operated in parallel up to the CF-IVT product, treated CF-IVT product, drug substance, or aqueous phase such that multiple CF-IVT products, treated CF-IVT products, drug substances, or aqueous phases may be combined, in-line or off- line, at any desired ratio and then further processed, in-line or off-line, to obtain a combined LNP formulation as described above. In some embodiments, multiple mSKIDs may be operated in parallel up to the quenched LNP dispersion, RNA drug product, or final RNA drug product such that multiple quenched LNP dispersions, RNA drug products, or final RNA drug products may be combined, in-line or off-line, at any desired ratio and further processed, in-line or off-line, to obtain a mixed LNP formulation, as described above. As described above, the rate of fluid entry into a vessel (e.g., APV, CCV, BPS, or PLV) matches its corresponding rate of fluid exit. In some aspects, e.g., during start-up or troubleshooting of downstream operations, the rate of fluid entry into any vessel may briefly not match its corresponding rate of fluid exit. As described above, desired mean residence time within a vessel is typically a minimal residence time, e.g. from about 0 sec to 5 min. In some aspects, e.g., during start-up or troubleshooting of downstream operations, mean residence time within a vessel may be longer, e.g., from about 5 min to an hour, or more. In some aspects, a vessel may be pressure regulated or vented to atmosphere. In some aspects, a length of tubing or piping to provide a desired incubation time may be replaced with a vessel and downstream pump with rate of fluid entry matching rate of fluid exit. In some aspects, sensors to measure in-line pressure, flow rate, pH, conductivity, and UV absorbance may be placed at or near any or all pump outlets and / or component inlets. In some aspects, filtering devices (e.g. particulate filtering, sterile filtering, or bioburden reduction filtering devices) may be placed at or near any or all pump outlets or component inlets. In some aspects, de-gassing devices may be placed at or near any or all pump outlets or component inlets. In some aspects, heat exchangers may be placed at or near any or all pump outlets or component inlets. In some embodiments, sampling tees and sampling valves can be included throughout the flow paths to allow for at-line or offline sampling for analysis of, for example, solution composition, RNA concentration, RNA quality (e.g., residual NTPs, residual DNA, residual enzymes, integrity, 5’ capping, dsRNA, RNA sequence identity, etc.), or LNP quality (e.g., LNP size, LNP polydispersity index [PDI] RNA concentration, RNA integrity, encapsulation efficiency, lipids content, LNP morphology, in vitro efficacy, immunogenicity, etc.). In some aspects, sampling tees and sampling valves can be included throughout the flow paths to allow for at-line sampling for other purposes such as parallel processing, process validation, validation of filtration performance, and / or for diversion of non-conforming material. In some aspects, the mSKIDs described herein comprise pumps, which may include piston pumps, syringe pumps, centrifugal pumps, peristaltic pumps, diaphragm pumps, screw pumps, and / or lobe pumps. In some aspects, the CF-IVT reactors comprise immobilized non-consumables, such as a CF- IVT reactor comprising immobilized enzymes, a CF-IVT reactor comprising immobilized DNA, or a CF-IVT reactor comprising immobilized enzymes and immobilized DNA. In some aspects, the CF-IVT reactors comprise a plastic or metallic housing and a substrate comprising membranes, microfluidic channels, resins, particles, and / or gels. In some aspects, the components in fluid communication are connected by tubing, and the tubing may include polyether ether ketone (PEEK) tubing, silicone tubing, thermoplastic elastomer (TPE) tubing, galvanized steel pipes, PVC pipes, and ABS pipes. In some aspects, the tubing and / or the pipes are connected by connections comprising finger-tight PEEK connectors, plastic quick- connect fittings, metal quick-connect fittings, aseptic connectors, sanitary flange connections, and threaded screw-tight connections. As described herein, in-line mixing devices may include tees, static mixing devices, helicoidal static mixing devices, and / or active mixing devices. In some aspects, in-line dispersion formation mixers may include tees, microfluidic mixers, coaxial mixers, and / or bifurcating mixers. In some aspects, sensors to measure In-line pressure comprise strain gauge pressure sensors, piezoresistive pressure sensors, piezoelectric pressure sensors, and pressure switches. In some aspects, sensors to measure flow rate comprise in-line and clamp-on ultrasonic flow sensors, Coriolis mass flow sensors, differential pressure flow sensors, thermal mass flow sensors, velocity flow sensors, and volumetric flow sensors. In some aspects, sensors to measure pH comprise glass bulb pH sensors and optical pH sensors. In some aspects, sensors to measure conductivity comprise conductivity sensors with and without temperature compensation. In some aspects, sensors to measure UV absorbance comprise single wave length and multiple wavelength UV and visible light spectrophotometers and / or variable pathlength spectrophotometers. As described herein, sensor readings and measurements may be visualized and recorded on a centralized automation system which may be used to monitor and / or control any and all desired process and equipment operating conditions and to maintain product quality attributes within desired ranges. Some process parameters necessary to verify desired process operating conditions and to control quality attributes may include, but are not limited to, in vitro reaction system residence time in the CF-IVT reactor, CF-IVT reactor temperature, SPTFF fluxes / pressures / loading, diluent flow rates, AP RNA concentration, AP flow rate, AP pressure, OP flow rate, and OP pressure. In some aspects, the mSKIDs described herein may be scaled to meet desired throughput while maintaining certain parameters, for example, in vitro transcription reaction system residence time in the CF-IVT reactor, CF-IVT reactor temperature, SPTFF fluxes / pressures / loading, diluent ratios, AP RNA concentration, N:P ratio (e.g., through control of AP RNA concentration and AP:OP ratio), and DP RNA concentration. In some aspects, the in vitro transcription reaction system residence time within the CF-IVT reactor is between 1 second and 3 hours at a temperature between about 20 °C and 50 °C. In some aspects, the CF-IVT reactor may be incubated at a desired temperature within for example, a water bath, a bead bath, a temperature-controlled chamber or room, a temperature controlled heating sleeve or wrapping, and insulating sleeve or wrapping. In some aspects, CF- IVT reactor temperature may be controlled by controlling the temperature of the in vitro transcription reaction system (e.g., through a heat exchanger) and where the CF-IVT reactor may be insulated to maintain the desired temperature. In some aspects, DS-SPTFF flux is maintained between at least, at most, or about 0.1 to 600 liters per meter squared per hour (LMH), or any range or value derivable therein. In a preferred embodiment, DS-SPTFF flux is maintained at most 120 LMH. In one embodiment, DS-SPTFF is maintained at 84 LMH. In some aspects, DP-SPTFF flux is maintained between at least, at most, or about 0.1 to 380 LMH, or any range or value derivable therein. In some aspects, DS-SPTFF transmembrane pressure (TMP) is maintained between at least, at most, or about 0.1 to 58 psi, e.g., 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 58 psi, or any range or value derivable therein. In a preferred embodiment, DS-SPTFF TMP is maintained at most about 20 psi. In one embodiment, DS-SPTFF is maintained at most about 5.5 psi. In some aspects, DP- SPTFF TMP is maintained between at least, at most, or about 0.1 to 50 psi, e.g., 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 psi, or any range or value derivable therein. In some aspects, DS-SPTFF loading is maintained between at least, at most, or about 0.1 to 2400 g-RNA / m2, or any range or value derivable therein. In some aspects, DP-SPTFF loading is maintained between at least, at most, or about 0.1 to 2400 g-RNA / m2, or any range or value derivable therein. In some aspects, the RNA in the aqueous phase is at a concentration from or from about 0.01 mg / mL to about 5 mg / mL, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mg / mL or any range or value derivable therein. In some aspects, the RNA is at a concentration higher than 5.0 mg / mL. In some aspects, the concentration of RNA in the aqueous phase may be controlled by, for example, the operation of the DS-SPTFF devices and delivery of an acidifying diluent into the RNA drug substance via an in-line mixing device. In some aspects, an LNP of the disclosure comprises or does not comprise a molar ratio of positively chargeable nitrogen of tertiary amine in the cationic lipid to negatively charged phosphates of RNA backbone (known as N:P ratio) of or of from about 2:1 to about 30:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 6:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 3:1. In some aspects, the N:P ratio may be controlled by, for example, the AP RNA concentration and AP:OP ratio. In some aspects, the total RNA concentration in the final DP is at a concentration from or from abou...

Claims

CLAIMS 1. A method for continuous production of an RNA molecule comprising: (a) incubating a circular double-stranded DNA (dsDNA) template with a restriction endonuclease to obtain a linearized dsDNA fragment; (b) incubating the linearized dsDNA fragment with an aminoallyl-dNTP and a DNA polymerase for a time period between about three and twenty-four hours at a temperature between about 30 and 45 °C to obtain a labeled composition; (c) purifying the labeled composition to obtain a labeled dsDNA fragment; (d) immobilizing the labeled dsDNA fragment within a substrate to obtain an immobilized DNA reactor; (e) flowing an in vitro transcription reaction system across the immobilized DNA reactor to obtain a continuous-flow in vitro transcription (CF-IVT) product comprising the RNA molecule, thereby continuously producing the RNA molecule.

2. The method of claim 1, further comprising: (f) incubating the CF-IVT product with an additive to obtain a treated CF-IVT product; (g) contacting the treated CF-IVT product with a diluent to obtain a diluted treated CF-IVT product; (h) contacting the diluted treated CF-IVT product with a continuous in-line purification device to obtain a drug substance.

3. The method of claim 2, further comprising: (i) contacting the drug substance with an aqueous solution to obtain an aqueous phase, wherein contacting the drug substance with the aqueous solution adjusts a concentration and a pH of the drug substance; (j) mixing the aqueous phase with an organic solvent comprising a lipid via an in-line mixer to obtain an RNA-loaded lipid nanoparticle (LNP) dispersion; (k) contacting the RNA-loaded LNP dispersion with a diluent to obtain a quenched LNP dispersion; (l) contacting the quenched LNP dispersion with a diluent to obtain a diluted quenched LNP dispersion;(m) contacting the diluted quenched LNP dispersion with a continuous in-line purification device to obtain a re-concentrated LNP dispersion; (n) contacting the re-concentrated LNP dispersion with a diluent to obtain a diluted LNP dispersion; (o) contacting the diluted LNP dispersion with a continuous in-line purification device to obtain a purified LNP dispersion; (p) filtering the purified LNP dispersion through an in-line filtration device to obtain a filtered purified LNP dispersion; (q) contacting the filtered purified LNP dispersion with a diluent to obtain a diluted, filtered purified LNP dispersion; and (r) filtering the diluted, filtered purified LNP dispersion through an in-line filtration device thereby continuously producing an RNA drug product.

4. The method of any one of claim 1 to 3, wherein the aminoallyl-dNTP comprises aminoallyl-dUTP.

5. The method of any one of claims 2 to 4, wherein the additive is selected from the group consisting of proteinase K, ethylenediaminetetraacetic acid (EDTA), and DNaseI.

6. The method of any one of claims 1 to 5, wherein a yield of the CF-IVT product is measured in-line via a pH sensor.

7. The method of any one of claims 1 to 6, wherein late migrating species of the CF-IVT product are reduced by up to 60% compared to an IVT product obtained by non- continuous production.

8. The method of any one of claims 1 to 7, wherein the circular dsDNA template comprises a synthetic dsDNA template.

9. The method of any one of claims 1 to 8, wherein the substrate comprises an N- hydroxysuccinimide-activated resin-packed column or a carbonylimidazole-activated resin-packed column.

10. The method of any one of claims 1 to 9, wherein the in vitro transcription reaction system comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, N1-Me-pUTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

11. The method of any one of claims 1 to 10, wherein flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a residence time of between 100 minutes and 2 minutes at a temperature between about 20 and 45 °C.

12. The method of any one of claims 1 to 11, wherein flowing the in vitro transcription reaction system across the immobilized DNA reactor comprises a time period between 10 minutes and 1,200 minutes.

13. The method of any one of claims 1 to 12, wherein following the step of flowing the in vitro transcription reaction system across the immobilized DNA reactor, the immobilized DNA reactor remains functional for at least 14, 20, 42, or 64 days.

14. The method of any one of claims 2 to 13, wherein the continuous in-line purification device is selected from the group consisting of an oligo(dT) column and a single-pass tangential flow filtration (SPTFF) device.

15. The method of claim 14, wherein the oligo(dT) column comprises a resin-packed column or a monolith column.

16. The method of any one of claims 3 to 15, wherein the aqueous solution comprises an acidifying solution.

17. The method of any one of claims 2 to 16, wherein the diluent is selected from the group consisting of a HEPES-buffered diluent, a Tris-buffered diluent, and water.

18. The method of any one of claims 16 or 17, wherein the acidifying solution comprises a low pH citrate solution.

19. The method of any one of claims 3 to 18, wherein contacting the drug substance with the aqueous solution comprises passing the purified RNA molecule through a device selected from the group consisting of a continuous in-line purification device, an in-line mixing device, an in-line UV absorbance sensor, and a surge vessel.

20. The method of any one of claims 3 to 19, wherein the lipid is selected from the group consisting of a cationic lipid, a phospholipid, a sterol, and a polymer-conjugated lipid.

21. The method of any one of claims 3 to 20, wherein contacting the RNA-loaded LNP dispersion with a diluent comprises passing the RNA-loaded LNP dispersion through an in-line mixing device.

22. The method of any one of claims 3 to 21, wherein the in-line filtration device is selected from the group consisting of a bioburden reduction filtration device and a sterile filtration device.

23. The method of any one of claims 2 to 22, wherein the steps of contacting the treated CF-IVT product with a diluent and contacting the diluted treated CF-IVT product with a continuous in-line purification device are repeated one or more times.

24. The method of any one of claims 3 to 23, wherein the steps of contacting the re- concentrated LNP dispersion with a diluent and contacting the diluted LNP dispersion with a continuous in-line purification device are repeated one or more times.

25. The method according to any one of claims 1 to 24, wherein the RNA molecule is selected from the group consisting of mRNA, modRNA, and saRNA.

26. The method according to any one of claims 1 to 25, wherein the RNA molecule comprises a vaccine RNA molecule or a therapeutic RNA molecule.

27. The method according to any one of claims 1 to 26, wherein the DNA polymerase comprises phi29 DNA polymerase or a Klenow fragment DNA polymerase.

28. The method of any one of claims 1-27, wherein the immobilized DNA reactor further comprises in-line sensors selected from the group consisting of an in-line pH sensor, an in-line UV absorbance sensor, an in-line conductivity sensor, and an in-line pressure sensor.

29. The method of any one of claims 14-28, wherein the single-pass tangential flow filtration (SPTFF) device further comprises in-line sensors selected from the group consisting of a feed pressure sensor, a permeate pressure sensor, and a retentate pressure sensor.

30. The method of any one of claims 3-29, wherein contacting the drug substance with an aqueous solution further comprises an in-line UV absorbance sensor.