CN116710074A - Lipid nanoparticle manufacturing method and compositions derived therefrom - Google Patents

Abstract

Disclosed herein are methods of increasing the efficacy of nucleic acid loaded lipid nanoparticles (naLNP) by certain novel and unexpectedly superior LNP manufacturing techniques. Also disclosed are pharmaceutical compositions containing naLNP made according to the methods of making described herein. The methods disclosed herein overcome the major technical challenges and high costs associated with previous LNP manufacturing techniques. Thus, the methods disclosed herein greatly improve the industrial production of LNP in an unexpected manner, thereby providing a more efficient naLNP for nucleic acid delivery. In particular, the application disclosed herein is a method showing an increase in naLNP potency due to an increase in the mixed concentration of lipid and mRNA during assembly.

Description

Lipid nanoparticle manufacturing method and compositions derived therefrom

Cross Reference to Related Applications

The application requires U.S. provisional application No. 63/091,616 filed on 10/14/2020; U.S. provisional application No. 63/179,885, filed on 26, 4, 2021; U.S. provisional application No. 63/091,603, filed on 10/14/2020; and U.S. provisional application No. 63/179,872, filed on 26, 4, 2021, each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention is in the field of nanoparticle manufacture for delivery of drug nucleic acid payloads.

Background

Delivery of bioactive agents (including therapeutically relevant compounds) to a subject is often hampered by the difficulty of the compound reaching the target cell or tissue. In particular, the transport of many bioactive agents into living cells is greatly limited by the complex membrane systems of the cells. These limitations can result in the need to use much higher concentrations of bioactive agents than are required to achieve the results, thereby increasing the risk of toxic and side effects. One solution to this problem is to use specific carrier molecules and carrier compositions that allow selective entry into cells. Lipid carriers, biodegradable polymers, and various conjugate systems can be used to improve delivery of bioactive agents to cells.

One class of bioactive agents that is particularly difficult to deliver to cells are biologic therapeutic agents (including peptides, proteins, nucleosides, nucleotides, polynucleotides, nucleic acids, and derivatives such as mRNA, RNAi/siRNA, and self-replicating RNA agents). In general, nucleic acids are stable in cells or body fluids for only a limited duration. In particular, the development of CRISPR/CAS9, RNA interference, RNAi therapies, mRNA therapies, RNA drugs, antisense therapies, gene therapies, and nucleic acid vaccines (e.g., RNA vaccines) has increased the need for effective means of introducing active nucleic acid agents into cells. For these reasons, compositions that can stabilize and deliver nucleic acid-based agents into cells are of interest.

The most well studied method for improving the transport of foreign nucleic acids into cells involves the use of viral vectors or formulations with cationic lipids. Viral vectors can be used to efficiently transfer genes into some cell types, but they are generally not used to introduce chemically synthesized molecules into cells.

An alternative approach is to use a delivery composition incorporating a cationic lipid that interacts with the bioactive agent at one part and interacts with the membrane system at another part. Depending on the composition and method of preparation, such compositions are reported to provide liposomes, micelles, lipid complexes or lipid nanoparticles (for reviews see Felgner,1990,Advanced Drug Delivery Reviews,5,162-187;Felgner,1993,J.Liposome Res, 3-16; gallas,2013, chem. Soc. Rev.,42,7983-7997; falsini,2013, J.Med. Chem. Dx. Doi. Org/10.1021/jm400791q; and references therein).

Since the first description of liposomes by Bangham (J.mol. Biol.13, 238-252) in 1965, there has been continued attention and effort to develop lipid-based carrier systems for delivery of bioactive agents (Allen, 2013,Advanced Drug Delivery Reviews,65,36-48). The method of introducing functional nucleic acids into cultured cells by using positively charged liposomes is described first by Philip Felgner et al Proc.Natl.Acad.Sci., USA,84,7413-7417 (1987). The method was later demonstrated in vivo by K.L. Brigham et al, am.J.Med.Sci.,298,278-281 (1989).

Recently, lipid nanoparticle formulations have been developed and show efficacy in vitro and in vivo. (Falsini, 2013, J.Med. Chem. Dx. Doi. Org/10.1021/jm400791q; morrissey,2005, nat. Biotech.,23,1002-1007; zimmerman,2006, nature,441, 111-114; jayaraman,2012, angew. Chem. Int. Ed.,51, 8529-8533.) lipid formulations are attractive carriers because they can protect biomolecules from degradation while improving their cellular uptake. Among the various classes of lipid formulations, cationic lipid-containing formulations are commonly used to deliver polyanions (e.g., nucleic acids). Such formulations may be formed using only cationic lipids and optionally include other lipids and amphiphilic molecules such as phosphatidylethanolamine. It is well known in the art that the composition of lipid formulations, as well as the method of their preparation, all affect the structure and size of the resulting nanoparticle or aggregate (Leung, 2012,J.Phys Chem.C,116,18440-18450).

Various methods of formulating LNP systems containing genetic drugs have been developed. These methods include mixing a preformed LNP with a nucleic acid in the presence of ethanol or mixing a lipid dissolved in ethanol with an aqueous medium containing the nucleic acid and producing an LNP having a diameter of 100nm or less and a nucleic acid encapsulation efficiency of 65% -95%. These methods all rely on the presence of cationic lipids to achieve encapsulation of the Oligonucleotides (OGNs) and poly (ethylene glycol) (PEG) to inhibit aggregation and form larger structures. The properties of the resulting LNP system, including size and OGN encapsulation efficiency, are sensitive to various formulation parameters such as ionic strength, lipid and ethanol concentrations, pH, nucleic acid concentration, and mixing ratio. In general, parameters such as relative lipid and nucleic acid concentrations at the time of mixing and mixing ratios are difficult to control using current formulation procedures, resulting in variations in the characteristics of the LNP produced inside the formulation as well as between formulations.

Among the covd-19 vaccines, several are immunogens encoded based on mRNA delivered in Lipid Nanoparticles (LNP). This higher proportion of mRNA vaccine is due to its rapid implementation and excellent efficacy in animal models. In mRNA vaccines, the immunogen is encoded in an mRNA sequence that is typically substituted with an immunosilent nucleoside. In addition to nucleoside substitutions, mRNA design often involves codon optimization, UTR and polyA tail design, 5' cap selection and purification to remove double stranded RNA contaminants that can activate the innate immunosensor to inhibit translation of the delivered mRN.

The antibody titers in the covd-19 mRNA vaccinated patients were higher than the convalescence serum, while the neutralization titers were comparable to the convalescence in the published trial. In both experiments cd4+ and cd8+ T cell responses were present and Th2 components were absent, th2 components being critical due to the potential role of Th2 responses in vaccine-related enhanced respiratory diseases.

However, local and systemic adverse events are common and more frequent as severity increases after the second vaccination. Although there were no serious life threatening adverse events, in some trials, the highest dose was stopped due to serious local and systemic adverse events. Self-amplified mRNA LNP is effective at lower doses, but has additional safety concerns.

FDA guidelines suggest that success in phase 3 trials requires 50% protection compared to placebo based on the endpoint of severe covd-19 disease. Vaccines will need to be manufactured cheaply to obtain about 150 hundred million doses and supply most people worldwide. Meeting a combination of these 3 criteria would be challenging. For example, 50% protection is provided for 1 year, billions of doses are manufactured annually and are available and willing to vaccinate the global population.

If the current dose is successful in a clinical trial, the increased efficacy may reduce the dose and adverse effects while maintaining efficacy, and increase the ability to vaccinate worldwide by reducing costs and increasing manufacturing capacity. Alternatively, if the current dose fails in a clinical trial, the increased efficacy at these same doses may increase protection. Thus, there is a great need to increase efficacy by protecting mRNA vaccines through payload mRNA expression, immunogenicity, and by controlling LNP assembly.

Despite advances in the development of methods for LNP systems containing genetic drugs, there remains a need for methods for preparing lipid nanoparticles containing therapeutic materials, and improved lipid nanoparticles containing therapeutic materials. The present invention seeks to meet this need and provide further related advantages.

Disclosure of Invention

This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary merely illustrates many different embodiments. References to one or more representative features of a given embodiment are likewise exemplary. Such embodiments may generally exist with or without the mentioned features; likewise, those features may be applied to other embodiments of the presently disclosed subject matter, whether or not listed in this summary. This summary does not list or suggest all possible combinations of such features in order to avoid undue repetition.

One aspect of the invention relates to a method for preparing a lipid nanoparticle comprising nucleic acid ("naLNP"), providing a nucleic acid solution comprising at least one nucleic acid at a concentration of nucleic acid; providing a lipid solution comprising at least one lipid at a lipid concentration; and combining a portion of the nucleic acid solution and a portion of the lipid solution to produce a mixed solution comprising a mixed nitrogen-phosphate ratio and a lipid: nucleic acid ratio; and adjusting the pH in the mixed solution to a physiological pH to obtain a pH-adjusted mixed solution; and obtaining the naLNP from the pH adjusted mixed solution; and wherein the naLNP has greater potency than a reference lipid nanoparticle ("refLNP"), wherein the refLNP comprises at least one lipid and at least one nucleic acid and is prepared by a reference LNP manufacturing method.

In one embodiment, a portion of the nucleic acid solution and a portion of the lipid solution are combined in step (c) in a volume ratio selected from the group consisting of: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, and 7:1. In another embodiment, the naLNP has an average diameter in the range of about 40 to about 150 nanometers. In another embodiment, the naLNP has an average diameter in the range of about 50 to about 100 nanometers. In yet another embodiment, the naLNP has a nucleic acid encapsulation efficiency of about 40% to about 100%. In yet another embodiment, the naLNP has a nucleic acid encapsulation efficiency of about 50% to about 85%. In another embodiment, the naLNP has a nucleic acid encapsulation efficiency of about 60% to about 85%. In yet another embodiment, the naLNP has a nucleic acid encapsulation efficiency of about 68% to about 83%.

In yet another embodiment, the naLNP has a lower nucleic acid encapsulation efficiency than refLNP. In another embodiment, at least one nucleic acid is DNA or RNA. In still further embodiments, at least one nucleic acid is RNA. In another embodiment, at least one nucleic acid is mRNA. In another embodiment, the at least one nucleic acid is an mRNA encoding at least one open reading frame. In yet another embodiment, the at least one nucleic acid is an mRNA encoding at least one open reading frame encoding an immunogen. In another embodiment, the nucleic acid solution comprises a buffer. In another embodiment, the nucleic acid concentration is at least or about 0.21 to about 3mg/ml. In yet another embodiment, the nucleic acid concentration is at least or about 0.23 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.25 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.28 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.29 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.30 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.40 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.50 to about 3mg/ml. In yet another embodiment, the nucleic acid concentration is at least or about 0.60 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.70 to about 3mg/ml. In still other embodiments, the nucleic acid concentration is at least or about 1 to about 3mg/ml.

In yet another embodiment, the lipid solution comprises an organic solvent selected from the group consisting of: methanol, ethanol, acetone, benzene and toluene. In another embodiment, the lipid solution is selected from the group consisting of: MC3, KC2, DLin, DODMA, DODAP, formula I, formula II, and combinations thereof. In yet another embodiment, the at least one lipid in the lipid solution is selected from the group consisting of: MC3, KC2, DLin, DODMA, DODAP, and combinations thereof. In yet another embodiment, at least one lipid in the lipid solution is a cationic lipid having a pKa. In yet another embodiment, at least one lipid in the lipid solution is an ionizable cationic lipid having a pKa. In yet another embodiment, the mixed solution has a pH of about 0 to about 2 pH units below the pKa of the lipid in refLNP. In yet another embodiment, the mixed solution has a pH of about 0.5 to about 1.5 pH units below the pKa of the lipid in refLNP. In still further embodiments, the mixed solution has a pH of about 0.75 to about 1.25 pH units below the pKa of the lipids in refLNP. In another embodiment, the lipid concentration is at least or about 1 mM to about 200 mM. In yet another embodiment, the lipid concentration is at least or about 10 mM to about 150 mM. In another embodiment, the lipid concentration is at least or about 50 mM to about 100mM. In another embodiment, the mixed liquid nitrogen-phosphate ratio is at least or about 2 to at least or about 10.

In yet another embodiment, the mixed solution lipid to nucleic acid weight ratio is at least or about 1:0, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 15:1, 17:1, 18:1, 20:1, 25:1, 30:1, 35:1, 40:1, or 50:1. In yet another embodiment, refLNP is prepared using a reference nucleic acid concentration of less than 0.21 mg/ml. In another embodiment, refLNP is prepared using a reference lipid concentration of less than 10.5 mM. In another embodiment, ref LNP is prepared using a reference nucleic acid concentration of less than 0.21mg/ml and a reference lipid concentration of less than 10.5 mM. In yet another embodiment, the potency is about 1.5 times greater than refLNP.

In another embodiment, the potency is about 2-fold higher than refLNP. In another embodiment, the potency is about 3-fold higher than refLNP. In yet another embodiment, the potency is about 4-fold higher than refLNP. In another embodiment, the potency is at least or about 5-fold higher than refLNP. In yet another embodiment, the potency is at least or about 6-fold higher than refLNP. In still other embodiments, the potency is at least or about 7-fold higher than refLNP. In another embodiment, the potency is at least or about 8-fold higher than refLNP. In another embodiment, the potency is at least or about 9-fold higher than refLNP. In yet another embodiment, the potency is at least or about 10-fold higher than refLNP. In another embodiment, the potency is at least or about 11-fold higher than refLNP. In still other embodiments, the potency is at least or about 12-fold higher than refLNP. In yet another embodiment, the potency is at least or about 13-fold higher than refLNP. In yet another embodiment, the potency is at least or about 14-fold higher than refLNP. In yet another embodiment, the potency is at least or about 15-fold higher than refLNP. In another embodiment, the potency is at least or about 20-fold higher than refLNP. In yet another embodiment, the potency is at least or about 25-fold higher than refLNP. In another embodiment, the potency is at least or about 50-fold higher than refLNP.

Another aspect of the invention relates to a composition comprising at least one ionizable lipid at a concentration of about, equal to, or greater than 5.25 mM; a solution of at least one nucleic acid at a concentration of about, equal to, or greater than 0.21 mg/ml; wherein the acid to lipid ratio is in the range of about 2 to about 10; and the nucleic acid-bearing lipid nanoparticle ("naLNP") comprises at least one ionizable lipid and at least one nucleic acid; wherein naLNP at physiological pH has greater potency than a reference lipid nanoparticle ("refLNP") formed with the same at least one ionizable lipid and the same at least one nucleic acid in a reference LNP manufacturing process.

In one embodiment of this aspect of the invention, the naLNP has an average diameter in the range of about 40 to about 150 nanometers. In another embodiment, the naLNP has an average diameter in the range of about 50 to about 100 nanometers. In another embodiment, the naLNP has a nucleic acid encapsulation efficiency of about 40% to about 90%. In yet another embodiment, the naLNP has a nucleic acid encapsulation efficiency of about 50% to about 85%. In yet another embodiment, the naLNP has a nucleic acid encapsulation efficiency of about 60% to about 85%. In yet another embodiment, the naLNP has a nucleic acid encapsulation efficiency of about 68% to about 83%. In yet another embodiment, the naLNP has a lower nucleic acid encapsulation efficiency than refLNP. In yet another embodiment, at least one nucleic acid is DNA or RNA. In another embodiment, at least one nucleic acid is RNA. In another embodiment, at least one nucleic acid is mRNA. In yet another embodiment, the at least one nucleic acid is an mRNA encoding at least one open reading frame. In yet another embodiment, the at least one nucleic acid is an mRNA encoding at least one open reading frame encoding an immunogen.

In another embodiment, the nucleic acid solution comprises a buffer. In another embodiment, the nucleic acid concentration is at least or about 0.21 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.23 to about 3mg/ml. In still other embodiments, the nucleic acid concentration is at least or about 0.25 to about 3mg/ml. In yet another embodiment, the nucleic acid concentration is at least or about 0.28 to about 3mg/ml. In yet another embodiment, the nucleic acid concentration is at least or about 0.29 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.30 to about 3mg/ml. In yet another embodiment, the nucleic acid concentration is at least or about 0.40 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.50 to about 3mg/ml. In another embodiment, the nucleic acid concentration is at least or about 0.60 to about 3mg/ml. In yet another embodiment, the nucleic acid concentration is at least or about 0.70 to about 3mg/ml. In yet another embodiment, the nucleic acid concentration is at least or about 1 to about 3mg/ml.

In another embodiment, the solution comprises an organic solvent selected from the group consisting of: methanol, ethanol, acetone, benzene and toluene. In another embodiment of this aspect of the invention, the at least one lipid is selected from the group consisting of: MC3, KC2, DLin, DODMA, DODAP, formula I, formula II, and combinations thereof. In yet another embodiment, the at least one lipid is selected from the group consisting of: MC3, KC2, DLin, DODMA, DODAP, and combinations thereof. In yet another embodiment, at least one lipid is a cationic lipid having a pKa. In yet another embodiment, the at least one lipid is an ionizable cationic lipid having a pKa. In yet another embodiment, the mixed solution has a pH of about 0 to about 2 pH units below the pKa of the lipid in refLNP. In yet another embodiment, the mixed solution has a pH of about 0.5 to about 1.5 pH units below the pKa of the lipid in refLNP. In yet another embodiment, the mixed solution has a pH of about 0.75 to about 1.25 pH units below the pKa of the lipid in refLNP. In yet another embodiment, the lipid concentration is at least or about 1mM to about 200mM. In yet another embodiment, the lipid concentration is at least or about 10mM to about 150mM. In another embodiment, the lipid concentration is at least or about 50mM to about 100mM. In another embodiment, the mixed liquid nitrogen-phosphate ratio is at least or about 2 to at least or about 10. In yet another embodiment, the mixed solution lipid to nucleic acid weight ratio is at least or about 1:0, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 15:1, 17:1, 18:1, 20:1, 25:1, 30:1, 35:1, 40:1, or 50:1.

In yet another embodiment, refLNP is prepared using a reference nucleic acid concentration of less than 0.21 mg/ml. In another embodiment, refLNP is prepared using a reference lipid concentration of less than 10.5 mM. In yet another embodiment, refLNP is prepared using a reference lipid concentration of less than 10.5mM and a reference nucleic acid concentration of less than 0.21 mg/ml. In yet another embodiment, the naLNP is about 1.5 times more potent than refLNP. In another embodiment, the potency is about 2-fold higher than refLNP. In yet another embodiment, the potency is about 3-fold higher than refLNP. In another embodiment, the potency is about 4-fold higher than refLNP. In yet another embodiment, the potency is at least or about 5-fold higher than refLNP. In yet another embodiment, the potency is at least or about 6-fold higher than refLNP. In another embodiment, the potency is at least or about 7-fold higher than refLNP. In still other embodiments, the potency is at least or about 8-fold higher than refLNP. In yet another embodiment, the potency is at least or about 9-fold higher than refLNP. In another embodiment, the potency is at least or about 10-fold higher than refLNP. In yet another embodiment, the potency is at least or about 11-fold higher than refLNP. In another embodiment, the potency is at least or about 12-fold higher than refLNP. In still other embodiments, the potency is at least or about 13-fold higher than refLNP. In another embodiment, the potency is at least or about 14-fold higher than refLNP. In yet another embodiment, the potency is at least or about 15-fold higher than refLNP. In another embodiment, the potency is at least or about 20-fold higher than refLNP. In another embodiment, the potency is at least or about 25-fold higher than refLNP. In another embodiment, the potency is at least or about 50-fold higher than refLNP.

Another aspect of the invention relates to a pharmaceutical composition comprising naLNP prepared according to the method described above. In one embodiment, the pharmaceutical composition is a vaccine. In another embodiment, the vaccine is a prophylactic vaccine. In a further embodiment, the vaccine is a therapeutic vaccine. In another embodiment, the vaccine is for the treatment or prevention of an infectious disease. In another embodiment, the vaccine is for the treatment or prevention of covd-19. In another embodiment, the vaccine is for treating or preventing a coronavirus infection. In another embodiment, the composition comprises a bioactive agent selected from the group consisting of: peptides, antibodies, antibody fragments, and small molecule therapeutics.

Drawings

A more complete understanding of the presently disclosed subject matter may be obtained by reference to the drawings, which are considered in connection with the following detailed description. The embodiments illustrated in the figures are intended to be exemplary only and should not be taken as limiting the presently disclosed subject matter to the illustrated embodiments. The figures described below relate to experimental results from the various embodiments described below.

Fig. 1A: firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plate as positive enzymesAn activity control (data not shown) was used to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 1A corresponds to the procedure outlined in example 1A.

Fig. 1B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 1B corresponds to the procedure outlined in example 1B.

Fig. 1C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 1C corresponds to the procedure outlined in example 1C.

Fig. 2A: firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 2A corresponds to the procedure outlined in example 2A.

Fig. 2B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 2B corresponds to the procedure outlined in example 2B.

Fig. 2C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 2C corresponds to the procedure outlined in example 2C.

Fig. 3A: firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 3A corresponds to the procedure outlined in example 3A.

Fig. 3B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 3B corresponds to the procedure outlined in example 3B.

Fig. 3C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 3C corresponds to the procedure outlined in example 3C.

Fig. 3D: toxicity assay based on prest Blue HS viability reagent. After 24 hours transfection, the transfected cells were incubated with pre-warmed prest Blue HS reagent (10% v/v) for 15 minutes at 37 ℃. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 540/Em 590). The embodiment shown in fig. 3D corresponds to the procedure outlined in example 3D.

FIG. 4A firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 107RLU +.Linearity in mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 4A corresponds to the procedure outlined in example 4A.

Fig. 4B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 4B corresponds to the procedure outlined in example 4B.

Fig. 4C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 4C corresponds to the procedure outlined in example 4C.

Fig. 4D: toxicity assay based on prest Blue HS viability reagent. After 24 hours transfection, the transfected cells were incubated with pre-warmed prest Blue HS reagent (10% v/v) for 15 minutes at 37 ℃. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 540/Em 590). The embodiment shown in fig. 4D corresponds to the procedure outlined in example 4D.

Fig. 5A: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 5A corresponds to the procedure outlined in example 5A.

Fig. 5B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 5B corresponds to the procedure outlined in example 5B.

FIG. 6A. Firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 6A corresponds to the procedure outlined in example 6A.

Fig. 6B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 6B corresponds to the procedure outlined in example 6B.

Fig. 6C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 6C corresponds to the procedure outlined in example 6C.

Fig. 6D: and (5) pH measurement. Measurements were made before and after 4 hours of dialysis against 1x DPBS ph 7.4. The embodiment shown in fig. 6D corresponds to the procedure outlined in example 6D.

FIG. 7A firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 7A corresponds to the procedure outlined in example 7A.

Fig. 7B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 7B corresponds to the procedure outlined in example 7B.

Fig. 7C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 7C corresponds to the procedure outlined in example 7C.

Fig. 7D: and (5) pH measurement. Measurements were made before and after 4 hours of dialysis against 1x DPBS ph 7.4. Note here that these LNPs were formulated using 100mM NaOAc compared to example 6. Increasing the concentration of sodium acetate buffer in the formulation keeps the pH lower due to the higher buffering capacity, thereby producing a lower pH prior to LNP dialysis. The embodiment shown in fig. 7D corresponds to the procedure outlined in example 7D.

FIG. 8A firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, fluorescence was observed in fireflyThe transfected cells were conditioned to room temperature 30 minutes prior to the luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 8A corresponds to the procedure outlined in example 8A.

Fig. 8B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 8B corresponds to the procedure outlined in example 8B.

Fig. 8C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 8C corresponds to the procedure outlined in example 8C.

Fig. 8D: toxicity assay based on prest Blue HS viability reagent. After 24 hours transfection, the transfected cells were incubated with pre-warmed prest Blue HS reagent (10% v/v) for 15 minutes at 37 ℃. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 540/Em 590). The embodiment shown in fig. 8D corresponds to the procedure outlined in example 8D.

Fig. 8E: and (5) pH measurement. Measurements were made before and after 4 hours of dialysis against 1x DPBS ph 7.4. The embodiment shown in fig. 8E corresponds to the procedure outlined in example 8E.

Fig. 9A: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 9A corresponds to the procedure outlined in example 9A.

Fig. 9B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 9B corresponds to the procedure outlined in example 9B.

Fig. 9C: firefly luciferase expression in vivo in IM administration. 5ug of encapsulated mRNA was injected into mice in intramuscular (I.M.), intradermal (I.D.), and intravenous (I.V.) injections. The ROI was calculated using the IVIS system. Imaging was performed at 4 and 20 hours. The embodiment shown in fig. 9C corresponds to the procedure outlined in example 9C.

Fig. 10A: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 10A corresponds to the procedure outlined in example 10A.

Fig. 10B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 10B corresponds to the procedure outlined in example 10B.

Fig. 10C: and (5) pH measurement. Measurements were made prior to dialysis. The embodiment shown in fig. 10C corresponds to the procedure outlined in example 10C.

FIG. 11A firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 _-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 11A corresponds to the procedure outlined in example 11A.

Fig. 11B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 11B corresponds to the procedure listed in example 11B.

Fig. 11C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 11C corresponds to the procedure outlined in example 11C.

Fig. 11D: and (5) pH measurement. Measurements were made before and after 4 hours of dialysis against 1x DPBS ph 7.4. The embodiment shown in fig. 11D corresponds to the procedure outlined in example 11D.

FIG. 12A firefly luciferase assay for mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curve was prepared in 5-fold serial dilutions in 10% EMEM And (5) preparing. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 12A corresponds to the procedure outlined in example 12A.

Fig. 12B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 12B corresponds to the procedure outlined in example 12B.

Fig. 12C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 12C corresponds to the procedure outlined in example 12C.

Fig. 12D: and (5) pH measurement. Measurements were made before (well 1) and after 4 hours of dialysis against 1x DPBS ph 7.4. The embodiment shown in fig. 12D corresponds to the procedure outlined in example 12D.

FIG. 13A firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 13A corresponds to the procedure outlined in example 13A.

Fig. 13B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 13B corresponds to the procedure outlined in example 13B.

Fig. 13C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 13C corresponds to the procedure outlined in example 13C.

FIG. 14A firefly luciferase assay of mRNA delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 14A corresponds to the procedure outlined in example 14A.

Fig. 14B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 14B corresponds to the procedure outlined in example 14B.

Fig. 14C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 14C corresponds to the procedure outlined in example 14C.

Fig. 15A: initial mRNA concentration, initial lipid mixture concentration, initial sodium acetate concentration yielding the highest efficacy for each particular initial mRNA concentration tested in examples 13 and 14 (fig. 13 and 14). The embodiment shown in fig. 15A corresponds to the procedure outlined in example 15A.

FIG. 15B mRNA delivery efficiency combining examples 13 and 14 of optimal sodium acetate concentration at each particular initial mRNA concentrationFirefly luciferase assay. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence. The embodiment shown in fig. 15B corresponds to the procedure outlined in example 15B.

Fig. 15C: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528). The embodiment shown in fig. 15C corresponds to the procedure outlined in example 15C.

Fig. 15D: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing. The embodiment shown in fig. 15D corresponds to the procedure outlined in example 15D.

FIG. 16A firefly luciferase assay of mRNA delivery efficiency as listed in example 16.

Fig. 16B: ribogreen assay of mRNA encapsulation efficiency as listed in example 16.

Fig. 16C: LNP-sized dynamic light scattering (PDI on the right side of the y-axis for red dots) as listed in example 16.

Fig. 17A: firefly luciferase assay of mRNA delivery efficiency as listed in example 17.

Fig. 17B: ribogreen assay of mRNA encapsulation efficiency as listed in example 17.

Fig. 17C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis) as listed in example 17.

Fig. 18A: firefly luciferase assay of mRNA delivery efficiency as listed in example 18.

Fig. 18B: ribogreen assay of mRNA encapsulation efficiency as listed in example 18.

Fig. 18C: LNP-sized dynamic light scattering (PDI on the right side of the y-axis for red dots) as listed in example 18.

Fig. 19A: firefly luciferase assay of mRNA delivery efficiency as listed in example 19.

Fig. 19B: ribogreen assay of mRNA encapsulation efficiency as listed in example 19.

Fig. 19C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis) as listed in example 19.

Fig. 20A: ribogreen assay of mRNA encapsulation efficiency as listed in example 20.

Fig. 20B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis) as listed in example 20.

Fig. 20C: firefly luciferase expression in vivo in IM administration as set forth in example 20.

Fig. 21A: in vivo immunogenicity endpoint ELISA anti-RBD titers as listed in example 21.

Fig. 21B: in vivo immunogenicity FRNT50 titers determined by pseudo-neutralization as set forth in example 21.

Fig. 22A: in vivo protection against viral challenge-survival ratio, body weight and temperature in the challenge model as listed in example 22.

Fig. 22B: body weight and temperature in the challenge model as set forth in example 22.

Fig. 23A: ribogreen assay of mRNA encapsulation efficiency as listed in example 23.

Fig. 23B: LNP-sized dynamic light scattering (red dots are PDI on the right side of the y-axis) as listed in example 23.

Fig. 23C: in vivo firefly luciferase expression in IM and IV administration as listed in example 23.

Fig. 24A: firefly luciferase assay of mRNA delivery efficiency as listed in example 24.

Fig. 24B: ribogreen assay of mRNA encapsulation efficiency as listed in example 24.

Fig. 24C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis) as listed in example 24.

Fig. 25A: firefly luciferase assay of mRNA delivery efficiency as set forth in example 25.

Fig. 25B: ribogreen assay of mRNA encapsulation efficiency as listed in example 25.

Fig. 25C: LNP-sized dynamic light scattering (PDI on the right side of the y-axis for red dots) as listed in example 25.

Fig. 26A: firefly luciferase assay of mRNA delivery efficiency as set forth in example 26.

Fig. 26B: ribogreen assay of mRNA encapsulation efficiency as listed in example 26.

Fig. 26C: LNP-sized dynamic light scattering (PDI on the right side of the y-axis for red dots) as listed in example 26.

Fig. 27A: firefly luciferase assay of mRNA delivery efficiency as set forth in example 27.

Fig. 27B: ribogreen assay of mRNA encapsulation efficiency as listed in example 27.

Fig. 27C: LNP-sized dynamic light scattering (red dots are PDI on the right side of the y-axis) as listed in example 27.

Fig. 27D: in vivo and ex vivo firefly luciferase expression by IM administration of LNP mixed at 1.5mg/ml as listed in example 27.

Fig. 27E: in vivo and ex vivo firefly luciferase expression administered as IV of 1.5mg/ml mixed LNP as set forth in example 27.

Fig. 28A: firefly luciferase assay of mRNA delivery efficiency as listed in example 28.

Fig. 28B: ribogreen assay of mRNA encapsulation efficiency as listed in example 28.

Fig. 28C: LNP-sized dynamic light scattering (PDI on the right side of the y-axis for red dots) as listed in example 28.

Fig. 28D: in vivo and ex vivo firefly luciferase expression by IM administration of LNP mixed at 1.5mg/ml as listed in example 28.

Fig. 28E: in vivo and ex vivo firefly luciferase expression administered as IV of 1.5mg/ml mixed LNP as listed in example 28.

Fig. 29A: firefly luciferase assay of mRNA delivery efficiency as listed in example 29.

Fig. 29B: ribogreen assay of mRNA encapsulation efficiency as listed in example 29.

Fig. 29C: LNP-sized dynamic light scattering (red dots are PDI on the right side of the y-axis) as listed in example 29.

Fig. 30A: firefly luciferase expression in vivo at the injection site in IM administration as set forth in example 30.

Fig. 30B: ex vivo firefly luciferase expression in IM administration as set forth in example 30.

Fig. 31A: firefly luciferase expression in vivo at the injection site in IM administration as set forth in example 31.

Fig. 31B: ex vivo firefly luciferase expression in IM administration as set forth in example 31.

Fig. 32A: firefly luciferase assay of mRNA delivery efficiency as set forth in example 32.

Fig. 32B: ribogreen assay of mRNA encapsulation efficiency as listed in example 32.

Fig. 32C: LNP-sized dynamic light scattering (PDI on the right side of the y-axis for red dots) as listed in example 32.

Fig. 33A: firefly luciferase assay of mRNA delivery efficiency as listed in example 33.

Fig. 33B: ribogreen assay of mRNA encapsulation efficiency as listed in example 33.

Fig. 33C: LNP-sized dynamic light scattering (red dots are PDI on the right side of the y-axis) as listed in example 33.

Fig. 33D: firefly luciferase expression in vivo in IM administration of LNP mixed at 1.5mg/ml as listed in example 33.

Fig. 33E: ex vivo firefly luciferase expression administered at IV of 1.5mg/ml mixed LNP as listed in example 33.

Figure 34 shows that LNP potency and endosomal internalization increased as lipid and mRNA concentrations increased during microfluidic mixing. A) KC2 LNP assembled at higher concentrations produced higher Fluc expression in vitro at the same dose of 25-200ng per well containing 12k hek293 cells. B) LNP produced at higher mix concentrations (total lipid concentration in mM at mix shown above animals) and diluted to a constant 5 μg dose in 50 μl of IM injection was more effective (color bars are emissivity in 107p/sec/cm 2/sr). C) Zeta potential measurements revealed a greater increase in protonation when the pH was reduced from 7.4 to 5 for LNP prepared by high concentration mixing, indicating greater endosomal release.

Detailed Description

Disclosed herein are methods of increasing the efficacy of nucleic acid-loaded lipid nanoparticles by certain novel and unexpectedly superior LNP manufacturing techniques. Also disclosed are pharmaceutical compositions containing LNPs manufactured according to the manufacturing methods described herein.

The methods disclosed herein overcome the major technical challenges and high costs associated with previous LNP manufacturing techniques. Thus, the methods disclosed herein greatly improve the industrial production of LNP in an unexpected manner, thereby providing a more efficient LNP for nucleic acid delivery.

One embodiment of the invention disclosed herein is a method showing increased LNP efficacy due to increased mixed concentrations of lipids and mRNA during assembly.

The methods disclosed herein are applicable to any ionizable lipid and nucleic acid payload. While not wishing to be bound by any particular mechanism of action, it is believed that increasing LNP potency is mediated by increasing endosomal release and subsequent dissociation of mRNA from ionizable lipids. Furthermore, differences in mixing concentration, changes in charge state, distribution or state of LNP components, in a manner that promotes endosomal escape and mRNA release, result in changes in ultrastructural and ionization properties (ionizable lipids or cholesterol are closer to the LNP periphery, ionizable lipid-mRNA interactions and internal structure changes, a greater proportion of non-protonated ionizable lipids available for endosomal ionization, multilamellar, terminal structure). Preferably, LNPs formed by the methods disclosed herein that deliver nucleic acids (e.g., mRNA encoded immunogens) are more effective, e.g., provide greater protection against viral challenge, than LNPs formed at the same dose at the current low concentrations.

While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to aid in the description of the presently disclosed subject matter.

Unless defined otherwise below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques as generally understood in the art, including variations of those techniques or substitution of equivalent techniques that would be apparent to one of ordinary skill in the art. While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to aid in the description of the presently disclosed subject matter. Accordingly, unless defined otherwise, all technical and scientific terms and any abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art of the presently disclosed subject matter. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used in the practice of the presently disclosed subject matter, the specific compositions, methods, kits, and means for communicating information are described herein. It should be understood that the particular compositions, methods, kits, and means for communicating information described herein are merely exemplary and that the presently disclosed subject matter is not intended to be limited to only those embodiments.

Following long-standing patent law convention, the terms "a" and "an" as used in the present application, including the claims, refer to "one or more". For example, in some embodiments, the phrases "LNP", "nucleic acid" refer to one or more LNPs or nucleotides, respectively.

It should be understood that for all numerical boundaries in this disclosure describing a certain parameter, such as "about," "at least," "less than," and "greater than," the description necessarily also encompasses any range defined by the recited values. Thus, for example, a description of "at least 1, 2, 3, 4, or 5" also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, and so forth.

The term "about" as used herein to refer to a measurable amount such as a weight, time, dose (e.g., therapeutic dose), etc., is intended to encompass a variation of +/-20% in some embodiments, +/-10% in some embodiments, +/-5% in some embodiments, +/-1% in some embodiments, +/-0.1% in some embodiments, +/-0.5% in some embodiments, and +/-0.01% in some embodiments, as compared to a specified amount, as such variation is suitable for performing the disclosed methods.

As used herein, the term "and/or" when used in the context of a list of entities means that the entities exist alone or in all possible combinations and sub-combinations. Thus, for example, the phrase "A, B, C and/or D" includes A, B, C and D alone, and includes any and all combinations and subcombinations of A, B, C and D. It should be further appreciated that for each of the plurality of possible options listing a given element (i.e., for all "Markush" groups of optional components for any element and the like), the optional components may be present alone or in any combination or subcombination of the optional components in some embodiments. Each combination and sub-combination is implicitly contemplated in the list of forms and is solely for convenience and does not list each such combination or sub-combination. In addition, it is to be further understood that all statements of "or" are to be understood as "and/or" unless the context clearly requires that the listed components are to be considered as merely alternatives (e.g., if the components are mutually exclusive and/or not combinable with each other in a given context).

I. Definition of the definition

The term "lipid" refers to a group of organic compounds that are fatty acid esters and are characterized as insoluble in water, but soluble in many organic solvents. Lipids are generally classified into at least three categories: (1) "simple lipids" including fats and oils and waxes; (2) "complex lipids" including phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

The term "lipid nanoparticle" or "LNP" as used herein refers to particles comprising a plurality, i.e. more than one lipid molecule, physically associated with each other by intermolecular forces. In one embodiment, the LNP is loaded with a nucleic acid payload. LNP may have one or more different types of lipids. The lipid nanoparticle may be, for example, a microsphere (including unilamellar and multilamellar vesicles, e.g., "liposomes" -lamellar phase lipid bilayers, in some embodiments substantially spherical, and may comprise an aqueous core in more particular embodiments, e.g., comprising a substantial portion of an RNA molecule), a dispersed phase in an emulsion, micelles in suspension, or an internal phase.

In some embodiments, the lipid nanoparticle has a size of about 1 to about 2,500nm, about 10 to about 1,500nm, about 20 to about 1,000nm, in one embodiment about 50 to about 600nm, in one sub-embodiment about 50 to about 400nm, in one sub-embodiment about 50 to about 250nm, and in one sub-embodiment about 50 to about 150 nm. Unless indicated otherwise, all sizes mentioned herein are the average size (diameter) of the fully formed nanoparticles as measured by dynamic light scattering on Malvern Zetasizer. The nanoparticle samples were diluted in Phosphate Buffered Saline (PBS) to give a count rate of about 200-400kcts. The data are presented as a number weighted average obtained by the transformation of the intensity weighted average. The number weighted average is preferred because it corresponds most closely to the physical diameter of the particles as measured by electron microscopy.

As used herein, "LNP lipid" refers to the individual lipid molecules that form LNP. In certain embodiments, the LNP lipid is an ionizable cationic lipid.

As used herein, the term "cationic lipid" refers to a lipid that is cationic or becomes cationic (protonated) when the pH is reduced below the pKa of the ionizable groups of the lipid present in the LNP (i.e., the pKa of the ionizable lipid in the lipid environment of the LNP that is different from the pKa of the ionizable lipid in the aqueous medium), but is progressively more neutral at higher pH values. At pH values below pKa, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "cationic lipid" includes zwitterionic lipids that exhibit a positive charge upon a decrease in pH. Notably, most helper lipids, such as DSPC, are zwitterionic but not cationic, as they have phosphate groups that balance any cationic charge.

The term "cationic lipid" also refers to any of a number of lipid materials that carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N-dioleoyl-N, N-dimethyl ammonium chloride (DODAC); n- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride (DOTMA); n, N-distearyl-N, N-dimethyl ammonium bromide (DDAB); n- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride (DOTAP); 3- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol) and N- (1, 2-dimyristoxyprop-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). In addition, many commercially available cationic lipid formulations are available for use in the present invention. These formulations include, for example, (commercial cationic liposomes comprising DOTMA and 1, 2-dioleoyl-sn-3-phosphoethanolamine (DOPE), obtained from GIBCO/BRL, grand Island, n.y.); />(commercially available cationic liposomes comprising N- (1- (2, 3-dioleoyloxy) propyl) -N- (2- (spermidine carboxamide) ethyl) -N, N-dimethyl-ammonium trifluoroacetate (DOSPA) and (DOPE), obtained from GIBCO/BRL); and->(commercially available cationic lipids comprising dioctadecyl amidoglycyl carboxy spermine (DOGS) in ethanol, obtained from Promega Corp., madison, wis.). The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLenDMA).

In some embodiments, the "LNP lipid" is MC3, DLin, and/or KC2, as shown below. The table highlights the pKa of the ionizable lipids measured in LNP (TNS pKa) compared to pKa in aqueous medium predicted by commercial software ACDLabs persistence:

in some embodiments, the invention encompasses LNP lipids as compounds encompassed by formula I:

wherein each R is ¹ And each R ² Independently selected from the group consisting of: H. optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ₆ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ An arylalkyl group; optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group, an optionally substituted amine; or R is ¹ And R is ² Can together form a cycloalkyl or heterocycloalkyl ring, wherein each R ³ And R is ⁴ Independently selected from the group consisting of: optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl; wherein each R is ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ⁹ And R is ¹⁰ Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl; wherein each of w, x, y and z is independently an integer from 0 to 10; wherein each Q is independently an atom selected from O, NH and S; wherein each m is an integer from 0 to 8; and wherein L is ¹ And L ² Independently selected from the group consisting of: -C (=o) -; OC (=o); -NH-C (=o) -; -C (=o) NH-; -SO-; -SO ₂ -；-SO ₃ -；-NSO ₂ -；-SO ₂ N-；-NH((C ₁ -C ₈ ) An alkyl group); -N ((C) ₁ -C ₈ ) Alkyl group ₂ ；-NH((C ₆ ) An aryl group); -N ((C) ₆ ) Aryl group ₂ ；-C(＝O)R ¹ -；-CO((C ₁ -C ₈ ) An alkyl group); CO ((C) ₆ ) An aryl group); -CO ₂ ((C ₁ -C ₈ ) An alkyl group); -CO ₂ ((C ₆ ) An aryl group); -SO ₂ ((C ₁ -C ₈ ) An alkyl group); and-SO ₂ ((C ₆ ) Aryl).

In other embodiments, the invention encompasses LNP lipids as compounds encompassed by formula II:

wherein each R is ¹ And each R ² Independently selected from the group consisting of: H. optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ₆ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ An arylalkyl group; optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group, an optionally substituted amine; or R is ¹ And R is ² May together form a cycloalkyl or heterocycloalkyl ring, wherein if Q is S or O, R is attached to S or O ¹ Is an electron pair; wherein each R is ³ And R is ⁴ Independently selected from the group consisting of: optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl; wherein each R is ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ⁹ And R is ¹⁰ Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl, wherein each of x, y and z is independently an integer from 0 to 10; wherein G and Q are each independently an atom selected from CH, O, N and S;

Wherein each of m and n is an integer from 0 to 8; and wherein L is ¹ And L ² Independently selected from the group consisting of: -C (=o) -; OC (=o) -; -C (=o) O-; -NH-C(＝O)-；-C(＝O)NH-；-SO-；-SO ₂ -；-SO ₃ -；-NSO ₂ -；-SO ₂ N-；-NH((C ₁ -C ₈ ) An alkyl group); -N ((C) ₁ -C ₈ ) Alkyl group ₂ ；-NH((C ₆ ) An aryl group); -N ((C) ₆ ) Aryl group ₂ ；-C(＝O)R ¹ -；-CO((C ₁ -C ₈ ) An alkyl group); CO ((C) ₆ ) An aryl group); -CO ₂ ((C ₁ -C ₈ ) An alkyl group); -CO ₂ ((C ₆ ) An aryl group); -SO ₂ ((C ₁ -C ₈ ) An alkyl group); and-SO ₂ ((C ₆ ) Aryl).

In another embodiment, the invention encompasses LNP lipids as compounds encompassed by formula III:

wherein each R is ¹ And each R ² Independently selected from the group consisting of: H. optionally substituted C ₁ -C ₁₂ Alkyl, optionally substituted C ₂ -C ₁₂ Alkenyl, optionally substituted C ₂ -C ₁₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ⁶ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ An arylalkyl group; optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group, an optionally substituted amine; or R is ¹ And R is ² May together form a cycloalkyl or heterocycloalkyl ring, wherein if Q is S or O, R is attached to S or O ¹ Is an electron pair;

wherein each R is ³ And R is ⁴ Independently selected from the group consisting of: optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl;

wherein each R is ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ 、R ¹¹ 、R ¹² 、R ¹³ 、R ¹⁴ 、R ¹⁵ And R is ¹⁶ Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ An alkynyl group, an amino group,

wherein u, v, w, x, y and z are each independently integers from 0 to 20;

wherein each Q is independently an atom selected from O, NH, S, or a disulfide bond;

wherein each m is an integer from 0 to 4, preferably 0, 1 or 2; and is also provided with

Wherein each L ¹ And L ² Independently selected from the group consisting of: -C (=o) -; OC (=o) -; -OC (=o) O-; -C (=o) O-; -C (=o) O (CR ⁵ R ⁶ R ⁷ )；-NH-C(＝O)-；-C(＝O)NH-；-SO-；-SO ₂ -；-SO ₃ -；-NSO ₂ -；-SO ₂ N-；-NH((C ₁ -C ₈ ) An alkyl group); -N ((C) ₁ -C ₈ ) Alkyl group ₂ ；-NH((C ₆ ) An aryl group); -N ((C) ₆ ) Aryl group ₂ ；-NHC(＝O)NH-；-NHC(＝O)O-；-OC(＝O)NH-；-NHC(＝O)NR ¹ -；-NHC(＝O)O-；-OC(＝O)NR ¹ -；-C(＝O)R ¹ -；-CO((C ₁ -C ₈ ) An alkyl group); CO ((C) ₆ ) An aryl group); -CO ₂ ((C ₁ -C ₈ ) An alkyl group); -CO ₂ ((C ₆ ) An aryl group); -SO ₂ ((C ₁ -C ₈ ) An alkyl group); and-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, R ¹ H.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ² H.

In certain embodiments, R ² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl group

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ² Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ³ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ An alkyl group.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkenyl groups.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ An alkyl group.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkenyl groups.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁵ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁵ H.

In certain embodiments, R ⁵ Is OH.

In certain embodiments, R ⁵ Is halo.

In certain embodiments, R ⁵ Is phenyl.

In certain embodiments, R ⁵ Is benzyl.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁶ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or (b)Substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁶ H.

In certain embodiments, R ⁶ Is OH.

In certain embodiments, R ⁶ Is halo.

In certain embodiments, R ⁶ Is phenyl.

In certain embodiments, R ⁶ Is benzyl.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H.

In certain embodiments, R ⁷ Is OH.

In certain embodiments, R ⁷ Is halo.

In certain embodiments, R ⁷ Is phenyl.

In certain embodiments, R ⁷ Is benzyl.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H.

In certain embodiments, R ⁸ Is OH.

In certain embodiments, R ⁸ Is halo.

In certain embodiments, R ⁸ Is phenyl.

In certain embodiments, R ⁸ Is benzyl.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H.

In certain embodiments, R ⁹ Is OH.

In certain embodiments, R ⁹ Is halo.

In certain embodiments, R ⁹ Is phenyl.

In certain embodiments, R ⁹ Is benzyl.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H.

In certain embodiments, R ¹⁰ Is OH.

In certain embodiments, R ¹⁰ Is halo.

In certain embodiments, R ¹⁰ Is phenyl.

In certain embodiments, R ¹⁰ Is benzyl.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H.

In certain embodiments, R ¹¹ Is OH.

In certain embodiments, R ¹¹ Is halo.

In some embodimentsIn the scheme, R ¹¹ Is phenyl.

In certain embodiments, R ¹¹ Is benzyl.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹² H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹² H.

In certain embodiments, R ¹² Is OH.

In certain embodiments, R ¹² Is halo.

In certain embodiments, R ¹² Is phenyl.

In certain embodiments, R ¹² Is benzyl.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H.

In certain embodiments, R ¹³ Is OH.

In certain embodiments, R ¹³ Is halo.

In certain embodiments, R ¹³ Is phenyl.

In certain embodiments, R ¹³ Is benzyl.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H.

In certain embodiments, R ¹⁴ Is OH.

In certain embodiments, R ¹⁴ Is halo.

In certain embodiments, R ¹⁴ Is phenyl.

In certain embodiments, R ¹⁴ Is benzyl.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H.

In certain embodiments, R ¹⁵ Is OH.

In certain embodiments, R ¹⁵ Is halo.

In certain embodiments, R ¹⁵ Is phenyl.

In certain embodiments, R ¹⁵ Is benzyl.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H.

In certain embodiments, R ¹⁶ Is OH.

In certain embodiments, R ¹⁶ Is halo.

In certain embodiments, R ¹⁶ Is phenyl.

In certain embodiments, R ¹⁶ Is benzyl.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups. In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups. In certain embodiments, u is 0.

In certain embodiments, u is 1.

In certain embodiments, u is 2.

In certain embodiments, u is 3.

In certain embodiments, u is 4.

In certain embodiments, u is 5.

In certain embodiments, u is 6.

In certain embodiments, u is 7.

In certain embodiments, u is 8.

In certain embodiments, u is 9.

In certain embodiments, u is 10.

In certain embodiments, u is 11.

In certain embodiments, u is 12.

In certain embodiments, u is 13.

In certain embodiments, u is 14.

In certain embodiments, u is 15.

In certain embodiments, u is 16.

In certain embodiments, u is 17.

In certain embodiments, u is 18.

In certain embodiments, u is 19.

In certain embodiments, u is 20.

In certain embodiments, v is 0.

In certain embodiments, v is 1.

In certain embodiments, v is 2.

In certain embodiments, v is 3.

In certain embodiments, v is 4.

In certain embodiments, v is 5.

In certain embodiments, v is 6.

In certain embodiments, v is 7.

In certain embodiments, v is 8.

In certain embodiments, v is 9.

In certain embodiments, v is 10.

In certain embodiments, v is 11.

In certain embodiments, v is 12.

In certain embodiments, v is 13.

In certain embodiments, v is 14.

In certain embodiments, v is 15.

In certain embodiments, v is 16.

In certain embodiments, v is 17.

In certain embodiments, v is 18.

In certain embodiments, v is 19.

In certain embodiments, v is 20.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, x is 0. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. In certain embodiments, x is 4. In certain embodiments, x is 5. In certain embodiments, x is 6. In certain embodiments, x is 7. In certain embodiments, x is 8. In certain embodiments, x is 9. In certain embodiments, x is 10.

In certain embodiments, x is 11.

In certain embodiments, x is 12.

In certain embodiments, x is 13.

In certain embodiments, x is 14.

In certain embodiments, x is 15.

In certain embodiments, x is 16.

In certain embodiments, x is 17.

In certain embodiments, x is 18.

In certain embodiments, x is 19.

In certain embodiments, x is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments, L ¹ Is a key.

In certain embodiments, L ¹ is-C (=o) -.

In certain embodiments, L ¹ is-OC (=O) O-.

In certain embodiments, L ¹ is-NH-C (=O) -.

In certain embodiments, L ¹ is-SO-.

In certain embodiments, L ¹ is-SO ₂ -。

In certain embodiments, L ¹ OC (=o).

In certain embodiments, L ¹ is-C (=O) O-.

In certain embodiments, L ¹ is-C (=O) NH-.

In certain embodiments, L ¹ is-SO ₃ -。

In certain embodiments, L ¹ is-NSO ₂ -。

In certain embodiments, L ¹ is-SO ₂ N。

In certain embodiments, L ¹ is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ¹ is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ¹ Is dioxopyrrolidine-dione.

In certain embodiments, L ¹ is-C (=O) R ¹ -。

In certain embodiments, L ¹ is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ¹ is-C (=O) O (CR) ¹ R ² R ³ ) In certain embodiments, L ¹ is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl). In certain embodiments, L ¹ is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² Is a key.

In certain embodiments, L ² is-C (=o) -.

In certain embodiments, L ² is-OC (=O) O-.

In certain embodiments, L ² is-NH-C (=O) -.

In certain embodiments, L ² is-SO-.

In certain embodiments, L ² is-SO ₂ -。

In certain embodiments, L ² OC (=o).

In certain embodiments, L ² is-C (=O) O-.

In certain embodiments, L ² is-C (=O) NH-.

In certain embodiments, L ² is-SO ₃ -。

In certain embodiments, L ² is-NSO ₂ -。

In certain embodiments, L ² is-SO ₂ N。

In certain embodiments, L ² is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ² is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ² is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ² Is dioxopyrrolidine-dione.

In certain embodiments, L ² is-C (=O) R ¹ -。

In certain embodiments, L ² is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is NH.

In certain embodiments, Q is a disulfide bond.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

In another embodiment, the invention encompasses LNP lipids as compounds encompassed by formula IV:

wherein each R is ¹ And each R ² Independently selected from the group consisting of: H. optionally substituted C ₁ -C ₁₂ Alkyl, optionally substituted C ₂ -C ₁₂ Alkenyl, optionally substituted C ₂ -C ₁₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ⁶ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ An arylalkyl group; optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group, an optionally substituted amine; or R is ¹ And R is ² May together form a cycloalkyl or heterocycloalkyl ring, wherein if Q is S or O, R is attached to S or O ¹ Is an electron pair;

wherein each R is ⁵ 、R ⁶ 、R ^5’ 、R ^6’ 、R ^5” And R is ^6” Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ An alkynyl group, an amino group,

wherein each R is ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ 、R ¹¹ 、R ¹² 、R ¹³ 、R ¹⁴ 、R ¹⁵ And R is ¹⁶ Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ An alkynyl group, an amino group,

wherein u, v, w, y and z are each independently integers from 0 to 20;

wherein each Q is independently an atom selected from O, NH, S, or a disulfide bond; and is also provided with

Wherein each L ¹ And L ² Independently selected from the group consisting of: -C (=o) -; OC (=o) -; -OC (=o) O-; -C (=o) O-; -C (=o) O (CR ⁵ R ⁶ R ⁷ ) _m ；-NH-C(＝O)-；-C(＝O)NH-；-SO-；-SO ₂ -；-SO ₃ -；-NSO ₂ -；-SO ₂ N-；-NH((C ₁ -C ₈ ) An alkyl group); -N ((C) ₁ -C ₈ ) Alkyl group ₂ ；-NH((C ₆ ) An aryl group); -N ((C) ₆ ) Aryl group ₂ ；-NHC(＝O)NH-；-NHC(＝O)O-；-OC(＝O)NH-；-NHC(＝O)NR ¹ -；-NHC(＝O)O-；-OC(＝O)NR ¹ -；-C(＝O)R ¹ -；-CO((C ₁ -C ₈ ) An alkyl group); CO ((C) ₆ ) An aryl group); -CO ₂ ((C ₁ -C ₈ ) An alkyl group); -CO ₂ ((C ₆ ) An aryl group); -SO ₂ ((C ₁ -C ₈ ) An alkyl group); and-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, R ¹ H.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ¹ Is substituted or unsubstitutedC ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ² H.

In certain embodiments, R ² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl group

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ² Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, each R ⁵ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substitutedOr unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁵ H.

In certain embodiments, R ⁵ Is OH.

In certain embodiments, R ⁵ Is halo.

In certain embodiments, R ⁵ Is phenyl.

In certain embodiments, R ⁵ Is benzyl.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁶ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁶ H.

In certain embodiments, R ⁶ Is OH.

In certain embodiments, R ⁶ Is halo.

In certain embodiments, R ⁶ Is phenyl.

In certain embodiments, R ⁶ Is benzyl.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In some embodimentsIn the scheme, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H.

In certain embodiments, R ⁷ Is OH.

In certain embodiments, R ⁷ Is halo.

In certain embodiments, R ⁷ Is phenyl.

In certain embodiments, R ⁷ Is benzyl.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H.

In certain embodiments, R ⁸ Is OH.

In certain embodiments, R ⁸ Is halo.

In certain embodiments, R ⁸ Is phenyl.

In certain embodiments, R ⁸ Is benzyl.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H.

In certain embodiments, R ⁹ Is OH.

In certain embodiments, R ⁹ Is halo.

In certain embodiments, R ⁹ Is phenyl.

In certain embodiments, R ⁹ Is benzyl.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H.

In certain embodiments, R ¹⁰ Is OH.

In certain embodiments, R ¹⁰ Is halogenA base.

In certain embodiments, R ¹⁰ Is phenyl.

In certain embodiments, R ¹⁰ Is benzyl.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H.

In certain embodiments, R ¹¹ Is OH.

In certain embodiments, R ¹¹ Is halo.

In certain embodiments, R ¹¹ Is phenyl.

In certain embodiments, R ¹¹ Is benzyl.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹² H.

In certain embodiments, R ¹² Is OH.

In certain embodiments, R ¹² Is halo.

In certain embodiments, R ¹² Is phenyl.

In certain embodiments, R ¹² Is benzyl.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H.

In certain embodiments, R ¹³ Is OH.

In certain embodiments, R ¹³ Is halo.

In certain embodiments, R ¹³ Is phenyl.

In certain embodiments, R ¹³ Is benzyl.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H.

In certain embodiments, R ¹⁴ Is OH.

In certain embodiments, R ¹⁴ Is halo.

In certain embodiments, R ¹⁴ Is phenyl.

In certain embodiments, R ¹⁴ Is benzyl.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H.

In certain embodiments, R ¹⁵ Is OH.

In certain embodiments, R ¹⁵ Is halo.

In certain embodiments, R ¹⁵ Is phenyl.

In certain embodiments, R ¹⁵ Is benzyl.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H.

In certain embodiments, R ¹⁶ Is OH.

In certain embodiments, R ¹⁶ Is halo.

In certain embodiments, R ¹⁶ Is phenyl.

In certain embodiments, R ¹⁶ Is benzyl.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, u is 0.

In certain embodiments, u is 1.

In certain embodiments, u is 2. In certain embodiments, u is 3. In certain embodiments, u is 4. In certain embodiments, u is 5. In certain embodiments, u is 6. In certain embodiments, u is 7. In certain embodiments, u is 8.

In certain embodiments, u is 9.

In certain embodiments, u is 10.

In certain embodiments, u is 11.

In certain embodiments, u is 12.

In certain embodiments, u is 13.

In certain embodiments, u is 14.

In certain embodiments, u is 15.

In certain embodiments, u is 16.

In certain embodiments, u is 17.

In certain embodiments, u is 18.

In certain embodiments, u is 19.

In certain embodiments, u is 20.

In certain embodiments, v is 0.

In certain embodiments, v is 1.

In certain embodiments, v is 2.

In certain embodiments, v is 3.

In certain embodiments, v is 4.

In certain embodiments, v is 5.

In certain embodiments, v is 6.

In certain embodiments, v is 7.

In certain embodiments, v is 8.

In certain embodiments, v is 9.

In certain embodiments, v is 10.

In certain embodiments, v is 11.

In certain embodiments, v is 12.

In certain embodiments, v is 13.

In certain embodiments, v is 14.

In certain embodiments, v is 15.

In certain embodiments, v is 16.

In certain embodiments, v is 17.

In certain embodiments, v is 18.

In certain embodiments, v is 19.

In certain embodiments, v is 20.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, y is 0. In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4. In certain embodiments, y is 5. In certain embodiments, y is 6. In certain embodiments, y is 7. In certain embodiments, y is 8. In certain embodiments, y is 9. In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments, L ¹ Is a key.

In certain embodiments, L ¹ is-C (=o) -.

In certain embodiments, L ¹ is-OC (=O) O-.

In certain embodiments, L ¹ is-NH-C (=O) -.

In certain embodiments, L ¹ is-SO-.

In certain embodiments, L ¹ is-SO ₂ -。

In certain embodiments, L ¹ OC (=o).

In certain embodiments, L ¹ is-C (=O) O-.

In certain embodiments, L ¹ is-C (=O) NH-.

In certain embodiments, L ¹ is-SO ₃ -。

In certain embodiments, L ¹ is-NSO ₂ -。

In certain embodiments, L ¹ is-SO ₂ N。

In certain embodiments, L ¹ is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ¹ is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ¹ Is dioxopyrrolidine-dione.

In certain embodiments, L ¹ is-C (=O) R ¹ -。

In certain embodiments, L ¹ is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ¹ is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² Is a key.

In certain embodiments, L ² is-C (=o) -.

In certain embodiments, L ² is-OC (=O) O-.

In certain embodiments, L ² is-NH-C (=O) -.

In certain embodiments, L ² is-SO-.

In certain embodiments, L ² is-SO ₂ -。

In certain embodiments, L ² OC (=o).

In certain embodiments, L ² is-C (=O) O-.

In certain embodiments, L ² is-C (=O) NH-.

In certain embodiments, L ² is-SO ₃ -。

In certain embodiments, L ² is-NSO ₂ -。

In certain embodiments, L ² is-SO ₂ N。

In certain embodiments, L ² is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ² is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ² is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ² Is dioxopyrrolidine-dione.

In certain embodiments, L ² is-C (=O) R ¹ -。

In certain embodiments, L ² is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ (CR ¹ R ² R ³ )。

In certain embodiments, L ² is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is NH.

In certain embodiments, Q is a disulfide bond.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

In certain embodiments, the LNP lipid has the following structure:

in certain embodiments, the LNP lipid has the following structure:

/>

in certain embodiments, the LNP lipid has the following structure:

in certain embodiments, the LNP lipid has the following structure:

in certain embodiments, the LNP lipid has the following structure:

in certain preferred embodiments, the LNP lipid has the following structure:

in certain preferred embodiments, the LNP lipid has the following structure:

In certain preferred embodiments, the LNP lipid has the following structure:

in certain preferred embodiments, the LNP lipid has the following structure:

/>

in certain preferred embodiments, the LNP lipid has the following structure:

in certain preferred embodiments, the LNP lipid has the following structure:

in certain embodiments, the LNP lipid is selected from the structures in table 2 below:

table 2 in another embodiment, the present invention encompasses the ionizable lipids of formula V:

wherein each R is ¹ And each R ² Independently selected from the group consisting of: H. optionally substituted C ₁ -C ₁₂ Alkyl, optionally substituted C ₂ -C ₁₂ Alkenyl, optionally substituted C ₂ -C ₁₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ⁶ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ An arylalkyl group; optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group, an optionally substituted amine; or R is ¹ And R is ² May together form a cycloalkyl or heterocycloalkyl ring, wherein if Q is S or O, R is attached to S or O ¹ Is an electron pair;

wherein each R is ³ 、R ⁴ 、R ¹³ And R is ¹⁴ Independently selected from the group consisting of: optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl;

wherein each R is ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ 、R ¹⁵ And R is ¹⁶ Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ An alkynyl group, an amino group,

wherein each of w, x, y and z is independently an integer from 0 to 10;

wherein each Q is independently an atom selected from O, NH, S, or a disulfide bond;

wherein each m is an integer from 0 to 4, preferably 0, 1 or 2; and is also provided with

Wherein each L ¹ And L ² Independently selected from the group consisting of: -C (=o) -; OC (=o) -; -OC (=o) O-; -C (=o) O-; -C (=o) O (CR ⁶ R ⁷ ) _m ；-NH-C(＝O)-；-C(＝O)NH-；-SO-；-SO ₂ -；-SO ₃ -；-NSO ₂ -；-SO ₂ N-；-NH((C ₁ -C ₈ ) An alkyl group); -N ((C) ₁ -C ₈ ) Alkyl group ₂ ；-NH((C ₆ ) An aryl group); -N ((C) ₆ ) Aryl group ₂ ；-NHC(＝O)NH-；-NHC(＝O)O-；-OC(＝O)NH-；-NHC(＝O)NR ¹ -；-NHC(＝O)O-；-OC(＝O)NR ¹ -；-C(＝O)R ¹ -；-CO((C ₁ -C ₈ ) An alkyl group); CO ((C) ₆ ) An aryl group); -CO ₂ ((C ₁ -C ₈ ) An alkyl group); -CO ₂ ((C ₆ ) An aryl group); -SO ₂ ((C ₁ -C ₈ ) An alkyl group); and-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, R ¹ H.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ² H.

In certain embodiments, R ² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl group

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ² Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ³ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ An alkyl group.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkenyl groups.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ An alkyl group.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkenyl groups.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁵ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁵ H.

In certain embodiments, R ⁵ Is OH.

In certain embodiments, R ⁵ Is halo.

In certain embodiments, R ⁵ Is phenyl.

In certain embodiments, R ⁵ Is benzyl.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁶ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁶ H.

In certain embodiments, R ⁶ Is OH.

In certain embodiments, R ⁶ Is halo.

In certain embodiments, R ⁶ Is phenyl.

In certain embodiments, R ⁶ Is benzyl.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H.

In certain embodiments, R ⁷ Is OH.

In certain embodiments, R ⁷ Is halo.

In certain embodiments, R ⁷ Is phenyl.

In certain embodiments, R ⁷ Is benzyl.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H, OH is halogen, phenyl, benzyl, substitutedOr unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H.

In certain embodiments, R ⁸ Is OH.

In certain embodiments, R ⁸ Is halo.

In certain embodiments, R ⁸ Is phenyl.

In certain embodiments, R ⁸ Is benzyl.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H.

In certain embodiments, R ⁹ Is OH.

In certain embodiments, R ⁹ Is halo.

In certain embodiments, R ⁹ Is phenyl.

In certain embodiments, R ⁹ Is benzyl.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H.

In certain embodiments, R ¹⁰ Is OH.

In certain embodiments, R ¹⁰ Is halo.

In certain embodiments, R ¹⁰ Is phenyl.

In certain embodiments, R ¹⁰ Is benzyl.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5. In certain embodiments, w is 6. In certain embodiments, w is 7. In certain embodiments, w is 8. In certain embodiments, w is 9. In certain embodiments, w is 10. In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, x is 0. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. In certain embodiments, x is 4.

In certain embodiments, x is 5.

In certain embodiments, x is 6.

In certain embodiments, x is 7.

In certain embodiments, x is 8.

In certain embodiments, x is 9.

In certain embodiments, x is 10.

In certain embodiments, x is 11.

In certain embodiments, x is 12.

In certain embodiments, x is 13.

In certain embodiments, x is 14.

In certain embodiments, x is 15.

In certain embodiments, x is 16.

In certain embodiments, x is 17.

In certain embodiments, x is 18.

In certain embodiments, x is 19.

In certain embodiments, x is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments, L ¹ Is a key.

In certain embodiments, L ¹ is-C (=o) -.

In certain embodiments, L ¹ is-OC (=O) O-.

In certain embodiments, L ¹ is-NH-C (=O) -.

In certain embodiments, L ¹ is-SO-.

In certain embodiments, L ¹ is-SO ₂ -。

In certain embodiments, L ¹ OC (=o).

In certain embodiments, L ¹ is-C (=O) O-.

In certain embodiments, L ¹ is-C (=O) NH-.

In certain embodiments, L ¹ is-SO ₃ -。

In certain embodiments, L ¹ is-NSO ₂ -。

In certain embodiments, L ¹ is-SO ₂ N。

In certain embodiments, L ¹ is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ¹ is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ¹ Is dioxopyrrolidine-dione.

In certain embodiments, L ¹ is-C (=O) R ¹ -。

In certain embodiments, L ¹ is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ¹ is-C (=O) O (CR) ¹ R ² R ³ ) In certain embodiments, L ¹ is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl). In certain embodiments, L ¹ is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² Is a key.

In certain embodiments, L ² is-C (=o) -.

In certain embodiments, L ² is-OC (=O) O-.

In certain embodiments, L ² is-NH-C (=O) -.

In certain embodiments, L ² is-SO-.

In certain embodiments, L ² is-SO ₂ -。

In certain embodiments, L ² OC (=o).

In some implementationsIn the scheme, L ² is-C (=O) O-.

In certain embodiments, L ² is-C (=O) NH-.

In certain embodiments, L ² is-SO ₃ -。

In certain embodiments, L ² is-NSO ₂ -。

In certain embodiments, L ² is-SO ₂ N。

In certain embodiments, L ² is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ² is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ² is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ² Is dioxopyrrolidine-dione.

In certain embodiments, L ² is-C (=O) R ¹ -。

In certain embodiments, L ² is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is NH.

In certain embodiments, Q is a disulfide bond.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

In another embodiment, the present invention encompasses the ionizable lipids of formula VI:

wherein each R is ¹ And each is provided withR ² Independently selected from the group consisting of: H. optionally substituted C ₁ -C ₁₂ Alkyl, optionally substituted C ₂ -C ₁₂ Alkenyl, optionally substituted C ₂ -C ₁₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ⁶ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ An arylalkyl group; optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group, an optionally substituted amine; or R is ¹ And R is ² May together form a cycloalkyl or heterocycloalkyl ring, wherein if Q is S or O, R is attached to S or O ¹ Is an electron pair;

wherein each R is ³ 、R ⁴ 、R ²³ And R is ²⁴ Independently selected from the group consisting of: optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl;

wherein each R is ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ¹¹ 、R ¹² 、R ¹³ 、R ¹⁷ 、R ¹⁸ 、R ³⁴ 、R ³⁵ 、R ³⁶ Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ An alkynyl group, an amino group,

wherein u, v, w, x, y and z are each independently integers from 0 to 20;

wherein each Q is independently an atom selected from O, NH, S, or a disulfide bond;

wherein each m is an integer from 0 to 4, preferably 0, 1 or 2; and is also provided with

Wherein each L ¹ And L ² Independently selected from the group consisting of: -C (=o) -; OC (=o) -; -OC(＝O)O-；-C(＝O)O-；-C(＝O)O(CR ⁶ R ⁷ ) _m ；-NH-C(＝O)-；-C(＝O)NH-；-SO-；-SO ₂ -；-SO ₃ -；-NSO ₂ -；-SO ₂ N-；-NH((C ₁ -C ₈ ) An alkyl group); -N ((C) ₁ -C ₈ ) Alkyl group ₂ ；-NH((C ₆ ) An aryl group); -N ((C) ₆ ) Aryl group ₂ ；-NHC(＝O)NH-；-NHC(＝O)O-；-OC(＝O)NH-；-NHC(＝O)NR ¹ -；-NHC(＝O)O-；-OC(＝O)NR ¹ -；-C(＝O)R ¹ -；-CO((C ₁ -C ₈ ) An alkyl group); CO ((C) ₆ ) An aryl group); -CO ₂ ((C ₁ -C ₈ ) An alkyl group); -CO ₂ ((C ₆ ) An aryl group); -SO ₂ ((C ₁ -C ₈ ) An alkyl group); and-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, R ¹ H.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ² H.

In certain embodiments, R ² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl group

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ² Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ³ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ An alkyl group.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkenyl groups.

In a preferred embodiment, R ³ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ An alkyl group.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkenyl groups.

In a preferred embodiment, R ⁴ Is substituted or unsubstituted-C (=O) O-C ₁ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁵ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁵ H.

In certain embodiments, R ⁵ Is OH.

In certain embodiments, R ⁵ Is halo.

In certain embodiments, R ⁵ Is phenyl.

In certain embodiments, R ⁵ Is benzyl.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁶ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁶ H.

In certain embodiments, R ⁶ Is OH.

In certain embodiments, R ⁶ Is halo.

In certain embodiments, R ⁶ Is phenyl.

In certain embodiments, R ⁶ Is benzyl.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H.

In certain embodiments, R ⁷ Is OH.

In certain embodiments, R ⁷ Is halo.

In certain embodiments, R ⁷ Is phenyl.

In certain embodiments, R ⁷ Is benzyl.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H.

In certain embodiments, R ⁸ Is OH.

In certain embodiments, R ⁸ Is halo.

In certain embodiments, R ⁸ Is phenyl.

In certain embodiments, R ⁸ Is benzyl.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H.

In certain embodiments, R ⁹ Is OH.

In certain embodiments, R ⁹ Is halo.

In certain embodiments, R ⁹ Is phenyl.

In certain embodiments, R ⁹ Is benzyl.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H.

In certain embodiments, R ¹⁰ Is OH.

In certain embodiments, R ¹⁰ Is halo.

In certain embodiments, R ¹⁰ Is benzeneA base.

In certain embodiments, R ¹⁰ Is benzyl.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H.

In certain embodiments, R ¹¹ Is OH.

In certain embodiments, R ¹¹ Is halo.

In certain embodiments, R ¹¹ Is phenyl.

In certain embodiments, R ¹¹ Is benzyl.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹² H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹² H.

In certain embodiments, R ¹² Is OH.

In certain embodiments, R ¹² Is halo.

In certain embodiments, R ¹² Is phenyl.

In certain embodiments, R ¹² Is benzyl.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H.

In certain embodiments, R ¹³ Is OH.

In certain embodiments, R ¹³ Is halo.

In certain embodiments, R ¹³ Is phenyl.

In certain embodiments, R ¹³ Is benzyl.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H, OH is halogen radicalPhenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H.

In certain embodiments, R ¹⁴ Is OH.

In certain embodiments, R ¹⁴ Is halo.

In certain embodiments, R ¹⁴ Is phenyl.

In certain embodiments, R ¹⁴ Is benzyl.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H.

In certain embodiments, R ¹⁵ Is OH.

In certain embodiments, R ¹⁵ Is halo.

In certain embodiments, R ¹⁵ Is phenyl.

In certain embodiments, R ¹⁵ Is benzyl.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁵ Is substituted or unsubstitutedC ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H.

In certain embodiments, R ¹⁶ Is OH.

In certain embodiments, R ¹⁶ Is halo.

In certain embodiments, R ¹⁶ Is phenyl.

In certain embodiments, R ¹⁶ Is benzyl.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups. In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups. In certain embodiments, u is 0.

In certain embodiments, u is 1.

In certain embodiments, u is 2.

In certain embodiments, u is 3.

In certain embodiments, u is 4.

In certain embodiments, u is 5.

In certain embodiments, u is 6.

In certain embodiments, u is 7.

In certain embodiments, u is 8.

In certain embodiments, u is 9.

In certain embodiments, u is 10.

In certain embodiments, u is 11.

In certain embodiments, u is 12.

In certain embodiments, u is 13.

In certain embodiments, u is 14.

In certain embodiments, u is 15.

In certain embodiments, u is 16.

In certain embodiments, u is 17.

In certain embodiments, u is 18.

In certain embodiments, u is 19.

In certain embodiments, u is 20.

In certain embodiments, v is 0.

In certain embodiments, v is 1.

In certain embodiments, v is 2.

In certain embodiments, v is 3.

In certain embodiments, v is 4.

In certain embodiments, v is 5.

In certain embodiments, v is 6.

In certain embodiments, v is 7.

In certain embodiments, v is 8.

In certain embodiments, v is 9.

In certain embodiments, v is 10.

In certain embodiments, v is 11.

In certain embodiments, v is 12.

In certain embodiments, v is 13.

In certain embodiments, v is 14.

In certain embodiments, v is 15.

In certain embodiments, v is 16.

In certain embodiments, v is 17.

In certain embodiments, v is 18.

In certain embodiments, v is 19.

In certain embodiments, v is 20.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, x is 0. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. In certain embodiments, x is 4. In certain embodiments, x is 5. In certain embodiments, x is 6. In certain embodiments, x is 7. In certain embodiments, x is 8. In certain embodiments, x is 9. In certain embodiments, x is 10.

In certain embodiments, x is 11.

In certain embodiments, x is 12.

In certain embodiments, x is 13.

In certain embodiments, x is 14.

In certain embodiments, x is 15.

In certain embodiments, x is 16.

In certain embodiments, x is 17.

In certain embodiments, x is 18.

In certain embodiments, x is 19.

In certain embodiments, x is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments, L ¹ Is a key.

In certain embodiments, L ¹ is-C (=o) -.

In certain embodiments, L ¹ is-OC (=O) O-.

In some embodimentsIn the case of L ¹ is-NH-C (=O) -.

In certain embodiments, L ¹ is-SO-.

In certain embodiments, L ¹ is-SO ₂ -。

In certain embodiments, L ¹ OC (=o).

In certain embodiments, L ¹ is-C (=O) O-.

In certain embodiments, L ¹ is-C (=O) NH-.

In certain embodiments, L ¹ is-SO ₃ -。

In certain embodiments, L ¹ is-NSO ₂ -。

In certain embodiments, L ¹ is-SO ₂ N。

In certain embodiments, L ¹ is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ¹ is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ¹ Is dioxopyrrolidine-dione.

In certain embodiments, L ¹ is-C (=O) R ¹ -。

In certain embodiments, L ¹ is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ¹ is-C (=O) O (CR) ¹ R ² R ³ ) In certain embodiments, L ¹ is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl). In certain embodiments, L ¹ is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² Is a key.

In certain embodiments, L ² is-C (=o) -.

In certain embodiments, L ² is-OC (=O) O-.

In certain embodiments, L ² is-NH-C (=O) -.

In certain embodiments, L ² is-SO-.

In certain embodiments, L ² is-SO ₂ -。

In certain embodiments, L ² OC (=o).

In certain embodiments, L ² is-C (=O) O-.

In certain embodiments, L ² is-C (=O) NH-.

In certain embodiments, L ² is-SO ₃ -。

In certain embodiments, L ² is-NSO ₂ -。

In certain embodiments, L ² is-SO ₂ N。

In certain embodiments, L ² is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ² is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ² is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ² Is dioxopyrrolidine-dione.

In certain embodiments, L ² is-C (=O) R ¹ -。

In certain embodiments, L ² is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is NH.

In certain embodiments, Q is a disulfide bond.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

In another embodiment, the present invention encompasses the ionizable lipids of the invention of formula VII:

wherein each R is ¹ And each R ² Independently selected from the group consisting of: H. optionally substituted C ₁ -C ₁₂ Alkyl, optionally substituted C ₂ -C ₁₂ Alkenyl, optionally substituted C ₂ -C ₁₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ⁶ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ An arylalkyl group; optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group, an optionally substituted amine; or R is ¹ And R is ² Together may form a 3-7 membered heterocycloalkyl or heteroaryl ring;

wherein each R is ⁵ 、R ⁶ 、R ^5’ 、R ^6’ 、R ^5” And R is ^6” Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substitutedC of (2) ₂ -C ₂₂ An alkynyl group, an amino group,

wherein each R is ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ 、R ¹¹ 、R ¹² 、R ¹³ 、R ¹⁴ 、R ¹⁵ And R is ¹⁶ Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ An alkynyl group, an amino group,

wherein u, v, w, y and z are each independently integers from 0 to 20;

wherein each Q is independently an atom selected from O, NH, S, or a disulfide bond; and is also provided with

Wherein each L ¹ And L ² Independently selected from the group consisting of: -C (=o) -; OC (=o) -; -OC (=o) O-; -C (=o) O-; -C (=o) O (CR ⁵ R ⁶ R ⁷ ) _m ；-NH-C(＝O)-；-C(＝O)NH-；-SO-；-SO ₂ -；-SO ₃ -；-NSO ₂ -；-SO ₂ N-；-NH((C ₁ -C ₈ ) An alkyl group); -N ((C) ₁ -C ₈ ) Alkyl group ₂ ；-NH((C ₆ ) An aryl group); -N ((C) ₆ ) Aryl group ₂ ；-NHC(＝O)NH-；-NHC(＝O)O-；-OC(＝O)NH-；-NHC(＝O)NR ¹ -；-NHC(＝O)O-；-OC(＝O)NR ¹ -；-C(＝O)R ¹ -；-CO((C ₁ -C ₈ ) An alkyl group); CO ((C) ₆ ) An aryl group); -CO ₂ ((C ₁ -C ₈ ) An alkyl group); -CO ₂ ((C ₆ ) An aryl group); -SO ₂ ((C ₁ -C ₈ ) An alkyl group); and-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, R ¹ H.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In some implementationsIn the scheme, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ² H.

In certain embodiments, R ² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl group

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ² Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, each R ⁵ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁵ H.

In certain embodiments, R ⁵ Is OH.

In certain embodiments, R ⁵ Is halo.

In certain embodiments, R ⁵ Is phenyl.

In certain embodiments, R ⁵ Is benzyl.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁶ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁶ H.

In certain embodiments, R ⁶ Is OH.

In certain embodiments, R ⁶ Is halo.

In certain embodiments, R ⁶ Is phenyl.

In certain embodiments, R ⁶ Is benzyl.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H.

In certain embodiments, R ⁷ Is OH.

In certain embodiments, R ⁷ Is halo.

In certain embodiments, R ⁷ Is phenyl.

In certain embodiments, R ⁷ Is benzyl.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H.

In certain embodiments, R ⁸ Is OH.

In certain embodiments, R ⁸ Is halo.

In certain embodiments, R ⁸ Is phenyl.

In certain embodiments, R ⁸ Is benzyl.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H.

In certain embodiments, R ⁹ Is OH.

In certain embodiments, R ⁹ Is halo.

In certain embodiments, R ⁹ Is phenyl.

In certain embodiments, R ⁹ Is benzyl.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H.

In certain embodiments, R ¹⁰ Is OH.

In certain embodiments, R ¹⁰ Is halo.

In certain embodiments, R ¹⁰ Is phenyl.

In certain embodiments, R ¹⁰ Is benzyl.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H.

In certain embodiments, R ¹¹ Is OH.

In certain embodiments, R ¹¹ Is halo.

In certain embodiments, R ¹¹ Is phenyl.

In certain embodiments, R ¹¹ Is benzyl.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹² H.

In certain embodiments, R ¹² Is OH.

In certain embodiments, R ¹² Is halo.

In certain embodiments, R ¹² Is phenyl.

In certain embodiments, R ¹² Is benzyl.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H, OH is selected from the group consisting of halogen, phenyl, benzyl, etc,Substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H.

In certain embodiments, R ¹³ Is OH.

In certain embodiments, R ¹³ Is halo.

In certain embodiments, R ¹³ Is phenyl.

In certain embodiments, R ¹³ Is benzyl.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H.

In certain embodiments, R ¹⁴ Is OH.

In certain embodiments, R ¹⁴ Is halo.

In certain embodiments, R ¹⁴ Is phenyl.

In certain embodiments, R ¹⁴ Is benzyl.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H.

In certain embodiments, R ¹⁵ Is OH.

In certain embodiments, R ¹⁵ Is halo.

In certain embodiments, R ¹⁵ Is phenyl.

In certain embodiments, R ¹⁵ Is benzyl.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H.

In certain embodiments, R ¹⁶ Is OH.

In certain embodiments, R ¹⁶ Is halo.

In certain embodiments, R ¹⁶ Is phenyl.

In certain embodiments, R ¹⁶ Is benzyl.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, u is 0.

In certain embodiments, u is 1.

In certain embodiments, u is 2. In certain embodiments, u is 3. In certain embodiments, u is 4. In certain embodiments, u is 5. In certain embodiments, u is 6. In certain embodiments, u is 7. In certain embodiments, u is 8.

In certain embodiments, u is 9.

In certain embodiments, u is 10.

In certain embodiments, u is 11.

In certain embodiments, u is 12.

In certain embodiments, u is 13.

In certain embodiments, u is 14.

In certain embodiments, u is 15.

In certain embodiments, u is 16.

In certain embodiments, u is 17.

In certain embodiments, u is 18.

In certain embodiments, u is 19.

In certain embodiments, u is 20.

In certain embodiments, v is 0.

In certain embodiments, v is 1.

In certain embodiments, v is 2.

In certain embodiments, v is 3.

In certain embodiments, v is 4.

In certain embodiments, v is 5.

In certain embodiments, v is 6.

In certain embodiments, v is 7.

In certain embodiments, v is 8.

In certain embodiments, v is 9.

In certain embodiments, v is 10.

In certain embodiments, v is 11.

In certain embodiments, v is 12.

In certain embodiments, v is 13.

In certain embodiments, v is 14.

In certain embodiments, v is 15.

In certain embodiments, v is 16.

In certain embodiments, v is 17.

In certain embodiments, v is 18.

In certain embodiments, v is 19.

In certain embodiments, v is 20.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, y is 0. In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4. In certain embodiments, y is 5. In certain embodiments, y is 6. In certain embodiments, y is 7. In certain embodiments, y is 8. In certain embodiments, y is 9. In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments, L ¹ Is a key.

In certain embodiments, L ¹ is-C (=o) -.

In certain embodiments, L ¹ is-OC (=O) O-.

In certain embodiments, L ¹ is-NH-C (=O) -.

In certain embodiments, L ¹ is-SO-.

In certain embodiments, L ¹ is-SO ₂ -。

In certain embodiments, L ¹ OC (=o).

In certain embodiments, L ¹ is-C (=O) O-.

In certain embodiments, L ¹ is-C (=O) NH-.

In certain embodiments, L ¹ is-SO ₃ -。

In certain embodiments, L ¹ is-NSO ₂ -。

In certain embodiments, L ¹ is-SO ₂ N。

In certain embodiments, L ¹ is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ¹ is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ¹ Is dioxopyrrolidine-dione.

In certain embodiments, L ¹ is-C (=O) R ¹ -。

In certain embodiments, L ¹ is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ¹ is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² Is a key.

In certain embodiments, L ² is-C (=o) -.

In certain embodiments, L ² is-OC (=O) O-.

In some embodimentsIn the case of L ² is-NH-C (=O) -.

In certain embodiments, L ² is-SO-.

In certain embodiments, L ² is-SO ₂ -。

In certain embodiments, L ² OC (=o).

In certain embodiments, L ² is-C (=O) O-.

In certain embodiments, L ² is-C (=O) NH-.

In certain embodiments, L ² is-SO ₃ -。

In certain embodiments, L ² is-NSO ₂ -。

In certain embodiments, L ² is-SO ₂ N。

In certain embodiments, L ² is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ² is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ² is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ² Is dioxopyrrolidine-dione.

In certain embodiments, L ² is-C (=O) R ¹ -。

In certain embodiments, L ² is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ (CR ¹ R ² R ³ )。

In certain embodiments, L ² is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is NH.

In certain embodiments, Q is a disulfide bond.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

In another embodiment, the present invention encompasses the ionizable lipids of formula VIII:

wherein the method comprises the steps of

R ₁ And R is ₂ Each independently selected from the group consisting of: H. optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ₆ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ Arylalkyl or optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group;

R ₃ and R is ₄ Each independently is optionally substituted C ₁₀ -C ₂₂ Alkyl, optionally substituted C ₁₀ -C ₂₂ Alkenyl, optionally substituted C ₁₀ -C ₂₂ Alkynyl groups or together form a 3-7 membered heterocycloalkyl or heteroaryl ring;

x is OH, or NR ₁ R ₂ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

Z is an integer of 0 to 5.

In certain embodiments, the ionizable lipid of formula VIII is selected from the group consisting of:

in certain embodiments, the ionizable lipid of formula VIII is selected from the group consisting of:

in certain embodiments, the ionizable lipid of formula VIII is selected from the group consisting of:

in certain embodiments, the ionizable lipid of formula VIII is selected from the group consisting of:

in certain embodiments, the ionizable lipid of formula VIII is selected from the group consisting of:

in certain embodiments, the ionizable lipid of formula VIII is selected from the group consisting of:

in certain embodiments, the ionizable lipid of formula VIII is selected from the group consisting of:

in certain embodiments, the ionizable lipid of formula VIII is selected from the group consisting of:

in another embodiment, the present invention encompasses the ionizable lipids of formula IX:

Wherein each R is ¹ And each R ² Independently selected from the group consisting of: H. electron pair, optionally substituted C ₁ -C ₁₂ Alkyl, optionally substituted C ₂ -C ₁₂ Alkenyl, optionally substituted C ₂ -C ₁₂ Alkynyl, optionally substituted C ₃ -C ₆ Cycloalkyl, optionally substituted C ₄ -C ₆ Heterocycloalkyl, optionally substituted C ₄ -C ₆ Alkylcycloalkyl, optionally substituted C ₄ -C ₆ Aryl, optionally substituted C ₃ -C ⁶ Heteroaryl, optionally substituted C ₄ -C ₈ Aryloxy, optionally substituted C ₇ -C ₁₀ An arylalkyl group; optionally substituted C ₅ -C ₁₀ A heteroarylalkyl group, an optionally substituted amine; or R is ¹ And R is ² Together may form a 3-7 membered heterocycloalkyl or heteroaryl ring;

wherein each R is ⁵ 、R ⁶ 、R ^5’ 、R ^6’ 、R ^5” And R is ^6” Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ An alkynyl group, an amino group,

wherein each R is ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ 、R ¹¹ 、R ¹² 、R ¹³ 、R ¹⁴ 、R ¹⁵ And R is ¹⁶ Independently selected from the group consisting of: H. OH, halo, phenyl, benzyl, optionally substituted C ₁ -C ₂₂ Alkyl, optionally substituted C ₂ -C ₂₂ Alkenyl, optionally substituted C ₂ -C ₂₂ An alkynyl group, an amino group,

wherein u, v, w, y and z are each independently integers from 0 to 20;

wherein X is O, S or N; and is also provided with

Wherein each L ¹ And L ² Independently selected from the group consisting of: -C (=o) -; OC (=o) -; -OC (=o) O-; -C (=o) O-; -C (=o) O (CR ⁵ R ⁶ R ⁷ ) _m ；-NH-C(＝O)-；-C(＝O)NH-；-SO-；-SO ₂ -；-SO ₃ -；-NSO ₂ -；-SO ₂ N-；-NH((C ₁ -C ₈ ) An alkyl group); -N ((C) ₁ -C ₈ ) Alkyl group ₂ ；-NH((C ₆ ) An aryl group); -N ((C) ₆ ) Aryl group ₂ ；-NHC(＝O)NH-；-NHC(＝O)O-；-OC(＝O)NH-；-NHC(＝O)NR ¹ -；-NHC(＝O)O-；-OC(＝O)NR ¹ -；-C(＝O)R ¹ -；-CO((C ₁ -C ₈ ) An alkyl group); CO ((C) ₆ ) An aryl group); -CO ₂ ((C ₁ -C ₈ ) An alkyl group); -CO ₂ ((C ₆ ) An aryl group); -SO ₂ ((C ₁ -C ₈ ) An alkyl group); and-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, R ¹ H.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In some casesIn embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ¹ Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, R ² H.

In certain embodiments, R ² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups

In certain embodiments, R ² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl group

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Cycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ A heterocycloalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Alkylcycloalkyl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₆ Aryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₃ -C ₆ Heteroaryl groups.

In certain embodiments, R ² Is substituted or unsubstituted C ₄ -C ₈ An aryloxy group.

In certain embodiments, R ² Is substituted or unsubstituted C ₇ -C ₁₀ An arylalkyl group.

In certain embodiments, R ² Is substituted or unsubstituted C ₅ -C ₁₀ A heteroarylalkyl group.

In certain embodiments, each R ⁵ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁵ H.

In certain embodiments, R ⁵ Is OH.

In certain embodiments, R ⁵ Is halo.

In certain embodiments, R ⁵ Is phenyl.

In certain embodiments, R ⁵ Is benzyl.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, each R ⁶ Independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁶ H.

In certain embodiments, R ⁶ Is OH.

In certain embodiments, R ⁶ Is halo.

In certain embodiments, R ⁶ Is phenyl.

In certain embodiments, R ⁶ Is benzyl.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁷ H.

In certain embodiments, R ⁷ Is OH.

In certain embodiments, R ⁷ Is halo.

In certain embodiments, R ⁷ Is phenyl.

In certain embodiments, R ⁷ Is benzyl.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁷ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁸ H.

In certain embodiments, R ⁸ Is OH.

In certain embodiments, R ⁸ Is halo.

In certain embodiments, R ⁸ Is phenyl.

In certain embodiments, R ⁸ Is benzyl.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁸ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ⁹ H.

In certain embodiments, R ⁹ Is OH.

In certain embodiments, R ⁹ Is halo.

In certain embodiments, R ⁹ Is phenyl.

In certain embodiments, R ⁹ Is benzyl.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ⁹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁰ H.

In certain embodiments, R ¹⁰ Is OH.

In certain embodiments, R ¹⁰ Is halo.

In certain embodiments, R ¹⁰ Is phenyl.

In certain embodiments, R ¹⁰ Is benzyl.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁰ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H.

In certain embodiments, R ¹¹ Is OH.

In certain embodiments, R ¹¹ Is halo.

In certain embodiments, R ¹¹ Is phenyl.

In certain embodiments, R ¹¹ Is benzyl.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹¹ To take outSubstituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹¹ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹¹ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹² H.

In certain embodiments, R ¹² Is OH.

In certain embodiments, R ¹² Is halo.

In certain embodiments, R ¹² Is phenyl.

In certain embodiments, R ¹² Is benzyl.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹² Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹³ H.

In certain embodiments, R ¹³ Is OH.

In certain embodiments, R ¹³ Is halo.

In certain embodiments, R ¹³ Is phenyl.

In certain embodiments, R ¹³ Is benzyl.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹³ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁴ H.

In certain embodiments, R ¹⁴ Is OH.

In certain embodiments, R ¹⁴ Is halo.

In certain embodiments, R ¹⁴ Is phenyl.

In certain embodiments, R ¹⁴ Is benzyl.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁴ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁵ H.

In certain embodiments, R ¹⁵ Is OH.

In certain embodiments, R ¹⁵ Is halo.

In certain embodiments, R ¹⁵ Is phenyl.

In certain embodiments, R ¹⁵ Is benzyl.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁵ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H, OH, halo, phenyl, benzyl, substituted or unsubstituted C ₁ -C ₂₂ Alkyl, substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups; or substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, R ¹⁶ H.

In certain embodiments, R ¹⁶ Is OH.

In certain embodiments, R ¹⁶ Is halo.

In certain embodiments, R ¹⁶ Is phenyl.

In certain embodiments, R ¹⁶ Is benzyl.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₁ -C ₂₂ An alkyl group.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkenyl groups.

In certain embodiments, R ¹⁶ Is substituted or unsubstituted C ₂ -C ₂₂ Alkynyl groups.

In certain embodiments, u is 0.

In certain embodiments, u is 1.

In certain embodiments, u is 2. In certain embodiments, u is 3. In certain embodiments, u is 4. In certain embodiments, u is 5. In certain embodiments, u is 6. In certain embodiments, u is 7. In certain embodiments, u is 8.

In certain embodiments, u is 9.

In certain embodiments, u is 10.

In certain embodiments, u is 11.

In certain embodiments, u is 12.

In certain embodiments, u is 13.

In certain embodiments, u is 14.

In certain embodiments, u is 15.

In certain embodiments, u is 16.

In certain embodiments, u is 17.

In certain embodiments, u is 18.

In certain embodiments, u is 19.

In certain embodiments, u is 20.

In certain embodiments, v is 0.

In certain embodiments, v is 1.

In certain embodiments, v is 2.

In certain embodiments, v is 3.

In certain embodiments, v is 4.

In certain embodiments, v is 5.

In certain embodiments, v is 6.

In certain embodiments, v is 7.

In certain embodiments, v is 8.

In certain embodiments, v is 9.

In certain embodiments, v is 10.

In certain embodiments, v is 11.

In certain embodiments, v is 12.

In certain embodiments, v is 13.

In certain embodiments, v is 14.

In certain embodiments, v is 15.

In certain embodiments, v is 16.

In certain embodiments, v is 17.

In certain embodiments, v is 18.

In certain embodiments, v is 19.

In certain embodiments, v is 20.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, y is 0. In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4. In certain embodiments, y is 5. In certain embodiments, y is 6. In certain embodiments, y is 7. In certain embodiments, y is 8. In certain embodiments, y is 9. In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments, L ¹ Is a key.

In certain embodiments, L ¹ is-C (=o) -.

In certain embodiments, L ¹ is-OC (=O) O-.

In certain embodiments, L ¹ is-NH-C (=O) -.

In certain embodiments, L ¹ is-SO-.

In certain embodiments, L ¹ is-SO ₂ -。

In certain embodiments, L ¹ OC (=o).

In certain embodiments, L ¹ is-C (=O) O-.

In certain embodiments, L ¹ is-C (=O) NH-.

In certain embodiments, L ¹ is-SO ₃ -。

In certain embodiments, L ¹ is-NSO ₂ -。

In certain embodiments, L ¹ is-SO ₂ N。

In certain embodiments, L ¹ is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ¹ is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ¹ Is dioxopyrrolidine-dione.

In certain embodiments, L ¹ is-C (=O) R ¹ -。

In certain embodiments, L ¹ is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ¹ is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ¹ is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ¹ is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² Is a key.

In certain embodiments, L ² is-C (=o) -.

In certain embodiments, L ² is-OC (=O) O-.

In certain embodiments, L ² is-NH-C (=O) -.

In certain embodiments, L ² is-SO-.

In certain embodiments, L ² is-SO ₂ -。

In certain embodiments, L ² OC (=o).

In certain embodiments, L ² is-C (=O) O-.

In certain embodiments, L ² is-C (=O) NH-.

In certain embodiments, L ² is-SO ₃ -。

In certain embodiments, L ² is-NSO ₂ -。

In certain embodiments, L ² is-SO ₂ N。

In certain embodiments, L ² is-NH ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-N ((C) ₁ -C ₈ ) Alkyl group ₂ 。

In certain embodiments, L ² is-NH ((C) ₆ ) Aryl).

In certain embodiments, L ² is-N ((C) ₆ ) Aryl group ₂ 。

In certain embodiments, L ² Is dioxopyrrolidine-dione.

In certain embodiments, L ² is-C (=O) R ¹ -。

In certain embodiments, L ² is-CO ((C) ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ((C) ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-CO ₂ ((C ₆ ) Aryl).

In certain embodiments, L ² is-CO ₂ (CR ¹ R ² R ³ )。

In certain embodiments, L ² is-SO ₂ ((C ₁ -C ₂₂ ) Alkyl).

In certain embodiments, L ² is-SO ₂ ((C ₆ ) Aryl).

In certain embodiments, Q is CH.

In certain embodiments, X is O.

In certain embodiments, X is S.

In certain embodiments, X is N.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

"naLNP" as used herein refers to lipid nanoparticles with a nucleic acid payload produced according to the methods disclosed herein. naLNP has significantly greater efficacy relative to the reference LNP.

As used herein, "reference LNP or refLNP" refers to lipid nanoparticles formed with one or more LNP lipids using a reference LNP manufacturing method. In one embodiment, the reference LNP is a 50-100nm diameter LNP comprising nucleic acid and 4 lipids: ionizable lipids with amine groups (50%), zwitterionic helper lipids-1, 2-distearoyl-sn-glycerol-3-phosphorylcholine (DSPC) (10%), cholesterol (38.5%) and pegylated lipids-1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) (1.5%) (molar ratio in brackets). Hydrophilic DMG-PEG forms the outer shell of LNP.

The "reference LNP" is defined by the fact that it is generated by the "reference LNP manufacturing method".

In one embodiment, the "reference manufacturing method" may be summarized as follows:

nucleic acid solution: mRNA at a reference nucleic acid concentration of less than 0.20mg/ml is prepared in a buffer at a concentration and pH, i.e.in 50mM citrate pH 5 or 25mM sodium acetate pH 4, or 10-50mM citrate pH 4.

Lipid solution: preparing a lipid mixture in ethanol at a reference lipid concentration corresponding to the desired NP ratio (5.67) and/or lipid/mRNA weight ratio (10:1 to 30:1)

Assembled refLNP: combining portions of the nucleic acid solution and portions of the lipid solution produces a single mixed solution in the buffer.

Dialyzing refLNP in the mixed solution to physiological pH.

Measurement of refLNP size using light scattering

Measurement of RNA encapsulation in refLNP using Ribogreen assay

Select refLNP with >70% higher encapsulation and diameter of 50-100nm

"mixing" as used herein preferably refers to turbulent mixing ("T-mixing"), vortex mixing ("V-mixing"), microfluidic mixing, or both. See, for example, the blends described in U.S. publication No. 20200306191.

In one embodiment, the reference LNP manufacturing process is the process described in Hassett et al, mol. Ther. -Nucleic Acids 2019,15,1-11. In another embodiment, the "reference manufacturing method" is as described in U.S. application No. 20190032087. In one embodiment, the reference method comprises: (a) Introducing a first fluid stream comprising a therapeutic agent (e.g., a nucleic acid) in a first solvent into the microchannel; wherein the microchannel has a first region adapted to flow one or more fluid streams introduced into the microchannel and a second region to mix the contents of the one or more fluid streams; (b) Introducing a second liquid stream comprising LNP-forming material (e.g., reference LNP lipids) in a second solvent into the microchannel to provide first and second liquid streams flowing under laminar flow conditions, wherein the lipid particle-forming material comprises ionizable lipids, and wherein the first and second solvents are the same or different; (c) Flowing one or more first fluid streams and one or more second fluid streams from a first region of the microchannel into a second region of the microchannel; and (d) mixing the contents of the one or more first fluid streams and the one or more second fluid streams in the second region of the microchannel to provide a third fluid stream comprising lipid particles having encapsulated therapeutic agents.

The contents of the first and second streams may be mixed by chaotic convection in a microfluidic channel or by nano-precipitation in a T-mixer.

In one embodiment, mixing the contents of the one or more first fluid streams and the one or more second fluid streams includes varying the concentration or relative mixing rate of the one or more first fluid streams and the one or more second fluid streams. In the above embodiments, unlike known methods, the method does not include dilution after mixing.

In order to further stabilize the third fluid stream containing lipid particles with encapsulated therapeutic agents, the method may include, but need not further include, diluting the third fluid stream with an aqueous buffer. In one embodiment, diluting the third liquid stream comprises flowing the third liquid stream and an aqueous buffer into the second mixing structure. In another embodiment, an aqueous buffer comprising lipid particles having an encapsulated therapeutic agent is dialyzed to reduce the amount of the second solvent.

The first liquid stream comprises the therapeutic agent in the first solvent. Suitable first solvents include solvents in which the therapeutic agent is soluble and which are miscible with the second solvent. Suitable first solvents include aqueous buffers. Representative first solvents include citrate and acetate buffers. The first solvent may be water alone if the second solvent comprises a protonating agent such as HCl to protonate the ionizable lipids.

The second liquid stream comprises lipid particle forming material in a second solvent. Suitable second solvents include solvents in which the ionizable lipid is soluble and which are miscible with the first solvent. Suitable second solvents include 1, 4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethylsulfoxide, dimethylformamide, acids and alcohols. Representative second solvents include >50% aqueous ethanol.

In some embodiments, the lipid particles of the present invention are advantageously formed in a microfluidic process that utilizes relatively rapid mixing and higher flow rates. Rapid mixing provides lipid particles with the advantageous properties mentioned above, including size, homogeneity and encapsulation efficiency. The mixing rate used to practice the method of the present invention is in the range of about 100 musec to about 10 msec. Representative mixing rates include from about 1 to about 5msec. Although hydrodynamic flow focusing methods operate at relatively low flow rates (e.g., 5 to 100 μl/min) and relatively low liposome volumes, the methods of the present invention can operate at relatively high flow rates and relatively high liposome volumes. In certain embodiments, for processes incorporating a single mixing zone (i.e., mixer), the flow rate is from about 1 to about 100mL/min. For the present method using an array of mixers (e.g., 10 mixers), a flow rate of 40mL/min (flow rate of 400mL/min for 100 mixers) is used. Thus, the process of the present invention can be easily scaled to provide the amount of lipid particles required for demanding production requirements.

The LNP compositions of the invention disclosed herein can comprise one or more bioactive agents including, but not limited to, antibodies (e.g., monoclonal, chimeric, humanized, nanobodies, fragments thereof, etc.), cholesterol, hormones, peptides, proteins, chemotherapeutic agents, and other types of anti-neoplastic agents, low molecular weight drugs, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, and enzymatic nucleic acids. Various methods of loading bioactive agents into lipid compositions, such as liposomes and lipid nanoparticles, can be utilized in the art, including both passive and active loading methods. The exact method used may be selected based on a variety of factors including, but not limited to, for example, the bioactive agent to be loaded, the storage method to be used once loaded, the size of the resulting particles, and the intended dosing regimen. Methods include, for example, mechanically mixing the drug and lipid at the time of liposome formation or reconstitution, dissolving all components in an organic solvent and concentrating them into a dry film, forming a pH or ionic gradient to pull the active agent into the liposome interior, establishing transmembrane potential and ionophore-mediated loading. See, for example, PCT publication No. WO 95/08986, U.S. Pat. Nos. 5,837,282; 5,837,282 and 7,811,602.

The term "nucleic acid" refers to ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phospho-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single-stranded, double-stranded, or optionally contain portions of both double-and single-stranded sequences. In some embodiments, "nucleic acids" include antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA RNA compositions, alleles, aptamers, ribozymes, baits, and analogs thereof, plasmids, and other types of expression vectors and small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNA), messenger ribonucleic acids (messenger RNA, mRNA), short interfering RNAs (siRNA), double-stranded RNAs (dsRNA), micro-RNAs (miRNA) and short hairpin RNA (shRNA) molecules, peptide Nucleic Acids (PNA), locked nucleic acid ribonucleotides (LNA), morpholino nucleotides, threose Nucleic Acids (TNA), glycol Nucleic Acids (GNA), siRNAs (small internal segment interfering RNA), aiRNAs (asymmetric interfering RNA), and siRNAs with 1, 2 or more mismatches between the sense and antisense strands of related cells and/or tissues such as cell cultures, subjects, or organisms. Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified. In one embodiment the bioactive agent is an RNAi agent, a short interfering nucleic acid (siNA), a short interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microrna (miRNA), or a short hairpin RNA (shRNA) molecule. In one embodiment the bioactive agent is an RNA interference (RNAi) agent suitable for mediating RNAi.

As used herein, the term "nucleic acid" is also intended to include any oligonucleotide or polynucleotide. Fragments containing up to 50 nucleotides are commonly referred to as oligonucleotides, and longer fragments are referred to as polynucleotides. In a particular embodiment, the oligonucleotides of the invention are 20-50 nucleotides in length. In the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers composed of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and inter-sugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers comprising non-naturally occurring monomers or functionally similar parts thereof. Such modified or substituted oligonucleotides are generally preferred over the natural form because of the following properties: such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases. Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribose oligonucleotides consist of 5-carbon sugars (known as deoxyribose) covalently linked to a phosphate at the 5 'and 3' carbons of this sugar, thereby forming alternating unbranched polymers. Ribooligonucleotides consist of similar repeat structures in which the 5-carbon sugar is ribose. The nucleic acids present in the lipid particles according to the invention include any form of known nucleic acids. The nucleic acid used herein may be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double stranded RNAs include siRNA and other RNA interfering agents. Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNAs, and triplex-forming oligonucleotides.

In another embodiment the bioactive agent is mRNA. In one embodiment, the nucleic acid is mRNA encoding a COVID-19 protein or peptide.

As used herein, the terms "Pyr", "Pyrd" and "Pyrd" are used interchangeably and refer to pyridine or pyridinyl substituents.

As referred to herein, "efficacy" refers to the ability of an LNP to deliver a nucleic acid payload to a cell or tissue, wherein the LNP internalizes into the cell (or cells in the tissue) and is released from the endosome to the cytoplasm, whereupon the nucleic acid payload is released from the lipid and becomes bioavailable. Efficacy may be measured by any of a number of methods known to those of skill in the art. For example, it may be measured in terms of cellular uptake, nucleic acid payload transcription, nucleic acid payload translation, or production of a polypeptide encoded by a nucleic acid payload. When the nucleic acid payload of an LNP is intended to function in a gene expression inhibition manner such as, for example, RNAi, LNP efficacy can be measured in terms of achieving a target gene "knockdown" by reducing the transcription rate of the target gene, the duration of the mRNA transcript half-life of the gene target, or translation of the mRNA transcript of the target gene. Additional assays for measuring efficacy depend on measuring LNP immunogenicity and LNP systemic distribution. All such assays and their permutations are well known in the art.

In one embodiment, the experiment measuring the effectiveness of the LNP is performed in parallel with the reference LNP. Such experiments may use standardized nucleic acid payloads such as, for example, reporter genes. A reporter gene (often referred to simply as a reporter gene) is a gene that a researcher connects to the regulatory sequences of another gene of interest in bacteria, cell cultures, animals or plants. Such genes are referred to as reporter genes because of their property to confer on the organism expressing them easy to identify and measure, or because they are selective markers. A reporter gene may be used as an indication of whether a certain gene is taken up or expressed in a cell or population of organisms. Typical reporter genes are lacZ, cat, gfp, rfp, luc, which encode β -galactosidase, chloramphenicol acetyl transferase, green fluorescent protein, red fluorescent protein, luciferase, respectively, useful in corresponding histochemistry, acetylation, fluorescence, spectrophotometry, and bioluminescence assays. All such assays and their permutations are well known in the art.

In one embodiment, LNP efficacy is measured in vitro or in vivo at a known dose or doses by luciferase reporter activity. Relative LNP efficacy was determined in vitro or in vivo by a luciferase activity assay. See also, for example, U.S. patent No. 10,221,127.

LNP efficacy can also be measured in terms of a desired biological response of a nucleic acid payload, including, for example, a therapeutic or prophylactic effect or impact on the mechanism of action that produces the response. In some embodiments, LNP efficacy is measured by the ability of LNP to carry mRNA encoding an immunogen, e.g., after administration, to induce the immune system to produce polypeptides of secreted cognate IgG antibodies.

"increased LNP efficacy" refers to the extent to which the nalNP of the present invention has greater efficacy than the reference LNP. In certain embodiments, the LNP of the invention disclosed herein has an LNP potency increase of about or at least 1.25, 1.50, 1.75, 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, 60, 70, 80, 90, 100, 150, 200, 400, 500, 750, 1000, 10,000 times greater than the reference LNP in the same assay delivering the same nucleic acid cargo.

As used herein, "organic solvent" refers to a type of Volatile Organic Compound (VOC). VOCs are organic chemicals that evaporate at room temperature and are commonly used in the art to dissolve certain materials and substances in the manufacture of pharmaceutical products. Organic solvents are generally used in the manufacture: aromatic compounds such as benzene and toluene, alcohols such as methanol or ethanol, esters and ethers, ketones such as acetone, amines, nitrated and halogenated hydrocarbons.

As used herein, "nitrogen-phosphate" or "NP" ratio is defined as the molar ratio of ionizable lipids to moles of phosphate on the nucleic acid backbone. It should be noted that the number of moles is the total number of moles in the mixed solution, rather than the concentration in the initial nucleic acid or lipid solution that can be mixed in different volume ratios. Note the case of more than one amine per ionizable lipid. Under mixing conditions, typically only one amine is protonated so that the NP ratio is most naturally defined as one amine per ionizable lipid, even when more than one amine is present, as electrostatic binding involves only charged groups. However, some ionizable lipids may have two or more amines that are charged during mixing.

As used herein, a "lipid/nucleic acid weight ratio" is a weight ratio that can be used in place of the NP ratio. The conversion between NP ratio and weight ratio involves the number of bases of the molar mass of ionizable lipids and nucleic acids divided by the molar mass of each phosphate.

In one embodiment, the amount of nucleic acid is expressed by the LNP molar ratio of amine on the ionizable lipid to phosphate groups on the nucleic acid backbone and is typically from about 3 to about 6. In one embodiment, the pKa of the LNP is in the range of about 6 to about 7, corresponding to the pH in early endosomes. The link between pKa of ionizable lipids in LNP and gene silencing efficiency demonstrates that LNP pKa in the range of about 6 to about 7 produces greater silencing of ionizable lipid DLinDMA and is associated with promotion of lipid structure of membrane that can interfere with endosomes. The pKa of LNP can be measured using the fluorescence-enhanced pH dependence of the anionic dye TNS.

As used herein, "cholesterol" refers to a bioactive organic compound having four rings arranged in a particular molecular configuration. The steroid core structure typically consists of seventeen carbon atoms bonded into four "fused" rings: three six-membered cyclohexane rings (rings A, B and C) and one five-membered cyclopentane ring (ring D). Steroids vary depending on the functional group attached to the tetracyclic core and the oxidation state of the ring. Sterols are steroid forms having a hydroxyl group in the third position and a skeleton derived from cholestane. Steroids may also be modified more thoroughly, such as by altering the ring structure, e.g., cutting one ring. Cleavage of ring B produces a ring-opened steroid, one of which is vitamin D3. Examples include lipid cholesterol, the sex hormones estradiol and testosterone, [4]:10-19 and the anti-inflammatory drug dexamethasone. Many steroids are present in plants, animals and fungi. The steroid is preferably produced in the cell from a sterol lanosterol (inverse keratin) or cycloartenol (plant). Lanosterol and cycloartenol are derived from cyclization of the triterpene squalene. In some embodiments, the LNP compositions disclosed herein contain cholesterol derivatives, e.g., dihydro-cholesterol, enantios-cholesterol, epi-cholesterol, chain sterols, cholestanol, cholestanone, cholestenone, cholesteryl-2 '-hydroxyethyl ether, cholesteryl-4' -hydroxybutyl ether, 3. Beta. - [ N- (N 'N' -dimethylaminoethyl) carbamoyl-cholesterol (DC-Chol), 24 (S) -hydroxycholesterol, 25 (R) -27-hydroxycholesterol, 22-oxidized cholesterol, 23-oxidized cholesterol, 24-oxidized cholesterol, cycloartenol, 22-ketosterols, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol 25-hydroxycholesterol, 7-dehydrocholesterols, 5.alpha. -cholest-7-ene-3.beta. -ol, 3,6, 9-octan-1-ol-cholesteryl-3 e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanosterol, photosterol, gu Gaihua alcohol, calciferol, coprol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, turnip sterol, tomato base, tomato glycoside, ursolic acid, chenodeoxycholic acid, mycosterol, diosgenin, fucosterol, fecal sterol or fecal sterol, or salts or esters thereof. In some embodiments, the cholesterol or cholesterol derivative is cholesterol, cholesterol succinic acid, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, cholesterol acetate, cholesterol oleate, cholesterol linoleate, cholesterol myristate, cholesterol palmitate, cholesterol arachidate, cholesterol phosphorylcholine, and sodium cholate. Other exemplary steroids are disclosed in U.S. publication No. 20200129445.

As used herein, "lipid encapsulation" refers to lipid nanoparticles that provide an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), by complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the LNP.

LNP manufacturing method

The prior art has heretofore focused on the identity and structure of ionizable lipids as the primary parameters that determine the efficacy of LNP. Secondary parameters include the effect of distinguishing other lipids, such as DSPE, from different cholesterol analogues of DSPC or cholesterol and the molar ratio of 4 lipids.

In addition, the prior art shows that those skilled in the art consider that buffer concentrations should be high enough and pH low enough to highly protonate the ionizable lipids, encapsulating the maximum amount of mRNA (close to 100%) and thus will maximize LNP efficacy.

Furthermore, the prior art teaches that operating mixed solutions with high concentrations of nucleic acids and lipids results in a range of technical problems including insolubility, undesired precipitation, poor mixing, uneven LNP size and different nucleic acid encapsulation rates. In addition, nucleic acid solutions, especially reporter mRNA solutions, having a concentration of greater than 1mg/ml are not commercially available. Even if high concentration solutions are commercially available, performing high concentration mixing several hundred times to test individual concentrations is cost prohibitive. In view of these general assumptions, scientists involved in LNP manufacturing focus on using mixed solutions with low concentrations of nucleic acids and lipids to avoid high test costs, improper precipitation, LNP size non-uniformity, and LNP nucleic acid encapsulation non-uniformity, it is believed that mRNA doses can be adjusted later by concentrating the resulting LNP in solution during downstream processing steps. However, this does not address the fundamental problem of this prior art procedure to produce a low potency LNP.

For example, 0.5ml of high concentration mRNA solution at a concentration of >2mg/ml costs at least $1,500 per mixture to date. To achieve the naLNP disclosed herein, the skilled artisan uses >100 mixtures at a cost of at least $150,000, solely for the preparation of the nucleic acid solutions disclosed herein, without regard to other costs. In addition to the expense of producing a highly concentrated nucleic acid solution, high concentrations have heretofore been considered to result in insolubility, aggregation, precipitation, and/or poor mixing.

In addition, applicants decided that using high nucleic acid concentrations resulted in lower encapsulation efficiency, e.g., about 70% in naLNP compared to about 90% in refLNP. However, the inventors have unexpectedly found that even with substantially lower encapsulation efficiency, the naLNP disclosed herein has greater LNP efficacy than refLNP.

Applicants developed highly controlled and reproducible in vitro FLuc assays that allow comparison of experimental results to determine naLNP efficacy under the same conditions (cell numbers, etc.), internal recombinant quatileum standards, and the same refLNP for each experiment.

The applicant further believes that increasing endosomal release and/or looser binding of mRNA by the unprotonated portion of the ionizable lipid favors better endosomal release and/or that a more peripheral distribution of ionizable lipid or nucleic acid, e.g. mRNA, in the LNP is heretofore unknown.

However, the following unexpected findings are disclosed herein: LNP efficacy is strongly affected by the absolute concentration of nucleic acid: lipid mixture while maintaining a constant NP or weight ratio.

The data presented herein indicate that both the absolute concentration of nucleic acid and the buffer concentration and pH at the time of mixing strongly influence LNP efficacy and that these two influence interactions. A significant variable mediating this interaction is the level of protonation of the ionizable lipids during mixing, which should not be maximized in order to maximize encapsulation, as previously thought. Indeed, according to the methods disclosed herein, the level of protonation of the ionizable lipids should be at a level that maximizes the effectiveness of the LNP.

In a general embodiment, the present invention encompasses an enhanced method of manufacturing lipid nanoparticles, wherein lipid nanoparticle delivery efficiency is dependent on ionizable lipid ionization and alkyl tail structure.

In certain embodiments, LNP with high delivery efficiency is of paramount importance in the success of current COVD-19mRNA vaccines from BioNTech/Pfizer and Moderna. In certain embodiments, the LNP in these vaccines contains 4 lipids, namely the ionizable lipids, helper lipids DSPC, cholesterol, and PEG lipids, which automatically assemble with mRNA sequences into nanoparticles with diameters of about 60 nm. In certain embodiments, the protonated form of the ionizable lipid electrostatically binds to the anionic phosphate backbone of the mRNA to encapsulate it in the LNP, while the DSPC forms a peripheral bilayer containing a PEG-lipid tail with a hydrophilic PEG domain toward the aqueous medium. In certain embodiments, the ionizable lipid functions to facilitate endosomal release by protonating when the endosomal pH drops below 7, then interacting with the endosomal membrane to turn it on and release mRNA. In certain embodiments, the ionizable lipid in the LNP has a pKa in the range of 5 to 7.4 to release RNA prior to endosome-lysosomal fusion. In certain embodiments, the alkyl tail of the ionizable lipid must also be incompatible with the endosomal bilayer to destabilize it. For the current ionizable lipids, this latter requirement is achieved by a tapered branching structure.

In certain embodiments, the invention also contemplates methods of rationally designing LNP ionization for endosomal release by predicting the pKa of the LNP from the structure of the ionizable lipid. In certain embodiments, the pKa of the LNP is lower than the pKa of the ionizable lipid due to the difference in proton solvation energy in the LNP as compared to the aqueous phase. In certain embodiments, this allows for rational design of novel ionizable lipids (C2C 4) with 3 protonatable nitrogen, providing increased endosomal protonation and mRNA translation. In certain embodiments, C24LNP was found to be 10X superior to MC3 LNP, which is a standard reference LNP, in terms of immunogenicity for mRNA encoding SARS-CoV-2 spike protein, and resulted in greater protection against infection. In certain embodiments, LNP rational design methods are extended by developing models that use ionizable lipid structures to predict LNP ionization and the ability of ionizable lipids to interfere with endosomal membranes and release mRNA.

In certain embodiments, the delivery efficiency is dependent on the mRNA-LNP manufacturing process. In certain embodiments, mRNA-LNP is manufactured via a self-assembly process using a microfluidic or larger scale T-mixer, wherein 4 lipids in ethanol are rapidly mixed with mRNA in a low pH buffer. In certain embodiments, formation of mRNA LNP occurs via electrostatic binding of the protonated cationic ionizable lipid to the anionic mRNA phosphate backbone, followed by separation of the lipid from the aqueous phase to form nanoparticles that are stabilized by a hydrophilic PEG interface. In certain embodiments, many factors may vary during the manufacturing process, including, but not limited to, absolute and relative concentrations of lipids and mRNA, buffer type, concentration and pH thereof, ratio of organic to aqueous phase, and flow rate. We found that increasing the absolute concentration of lipid and mRNA together, without changing the relative concentration, generally increased the delivery efficiency of the resulting mRNA LNP by 4X (fig. 34). In certain embodiments, the invention encompasses methods of increasing these mixed concentrations, e.g., by 6X to 75mM total lipid and 1.5mg/ml mRNA, resulting in an increase in delivery efficiency of 4X at the same dose in vitro and in vivo (fig. 34B). By measuring the zeta potential increase at pH7.4 to 5 we found that compared to LNP assembled under standard conditions These more potent LNPs exhibited a 2-fold greater increase in zeta potential (fig. 34C). Without being bound by theory, this result indicates that the improved efficacy is due in part to the greater level of unprotonated ionizable lipids in the LNP increasing endosomal internalization. In certain embodiments, mRNA LNP is assembled in a buffer-free environment. In certain embodiments, instead of using a low pH buffer in the mRNA solution to protonate the ionizable lipids after mixing, HCl is added to the lipid mixture prior to mixing with the mRNA in unbuffered water to pre-protonate the ionizable lipids. In certain embodiments, TLC-MS and ¹ h NMR was used to confirm that no lipid degradation occurred. In certain embodiments, C24 ionizable lipids with 3 nitrogens are used, and 0.25 protons are added per ionizable lipid, we average protonate 1 nitrogen on every 4 lipids such that the protonated nitrogen is at a 1:1 ratio to the phosphoric acid of the mRNA (NP ratio of 4). In certain embodiments, the bufferless assembly method results in greater efficacy in vivo for both IM and IV administration by IVIS than using buffer, both methods using higher lipid and mRNA mixed concentrations. In certain embodiments, the bufferless, high absolute lipid/mRNA concentration assembly method can thereby produce a more efficient LNP of 10X, but without altering the composition of the final formulation, as compared to current manufacturing methods using low concentration and low pH buffers. In certain embodiments, the pre-protonation method targets specific levels of ionizable lipids more precisely than buffers that are inherently fast and heterogeneous processes that require convection and diffusion to contact and protonate the lipids. In certain embodiments, the manufacturing process achieves a high potency LNP that is easily implemented at all manufacturing scales and using both microfluidic and T-mixing geometries.

In certain embodiments, the method of manufacture comprises the step of protonating the ionizable lipid prior to mixing with the unbuffered mRNA. In certain embodiments, the ionizable lipid can be protonated with HCL, DCL, a weak acid, ethanol, water, or any of the solvents described herein. In certain embodiments, protonation occurs separately prior to mixing with other lipids or with the entire lipid mixture.

In certain embodiments, hydrochloric acid is used to directly protonate the ionizable lipid stock itself prior to mixing with other lipids into a lipid mixture or to protonate the ionizable lipid once mixed in the lipid mixture. This unbuffered preprotonated ionizable lipid in the lipid mixture is then mixed with an unbuffered mRNA solution. In certain embodiments, 100% protonation means that for each mole of ionizable lipid in the solution, one mole of HCL is added to protonate the nitrogen in the ionizable lipid. To protonate multiple nitrogens in the multiprotein ionizable lipid, a corresponding mole of HCL is added to protonate each nitrogen.

In general embodiments, the improved methods for preparing naLNP disclosed herein can be summarized as follows:

nucleic acid solution: nucleic acid at a nucleic acid concentration is provided in a buffer at a buffer concentration and at a nucleic acid solution pH.

Lipid solution: corresponding to the required conditions: i) Lipid solution at lipid concentration at nitrogen-to-phosphate ("NP") ratio, or ii) lipid/nucleic acid weight ratio, provides lipid in an organic solvent.

Assembled naLNP: the nucleic acid solution and the lipid solution are partially combined into a mixed solution having a mixed buffer concentration and pH.

Those of skill in the art will understand that certain steps in the methods of the present invention are not necessarily performed in a certain order, and that other steps must be performed before other steps. In addition, different participants may perform the various steps of the overall method.

In some embodiments, the portion of the nucleic acid solution and the lipid solution in the mixed solution is at a volume ratio of about or at least 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:2, 3:1, 4:1, 4:3, 5:1, 5:3, 5:4, 6:1, 6:5, 7:1, 8:1, 9:10, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1.

LNP prepared according to the above method may then be further processed for use according to methods well known in the art. In some embodiments, such further processing involves one or more of the following:

Bringing the naLNP to physiological pH, e.g., via dialysis between about 4 to about 24 hours, or alternatively via filtration and exchange of buffers such as Repligen using tangential flowKR2i or KMPi system or CytivaFlux tangential flow filtration system.

The naLNP size is measured, for example, by light scattering.

RNA encapsulation is measured, for example, by Ribogreen assay.

Selecting a material having a high encapsulation, e.g., at least or about 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% encapsulation efficiency, or at least or about 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, 150, 175, 200, 250, 300, 350, 400, 450, 500nM, or have a minimum diameter of about 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, 100nM and a minimum diameter of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or the like, 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, 150, 175, 200, 250, 300, 350, 400, 450, 500 nM.

In some embodiments, the nucleic acid solution contains nucleic acid at a nucleic acid concentration of about or at least 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, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 milligrams per ml.

In some embodiments, the nucleic acid is present in about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more molecular species each encoding about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more open reading frames.

In some embodiments, the nucleic acid molecule can be about or at least 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, 50, 100, 200, 300, 400, 500, 750, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 15000, 20000, 30000, 40000, 50000, 75000, 100000 nucleotides in length.

In some embodiments, the nucleic acid solution contains a buffer of a concentration of about or at least 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, 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, 150, 175, 200, 250, 300, 350, 400, 450, 500 mM.

In some embodiments, the buffer is a salt buffer. In some embodiments, the buffer is citric acid, acetic acid, phosphoric acid, and boric acid. In some embodiments, the buffer is potassium acetate, magnesium acetate, or sodium acetate. In other embodiments, the buffer may be citric acid, MES, histidine, ADA, ACES, PIPES, MOPSO, BES, HEPES, DIPSO, TEA, AMPD, gly-Gly, TAPS, HEPBS, AMPD, TABS, AMP, CAPSO, CAPS, CABS, CHES, PBS, SSC, TAE, TBE, TE, or the like. See, e.g., "Acetate Buffer (pH 3.6 to 5.6) preparation," AAT Bioquest, inc,29sep.2020. Other suitable buffers are shown in the following table:

In some embodiments, the nucleic acid solution is at a pH of about or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14.

In some embodiments, the lipid solution contains an organic solvent benzene, toluene, an alcohol such as methanol, an ester, an ether, a ketone such as acetone, an amine, a nitrated and/or halogenated hydrocarbon, or a combination thereof. In one embodiment the organic solvent is ethanol. Preferably the solvent is volatile, non-toxic and/or acceptable for administration to humans in trace amounts.

In some embodiments, the lipid solution contains one or more total lipid concentrations of about or at least 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, 150, 175, 200, 250, 300, 350, 400, 450, 1000, 500, 750, 800 mM.

In some embodiments, the mixed solution has a mixed concentration of nucleic acids of about or at least 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, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 milligrams/ml.

In some embodiments, the mixed solution has a total lipid mixed concentration of about or at least 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, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 800 mM.

In some embodiments, the mixed solution has a mixed buffer concentration of about or at least 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, 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, 150, 175, 200, 250, 300, 350, 400, 450, 500 mM.

In some embodiments, the buffer, e.g., sodium acetate buffer, concentration in the mixed solution is optimized to maximize efficacy at any particular mixed concentration. For example, example 13 shows that LNP efficacy increases by 44% relative to the reference LNP when sodium acetate is reduced from 50mM to 25mM at 1.5mg/ml mRNA in the nucleic acid solution. Reducing the sodium acetate concentration from 25mM to 10mM increases LNP efficacy by about 2.2X at an mRNA concentration of 0.25mg/ml in the nucleic acid solution. The increase in the mixing concentration and the optimized decrease in the buffer concentration resulted in an encapsulation efficiency of about 70% lower than that typically obtained in the prior art method, wherein encapsulation was erroneously maximized by decreasing the mixing concentration and increasing the buffer concentration as in example 10. LNPs produced according to the methods disclosed herein are unexpectedly and significantly more efficient (e.g., 5-10X) than LNPs produced according to the reference LNP manufacturing methods, albeit with slightly lower encapsulation efficiency (e.g., 60% -80% compared to 80% -100%).

In some embodiments, the mixed solution is at a pH of about or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14.

In some embodiments the desired "nitrogen-phosphate" or "NP" ratio is about: 1:100, 1:75, 1:50, 1-25, preferably 2:10, more preferably 3:6.

In some embodiments the desired lipid/nucleic acid weight ratio is about: 1:1 to 1000:1, preferably 5:1-100:1, more preferably 10:1-30:1.

In some embodiments, increasing the mRNA concentration in the nucleic acid solution from 0.05mg/ml mRNA to 1.5mg/ml mRNA at a constant NP ratio of 4 in 50mM sodium acetate pH 4 results in an increase in LNP efficacy of about 10X compared to a 1.5mg/ml to 0.05mg/ml mixed concentration tested at the same dose in cells (fig. 8A) and animals (fig. 9C IM and IV administration). Low mRNA concentrations in the range of 0.05mg/ml to 0.1mg/ml LNP indicate LNP produced according to the reference LNP manufacturing method. Preferably, the method of the invention allows mixing to occur at about or above 0.20mg/ml of mRNA, at which point a greater increase in LNP efficacy is observed.

In one embodiment, the method of manufacture is set as follows:

nucleic acid solution: mRNA is prepared at a plurality of concentrations ranging from 0.05 to 3mg/ml in a single buffer, e.g.25 mM sodium acetate buffer, at pH 4. As a general rule, the buffer selection and pH herein should be selected to obtain a pH about 1 point lower than the pKa of the LNP produced during mixing to obtain about 70% encapsulation. For example, when 2 volumes of nucleic acid solution are mixed with 1 volume of lipid solution, mixing with 1.5mg/ml of mRNA in 25mM NaOAc at pH 4 at 75mM total lipid concentration (37.5 mM KC2 or MC3 concentration) to produce KC2 or MC3 of NP 4 with pKa = -6.5 results in a mixed solution consisting of 16.7mM sodium acetate, 67% H2O in 1mg/ml mRNA (3.1 mM phosphate group), 12.5mM KC2 and 33% Ethanol (ETOH) at a mixed pH of 5.5.

Lipid solution: for each of the mRNA solutions described immediately above in connection with nucleic acid solution preparation, a lipid mixture is prepared in ethanol or another suitable solvent at a plurality of concentrations corresponding to the desired NP ratio or lipid/mRNA weight ratio.

naLNP was assembled at the above-described multiple mixed concentrations with mRNA in a single buffer type, concentration and pH.

Bringing naLNP to physiological pH.

Measurement of naLNP size using light scattering

RNA encapsulation in naLNP was measured using Ribogreen assay

Accept naLNP with >40% encapsulation

Luciferase activity is measured in vitro or in vivo at a known dose or doses.

The relative naLNP potency compared to refLNP is determined by luciferase activity measured in vitro or in vivo.

In another embodiment, naLNP potency can be optimized at any given mixing concentration by adjusting mRNA buffer type, concentration, and pH during mixing to change mixing pH and ionizable lipid protonation level:

nucleic acid solution: mRNA was prepared at one concentration (i.e., in the range of 0.05 to 3mg/ml described above) in a range of buffer types (sodium acetate, sodium citrate, etc.), buffer concentrations (1-100 mM) and pH (3-7). For any particular mixing concentration, the selected buffer should produce an encapsulation efficiency that spans the range of 40% -90%, which may correspond approximately to that which may be achieved The level of protonation of the ionized lipids (40% -90%) and thus consists of 33% ETOH/67% H ₂ The pH of the O-buffer and thus the type of buffer, concentration and pH.

Lipid solution: for the mRNA solutions described directly above in connection with nucleic acid solution preparation, the lipid mixture is prepared in ethanol or another suitable solvent at a concentration corresponding to the desired NP ratio or lipid/mRNA weight ratio.

naLNP was assembled in a single mix concentration in the above-described multiple buffers.

Bringing naLNP to physiological pH.

Measurement of naLNP size using light scattering

RNA encapsulation in naLNP was measured using Ribogreen assay

Accept naLNP with >40% encapsulation

Luciferase activity is measured in vitro or in vivo at a known dose or doses.

Relative naLNP potency was determined by luciferase activity measured in vitro or in vivo.

For any particular naLNP formulation, an increase in potency of at least or about 10X relative to a reference LNP produced by a reference LNP manufacturing method can be obtained by appropriate optimization of the concentration of nucleic acid solutions and lipid solutions and thus mixed solutions and mRNA buffer concentration and pH according to the methods disclosed herein.

In some embodiments, the buffer is completely absent from the nucleic acid solution and the ionizable lipid is directly protonated by controlled addition of an acid, such as HCl, to the lipid solution lipid mixture, as shown in example 10. The latter achieves a defined level of protonation in the lipid mixture before mixing with the mRNA in water and can produce an encapsulation level (70%) similar to that found to be optimal when sodium acetate is used.

In one embodiment the two liquids are combined by mixing. For example, mixing is microfluidic mixing by chaotic convection. In another embodiment, T-junction mixing can be used on a larger scale, thereby producing LNP-like.

Microfluidic devices provide the ability to controllably and rapidly mix fluids on the nanoliter scale with precise control over temperature, residence time, and solute concentration. Control and rapid microfluidic mixing was previously applied to the synthesis of inorganic nanoparticles and microparticles and can outperform macro-scale systems for large-scale production of nanoparticles. Microfluidic two-phase microdroplet technology is used to create monodisperse polymer particles for drug delivery or to create larger vesicles for encapsulating cells, proteins or other biomolecules. In some embodiments, hydrodynamic flow focusing, a common microfluidic technique, is used to provide rapid mixing of reagents to produce monodisperse liposomes of controlled size. This technique has also proven suitable for the production of polymeric nanoparticles, where smaller, more monodisperse particles are obtained and the degree of encapsulation of small molecules is higher than in mass production methods.

In one embodiment, at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more lipids are dissolved in an organic solvent such as ethanol while the nucleic acid is in a pH 3-6, preferably 4 acetate buffer. The two streams meet in a common microfluidic channel and are forced to mix within a few milliseconds before being expelled into an aqueous receiving well. Upon mixing, two events occur: 1) While mixing with anionic mRNA, initially neutral ionizable lipids contact low pH buffers and become protonated, thus forming electrostatic bonds between cationic lipids and anionic nucleic acids; and 2) the lipid becomes insoluble in the main aqueous buffer and encapsulates the mRNA. The pH in the final well containing PBS is typically 6-6.5 due to a mixture of acetic acid, PBS and ionizable lipids, all of which are buffers with a certain buffering capacity and initial pH.

In some embodiments, naLNP dialyzes against PBS to raise the pH to about 7.4 and remove ethanol. LNP assembly continues during dialysis, when ionizable lipids with LNP pKa approaching 6.5 become progressively neutralized to ph7.4 and thus less soluble, triggering fusion of LNP, increasing in size and transforming the aqueous electronically transparent core into an electronically dense core containing mainly ionizable lipids and nucleic acids.

In one embodiment, the total amount of lipid provided by the present invention in the administered composition is from about 2 to about 100mg lipid/mg bioactive agent (e.g., RNA), in another embodiment from about 5 to about 25mg lipid/mg bioactive agent (e.g., RNA), in another embodiment from about 7 to about 25mg lipid/mg bioactive agent (e.g., RNA), and in one embodiment from about 10 to about 20mg lipid/mg bioactive agent (e.g., RNA).

Pharmaceutical compositions and methods

The LNP of the invention can be used to deliver therapeutic or prophylactic agents to cells in vitro or in vivo. In particular embodiments, the therapeutic agent is a nucleic acid, which is delivered to a cell using the nucleic acid-lipid particles of the invention. The methods and compositions can be readily adapted to deliver any suitable therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment.

In certain embodiments, the invention provides methods for introducing nucleic acids into cells. Preferred nucleic acids for introduction into cells are mRNA, siRNA, miRNA, immunostimulatory oligonucleotides, DNA plasmids, antisense and ribozymes. These methods may be performed by contacting the particles or compositions of the invention with a cell for a period of time sufficient for intracellular delivery to occur.

The nucleic acids for use in the present invention may be prepared according to any available technique. For mRNA, the primary method of preparation is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), and currently represents the most efficient method of producing long sequence-specific mRNA. In vitro transcription describes a template-directed process of synthesizing an RNA molecule from an engineered DNA template comprising an upstream phage promoter sequence (including, for example, but not limited to, sequences from T7, T3, and SP6 coliphage) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from a number of sources using suitable techniques well known in the art, including but not limited to plasmid DNA and polymerase chain reaction amplification (see Linpinsel, j.l and Conn, g.l., general protocols for preparation of plasmid DNA template and Bowman, j.c., azizi, b., lenz, t.k., ray, p., and Williams, l.d., RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v.941conn g.l. (ed.), new York, n.y. humana Press, 2012).

Transcription of RNA occurs in vitro using linearized DNA templates in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcripts. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax large-scale RNA production system (Promega), megaScript transcription kit (Life Technologies), and commercially available reagents, including RNA polymerase and rttp. methods for in vitro transcription of mRNA are well known in the art. (see, e.g., lock, R.,1972,In Vitro transcription,Ann Rev Biochem v.41 409-46; kamakaka, R.T., and Kraus, W.L.2001.In Vitro transmission. Current Protocols in Cell biology.2:11.6:11.6.1-11.6.17; beckert, B., and Mascoda, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H.works.), new York, N.Y. Humana Press,2010; brunelle, J.L., and Green, R., 3, chapter-In Vitro transcription from plasmid or PCR-amplified 201d DNA, methods in Enzymology v.530,101-114; all of which are incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from unwanted components of the transcription or related reactions, including non-incorporation of rNTPs, proteases, salts, short RNA oligonucleotides, etc. Techniques for isolating mRNA transcripts are well known in the art. Well known procedures include phenol/chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional non-limiting examples of purification procedures that may be used include size exclusion chromatography (Lukavsky, p.j. And Puglisi, j.d.,2004, large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, j.c., azizi, b., lenz, t.k., ray, p., and Williams, l.d., in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v.941conn g.l. (eds.), new York, n.y. humana Press, 2012). Purification can be performed using a variety of commercially available kits, including, but not limited to, the SV total classification system (SV Total Isolation System) (Promega) and the in vitro transcription purification and concentration kit (In Vitro Transcription Cleanup and Concentration Kit) (Norgen Biotek).

In addition, while reverse transcription may produce large amounts of mRNA, the product may contain one or more abnormal RNA impurities associated with undesired polymerase activity, which may need to be removed from the full-length mRNA preparation. These impurities include short RNAs produced by ineffective transcription initiation, double-stranded RNAs (dsRNA) produced by RNA-dependent RNA polymerase activity, chemotranscription initiated from RNA of RNA templates, and self-complementary 3' extensions. These contaminants with dsRNA structures have been demonstrated to produce undesirable immunostimulatory activity via interactions with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This in turn can greatly reduce mRNA translation, as protein synthesis is reduced during the innate cellular immune response. Thus, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, e.g., kariko, k., muramatsu, h., ludwig, j. And Weissman, d.,2011,Generating the optimal mRNA for therapy:HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, nucleic Acid Res, v.39e142; weissman, d., pari, n., muramatsu, h., and Kariko, k., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, p.h. co.) 2013). HPLC purification of mRNA has been reported to translate at much greater levels, especially in primary cells and in vivo.

A number of various modifications have been described in the art for altering the specific properties of in vitro transcribed mRNA and improving its utility. Such modifications include, but are not limited to, modifications of the 5 'and 3' ends of the mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' -end of the mature molecule that plays an important role in mediating the binding of mRNA Cap Binding Proteins (CBPs), which in turn are responsible for enhancing mRNA stability and mRNA translation efficiency in cells. Thus, the highest protein expression level is achieved with capped mRNA transcripts. The 5 '-cap contains a 5' -5 '-triphosphate linkage between the most 5' nucleotide and the guanine nucleotide. The binding guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the final and penultimate 5 '-nucleotide on the 2' -hydroxyl group.

A variety of different cap structures can be used to create a 5' -cap for in vitro transcription of synthetic mRNA. The 5' -capping of synthetic mRNA can be performed co-transcriptionally with a chemical cap analog (i.e., capping during in vitro transcription). For example, an anti-reverse cap analogue (ARCA) cap contains 5' -5' -triphosphate guanine-guanine linkages, wherein one guanine contains an N7 methyl group and a 3' -O-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process and synthetic cap analogs are not consistent with the 5' -cap structure of true cellular mRNA, potentially reducing translatable and cellular stability. Alternatively, synthetic mRNA molecules may be enzymatically capped post-transcriptionally. These molecules can produce a more realistic 5 '-cap structure that more accurately mimics, structurally or functionally, an endogenous 5' -cap with enhanced binding of the cap binding protein, increased half-life, reduced susceptibility to 5 'endonucleases, and/or reduced 5' uncapping. Many synthetic 5' -cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., grudzien-Nogalska, e., kowalska, j., su, w., kuhn, a.n., slepenkov, s.v., darynkiewicz, e., sahin, u., jeniey, j., and Rhoads, r.e., synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, p.h., code), 2013).

At the 3' -end, long-chain adenine nucleotides (poly-A tails) are typically added to the mRNA molecules during RNA processing. Immediately after transcription, the 3 'end of the transcript is cleaved to release the 3' hydroxyl group, and in a process known as polyadenylation, the poly-A polymerase adds an adenine nucleotide strand to the RNA. The poly-A tail is widely shown to enhance both translation efficiency and stability of mRNA (see Bernstein, P. And Ross, J.,1989, poly (A), poly (A) binding protein and the regulation of mRNA stability, trends Bio Sci.14.373-377; guhaniyogi, J. And Brewer, G.,2001,Regulation of mRNA stability in mammalian cells,Gene,v.265,11-23; dreyfus, M. And Regnier, P.,2002, the poly (A) tail of mRNAs: bodyguard in eukaryotes, scavenger in bacteria, cell, v.111, 611-613).

Poly (a) tailing of mRNA transcribed in vitro can be accomplished using a variety of methods including, but not limited to, cloning the Poly (T) sequence into a DNA template or post-transcriptional addition by use of a Poly (a) polymerase. The first case allows in vitro transcription of mRNA with a poly (A) tail of defined length, depending on the size of the poly (T) sequence, but requires additional manipulation of the template. The latter case involves the enzymatic addition of poly (A) tails to in vitro transcribed mRNA using a poly (A) polymerase that catalyzes the incorporation of adenine residues at the 3' end of the RNA, without additional manipulation of the DNA template, but results in mRNA having poly (A) tails of non-uniform length. The 5 '-capping and 3' -Poly (a) Tailing can be performed using a variety of commercial kits, including, but not limited to, the Poly (a) Polymerase Tailing kit (EpiCenter), the mMESSAGE mMACHINE T7 Ultra kit, and the Poly (a) Tailing kit (Life Technologies), as well as commercial reagents, various ARCA caps, poly (a) polymerase, and the like.

In addition to 5 'cap and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translational efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors within eukaryotes and trigger an effective innate immune response. The ability to distinguish between pathogenic and self DNA and RNA has been demonstrated to be based at least in part on structural and nucleoside modifications, as most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNAs lack these modifications, thereby rendering them immunostimulatory, which in turn can inhibit efficient mRNA translation, as outlined above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thereby alleviating such unwanted immunostimulatory activity and enhancing translational capacity (see, e.g., kariko, k. And Weissman, d.2007, naturally occurring nucleos ide modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, curr Opin Drug Disco v Devel, v.10 523-532; pari, n., muramatsu, h., weissman, d., kariko, k., in vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modu lation in Methods in Molecular Biology v.969 (Rabinovich, p.h. code), 2013); kariko, k, muramatsu, h, welsh, f.a., ludwig, j, kato, h, akira, s, weissman, d.,2008,Incorporation of PseudouridineInto mRNA Yields Superior Nonimmunogenic Vector With Increas ed Translational Capacity and Biological Stability,Mol Ther v.16,1833-1840. Modified nucleosides and nucleotides for synthesis of modified RNAs can be prepared for monitoring and utilization using general methods and procedures known in the art. A wide variety of nucleoside modifications are available which can be incorporated into in vitro transcribed mRNA alone or in combination with other modified nucleosides to some extent (see e.g. US 2012/0251618). In vitro synthesis of nucleoside modified mRNA has been reported to have reduced ability to activate immunosensors, with increased translational capacity.

Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5 'and 3' untranslated regions (UTRs). Simultaneously or independently, the optimization of UTRs (advantageously 5 'and 3' UTRs are obtainable from cellular or viral RNAs) has been demonstrated to increase mRNA stability and translation efficiency of in vitro transcribed mRNA (see, e.g., par di, n., muramatsu, h., weissman, d., kariko, k., in vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, p.h. code), 2013).

In addition to mRNA, other nucleic acid payloads may be used in the present invention. For oligonucleotides, methods of preparation include, but are not limited to, chemical synthesis and enzymatic, chemical cleavage of longer precursors, in vitro transcription as described above, and the like. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, e.g., gait, M.J. (eds.) Oligonucl eotide synthesis: a practical approach, oxford [ Oxfordshire ], washingt on, D.C.: IRL Press,1984; and Herdowijn, P. (eds.) Oligonucleotide syn thesis: methods and applications, methods in Molecular Biology, v.288 (Clifton, N.J.) Totowa, N.J.: humana Press,2005; all of which are incorporated herein by reference).

For plasmid DNA, the preparation for use in the present invention generally utilizes, but is not limited to, amplifying and isolating plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest. The presence of genes encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) in the plasmids of interest allows the selective growth of those bacteria containing the plasmids of interest in cultures containing the antibiotics. Methods of isolating plasmid DNA are widely used and well known in the art (see, e.g., heilig, J., elbing, K.L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular biology.41:II:1.7:1.7.1-1.7.16; rozkov, A., larsson, B., gillstrom, S., bjornestedt, R.and Schmidt, S.R. (2008), large-scale production of endotoxin-free plas mids for transient expression in mammalian cell culture. Biotechnol. Bioeng.,99:557-566; and U.S. Pat. No. 6197553B 1). Plasmid isolation can be performed using a variety of commercially available kits, including, but not limited to, plasmid Plus (Qiagen), genJET plasmidMaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits, as well as using commercially available reagents.

Typical applications include using well known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets. Alternatively, the application includes delivery of a DNA or mRNA sequence encoding a therapeutically useful polypeptide or gene editing component. In this way, therapies for genetic diseases are provided by supplying defective or deleted gene products. The methods of the invention may be practiced in vitro, ex vivo, or in vivo. For example, the compositions of the invention may also be used to deliver nucleic acids to cells in vivo using methods known to those of skill in the art.

The delivery of siRNA and its effectiveness in silencing gene expression by the lipid particles of the invention is described below.

For in vivo administration, the pharmaceutical composition is preferably administered parenterally (e.g., intra-articular, intravenous, intraperitoneal, subcutaneous, or intramuscular). In particular embodiments, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection. Other routes of administration include topical (skin, eye, mucosa), oral, pulmonary, intranasal, sublingual, rectal and vaginal.

In one embodiment, the invention provides a method of modulating the expression of a target polynucleotide or polypeptide. These methods generally comprise contacting a cell with an LNP of the invention, which LNP is associated with a nucleic acid capable of modulating expression of a target polynucleotide or polypeptide. As used herein, the term "modulate" refers to altering expression of a target polynucleotide or polypeptide. Modulation may mean increasing or enhancing, or it may mean decreasing or decreasing.

In a related embodiment, the invention provides a method of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, the method comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is selected from the group consisting of an siRNA, a microrna, an antisense oligonucleotide, and a plasmid capable of expressing the siRNA, the microrna, or the antisense oligonucleotide, and wherein the siRNA, the microrna, or the antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide or its complement.

In a further aspect, the invention provides a pharmaceutical composition comprising the lipid particle of the invention and a pharmaceutically acceptable carrier or diluent. Representative pharmaceutically acceptable carriers or diluents include solutions for intravenous injection (e.g., saline or dextrose). The composition may be in the form of a cream, ointment, gel, suspension or emulsion.

As used herein, "treatment" encompasses both improving, curative and prophylactic treatment. As used herein, "patient" means an animal, preferably a mammal, preferably a human, in need of treatment.

The term "therapeutically effective amount" refers to the amount of a compound of the invention and a biologically active agent (e.g., a therapeutic compound) required to treat or ameliorate a disease or condition of interest.

The term "immunologically effective amount" refers to the amount of a compound of the invention and RNA encoding an immunogen required to elicit an immune response that recognizes the immunogen (e.g., in the case of a pathogen). The term "immunogen" refers to any substance or organism that elicits an immune response when introduced into the body. The phrase "RNA encoding an immunogen" refers to a polynucleotide, such as messenger RNA or replicon (e.g., self-replicating RNA), that is capable of being translated into a polypeptide upon administration to a cell or organism according to the codon sequence of such RNA.

"proliferative disease" as used herein means any disease, disorder, trait, genotype or phenotype characterized by unregulated cell growth or replication, as known in the art. In one embodiment, the proliferative disease is cancer. In one embodiment, the proliferative disease is a tumor. In one embodiment, proliferative diseases include, but are not limited to, for example, liquid tumors such as, for example, leukemia, e.g., acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphoblastic Leukemia (ALL), multiple myeloma, and chronic lymphocytic leukemia; and solid tumors, e.g., AIDS-related cancers such as kaposi's sarcoma; breast cancer; bone cancer; brain cancer; head and neck cancer, non-hodgkin's lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gall bladder and bile duct cancer, retina cancer, esophagus cancer, gastrointestinal cancer, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, colorectal cancer, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, sarcoma, wilms' tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial cancer, renal cell carcinoma, gall bladder adenocarcinoma, endometrial sarcoma, multi-drug resistant cancer. In one embodiment, the proliferative disease includes neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet/dry age-related macular degeneration), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration. In one embodiment, the proliferative disease includes restenosis and polycystic kidney disease.

As used herein, "autoimmune disease" means any disease, disorder, trait, genotype, or phenotype characterized by autoimmunity, as known in the art. Autoimmune diseases include, but are not limited to, for example, multiple sclerosis, diabetes, lupus, scleroderma, fibromyalgia, transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, myasthenia gravis, lupus erythematosus, multiple sclerosis, and graves' disease.

By "infectious disease" is meant any disease, disorder or condition associated with an infectious agent such as a virus, bacterium, fungus, prion or parasite. The invention may be used to immunize against pathogens that cause infectious diseases, either actively or passively. Examples of such pathogens are given below.

By "neurological disease" is meant any disease, disorder or condition affecting the central or peripheral nervous system. Neurological diseases include, but are not limited to, diseases or conditions of the peripheral or central nervous system including, for example, alzheimer's Disease, aneurysms, brain injury, carpal tunnel syndrome, cerebral aneurysms, chronic pain, creutzfeldt-Jakob Disease (Creutzfeldt-Jakob Disease), epilepsy, huntington's Disease, meningitis, seizures, and other neurological diseases, conditions, and syndromes.

By "respiratory disease" is meant any disease or condition affecting the respiratory tract. Respiratory diseases include, but are not limited to, for example, asthma, chronic Obstructive Pulmonary Disease (COPD), allergic rhinitis, sinusitis, allergy, respiratory obstruction, respiratory distress syndrome, cystic fibrosis, pulmonary arterial hypertension or vasoconstriction, and emphysema.

By "cardiovascular disease" is meant a disease or condition affecting the heart and blood vessels. Cardiovascular diseases include, but are not limited to, for example, coronary Heart Disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, myocardial infarction (heart attack), cardiac arrhythmias, ischemia, and congestive heart failure.

As used herein, "ocular disease" means any disease, disorder, trait, genotype or phenotype of the eye and associated structures. Ocular diseases include, but are not limited to, for example, cystoid macular edema, diabetic retinopathy, latticework degeneration, retinal vein occlusion, retinal artery occlusion, macular degeneration (e.g., age-related macular degeneration such as wet AMD or dry AMD), toxoplasmosis, retinitis pigmentosa, conjunctival lacerations, corneal lacerations, glaucoma, and the like.

"metabolic disease" means any disease or disorder affecting a metabolic pathway. Metabolic diseases can lead to abnormal metabolic processes, congenital due to abnormalities in genetic enzymes (natural metabolic defects) or acquired due to diseases of endocrine organs or failure of metabolic vital organs such as the liver. In one embodiment, metabolic disorders include obesity, insulin resistance, and diabetes (e.g., type I and/or type II diabetes).

By "dermatological disorder" is meant any disease or condition of the skin, dermis, or any sub-structure therein (such as hair, hair follicles, etc.). Skin diseases, disorders, conditions, and traits may include psoriasis, atopic dermatitis, skin cancers such as melanoma and basal cell carcinoma, hair loss, hair removal, and pigmentation changes.

"auditory disease" means any disease or condition of the auditory system, including the ear, such as the inner ear, middle ear, outer ear, auditory nerve, and any substructures therein. Auditory diseases, disorders, conditions and traits may include hearing loss, deafness, tinnitus, dizziness, balance and movement disorders.

By "regenerative disease" is meant any disease or condition in which insufficient cellular or tissue production or regeneration in vivo or in vitro prevents establishment or restoration of proper organ function before or after injury, prevents or slows wound healing or regression of ulcerative lesions, accelerates aging, or prevents effective cell-based therapies. The term "messenger ribonucleic acid" (messenger RNA, mRNA) refers to a ribonucleic acid (RNA) molecule that mediates the transfer of genetic information to ribosomes in the cytoplasm where the molecule serves as a template for protein synthesis. Which is synthesized from the DNA template during the transcription process. See The American Dictionary of the English Language, fourth edition (updated 2009), houghton Mifflin Company.

In eukaryotes, mRNA is transcribed in vivo at the chromosome by the cellular enzyme RNA polymerase. During or after in vivo transcription, a 5 'cap (also known as an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is added to the 5' end of the mRNA in vivo. The 5' cap is a terminal 7-methylguanosine residue attached to the first transcription nucleotide via a 5' -5' -triphosphate linkage. In addition, most eukaryotic mRNA molecules have a poly (a) moiety ("poly (a)" tail) at the 3' end of the mRNA molecule. In vivo, eukaryotic cells add a poly (A) tail, often about 250 adenosine residues in length, after transcription (SEQ ID NO: 12). Thus, a typical mature eukaryotic mRNA has the structure starting at the 5' end with an mRNA cap nucleotide, followed by the 5' untranslated region (5 ' UTR) of the nucleotide, and then an open reading frame starting with the start codon of the AUG triplet, which is a nucleotide base of the coding sequence of the protein, and ending with the stop codon of the UAA, UAG, or UGA triplet, which can be a nucleotide base, followed by the 3' untranslated region (3 ' UTR) of the nucleotide and ending with a polyadenylation tail. Although the features of a typical mature eukaryotic mRNA are produced in eukaryotic cells in their native state, the same or structurally and functionally equivalent features can be produced in vitro using molecular biological methods. Thus, any RNA having a structure similar to that of a typical mature eukaryotic mRNA can serve as an mRNA and is within the scope of the term "messenger ribonucleic acid".

mRNA molecules typically have a size such that they can be encapsulated in the lipid nanoparticles of the present invention. Although the size of an mRNA molecule varies in nature depending on the nature of the mRNA species encoding a particular protein, the average size of an mRNA molecule is an average mRNA size of 500-10,000 bases.

DNA may exist in at least two forms, which have different sizes. The first form of DNA is a very large size polymer called a chromosome. Chromosomes contain genetic information for many or most proteins in cells and also contain information by which cells can control the replication of DNA molecules. The bacterial cell may contain one or more chromosomes. Eukaryotic cells typically contain more than one chromosome of the cell, each chromosome,

the second form of DNA is in a shorter size form. Many DNA molecules in the second form have a size such that they can be encapsulated in the lipid nanoparticle of the invention. Some of these shorter forms of DNA may be of a size suitable for encoding a protein. Examples of such second, shorter, useful forms of DNA include plasmids and other vectors. For a more complete description, see Alberts B et al (2007) Molecular Biology of the Cell, fourth edition, garland Science.

Plasmids are small DNA molecules that are physically separated from the chromosomal DNA within a cell and that can replicate independently. Plasmids are typically present in vivo as smaller circular, double-stranded DNA molecules. In nature, plasmids carry genes that can be transcribed and translated into proteins that can be beneficial to the survival of organisms (e.g., antibiotic resistance). In nature, plasmids can often be transferred from one organism to another by horizontal gene transfer. Artificial or recombinant plasmids are widely used in molecular biology to allow replication of recombinant DNA sequences and expression of useful proteins in host organisms. Plasmid sizes may vary from about 1 to over 25 kilobase pairs. Recombinant plasmids can be recombinantly produced to have a size such that they can be encapsulated in the lipid nanoparticles of the invention.

In molecular biology, a vector is a DNA molecule that serves as a vehicle to carry genetic material from one cell to another in vitro, or artificially from a biochemical reaction, in which DNA can be replicated and/or expressed. Vectors containing foreign DNA are referred to as recombinant vectors. Among the various types of vectors available are plasmids and viral vectors. For bacterial cells, insertion of the vector into the target cell is often referred to as transformation, for eukaryotic cells as transfection, but insertion of the viral vector is often referred to as transduction.

Viral vectors are typically recombinant viruses with modified viral DNA or RNA that has become non-infectious, but still contain viral promoters as well as transgenes, thereby allowing for translation of the transgene via the viral promoters. In some embodiments, the viral vector is designed to permanently incorporate the insert into the host genome (integration), and thereby leave a unique genetic marker in the host genome after incorporation of the transgene. The viral vector may be recombinantly produced to have a size such that it may be encapsulated in the lipid nanoparticle of the invention.

The term "short interfering nucleic acid" (siNA) as used herein refers to any nucleic acid molecule capable of inhibiting or down-regulating gene expression or viral replication by mediating RNA interference (RNAi) or gene silencing in a sequence-specific manner. Including short interfering RNAs (sirnas), micrornas (mirnas), short interfering oligonucleotides, and chemically modified short interfering nucleic acid molecules. siRNA is responsible for RNA interference, a sequence-specific post-transcriptional gene silencing process in animals and plants. siRNA is produced by ribonuclease III cleavage from longer double-stranded RNAs (dsRNA) homologous to or specific for a silencing gene target.

The term "RNA interference" (RNAi) is a post-transcriptional, target gene silencing technique that uses an RNAi agent to degrade messenger RNA (mRNA) containing the same or very similar sequence as the RNAi agent. See: zamore and Haley,2005, science,309,1519-1524; zamore et al, 2000, cell,101,25-33; elbashir et al, 2001, nature,411,494-498; and Kreutzer et al PCT publication WO 00/44895; fire, PCT publication WO 99/32619; mello and Fire PCT publication WO 01/29058; etc.

As used herein, RNAi is equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or expression genetics. For example, formulations containing the lipids of the present invention can be used in combination with siNA molecules to simultaneously epigenetically silence genes at post-transcriptional and/or pre-transcriptional levels. In non-limiting examples, modulation of gene expression by a siNA molecule may be produced by siNA-mediated cleavage of RNA (coding or non-coding RNA) via RISC or alternatively by translational inhibition, as is known in the art. In another embodiment, modulation of gene expression by siNA may result from transcriptional repression, such as reported, for example, by Janowski et al, 2005,Nature Chemical Biology,1,216-222.

The term "RNAi inhibitor" is any molecule that can down-regulate (e.g., reduce or inhibit) RNA interference function or activity in a cell or patient. RNAi inhibitors can down-regulate, reduce, or inhibit RNAi (e.g., RNAi-mediated cleavage of a target polynucleotide, translational inhibition, or transcriptional silencing) by interacting with or interfering with the function of any component of the RNAi pathway, including a protein component such as RISC, or a nucleic acid component such as a miRNA or siRNA. An RNAi inhibitor can be a siNA molecule, antisense molecule, aptamer, or small molecule that interacts with or interferes with the function of RISC, miRNA, or siRNA, or any other component of the RNAi pathway in a cell or patient. RNAi inhibitors can be used to modulate (e.g., up-regulate or down-regulate) expression of a target gene by inhibiting RNAi (e.g., RNAi-mediated cleavage of a target polynucleotide, translational inhibition, or transcriptional silencing). In one embodiment, the RNA inhibitor is used to up-regulate gene expression by interfering (e.g., reducing or preventing) with endogenous down-regulation or inhibition of gene expression via translational inhibition, transcriptional silencing, or RISC-mediated cleavage of a polynucleotide (e.g., mRNA). The RNAi inhibitors of the invention can thus be used to up-regulate gene expression by interfering with endogenous repression, silencing or inhibition mechanisms of gene expression to treat diseases or conditions resulting from loss of function. In various embodiments herein, the term "RNAi inhibitor" is used interchangeably with the term "siNA".

The term "enzymatic nucleic acid" as used herein refers to a nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target and also has enzymatic activity for specifically cleaving a target RNA, thereby inactivating the target RNA molecule. The complementary region allows sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus allows cleavage. 100% complementarity is preferred, but complementarity as low as 50% -75% is also suitable for use in the present invention (see, e.g., werner and Uhlenbeck,1995,Nucleic Acids Research,23,2092-2096; hammann et al 1999,Antisense and Nucleic Acid Drug Dev.,9, 25-31). The nucleic acid may be modified at the base, sugar and/or phosphate groups. The term enzymatic nucleic acid may be used interchangeably with phrases such as ribozymes, catalytic RNAs, enzymatic RNAs, catalytic DNAs, aptamer enzymes or aptamer-binding ribozymes, regulatable ribozymes, catalytic oligonucleotides, ribozymes, dnazymes, rnases, endoribonucleases, endonucleases, minienzymes, leader enzymes, oligoenzymes or dnazymes. All these terms describe nucleic acid molecules having enzymatic activity. A key feature of an enzymatic nucleic acid molecule is that it has a specific substrate binding site that is complementary to one or more target nucleic acid regions, and that it has a nucleotide sequence within or around the substrate binding site that confers nucleic acid cleavage and/or ligation activity to the molecule (see, e.g., cech et al, U.S. Pat. No. 4,987,071; cech et al, 1988,260JAMA 3030). The ribozymes and enzymatic nucleic acid molecules of the invention may be chemically modified, e.g., as described in the art and elsewhere herein.

The term "antisense nucleic acid" as used herein refers to a non-enzymatic nucleic acid molecule that binds to a target RNA and alters the activity of the target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; egollm et al 1993nature 365, 566) (for reviews see Stein and Cheng,1993Science 261,1004 and Woolf et al, U.S. patent No. 5,849,902). Antisense DNA can be chemically synthesized or expressed via the use of single stranded DNA expression vectors or equivalents thereof. The antisense molecules of the invention can be chemically modified, for example, as described in the art.

The term "rnase H activating region" as used herein refers to a region of a nucleic acid molecule (typically greater than or equal to 4-25 nucleotides in length, preferably 5-11 nucleotides in length) that is capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular rnase H enzymes (see, e.g., arow et al, U.S. patent No. 5,849,902; arow et al, U.S. patent No. 5,989,912). Rnase H enzyme binds to nucleic acid molecule-target RNA complex and cleaves target RNA sequence.

The term "2-5A antisense chimera" as used herein refers to an antisense oligonucleotide containing 5' -phosphorylated 2' -5' -linked adenylate residues. These chimeras bind to the target RNA in a sequence-specific manner and activate cellular 2-5A-dependent ribonucleases, which in turn cleave the target RNA (Torrenc et al, 1993Proc.Natl.Acad.Sci.USA 90,1300;Silverman et al, 2000,Methods Enzymol, 313,522-533; player and Torrenc, 1998, pharmacol. Ther.,78,55-113). The 2-5A antisense chimeric molecule can be chemically modified, for example as described in the art.

The term "triplex forming oligonucleotide" as used herein refers to an oligonucleotide that can bind to double stranded DNA in a sequence specific manner to form a triple helix. Formation of such triple helical structures has been shown to inhibit transcription of target genes (Duval-Valentin et al, 1992Proc.Natl.Acad.Sci.USA 89,504;Fox,2000,Curr.Med.Chem, 7,17-37; praseuth et al, 2000, biochem. Biophys. Acta,1489, 181-206). The triplex forming oligonucleotide molecules of the invention may be chemically modified, for example as described in the art.

The term "decoy RNA" as used herein refers to an RNA molecule or aptamer designed to preferentially bind to a predetermined ligand. Such binding may result in inhibition or activation of the target molecule. Decoy RNAs or aptamers can compete with naturally occurring binding targets to bind specific ligands. Similarly, decoy RNAs can be designed to bind to a receptor and block binding of an effector molecule or can be designed to bind to a target receptor and prevent interaction with the receptor. The bait molecules of the invention may be chemically modified, for example as described in the art.

The term "single stranded DNA" (ssDNA) as used herein refers to naturally occurring or synthetic deoxyribonucleic acid molecules comprising linear single strands, e.g., ssDNA may be a sense or antisense gene sequence or EST (expressed sequence tag).

The term "allelic enzyme" as used herein refers to an allosteric nucleic acid molecule, including, for example, U.S. patent No. 5,834,186; 5,741,679; 5,589,332; 5,871,914; and PCT publications WO 00/24931, WO 00/2626, WO 98/27104 and WO 99/29842.

The term "aptamer" as used herein means a polynucleotide composition that specifically binds to a target molecule, wherein the polynucleotide has a sequence that is different from the sequence normally recognized by the target molecule in a cell. Alternatively, an aptamer may be a nucleic acid molecule that binds to a target molecule, wherein the target molecule does not naturally bind to the nucleic acid. The target molecule may be any target molecule. The aptamer molecules of the invention may be chemically modified, for example as described in the art.

Pharmaceutical formulation of LNP composition

For pharmaceutical use, LNP compositions of the invention can be administered by enteral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), oral, intranasal, rectal, vaginal, buccal, nasopharyngeal, gastrointestinal, or sublingual administration. Administration may be systemic (e.g., IV) or local (e.g., IM, SC, TD, intranasal, or topical). Topical administration may involve, for example, catheterization, implantation, osmotic pumping, direct injection, skin/transdermal application, stent implantation, ear drops/eye drops or portal vein administration. Biopharmaceutical properties of the compound of formula (I), such as solubility and solution stability (at pH), permeability, etc., should be evaluated to select the most suitable dosage form and route of administration for treating the proposed indication.

The compositions of the present invention are typically, but not necessarily, administered in a formulation associated with one or more pharmaceutically acceptable excipients. The term "excipient" encompasses any ingredient other than the compounds of the present invention, other lipid components, and bioactive agents. Excipients may impart functional (e.g., drug release rate control) and/or non-functional (e.g., processing aids or diluents) characteristics to the formulation. The choice of excipient will depend to a large extent on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Typical pharmaceutically acceptable excipients include: diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; lubricants, for example silica, talc, stearic acid, its magnesium or calcium salt and/or polyethylene glycol; binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; disintegrants, for example starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or absorbents, colorants, flavors, and/or sweeteners.

In some embodiments, the naLNP is stored frozen and thawed prior to use. In some embodiments, stable for up to 2 weeks at 4 ℃. In some embodiments it is in the form of various cryopreservation solutions containing various sugars to maintain refrigeration upon injection.

The excipient may be an aqueous carrier, which may optionally contain buffers (e.g., PBS buffer) and/or sugars.

A thorough discussion of pharmaceutically acceptable excipients is available in Gennaro, remington: the Science and Practice of Pharmacy 2000, 20 th edition (ISBN: 0683306472).

The compositions of the present invention may be administered orally. Oral administration may involve swallowing to allow the compound to enter the gastrointestinal tract and/or buccal, lingual or sublingual administration to allow the compound to pass directly from the oral cavity into the blood stream.

The compositions of the present invention may be administered parenterally. The compounds and compositions of the present invention may be administered directly into the blood stream, subcutaneous tissue, muscle or internal organs. Suitable means for administration include intravenous, intra-arterial, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration. Suitable devices for administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques.

Parenteral formulations are typically aqueous or oily solutions. When the solution is aqueous, excipients such as sugar (including but not limited to dextrose, mannitol, sorbitol, and the like) salts, carbohydrates, and buffers (preferably pH 3 to 9), however, for some applications, it may be more suitably formulated as a sterile non-aqueous solution or in dry form for use in combination with a suitable vehicle such as sterile, pyrogen-free Water (WFI).

Parenteral formulations may include implants derived from degradable polymers such as polyesters (i.e., polylactic acid, polylactide-co-glycolide, polycaprolactone, polyhydroxybutyrate), polyorthoesters, and polyanhydrides. These formulations may be administered via a surgical incision into subcutaneous tissue, muscle tissue, or directly into a particular organ.

Preparation of parenteral formulations under sterile conditions, such as by lyophilization, can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

The solubility of the compounds and compositions used to prepare parenteral solutions can be increased by using suitable formulation techniques, such as the incorporation of co-solvents and/or solubility enhancers such as surfactants, micelle structures, and cyclodextrins.

The compositions of the invention may be administered intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (alone, in the form of a mixture, e.g. a dry mixture with lactose, or in the form of mixed component particles, e.g. mixed with phospholipids such as phosphatidylcholine), in the form of an aerosol spray from a pressurized container, pump, spray, nebulizer (preferably one using electrohydrodynamic to produce a fine mist) or nebulizer, with or without the use of a suitable propellant such as 1, 2-tetrafluoroethane or 1,2, 3-heptafluoropropane, or in the form of nasal drops. For intranasal use, the powder may comprise a bioadhesive, such as chitosan or cyclodextrin.

Pressurized containers, pumps, sprays, atomizers or nebulisers contain a solution or suspension of a compound of the invention comprising, for example, ethanol, aqueous ethanol or a suitable substitute for dispersing, solubilising or prolonging the release of a composition of the invention, a propellant as solvent and optionally a surfactant such as sorbitan trioleate, oleic acid or oligolactic acid.

The composition is micronized to a size suitable for delivery by inhalation (typically less than 5 microns) prior to use in a dry powder or suspension formulation. This may be achieved by any suitable comminution method such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization or spray drying.

Capsules (e.g. made of gelatin or hydroxypropyl methylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound or composition of the invention, a suitable powder base such as lactose or starch and a performance modifying agent such as I-leucine, mannitol or magnesium stearate. Lactose may be anhydrous or in the form of a monohydrate, the latter being preferred. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

Formulations for inhalation/intranasal administration may be formulated for immediate and/or modified release using PGLA, for example. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

Suitable formulations for transdermal application include a therapeutically effective amount of a compound or composition of the present invention in combination with a carrier. Advantageous carriers include absorbable pharmacologically acceptable solvents that aid in delivery through the skin of the host. Characteristically, the transdermal device is in the form of a bandage comprising a back component, a reservoir containing a compound optionally with a carrier, an optional rate controlling barrier to deliver the compound to the skin of the host at a controlled predetermined rate over an extended period of time, and means to secure the device to the skin.

The lipid compositions of the present invention are administered in any of a number of ways, including parenterally, intravenously, systemically, locally, orally, intratumorally, intramuscularly, subcutaneously, intraperitoneally, by inhalation, or any such delivery method. In one embodiment, the composition is administered parenterally, i.e., intra-articular, intravenous, intraperitoneal, subcutaneous, or intramuscular. In particular embodiments, the liposome composition is administered by intravenous infusion or intraperitoneal bolus injection.

The lipid compositions of the invention may be formulated into pharmaceutical compositions suitable for delivery to a subject. The pharmaceutical compositions of the invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, dextrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelators such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic, or slightly hypertonic with respect to the blood of the recipient, suspending agents, thickening agents, and/or preservatives. Alternatively, the compositions of the present invention may be formulated as a lyophilized product.

Suitable formulations for use in the present invention may be found, for example, in Pharmaceutical Sciences, mack Publishing Company, philiadelphia, pa., 17 th edition (1985). Often, the compositions comprise a solution of lipid nanoparticles suspended in an acceptable carrier, such as an aqueous carrier.

In one embodiment, the invention provides a pharmaceutical composition (i.e., formulation) comprising a lipid composition of the invention and a pharmaceutically acceptable carrier or excipient. In another embodiment, at least one other lipid component is present in the lipid composition. In another embodiment, the lipid composition is in the form of a liposome. In another embodiment, the lipid composition is in the form of a lipid nanoparticle. In another embodiment, the lipid composition is suitable for delivery to the liver. In another embodiment, the lipid composition is suitable for delivery to a tumor. In another embodiment, the lipid composition is suitable for topical delivery applications (eye, ear, skin, lung); delivery to muscle (intramuscular), fat or subcutaneous cells (subcutaneous administration). In another embodiment, the bioactive agent is RNA or DNA.

For immunization purposes, the compositions are typically prepared as injectable formulations and will be administered by injection (e.g., intramuscular injection).

The invention also provides delivery devices (e.g., syringes, nebulizers, inhalers, skin patches, etc.) containing the compositions of the invention. Such devices can be used to administer a pharmaceutical composition to a subject, for example, to a human for immunization.

Cells and organs targeted by pharmaceutical compositions

The compounds, compositions, methods and uses of the present invention may be used to deliver a bioactive agent to one or more of the following in a patient: liver or liver cells (e.g., hepatocytes); kidney or kidney cells; a tumor or tumor cell; CNS or CNS cells (central nervous system, e.g., brain and/or spinal cord); PNS or PNS cells (peripheral nervous system); lung or lung cells; a blood vessel or blood vessel cell; skin or skin cells (e.g., dermal cells and/or follicular cells); eye or ocular cells (e.g., macula, fovea, cornea, retina) and ear or ear cells (e.g., inner ear, middle ear, and/or outer ear cells).

The compounds, compositions, methods and uses of the invention are also useful for delivering bioactive agents (e.g., RNA encoding an immunogen) to cells of the immune system.

In one embodiment, the compounds, compositions, methods and uses of the invention are used to deliver a bioactive agent to a liver cell (e.g., a hepatocyte). In one embodiment, the compounds, compositions, methods and uses of the invention are used to deliver a bioactive agent to a tumor or tumor cell (e.g., a primary tumor or metastatic cancer cell). In another embodiment, the compounds, compositions, methods and uses are for delivering bioactive agents to skin fat, muscle and lymph nodes (i.e., subcutaneous administration).

For delivery of the bioactive agent to the liver or liver cells, in one embodiment, the composition of the invention is contacted with the liver or liver cells of the patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, portal intravenous injection, catheterization, stent implantation), as is generally known in the art, to facilitate delivery.

For delivery of the bioactive agent to the kidney or kidney cells, in one embodiment, the composition of the invention is contacted with the kidney or kidney cells of the patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, catheterization, stent implantation), as is generally known in the art, to facilitate delivery.

For delivery of the bioactive agent to the tumor or tumor cells, in one embodiment, the composition of the invention is contacted with the tumor or tumor cells of the patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, catheterization, stent implantation), as is generally known in the art, to facilitate delivery.

For delivery of a bioactive agent to the CNS or CNS cells (e.g., brain cells and/or spinal cord cells), in one embodiment, the composition of the invention is contacted with the CNS or CNS cells (e.g., brain cells and/or spinal cord cells) of a patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, catheterization, stent implantation, osmotic pump administration (e.g., intrathecal or ventricles)), as is generally known in the art, to facilitate delivery.

For delivery of the bioactive agent to PNS or PNS cells, in one embodiment, the composition of the present invention is contacted with PNS or PNS cells of a patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection), as is generally known in the art, to facilitate delivery.

For delivery of the bioactive agent to the lung or lung cells, in one embodiment, the composition of the invention is contacted with the patient's lung or lung cells, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct pulmonary administration to lung tissue and cells), as is generally known in the art, to facilitate delivery.

For delivery of the bioactive agent to the blood vessel or vascular cell, in one embodiment, the composition of the present invention is contacted with the blood vessel or vascular cell of the patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., clamping, catheterization, stent implantation), as is generally known in the art, to facilitate delivery.

For delivery of the bioactive agent to the skin or skin cells (e.g., dermal cells and/or follicular cells), in one embodiment, the composition of the present invention is contacted with the skin or skin cells (e.g., dermal cells and/or follicular cells) of the patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct skin application, iontophoresis), as is generally known in the art, to facilitate delivery.

For delivery of the bioactive agent to the eye or ocular cells (e.g., macula, fovea, cornea, retina), in one embodiment, the composition of the invention is contacted with the eye or ocular cells (e.g., macula, fovea, cornea, retina) of the patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eye drops, implants), as is generally known in the art, to facilitate delivery.

For delivery of the bioactive agent to the ear or ear cells (e.g., cells of the inner ear, middle ear, and/or outer ear), in one embodiment, as is generally known in the art, the composition of the invention is contacted with the ear or ear cells (e.g., inner ear, middle ear, and/or outer ear cells) of the patient, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection), to facilitate delivery.

To deliver a bioactive agent (e.g., RNA encoding an immunogen) to cells of the immune system (e.g., antigen presenting cells, including specialized antigen presenting cells), in one embodiment the compositions of the invention are delivered intramuscularly, and the immune cells can then infiltrate the delivery site and process the delivered RNA. Such immune cells may include macrophages (e.g., bone marrow derived macrophages), dendritic cells (e.g., bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myelogenous dendritic cells), monocytes (e.g., human peripheral blood mononuclear cells), and the like (see, e.g., WO 2012/006372).

V. immunization according to the invention

For immunization purposes, in some embodiments, the invention encompasses delivery of mRNA encoding an immunogen. Immunogens elicit an immune response that recognizes the immunogen and thus can be used to provide immunity against pathogens or against allergens or against tumor antigens. Immunization against diseases and/or infections caused by pathogens is preferred.

In certain embodiments, the naLNP has ancillary properties. For example, the naLNP disclosed herein can have specific T follicular helper cell helper activities that result in an effective antibody response. The asymmetric ionizable lipid LNP can act as a strong Th2 biasing adjuvant when delivered with protein subunit antigens. In some embodiments, the naLNP mRNA vaccine disclosed herein drives a Tfh bias response that stimulates proliferation and effective durable neutralizing antibody responses of Tfh and germinal center B cells.

RNA is delivered with the lipid composition of the invention (e.g., formulated as LNP). In some embodiments, the invention utilizes liposomes having an RNA encoding an immunogen encapsulated therein. Encapsulation within LNP protects RNA from rnase digestion. The encapsulation efficiency need not be 100%. The presence of external RNA molecules (e.g., on the outer surface of the liposome) or "naked" RNA molecules (RNA molecules that are not associated with LNP) is acceptable. Preferably, for compositions comprising liposomes and RNA molecules, at least half of the RNA molecules (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the RNA molecules) are encapsulated in naLNP.

The RNA molecule can also complex with LNP. For example, lipids need not form only liposomes (with an aqueous core). Some lipid nanoparticles may comprise a lipid core (e.g., a composition may comprise a mixture of liposomes having a lipid core and nanoparticles). In these cases, by non-covalent interactions (e.g., ionic interactions between negatively charged RNAs and cationic lipids), the RNA molecules can be encapsulated by the LNP with an aqueous core and complexed with the LNP with a lipid core. Encapsulation and complexation by LNP (whether with lipid or aqueous core) protects RNA from rnase digestion. The encapsulation/recombination efficiency need not be 100%. The presence of "naked" RNA molecules (RNA molecules that are not associated with liposomes) is acceptable. Preferably, for a composition comprising a population of LNPs and a population of RNA molecules, at least half of the population of RNA molecules (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the RNA molecules) is encapsulated in, or complexed with, the LNPs.

RNA molecules in pharmaceutical compositions

Following in vivo administration of the immunizing composition, the delivered RNA is released and translated inside the cell to provide the immunogen in situ. In certain embodiments, the RNA is plus ("+") stranded so that it can be translated by the cell without any intervening replication steps such as reverse transcription. It may also bind to TLR7 receptors expressed by immune cells, thereby eliciting an accessory effect. Additionally or alternatively, the RNA may bind other receptors, such as RIG I, MDA5, or RIG I and MDA5.

In certain embodiments, the RNA is self-replicating RNA. Self-replicating RNA molecules (replicons) can, when delivered to even vertebrate cells without any proteins, result in the production of multiple daughter RNAs by self-transcription (via their own produced antisense copies). Thus, in certain embodiments, the self-replicating RNA molecule is a (+) strand molecule that can be directly translated after delivery to a cell, and such translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in 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 the encoded immunogen, or may be transcribed to provide further transcripts of the same meaning as the delivered RNA, which are translated to provide in situ expression of the immunogen. The overall result of this series of transcriptions is a vast amplification of the number of replicon RNAs introduced and thus the encoded immunogens become the major polypeptide products of the host cell.

One suitable system for achieving self-replication is the use of an alphavirus-based RNA replicon. These (+) strand replicons translate after delivery to cells to give replicases (or replicase-transcriptases). Replicases translate into polyproteins that automatically cleave to provide replication complexes that produce genomic (-) strand copies of the (+) strand delivered RNA. These (-) strand transcripts can be transcribed on themselves to give further copies of the plus strand parent RNA and also give subgenomic transcripts encoding immunogens. Translation of the subgenomic transcripts thus results in situ expression of the immunogen by the infected cells. Suitable alphavirus replicons may use replicases from sindbis virus, semliki forest virus, eastern equine encephalitis virus, venezuelan equine encephalitis virus, etc. Mutant or wild-type viral sequences may be used, for example attenuated TC83 mutants of VEEV have been used in replicons.

Thus, it is preferred that the self-replicating RNA molecule encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule and (ii) an immunogen. The polymerase can be an alphavirus replicase, e.g., comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP 4.

Although the native alphavirus genome encodes a structural virion protein in addition to the non-structural replicase polyprotein, in certain embodiments, the self-replicating RNA molecules of the invention do not encode an alphavirus structural protein. Thus, a specific self-replicating RNA can result in the production of a self-genome RNA copy in a cell, but does not produce RNA-containing virions. The inability to produce these viral particles means that, unlike wild-type alphaviruses, self-replicating RNA molecules cannot perpetuate themselves in an infectious form. The alphavirus structural proteins necessary for perpetuation in wild-type viruses are not present in the self-replicating RNAs of the invention and are replaced in position by genes encoding immunogens of interest such that subgenomic transcripts encode immunogens rather than structural alphavirus virion proteins.

Self-replicating RNA molecules suitable for use in the present invention may therefore have two open reading frames. One open reading frame encodes a replicase, e.g., a first (5') open reading frame; the other open reading frame encodes an immunogen, e.g., a second (3') open reading frame. In some embodiments the RNA may have an additional (e.g., downstream) open reading frame, e.g., to encode a further immunogen (see below) or to encode an accessory polypeptide.

The self-replicating RNA molecule may have a 5' sequence that is compatible with the encoded replicase.

Self-replicating RNA molecules may have different lengths, but are typically 5000-25000 nucleotides in length, e.g., 8000-15000 nucleotides, or 9000-12000 nucleotides. Thus, the RNA is longer than that found in siRNA or conventional mRNA delivery. In some embodiments, the self-replicating RNA is greater than about 2000 nucleotides, such as greater than about: 9000. 12000, 15000, 18000, 21000, 24000 or more nucleotides in length.

The RNA molecule can have a 5' cap (e.g., 7-methylguanosine). This cap enhances in vivo translation of RNA.

The 5 'nucleotide of the RNA molecule suitable for use in the present invention may have a 5' triphosphate group. In capped RNA, this can be linked to 7-methylguanosine via a 5 '-to-5' bridge. 5' triphosphate can enhance RIG-I binding and thereby promote helper effects.

The RNA molecule may have a 3' poly a tail. It may also include a poly A polymerase recognition sequence (e.g., AAUAAA) near its 3' end.

RNA molecules suitable for use in the present invention are typically single stranded for immunization purposes. Single stranded RNA can elicit a helper effect, typically by binding to TLR7, TLR8, RNA helicase and/or PKR. RNA delivered in double stranded form (dsRNA) can bind to TLR3, and such receptors can also be triggered by dsRNA formed during replication of single stranded RNA or within the secondary structure of single stranded RNA.

RNA molecules for immunization purposes can be conveniently prepared by In Vitro Transcription (IVT). IVT may use a (cDNA) template produced in bacteria in plasmid form and propagated, or synthesized (e.g., by gene synthesis and/or Polymerase Chain Reaction (PCR) engineering methods). For example, a DNA-dependent RNA polymerase (such as phage T7, T3, or SP6 RNA polymerase) may be used to transcribe RNA from a DNA template. Appropriate capping and poly A addition reactions can be used as desired (although the poly-A of replicons is typically encoded within a DNA template). These RNA polymerases can have stringent requirements for transcribed 5' nucleotides and in some embodiments these requirements must be matched to the requirements of the encoded replicase to ensure that the IVT transcribed RNA can function effectively as a substrate for its own encoded replicase.

As discussed in WO2011/005799, self-replicating RNAs may include (in addition to any 5' cap structure) one or more nucleotides with modified nucleobases. For example, self-replicating RNA can include one or more modified pyrimidine nucleobases, such as pseudouridine and/or 5-methylcytosine residues. However, in some embodiments, the RNA does not comprise modified nucleobases and may not include modified nucleotides, i.e., all nucleotides in the RNA are standard A, C, G and U ribonucleotides (except any 5 'cap structure that may include 7' methylguanosine). In other embodiments, the RNA can include a 5 'cap comprising 7' methylguanosine, and the first 1, 2, or 3 5 'ribonucleotides can be methylated at the 2' position of the ribose.

The RNA used in the present invention desirably comprises only phosphodiester linkages between nucleosides for immunization purposes, but in some embodiments may contain phosphoramidate, phosphorothioate, and/or methylphosphonate linkages.

The invention includes embodiments in which multiple RNA species are formulated with the lipid compositions provided by the invention, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more species of RNA, including different classes of RNA (such as mRNA, siRNA, self-replicating RNA, and combinations thereof).

VII immunogens

In some embodiments, the RNA molecules used in the present invention encode polypeptide immunogens for immunization purposes. In these embodiments, following administration, the RNA is translated in vivo and the immunogen may elicit an immune response in the recipient. The immunogen may elicit an immune response against a pathogen (e.g., a bacterium, virus, fungus, or parasite), but in some embodiments it elicits an immune response against an allergen or tumor antigen. The immune response may include an antibody response (typically including IgG) and/or a cell-mediated immune response. Polypeptide immunogens will typically elicit an immune response that recognizes the corresponding pathogen (or allergen or tumor) polypeptide, but in some embodiments the polypeptide may act as a mimotope to elicit an immune response that recognizes a sugar. The immunogen is typically a surface polypeptide such as an adhesin, hemagglutinin, envelope glycoprotein, spike glycoprotein, or the like.

The RNA molecule may encode a single polypeptide immunogen or a plurality of polypeptides. Multiple immunogens may be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If the immunogen is expressed as a separate polypeptide from the mRNA, one or more of these polypeptides may have an upstream IRES or additional viral promoter element. Alternatively, multiple immunogens may be expressed by a multimeric protein that encodes a separate immunogen fused to a short autocatalytic protease (e.g., the hoof disease virus 2A protein), or expressed as an intein.

In certain embodiments, a polypeptide immunogen (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more immunogens) can be used alone or in combination with an RNA molecule, such as self-replicating RNA, that encodes one or more immunogens (e.g., the same or different polypeptide immunogens).

In some embodiments, the immunogen elicits an immune response against one of these bacteria:

neisseria meningitidis (Neisseria meningitidis): useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding proteins. Combinations of three useful polypeptides are disclosed in Giuliani et al (2006) Proc Natl Acad Sci USA (29): 10834-9.

Streptococcus pneumoniae (Streptococcus pneumoniae): useful polypeptide immunogens are disclosed in WO 2009/016515. These immunogens include, but are not limited to, the RrgB cilia subunit, the β -N-acetyl-hexosaminidase precursor (spr 0057), spr0096, the general stress protein GSP-781 (spr 2021, SP 2216), the serine/threonine kinase StkP (SP 1732), and the pneumococcal surface adhesin PsaA.

Streptococcus pyogenes (Streptococcus pyogenes): useful immunogens include, but are not limited to, the polypeptides disclosed in WO02/34771 and WO 2005/032582.

Moraxella catarrhalis (Moraxella catarrhalis).

Bordetella pertussis (Bordetella pertussis): pertussis immunogens including, but not limited to, pertussis toxin or toxoid (PT), filamentous Hemagglutinin (FHA), pertactin and lectins 2 and 3 may be used.

Staphylococcus aureus (Staphylococcus aureus): useful immunogens include, but are not limited to, the polypeptides disclosed in WO2010/119343, such as haemolysin, esxA, esxB, ferritin binding protein (sta 006) and/or sta011 lipoproteins.

Clostridium tetani (Clostridium tetani): a typical immunogen is tetanus toxoid.

Corynebacterium diphtheriae (Cornynebacterium diphtheriae): typical immunogens are diphtheria toxoids.

Haemophilus influenzae: useful immunogens include, but are not limited to, the polypeptides disclosed in WO2006/110413 and WO 2005/111066.

Pseudomonas aeruginosa (Pseudomonas aeruginosa)

Streptococcus agalactiae (Streptococcus agalactiae): useful immunogens include, but are not limited to, the polypeptides disclosed in WO 02/34771.

Chlamydia trachomatis (Chlamydia trachomatis): useful immunogens include, but are not limited to PepA, lcrE, artJ, dnaK, CT398, ompH-like, L7/L12, omcA, atoS, CT547, eno, htrA and MurG (e.g., as disclosed in WO 2005/002619). LcrE (WO 2006/138004) and HtrA (WO 2009/109860) are two preferred immunogens.

Chlamydia pneumoniae (Chlamydia pneumoniae): useful immunogens include, but are not limited to, the polypeptides disclosed in WO 02/02606.

Helicobacter pylori (Helicobacter pylori): useful immunogens include, but are not limited to CagA, vacA, NAP and/or urease (WO 03/018054).

Coli (Escherichia coli): useful immunogens include, but are not limited to, immunogens derived from enterotoxigenic E.coli (ETEC), entero-aggregating E.coli (EAggEC), broadly adherent E.coli (DAEC), enteropathogenic E.coli (EPEC), exoenteropathogenic E.coli (ExPEC), and/or enterohemorrhagic E.coli (EHEC). ExpEC strains include uropathogenic E.coli (UPEC) and meningitis/sepsis associated E.coli (MNEC). UPEC immunogens are disclosed in WO2006/091517 and WO 2008/020330. Useful MNEC immunogens are disclosed in WO 2006/089264. Several E.coli types of useful immunogens are AcfD (WO 2009/104092).

Bacillus anthracis (Bacillus anthracis)

Yersinia pestis (Yersinia pestis): useful immunogens include, but are not limited to, the immunogens disclosed in WO2007/049155 and WO 2009/031043.

Staphylococcus epidermidis (Staphylococcus epidermis)

Clostridium perfringens (Clostridium perfringens) or clostridium botulinum (Clostridium botulinum)

Legionella pneumophila (Legionella pneumophila)

Bonus Kokkera (Coxiella burnetiid)

Brucella such as Brucella abortus (B.abortus), brucella canis (B.canis), brucella melitensis (B.melitensis), lin Shubu Brucella (B.neotame), brucella melitensis (B.ovis), brucella suis (B.suis), brucella finpodiaceae (B.pinnipediae).

Francisella, such as Francisella new (F.novicida), francisella mirabilis (F.philipiragaia), francisella tularensis (F.tularensis)

Neisseria gonorrhoeae (Neisseria gonorrhoeae)

Pale dense screw (Treponema pallidum)

Haemophilus ducreyi (Haemophilus ducreyi)

Enterococcus faecalis (Enterococcus faecalis) or enterococcus faecium (Enterococcus faecium)

Staphylococcus saprophyticus (Staphylococcus saprophyticus)

Yersinia enterocolitica (Yersinia enterocolitica)

Mycobacterium tuberculosis (Mycobacterium tuberculosis)

Rickettsia (Rickettsia)

Listeria monocytogenes (Listeria monocytogenes)

Vibrio cholerae (Vibrio cholerae)

Salmonella typhi (Salmonella typhi)

Borrelia burgdorferi (Borrelia burgdorferi)

Porphyromonas gingivalis (Porphyromonas gingivalis)

Klebsiella (Klebsiella)

In some embodiments, the immunogen elicits an immune response against one of these viruses:

orthomyxovirus (Orthomyxovirus): useful immunogens may be derived from influenza A, B or C viruses, such as hemagglutinin, neuraminidase or matrix M2 proteins. When the immunogen is an influenza a virus hemagglutinin, it may be from any subtype, e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16.

Paramyxoviridae virus: immunogens include, but are not limited to, immunogens derived from pneumoviruses (e.g., respiratory syncytial virus, RSV), rubella viruses (e.g., mumps virus), paramyxoviruses (e.g., parainfluenza virus), metapneumoviruses, and measles viruses (e.g., measles virus).

Poxviridae family: immunogens include, but are not limited to, immunogens derived from orthopoxviruses such as Variola vera, including, but not limited to, heavy smallpox (Variola major) and light smallpox (Variola minor).

Picornavirus: immunogens include, but are not limited to, immunogens derived from picornaviruses, such as enteroviruses, rhinoviruses, hepaciviruses, cardioviruses, and aphtha viruses. In one embodiment, the enterovirus is a poliovirus, e.g., a type 1, type 2, and/or type 3 poliovirus. In another embodiment, the enterovirus is EV71 enterovirus. In another embodiment, the enterovirus is a coxsackie a or B virus.

Bunyavirus: immunogens include, but are not limited to, immunogens derived from an Orthobunyavirus (orthobiuniya virus), such as california encephalitis virus (California encephalitis virus), an intravenous virus, such as rift valley fever virus, or an internal rovirus (Nairovirus), such as crimia-congo hemorrhagic fever virus (Crimean-Congo hemorrhagic fever virus).

Hepadnavirus: immunogens include, but are not limited to, immunogens derived from hepadnaviruses, such as Hepatitis A Virus (HAV).

Filovirus: immunogens include, but are not limited to, immunogens derived from filoviruses, such as ebola viruses (including Zaire (Zaire), kotdi (Ivory Coast), leston (Reston) or Sudan (Sudan) ebola viruses) or Marburg viruses (Marburg viruses).

Togavirus: immunogens include, but are not limited to, immunogens derived from togaviruses, such as rubella, alphaviruses, or arteriviruses. This includes rubella virus.

Flaviviruses: immunogens include, but are not limited to, immunogens derived from flaviviruses such as tick-borne encephalitis (TBE) virus, dengue (type 1, type 2, type 3, or type 4) virus, yellow fever virus, japanese encephalitis virus, kosanol forest virus (Kyasanur Forest Virus), west Nile encephalitis virus (West Nile encephalitis virus), st.Louis encephalitis virus, russian spring-summer encephalitis virus (Russian spring-summer encephalitis virus), bowanus encephalitis virus (Powassan encephalitis virus).

Pestivirus: immunogens include, but are not limited to, immunogens derived from pestiviruses such as Bovine Viral Diarrhea (BVDV), classical Swine Fever (CSFV) or Borderline Disease (BDV).

Hepadnavirus: immunogens include, but are not limited to, immunogens derived from a hepadnavirus, such as hepatitis b virus. The composition may comprise hepatitis b virus surface antigen (HBsAg).

Other hepatitis viruses: the composition may include an immunogen from hepatitis c virus, hepatitis d virus, hepatitis e virus or hepatitis g virus.

Rhabdovirus: immunogens include, but are not limited to, immunogens derived from a rhabdovirus, such as rabies virus (e.g., rabies virus) and vesicular virus (VSV).

Caliciviridae family: immunogens include, but are not limited to, immunogens derived from the caliciviridae family, such as Norwalk Virus (Norwalk Virus), and Norwalk-like viruses, such as Hawaii Virus (Hawaii Virus) and snowmountain Virus.

Novel coronaviruses: immunogens include, but are not limited to, immunogens derived from covd-19, SARS coronavirus, avian Infectious Bronchitis (IBV), mouse Hepatitis Virus (MHV), SARS, MERS, and transmissible gastroenteritis virus (TGEV). In addition, immunogens from bats and pangolin coronaviruses having pandemic potential may be used. The coronavirus immunogen may be a spike polypeptide or other viral protein. Specific novel coronavirus epitopes are fully analyzed and described in Shrock et al, science, 9, 29 of 2020, which is incorporated herein by reference.

Retroviruses: immunogens include, but are not limited to, immunogens derived from cancer viruses, lentiviruses (e.g., HIV-1 or HIV-2), or foamy viruses.

Reovirus: immunogens include, but are not limited to, immunogens derived from orthoreovirus, rotavirus, circovirus or corrovirus (colotidvirus).

Parvovirus: immunogens include, but are not limited to, immunogens derived from parvovirus B19.

Herpes virus: immunogens include, but are not limited to, immunogens derived from human herpesviruses such as, by way of example only, herpes Simplex Virus (HSV) (e.g., HSV types 1 and 2), varicella-zoster virus (VZV), epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV 6), human herpesvirus 7 (HHV 7), and human herpesvirus 8 (HHV 8).

Milk polyposis virus: immunogens include, but are not limited to, immunogens derived from papillomaviruses and polyomaviruses. The (human) papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65, e.g. from one or more of serotypes 6, 11, 16 and/or 18.

Adenovirus: immunogens include immunogens derived from serotype 36 (Ad-36).

In some embodiments, the immunogen elicits an immune response against a virus that infects fish, such as: infectious Salmon Anemia Virus (ISAV), salmon Pancreatic Disease Virus (SPDV), infectious Pancreatic Necrosis Virus (IPNV), channel Catfish Virus (CCV), fish Lymphocystis Disease Virus (FLDV), infectious Hematopoietic Necrosis Virus (IHNV), koi herpesvirus, salmon picornaviridae-like virus (also known as picornaviridae-like virus of atlantic salmon), land-sealed salmon virus (LSV), atlantic Salmon Rotavirus (ASR), trout strawberry disease virus (TSD), silver salmon tumor virus (CSTV) or Viral Hemorrhagic Septicemia Virus (VHSV).

Fungal immunogens may be derived from dermatophytes, including: epidermophyton floccosum (Epidermophy ton floccusum), microsporum nodosum (Microsporum audouini), microsporum canis (Microsporum canis), qu Xiao spore bacteria (Microsporum distortum), microsporum equi (Microsporum equinum), microsporum gypseum (Microsporum gypsu m), microsporum dwarf (Microsporum nanum), trichophyton concentric (Trichophyton co ncentricum), trichophyton equi (Trichophyton equinum), trichophyton chicken (Trichophyt on gallinae), trichophyton gypseum (Trichophyton gypseum), trichophyton gracilii (Trichophyton megnini), trichophyton mentagrophytes (Trichophyton mentagrophytes), trichophyton kunucifolium (Trichophyton quinckeanum), trichophyton rubrum (Trichophyton rub rum), trichophyton schwanani (Trichophyton schoenleini), trichophyton clinopodiocum (Trichophyt on tonsurans), trichophyton verrucosum (Trichophyton verrucosum), trichophyton verrucosum (T. Verrucosum) white variants, trichophyton discus (var. Discoides), trichophyton flavum (var. Octopus), trichophyton purple trichophyte (Trichophyton violaceum) and/or Trichophyton compactum (Trichophyton faviforme); or from aspergillus fumigatus (Aspergillus fumigatus), aspergillus flavus (Aspergillus flavus), aspergillus niger (Aspergillus niger), aspergillus nidulans (As pergillus nidulans), aspergillus terreus (Aspergillus terreus), aspergillus salsa (Aspergillu s sydowii), aspergillus flavus (Aspergillus flavatus), aspergillus glaucescens (Aspergillus glau cus), candida capitis (Blastoschizomyces capitatus), candida albicans (Ca ndida albicans), candida enolase (Candida enolase), candida tropicalis (Candi da tropicalis), candida glabrata (Candida glabra), candida krusei (Candida glabra), candida parapsilosis (Candida parapsilosis), candida stari (Candida stellatoidea), candida krusei (Candida kusei), candida parakwsei, candida vinifera (Candida lusitaniae), candida pseudotropicalis (Candida pseudotropic alis), candida gilsonii (Candida guilliermondi), cladosporium kansuis (Cladospo rium carrionii), candida cruzi (Coccidioides immitis), candida dermatitis (Blas tomyces dermatidis), cryptococcus neoformans (Cryptococcus neoformans), candida clavicla (Geotrichum clavatum), histoplasma gondii (Geotrichum clavatum), candida krusei (673), candida stella sanguinea (Geotrichum clavatum), microcosmia (sori), and microcosmia (sori) and microcosmia (i) of the genus microcosmia (i) of the intestinal region of the human (i); less common are bradiolaspp, microsporum (Microsporum sp.), microsporum (Nosema sp.), piridia (Plastophosphora sp.), plastosporium (Trapleypopop), vittafama paracoccidiosporium (Paracoccidioides brasiliensis), pythium carbowax (Pneumocystis carinii), pythium (Pythiumn insidiosum), pythium ovale (Pityrosporum ovale), saccharomyces cerevisiae (Sacharomyces cerevisae), botrytis (Saccharomyces boulardii), schizosaccharomyces (Saccharomyces pombe), cyprologosporium (Scedosporium apiosperum), drynamia (Sporothrix schenckii), leucosporium white Jib (Trichosporon beigelii), toxoplasma (Toxoplasma gondii), penicillium marneffei (Penicillium marneffei), malassezia (Malassezia sp), plastosporium (Fonssporum), wallium (Wallium sp.), mortierella (Mortierella) and Mortierella (Mortierella, mortierella (Mortierella) and Mortierella (Mortierella) are preferably used in the genera, mortierella (Mortierella) and Mortierella (Mortierella) such as Mortierella (P.sp.) or Mortierella (P.sp.) and Mortierella (P.sp.) that are more commonly used in the genus Mortierella (P.sp.) and the genus Mortierella (P.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp., the genera streptococci (monoonia spp), rhizoctonia (rhizoctonia spp), paecilomyces (Paecilomyces spp), pithomyces (pithiomyces spp) and Cladosporium (cladospora spp.).

In some embodiments, the immunogen elicits an immune response against a parasite from the genus Plasmodium (Plasmodium), such as Plasmodium falciparum (P.falciparum), plasmodium vivax (P.vivax), plasmodium malariae (P.malarial) or Plasmodium ovale (P.ovale). The invention is therefore useful for immunization against malaria. In some embodiments, the immunogen elicits an immune response against a parasite from the family of fish lice (Caligidae), particularly a parasite from the genus common scab (leptophtheirus) and the genus fish lice (Caligus genera), e.g., sea lice such as salmon scab lice (Lepeophtheirus salmonis) or roger Lei Sai irfish lice (Caligus rogercresseyi).

In some embodiments, the immunogen elicits an immune response against: pollen allergens (tree, herb, weed, and grass pollen allergens); insect or arthropod allergens (inhalants, saliva and venom allergens, e.g., mite allergens, cockroach and biting midge allergens, hymenoptera venom allergens); animal hair and dander allergens (e.g., from dogs, cats, horses, rats, mice, etc.); and food allergens (e.g., gliadin). Important pollen allergens from trees, grasses and herbs are allergens derived from taxonomic fagales (Fag ales), oleaceae (Oleales), pinales (Pinales) and Platanaceae (Platanaceae), including but not limited to birch (Betula), alnus (Alnus), hazel (Cornus), horny tree (hornbeam (Carpinus)) and olive (Oleaceae), cedar (Cryptomeria) and juniper (Juniperus), penumbus (Platanus), poales (Poales), grass including ryegrass (Lolium), timothy (phyllum), poa pratensis (Poa), bermuda (Cynodon), festuca (Dactylis), erigeron (hollus), phalaris (Phalais), rye (Secale) and Sorghum (Sorghum), asterales (Asterales) and nettle (Urticales), including herbs of the genera cut grass (Urticales), artemisia (Art emilia) and Parietaria. Other important inhalation allergens are house dust mites from Dermatophagoides and myceliophthora (Euroglyphus), storage mites such as lepidophagoides (Lepidoglyphys), sweet mite (Glycyphagus) and Tyr phagus (Tyr phagus), allergens from cockroaches, midges and fleas such as Blatella (Blatella), periplaneta (Periplaneta), chironomus (Chironomus) and Ctenocephalides (Ctenocephalides), and allergens from mammals such as cats, dogs and horses, venom allergens including allergens derived from needle punching or biting insects such as those from the order of the taxonomic hymenoptera including bees (Apidae), wasps (Vespa) and ants (Formidae).

In some embodiments, the immunogen is a tumor antigen selected from the group consisting of: (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 and RAGE, BAGE, GAGE and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6 and MAGE-12 (which antigens may be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal and bladder tumors; (b) mutant antigens, e.g., p53 (associated with various solid tumors such as colorectal cancer, lung cancer, head and neck cancer), p21/Ras (associated with e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with e.g., melanoma), MUM1 (associated with e.g., melanoma), caspase-8 (associated with e.g., head and neck cancer), CIA 0205 (associated with e.g., bladder cancer), HLA-A2-R1701, β -catenin (associated with e.g., melanoma), TCR (associated with e.g., T-cell non-hodgkin lymphoma), BCR-abl (associated with e.g., chronic myelogenous leukemia), triose phosphate isomerase, KIA 0205, CDC-27 and LDLR-FUT, (c) overexpressed antigens, e.g., galactose protein 4 (associated with e.g., colorectal cancer), galactose protein 9 (associated with e.g., hodgkin disease), protease 3 (associated with e.g., chronic myelogenous leukemia), VVT 1 (associated with e.g., various leukemias), carbonic anhydrase (associated with e.g., renal cancer), aldolase a (associated with e.g. lung cancer), PRAME (associated with e.g. melanoma), HER-2/neu (associated with e.g. breast cancer, colon cancer, lung cancer and ovarian cancer), mammaglobin, alpha-fetoprotein (associated with e.g. liver cancer), KSA (associated with e.g. colorectal cancer), gastrin (associated with e.g. pancreatic cancer and stomach cancer), telomerase catalytic protein, MUC-1 (associated with e.g. breast cancer and ovarian cancer), G-250 (associated with e.g. renal cell carcinoma), p53 (associated with e.g. breast cancer, colon cancer) and carcinoembryonic antigen (associated with e.g. breast cancer, lung cancer and gastrointestinal cancer such as colorectal cancer); (d) Shared antigens, e.g., melanoma-melanocyte differentiation antigens such as MART-1/Melan a, gp 100, MC1R, melanocyte stimulating hormone receptor, tyrosinase-related protein-1/TRP 1, and tyrosinase-related protein-2/TRP 2 (associated with e.g., melanoma); (e) Prostate-associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P2, are associated with, for example, prostate cancer; (f) Immunoglobulin idiotypes (associated with, for example, myeloma and B-cell lymphoma). In certain embodiments, tumor immunogens include, but are not limited to, p15, hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, epstein Barr virus antigen, EBNA, human Papilloma Virus (HPV) antigens including E6 and E7, B-type and C-type hepatitis virus antigen, human T-cell lymphotropic virus antigen, TSP-180, p185erbB2, p180erbB-3, C-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, nuMa, K-Ras BTA, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791Tgp72, beta-HCG, BCA225, CA 125, CA 15-3 (CA 27.29 BCA), CA 195, CA 242, CA-50, 43, CD68KP1, CO-9, ga 5, mg-35, CA 72-35, CAM-37, CAM-35, and related proteins, CAL-37, CAC-35, and the like.

Vaccine composition

The pharmaceutical compositions of the invention, particularly compositions suitable for immunization, may include one or more small molecule immunopotentiators. For example, the composition can include a TLR2 agonist (e.g., pam3CSK 4), a TLR4 agonist (e.g., aminoalkyl glucosaminide phosphate, such as E6020), a TLR7 agonist (e.g., imiquimod), a TLR8 agonist (e.g., resiquimod), and/or a TLR9 agonist (e.g., IC 31). Any such agonist desirably has a molecular weight of <2000 Da. Such agonists may be encapsulated within or through the LNP in some embodiments, along with the RNA, but in other embodiments they are not encapsulated or complexed. In some embodiments, the adjuvant is, for example: montanide ISA-51 (Seppic inc., fairfield, n.j., united States of America); QS-21 (Aquila biopharmaceuticals.inc., framingham, mass. United States of America); arlacel A; oleic acid; tetanus helper peptides such as, but not limited to QYIKANSKFIGITEL (SEQ ID NO: 2376) and/or AQYIKANSKFIGITEL (SEQ ID NO: 2377), GM-CSF, cyclophosphamide, BCG, corynebacterium minium, levamisole (levamisole), A Ji Meizong (azimezone), iprone Li Song (isoprindione), dinitrochlorobenzene (dinichlorobenazene; DNCB), keyhole Limpet Hemocyanin (KLH), freund's adjuvant (complete and incomplete), mineral gel, aluminum hydroxide (alum), lysolecithin, complexing polyols, polyanions, peptides, oil emulsions, nucleic acids such as, but not limited to, soluble chain RNA, dinitrophenol, diphtheria Toxin (DT), toll-like receptors such as, but not limited to, TLR7, TLR8 and/or 9 agonists including, but not limited to, endotoxins such as Lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), and/or polyinosine-cytidine (LTI/L), polyanion, peptide, oil emulsions, nucleic acids such as, but not limited to soluble chain RNA, dinitrophenol, diphtheria Toxin (DT), toll-like receptor (TLR) such as, but not limited to endotoxin such as Lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), and/or polyinosine-cytidine (LTR/On/LTV, on, LTV, on C, RTV, on, C, RJ, C, RTV. United States of America); IMO-2055; glucopyranosyl Lipid A (GLA); QS-21 (saponin extracted from the bark of Quillaja saponaria, also known as Quillaja saponaria or Quillaja saponaria); resiquimod (TLR 7/8 agonist); CDX-1401 (a fusion protein consisting of a fully human monoclonal antibody with specificity for the dendritic cell receptor DEC-205 linked to the NY-ESO-1 tumor antigen); juvaris cationic lipid-DNA complex; vaxfectin; and combinations thereof. In one embodiment, an adjuvant derived from heterogeneous monophosphoryl lipid a (MPL) of salmonella minnesota (Salmonella minnesota) R595 is used to induce Th-1 type immune responses to heterologous proteins in animal and human vaccines. Exemplary monophosphoryl lipid a adjuvants are shown below:

The pharmaceutical compositions of the invention may have an osmolality of about or at least 100, 150, 175, 200, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 mOsm/kg. In some embodiments, the osmolality is between about 200mOsm/kg and 400mOsm/kg, such as between about 240-360mOsm/kg, or between about 290-310 mOsm/kg.

The pharmaceutical compositions of the invention may comprise one or more preservatives, such as thimerosal or 2 phenoxyethanol. Mercury-free compositions can be produced and preservative-free vaccines can be prepared.

The composition comprises an immunologically effective amount of a lipid composition described herein (e.g., liposomes and LNP), as well as any other components as desired. An immunologically effective amount refers to an amount that is administered to an individual that is effective for treatment (e.g., a prophylactic immune response against a pathogen) in a single dose or as part of a series of doses. This amount varies depending on: the health and physical condition of the individual to be treated, the age, the taxonomic group of the individual to be treated (e.g., non-human primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, and other relevant factors. The amounts are expected to fall within a relatively broad range that can be determined via routine experimentation. The compositions of the invention are generally expressed in terms of the amount of RNA per dose. The preferred dosage has 100 μg of RNA (e.g., 10-100 μg, such as about 10 μg, 25 μg, 50 μg, 75 μg, or 100 μg), but expression can be observed at much lower levels, e.g., 1 μg/dose, 100 ng/dose, 10 ng/dose, 1 ng/dose, etc.

The invention also provides delivery devices (e.g., syringes, nebulizers, inhalers, skin patches, etc.) containing the pharmaceutical compositions of the invention. Such devices can be used to administer the composition to a vertebrate subject.

Therapeutic methods and medical uses

LNP formulated RNAs and pharmaceutical compositions described herein are used in vivo to induce an immune response against an immunogen of interest.

The present invention provides methods for inducing an immune response in a vertebrate, the methods comprising administering an effective amount of a liposome-formulated or LNP-formulated RNA or pharmaceutical composition as described herein. The immune response is preferably protective and preferably involves antibody and/or cell mediated immunity. The composition may be used for both priming and strengthening purposes. Alternatively, the prime-boost immunization schedule may be a mix of RNA and the corresponding polypeptide antigen (e.g., RNA priming, protein boosting).

The invention also provides liposomes, LNPs, or pharmaceutical compositions for inducing an immune response in a vertebrate. The invention also provides the use of a liposome, LNP or pharmaceutical composition in the manufacture of a medicament for inducing an immune response in a vertebrate.

By inducing an immune response in a vertebrate by these uses and methods, the vertebrate can be protected from various diseases and/or infections, e.g., from bacterial and/or viral diseases as discussed above. The liposomes, LNP and compositions are immunogenic, and more preferably are vaccine compositions. Vaccines according to the invention may be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection) but are typically prophylactic.

The vertebrate is preferably a mammal, such as a human or larger veterinary mammal (e.g., horse, cow, deer, goat, pig). As used herein, "larger mammal" refers to a mammal having a typical or average adult weight of at least 5kg, preferably at least 7 kg. Such larger mammals may include, for example, humans, non-human primates, dogs, pigs, cows, deer, goats, and are intended to exclude smaller mammals such as mice, rats, guinea pigs, and other rodents.

When the vaccine is for prophylactic use, the human is preferably a child (e.g., a young child or infant) or adolescent; when the vaccine is for therapeutic use, the human is preferably adolescent or adult. Vaccines intended for children may also be administered to adults, for example, to assess safety, dose, immunogenicity, and the like.

Vaccines prepared according to the present invention are useful for the treatment of both children and adults. Thus, a human patient may be less than 1 year old, less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients receiving the vaccine are elderly (e.g.,. Gtoreq.50 years, > 60 years and preferably > 65 years), young (e.g.,. Ltoreq.5 years), hospitalized patients, health care workers, armed forces and military personnel, pregnant females, chronic patients or immunodeficiency patients. However, vaccines are not only suitable for use in these groups, but may be used in the general population. The compositions of the present invention are typically administered directly to a patient. Direct delivery may be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, or injection into the interstitial space of the tissue; sublingual injection is not commonly used for immunization purposes).

The invention may be used to induce systemic and/or mucosal immunity, preferably to elicit enhanced systemic and/or mucosal immunity.

The dose may be a single dose schedule or multiple dose schedules. Multiple doses may be used for the primary and/or booster immunization schedule. In multiple dose schedules, each dose may be administered by the same or different routes, e.g., parenteral priming and mucosal boosting, mucosal priming and parenteral boosting, etc. The multiple doses are typically administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one embodiment, multiple doses may be administered about 6 weeks, 10 weeks, and 14 weeks after birth, e.g., at 6 weeks, 10 weeks, and 14 weeks of age, as commonly used in the extended immunization program ("EPI") of the world health organization. In alternative embodiments, two primary doses are administered about two months apart, e.g., about 7, 8, or 9 weeks apart, followed by about 6 months to 1 year after the second primary dose, e.g., about 6, 8, 10, or 12 months after the second primary dose, with one or more booster doses. In another embodiment, three primary doses are administered about two months apart, e.g., about 7, 8, or 9 weeks apart, followed by about 6 months to 1 year after the third primary dose, e.g., about 6, 8, 10, or 12 months after the third primary dose, with one or more booster doses.

Examples

The following examples provide exemplary embodiments. In view of this disclosure and the level of ordinary skill in the art, those skilled in the art will recognize that the following embodiments are intended to be exemplary only and that many variations, modifications, and alterations may be used without departing from the scope of the presently disclosed subject matter. The data and disclosure of each of examples 1A-33E correspond to FIGS. 1A-33E.

Example 1 increasing lipid concentration from 6mM to 27mM and mRNA from 0.14mg/ml to 0.56mg/ml increased LNP delivery efficiency in vitro (study TRANS-10).

Overview: LNP was formulated using a total lipid concentration of 6 to 27mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and then serially diluted to reach 6 to 27mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 1 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.14 to 0.56mg/ml in 25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 200ng and 12k hek293 cells were transfected at the same 200ng dose but made at different concentrations in a microfluidic mixer. Increasing the mixing concentration resulted in an increase in delivery efficiency at the same dose, indicating that the concentration during mixing affects LNP structure and thus delivery efficiency. This finding is significant for commercial manufacture of mRNA vaccines.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 2: increasing lipid concentration from 6mM to 27mM and mRNA from 0.14mg/ml to 0.56mg/ml increased LNP delivery efficiency in vitro (study TRANS-12).

Overview: LNP was formulated using a total lipid concentration of 6 to 27mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and then serially diluted to reach 6 to 27mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 1 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.14 to 0.56mg/ml in 25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 200ng and 12k hek293 cells were transfected at the same 200ng dose but made at different concentrations in a microfluidic mixer. Increasing the mixing concentration resulted in an increase in delivery efficiency at the same dose, indicating that the concentration during mixing affects LNP structure and thus delivery efficiency. This finding is significant for commercial manufacture of mRNA vaccines.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 3: increasing lipid concentration from 3mM to 27mM and mRNA from 0.07mg/ml to 0.56mg/ml increased LNP delivery efficiency in vitro (study TRANS-14).

Overview: LNP was formulated using a total lipid concentration of 3 to 27mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and then serially diluted to 3 to 27mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 1 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.07 to 0.56mg/ml in 25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 200ng and 12k hek293 cells were transfected at the same 50-200ng dose but made at different concentrations in a microfluidic mixer. Increasing the mixing concentration resulted in an increase in delivery efficiency at the same dose, indicating that the concentration during mixing affects LNP structure and thus delivery efficiency. This finding is significant for commercial manufacture of mRNA vaccines.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: toxicity assay based on prest Blue HS viability reagent. After 24 hours transfection, the transfected cells were incubated with pre-warmed prest Blue HS reagent (10% v/v) for 15 minutes at 37 ℃. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 540/Em 590).

Example 4: increasing lipid concentration from 12.5mM to 50mM and mRNA from 0.25mg/ml to 1mg/ml increased LNP delivery efficiency in vitro (study TRANS-16).

Overview: LNP was formulated using a total lipid concentration of 12.5 to 50mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and then serially diluted to reach 12.5 to 50mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 3.6 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.25 to 1mg/ml in 25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 200ng and 12k hek293 cells were transfected at the same 50-200ng dose but made at different concentrations in a microfluidic mixer. Increasing the mixing concentration resulted in an increase in delivery efficiency at the same dose, indicating that the concentration during mixing affects LNP structure and thus delivery efficiency. This finding is significant for commercial manufacture of mRNA vaccines.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: toxicity assay based on prest Blue HS viability reagent. After 24 hours transfection, the transfected cells were incubated with pre-warmed prest Blue HS reagent (10% v/v) for 15 minutes at 37 ℃. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 540/Em 590).

Example 5: increasing lipid concentration from 50mM to 100mM and mRNA from 1mg/ml to 2mg/ml increased LNP delivery efficiency (study LNP-14 part II).

Overview: LNP was formulated using a total lipid concentration of 50 to 100mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively). To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 3.6 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 1 to 2mg/ml in 50mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour.

A: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 6: increasing lipid concentration from 50mM to 100mM and mRNA from 1mg/ml to 2mg/ml increased LNP delivery efficiency in vitro (study TRANS-25).

Overview: LNP was formulated using a total lipid concentration of 50 to 100mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively). To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 3.6 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 1 to 2mg/ml in 50mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 200ng and 12k hek293 cells were transfected at the same 25-200ng dose but made at different concentrations in a microfluidic mixer. Increasing the mixing concentration resulted in an increase in delivery efficiency at the same dose, indicating that the concentration during mixing affects LNP structure and thus delivery efficiency. This finding is significant for commercial manufacture of mRNA vaccines.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: and (5) pH measurement. Measurements were made before and after 4 hours of dialysis against 1x DPBS ph 7.4.

Sample of	pH before dialysis	pH after dialysis
			1mg/ml	6.05	7.38
1.5mg/ml	6.43	7.32
			2mg/ml	6.5	7.29

Example 7: increasing lipid concentration from 50mM to 100mM and mRNA from 1mg/ml to 2mg/ml increased LNP delivery efficiency in vitro (study TRANS-26).

Overview: LNP was formulated using a total lipid concentration of 50 to 100mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 100mM (25/5/19.25/0.75 mM, respectively). To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 3.6 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 1 to 2mg/ml in 100mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 200ng and 12k hek293 cells were transfected at the same 25-200ng dose but made at different concentrations in a microfluidic mixer. Increasing the mixing concentration resulted in an increase in delivery efficiency at the same dose, indicating that the concentration during mixing affects LNP structure and thus delivery efficiency. This finding is significant for commercial manufacture of mRNA vaccines.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: and (5) pH measurement. Measurements were made before and after 4 hours of dialysis against 1x DPBS ph 7.4. Note herein that these LNPs were formulated using 100mM NaOAc compared to example 6. Increasing the concentration of sodium acetate buffer in the formulation keeps the pH lower due to the higher buffering capacity, thereby producing a lower pH prior to LNP dialysis.

Example 8: increasing lipid concentration from 2.5mM to 100mM and mRNA from 0.05mg/ml to 2mg/ml increased LNP delivery efficiency in vitro (study TRANS-27).

Overview: LNP was formulated using a total lipid concentration of 2.5 to 100mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and then serially diluted to reach 2.5 to 50mM. To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 3.6 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration, reaching 0.05 to 2mg/ml in 50mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and discharged into 48. Mu.l of 1XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same 200ng dose but made at different concentrations in a microfluidic mixer. Increasing the mixing concentration resulted in an increase in delivery efficiency at the same dose, indicating that the concentration during mixing affects LNP structure and thus delivery efficiency. This finding is significant for commercial manufacture of mRNA vaccines.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 min and immediately introduced into a Cytation 5 cell imaging multimode reader (Biotek) for readingFluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: toxicity assay based on prest Blue HS viability reagent. After 24 hours transfection, the transfected cells were incubated with pre-warmed prest Blue HS reagent (10% v/v) for 15 minutes at 37 ℃. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 540/Em 590).

E: and (5) pH measurement. Measurements were made before and after 4 hours of dialysis against 1x DPBS ph 7.4.

Sample of	pH before dialysis	pH after dialysis
			0.05mg/ml	6.09	7.31
0.1mg/ml	6.12	7.35
			0.25mg/ml	6.44	7.33
0.5mg/ml	6.53	7.4
			1mg/ml	6.65	7.42
1.5mg/ml	6.67	7.41
			2mg/ml	6.08	7.35

Example 9: increasing lipid concentration from 5mM to 100mM and mRNA from 0.1mg/ml to 2mg/ml increased LNP delivery efficiency in vivo (study in vivo-3).

Overview: LNP was formulated using a total lipid concentration of 5 to 100mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and then serially diluted to reach 5 to 50mM. To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 3.6 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.1 to 2mg/ml in 50mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. 24 μl of lipid mixture and 48 μl of mRNA solution were mixed on set 5 and drained into 72 μl of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 144. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was concentrated to expel 5ug of encapsulated mRNA in 50ul for intramuscular administration. Increasing the mixing concentration resulted in an increase in delivery efficiency at the same dose, indicating that the concentration during mixing affects LNP structure and thus delivery efficiency. This finding is significant for commercial manufacture of mRNA vaccines.

A: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

C: firefly luciferase expression in vivo in IM administration. 5ug of encapsulated mRNA was injected into mice in intramuscular (I.M.), intradermal (I.D.), and intravenous (I.V.) injections. The ROI was calculated using the IVIS system. Imaging was performed at 4 and 20 hours.

Example 10: direct protonation of the ionizable lipids and mixing with mRNA in water allows mRNA to be encapsulated. Increasing the concentration of sodium acetate (NaOAc) buffer at pH4 increased mRNA encapsulation (study LNP-6 part II).

Overview: LNP was formulated using a total lipid concentration of 27.74mM containing MC 3/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The MC3 stock was protonated at 0%, 50% and 100% total amine using 1M HCl by counting the moles of amine on the ionizable lipid and adding 0%, 50%, 100% lipid to the number of moles of HCl. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively). Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 1 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock reached 0.56mg/ml in water, 5, 10, 25 and 50mM sodium acetate buffer pH4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. The results show that direct protonation of the ionizable lipid using HCl mixed with mRNA in water without the use of buffer results in a similar mRNA encapsulation level as when mixed in sodium acetate buffer at pH 4. In addition, a concentration of 25mM sodium acetate was required to maximize mRNA encapsulation when mixed in sodium acetate buffer at pH 4. This maximum encapsulation criterion was used in all prior art to determine buffer concentration and mixing conditions. The present invention shows that this procedure does not maximize LNP efficacy because buffer concentration needs to be optimized to maximize LNP efficacy in addition to absolute mix concentration.

A: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

C: and (5) pH measurement. Measurements were made prior to dialysis.

Sample of	pH before dialysis
		MC3 100% protonation in Water 0.56mg/ml	7.16
MC3 50% protonation in Water 0.56mg/ml	7.35
		MC3 0% protonation in Water 0.56mg/ml	7.5
MC 3.56 mg/ml in 5mM NaOAc	7.34
		MC 3.56 mg/ml in 10mM NaOAc	7.21
MC 3.56 mg/ml in 25mM NaOAc	6.84
		MC 3.56 mg/ml in 50mM NaOAc	6.29

Example 11: increasing lipid concentration from 50mM to 150mM and mRNA from 1mg/ml to 3mg/ml increases LNP delivery efficiency at lower sodium acetate concentrations. Increasing sodium acetate concentrations above 50mM reduced LNP efficacy at all concentrations in vitro (study TRANS-32).

Overview: LNP was formulated using a total lipid concentration of 50 to 120mM comprising KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively). To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. To prepare a total lipid concentration of 150mM (75/15/57.75/2.25 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 150mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 4.4 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 1 to 3mg/ml in 50, 100 and 150mM sodium acetate (NaOAc) buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer. Increasing the mixed concentration only at the lowest NaOAc concentration of 50mM resulted in an increase in delivery efficiency at the same dose. Higher NaOAc concentrations reduce LNP efficacy and even more so at higher mixed concentrations.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard points in the mg/ml range are includedControl positive enzyme activity in microwell plates (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: and (5) pH measurement. Measurements were made before and after 4 hours of dialysis against 1x DPBS ph 7.4.

Example 12: increasing the lipid concentration from 12.5mM to 75mM and the mRNA from 0.25mg/ml to 1.5mg/ml increases the LNP delivery efficiency of the ionizable lipids KC2, MC3 and BOD-ADDE-C2/C4-PipZ in vitro. Decreasing sodium acetate concentration increases LNP efficacy while decreasing mRNA encapsulation (study TRANS-33).

Overview: LNP was formulated using a total lipid concentration of 12.5 and 75mM containing several ionisables (KC 2/MC3/DL-ADDE-C2C 2-PipZ/BOD-ADDE-C2C 4-PipZ)/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and serial dilutions were then performed to reach 12.5mM. To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2/MC3/DL-ADDE-C2C 2-PipZ/BOD-ADDE-C2C 4-PipZ and cholesterol were first mixed and the same was true for DSPC and PEG to dissolve the two solutions in ethanol and then combined to achieve 75mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 4.4 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.25 to 1.5mg/ml in 25, 43 and 60mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and discharged into well 1 (W1 in the particle size diagram) containing 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH7.4 (D in the particle size diagram). LNP was then dialyzed against 1x DPBS pH7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer. KC2, MC3 and BOD-ADDE-C2C 4-PipZ ionizable lipids demonstrated a greater increase in potency at the higher concentration of 1.5mg/ml compared to the lower concentration of 0.25mg/ml, whereas DL-ADDE-C2C2-PipZ did not have the effect described above. The latter lipid has a much higher pKa (about 7.5) in LNP than the first 3 lipids (about 6.5) so that it is more highly protonated during mixing, possibly indicating that ideal mixing conditions including pH and protonation are not achieved for this lipid. KC2, MC3 and BOD-ADDE-C2C 4-PipZ LNP also increase in potency and may decrease mRNA encapsulation when sodium acetate concentration is reduced from 60mM to 25mM, while DL-ADDE-C2C2-PipZ does not show either effect.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: and (5) pH measurement. Measurements were made before (well 1) and after 4 hours of dialysis against 1x DPBS ph 7.4.

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Example 13: increasing lipid concentration from 12.5mM to 75mM and mRNA from 0.25mg/ml to 1.5mg/ml increased LNP delivery efficiency in vitro. Further optimization of sodium acetate concentration at any particular mixed concentration resulted in a further increase in LNP efficacy. (study TRANS-34).

Overview: LNP was formulated using a total lipid concentration of 12.5 to 75mM comprising several ionisable species KC2/DL-ADDE-C2C 2-PipZ/BOD-ADDE-C2C 4-PipZ)/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and serial dilutions were then performed to reach 12.5mM. To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2/DL-ADDE-C2C 2-PipZ/BODA-ADDE-C2C 4-PipZ and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 4.4 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.25 to 1.5mg/ml in 5, 10, 12.5, 25 and 50mM sodium acetate buffer pH 4, 5 or 6, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1XDPBS pH 7.4. LNP was then dialyzed against 1xdpbs pH7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer. LNP efficacy increases at higher mixed concentrations compared to lower mixed concentrations, but can be further increased at each concentration by adjusting the sodium acetate concentration. For example, at 1.5mg/ml mRNA, LNP efficacy increases by a further 44% when sodium acetate is reduced from 50mM to 25 mM. Decreasing sodium acetate concentration from 25mM to 10mM increases efficacy by 2.2X at an mRNA concentration of 0.25 mg/ml. The increase in the mixing concentration and the optimized decrease in the buffer concentration result in an encapsulation efficiency of about 70% lower than that typically obtained in the prior art, wherein encapsulation is erroneously maximized by decreasing the mixing concentration and increasing the buffer concentration. We have also found that increasing pH from 4 to 5 increases efficacy for BOD-ADDE-C2C 4-PipZ. For reasons explained in example 12, DL-ADDE-C2C2-PipZ acts differently.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: and (5) pH measurement. Measurements were made before (well 1) and after 4 hours of dialysis against 1x DPBS ph 7.4.

Example 14: increasing lipid concentration from 5mM to 150mM and mRNA from 0.1mg/ml to 3mg/ml increased LNP delivery efficiency in vitro. Optimizing sodium acetate concentration at any particular mixed concentration resulted in a further increase in LNP potency (TRANS-35)

Overview: LNP was formulated using a total lipid concentration of 50 to 150mM comprising ionizable KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and serial dilutions were then performed to achieve 5mM. To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. To prepare a total lipid concentration of 150mM (75/15/57.75/2.25 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 150mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 4.4 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.1 to 3mg/ml in 10, 20, 25 and 37.5mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH7.4 for 4x1 hour. Finally LNP was diluted so that 32ul contained 25-200ng and 12KHEK293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer. LNP efficacy increases at higher mixed concentrations compared to lower mixed concentrations. Each mixed concentration efficacy was optimized by adjusting the sodium acetate concentration. For example, at 1.5mg/ml mRNA, LNP efficacy increases by a further 44% when sodium acetate is reduced from 50mM to 25 mM. Decreasing sodium acetate concentration from 25mM to 10mM increases efficacy by 2.2X at an mRNA concentration of 0.25 mg/ml.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard points in the mg/ml range are included in the microControl positive enzyme activity in well plates (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 15: increasing lipid concentration from 5mM to 150mM and mRNA from 0.1mg/ml to 3mg/ml increased LNP delivery efficiency in vitro. Optimizing the sodium acetate concentration at any particular mixed concentration results in a further increase in LNP efficacy.

(TRANS-34/35)

Overview: LNP was formulated using a total lipid concentration of 50 to 150mM comprising ionizable KC 2/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and serial dilutions were then performed to achieve 5mM. To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. To prepare a total lipid concentration of 150mM (75/15/57.75/2.25 mM, respectively), KC2 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 150mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 4.4 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.1 to 3mg/ml in 10, 20, 25 and 37.5mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. Finally LNP was diluted so that 32ul contained 25-200ng and 12KHEK293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer. LNP efficacy increases at higher mixed concentrations compared to lower mixed concentrations. Each mixed concentration efficacy was optimized by adjusting the sodium acetate concentration. For example, at 1.5mg/ml mRNA, LNP efficacy increases by a further 44% when sodium acetate is reduced from 50mM to 25 mM. Decreasing sodium acetate concentration from 25mM to 10mM increases efficacy by 2.2X at an mRNA concentration of 0.25 mg/ml.

A. Optimum concentration of sodium acetate at pH 4 favoring highest efficacy at each mRNA concentration

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

C: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

D: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 16: increasing lipid concentration from 5mM to 100mM and mRNA from 0.1mg/ml to 2mg/ml increased LNP delivery efficiency in vitro. Optimizing the sodium acetate concentration at any particular mixed concentration results in a further increase in LNP efficacy.

(TRANS-36)

Overview: LNP was formulated using a total lipid concentration of 5 to 100mM comprising ionizable MC 3/DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (25/5/19.25/0.75 mM, respectively) and serial dilutions were then performed to achieve 5mM. To prepare a total lipid concentration of 75mM (37.5/7.5/28.88/1.13 mM, respectively), MC3 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (50/10/38.5/1.5 mM, respectively), MC3 and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 4.6 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.1 to 2mg/ml in 5-35mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. Finally LNP was diluted so that 32ul contained 25-200ng and 12KHEK293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer. LNP efficacy increases at higher mixed concentrations compared to lower mixed concentrations. Each mixed concentration efficacy was optimized by adjusting the sodium acetate concentration.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 min and immediately introduced into a Cytation 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 17: increasing lipid concentration from 5mM to 100mM and mRNA from 0.1mg/ml to 2mg/ml increased LNP delivery efficiency in vitro. Optimizing the sodium acetate concentration at any particular mixed concentration results in a further increase in LNP efficacy.

(TRANS-37)

Overview: LNP was formulated using a total lipid concentration of 5 to 100mM containing ionizable BOD-C2/C4-PipZ/DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 50mM (24/6.5/18.5/1 mM, respectively) and serial dilutions were then performed to achieve 5mM. To prepare a total lipid concentration of 75mM (36/9.75/27.75/1.5 mM, respectively), the ionisable species and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 75mM. To prepare a total lipid concentration of 100mM (48/13/37/2 mM, respectively), the ionisable and cholesterol were first mixed, and the same was true for DSPC and PEG, to dissolve the two solutions in ethanol, and then combined to achieve 100mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.1 to 2mg/ml in 5-30mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. Finally LNP was diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer. LNP efficacy increases at higher mixed concentrations compared to lower mixed concentrations. Each mixed concentration efficacy was optimized by adjusting the sodium acetate concentration.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, 30 minutes before firefly luciferase assay, willTransfected cells were conditioned to room temperature. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 18: increasing lipid concentration from 12.5mM to 75mM and mRNA from 0.25mg/ml to 1.5mg/ml increased LNP delivery efficiency in vitro. Optimizing the sodium acetate concentration at any particular mixed concentration results in a further increase in LNP efficacy.

(TRANS-41)

Overview: LNP was formulated using a total lipid concentration of 12.5-75mM comprising several ionisables/DSPC/cholesterol/PEG-DMG (47-50:10-13:37-38.5:1.5-2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 75mM, and then serial dilutions were performed to reach 12.5mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.25-1.5mg/ml in 15-25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant fluorescenceThe luciferase standard curve was prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 19: increasing lipid concentration from 12.5mM to 75mM and mRNA from 0.25mg/ml to 1.5mg/ml increased LNP delivery efficiency in vitro. Optimizing the sodium acetate concentration at any particular mixed concentration results in a further increase in LNP efficacy.

(TRANS-43)

Overview: LNP was formulated using a total lipid concentration of 12.5-75mM containing several ionisables/DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 75mM, and then serial dilutions were performed to reach 12.5mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.25-1.5mg/ml in 15mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12KHEK293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM.3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain 10 ⁷ Linearity of RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 20: increasing lipid concentration from 10mM to 75mM and mRNA from 0.2mg/ml to 1.5mg/ml increased LNP delivery efficiency in vivo. Optimizing the sodium acetate concentration at any particular mixed concentration results in a further increase in LNP efficacy. (in vivo-7)

Overview: LNP was formulated using a total lipid concentration of 10-75mM comprising several ionisables/DSPC/cholesterol/PEG-DMG (47-50:10-13:37-38.5:1.5-2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 75mM, and then serial dilutions were performed to reach 10mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.2-1.5mg/ml in 15-25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

A: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

C: firefly luciferase expression in vivo in IM administration. 0.5-5ug total mRNA was injected into mice as an intramuscular (i.m.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 and 24 hours. The ROI was calculated using the IVIS system.

Example 21: increasing lipid concentration from 10mM to 75mM and mRNA from 0.2mg/ml to 1.5mg/ml increased immunogenicity upon delivery of SARS-CoV-2 immunogen (in vivo 8)

Overview: LNP was formulated using a total lipid concentration of 10-75mM comprising several ionisables/DSPC/cholesterol/PEG-DMG (47-50:10-13:37-38.5:1.5-2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 75mM, and then serial dilutions were performed to reach 10mM. Codon optimized 2019-nCoV S-2P (Covid) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.2-1.5mg/ml in 15-25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

A: in vivo immunogenicity endpoint ELISA anti-RBD titers. 0.1, 0.25, 0.5, 1ug total mRNA was injected into mice as an intramuscular (i.m.) injection. Pre-reinforcement and post-reinforcement (after 3 weeks) are shown below.

B: in vivo immunogenicity FRNT50 titers determined by pseudo-neutralization. 0.1, 0.25, 0.5, 1ug total mRNA was injected into mice as an intramuscular (i.m.) injection. Pre-reinforcement and post-reinforcement (after 3 weeks) are shown below.

Example 22: in the challenge model, upon delivery of SARS-CoV-2 immunogen (9 in vivo), increasing lipid concentration from 10mM to 75mM and mRNA from 0.2mg/ml to 1.5mg/ml increased protection against lethal challenge, with 0.25ug of proprietary BODD C2C4 PipZ being 100% protective and with 0.5ug of MC3 standard reference being 100% protective.

Overview: LNP was formulated using a total lipid concentration of 10-75mM comprising several ionisables/DSPC/cholesterol/PEG-DMG (47-50:10-13:37-38.5:1.5-2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 75mM, and then serial dilutions were performed to reach 10mM. Codon optimized 2019-nCoV S-2P (Covid) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.2-1.5mg/ml in 15-25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

A: in vivo protection against viral challenge in challenge model—survival ratio, body weight and temperature. 0.1, 0.25, 0.5, 1ug total mRNA was injected into mice with pre-and post-boost (after 5 weeks) of intramuscular (i.m.) injection. Excitation using 5x 10 ⁴ Italian strain of PFU.

B: body weight and temperature in the model were stimulated. 0.1, 0.25, 0.5, 1ug total mRNA was injected into mice with pre-and post-boost (after 5 weeks) of intramuscular (i.m.) injection. Excitation using 5x 10 ⁴ Italian strain of PFU.

Example 23: high concentrations of lipid (77 mM) and mRNA (1.5 mg/ml) resulted in high LNP delivery efficiency in vivo. (in vivo-10)

Overview: LNP was formulated using a total lipid concentration of 77mM comprising several ionisables/DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a 77mM total lipid concentration. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration, reaching 1.5mg/ml in 15mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

A: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

B: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

C: firefly luciferase expression in vivo in IM and IV administration. 1ug of total mRNA was injected into mice with intramuscular (i.m.) and intravenous (i.v.) injections. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 and 24 hours. The ROI was calculated using the IVIS system.

Example 24: for rapid microfluidic mixing, increasing lipid concentration from about 16mM to about 120mM and mRNA from 0.2mg/ml to 1.5mg/ml increased LNP delivery efficiency in vitro. PEG-DMG was tested in comparison to PEG-DMA. (TRANS-47)

Overview: LNP was formulated using a total lipid concentration of about 12 to about 120mM containing ALC-0315 ionizable/DSPC/cholesterol/PEG-DMG (46:9.4:42.9:1.7 mol%) and each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of about 120 mM. Different stock solutions up to a lower total lipid concentration of about 16mM were prepared independently. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.2-1.5mg/ml in 25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and discharged into 48. Mu.l of 1XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 6x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 25: for rapid microfluidic mixing, increasing lipid concentration from 10.2mM to 77mM and mRNA from 0.2mg/ml to 1.5mg/ml increased LNP delivery efficiency in vitro. (TRANS-49)

Overview: LNP was formulated using a total lipid concentration of 10.2-77mM comprising several ionizable lipids and DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of 77mM, and then serial dilutions were performed to reach 10.2mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 0.2-1.5mg/ml in 15mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12KHEK293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 1.02cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 26: for rapid microfluidic mixing, high concentrations of lipid (77 mM) and mRNA (1.5 mg/ml) resulted in higher LNP delivery efficiency in vitro. (TRANS-52)

Overview: LNP was formulated using a total lipid concentration of 77mM comprising several ionisables and DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a 77mM total lipid concentration. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration, reaching 1.5mg/ml in 15mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 0.88cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 27: for rapid microfluidic mixing, increasing lipid concentration from about 10mM to about 77mM and mRNA from 0.2mg/ml to 1.5mg/ml showed LNP delivery efficiency at different sodium acetate concentrations in vitro. Directly protonating the ionizable lipid prior to mixing with mRNA in water improves the delivery efficiency in IM and IV injections in vivo, without any buffer. (in vivo-12v.2)

Overview: BOD C2/C4 PipZ (C24 PipZ) LNP was formulated using a total lipid concentration of 77mM with DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). The double lipid stock (ionizable/cholesterol, DSPC/PEG-DMG) was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a 77mM total lipid concentration and diluted to about 10mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 1.5mg/ml in 15-50mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Also, mRNA was diluted in water and the lipid mixture was protonated at different levels of 200% -25% to be mixed later under the same conditions as described above. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against sucrose buffer pH 7.5 for 6x1 hour. LNP was kept frozen at-80C for in vivo injection. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in sucrose buffer pH 7.5 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, in Zetasizer Nano ZS (Malvern Panalytical), viscosity of 1.1cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: in vivo and ex vivo firefly luciferase expression with IM administration of 1.5mg/ml mixed LNP. 1ug of total mRNA was injected into mice by intramuscular (I.M.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system. For ex vivo, organs are extracted and imaged immediately after in vivo imaging.

E in vivo and ex vivo firefly luciferase expression administered at an IV of 1.5mg/ml mixed LNP. 1ug of total mRNA was injected into mice by intravascular (I.V.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system. For ex vivo, organs are extracted and imaged immediately after in vivo imaging.

Example 28: for rapid microfluidic mixing, LNP/18PA and 1.5mg/ml mRNA mixed at 74mM total lipid showed reduced liver expression and increased spleen expression after IV injection. (TRANS-55)

Overview: MC3 was formulated using a total lipid concentration of 74mM with DSPC/cholesterol/PEG-DMG (50:10:38.5:1.5 mol%). The double lipid stock (ionizable/cholesterol, DSPC/PEG-DMG) was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a 77mM total lipid concentration. The lipid mixture was mixed with 18PA lipid to 0%, 15% and 30% total lipid. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration, reaching 1.5mg/ml in 25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of lipid mixture and 32. Mu.l of lmRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against sucrose buffer pH 7.5 for 6x1 hour. LNP was kept frozen at-80C for in vivo injection. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. However, the presence of the anion 18PA may increase the background signal. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in sucrose buffer pH 7.5 and transferred to a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in sucrose at 25 ℃, viscosity of 1.1cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: in vivo and ex vivo firefly luciferase expression with IM administration of 1.5mg/ml mixed LNP. 1ug of total mRNA was injected into mice by intramuscular (I.M.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system. For ex vivo, organs are extracted and imaged immediately after in vivo imaging.

E in vivo and ex vivo firefly luciferase expression administered at an IV of 1.5mg/ml mixed LNP. 1ug of total mRNA was injected into mice by intravascular (I.V.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system. For ex vivo, organs are extracted and imaged immediately after in vivo imaging.

Example 29: for rapid microfluidic mixing, approximately 25mM of the new lipid mixed with 0.5mg/ml mRNA showed higher LNP delivery efficiency in vitro. (TRANS-59)

Overview: LNP was formulated using a total lipid concentration of about 25mM containing several ionisables/DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). Each lipid was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a total lipid concentration of about 25 mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration, reaching 0.5mg/ml in 25mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 4x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in sucrose buffer pH 7.5 and transferred to a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in sucrose at 25 ℃, viscosity of 1.1cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 30: for rapid microfluidic mixing, LNP with a total lipid concentration of 77mM was assembled with mRNA at a concentration of 1.5mg/ml to demonstrate LNP delivery efficiency in vivo. (in vivo-11)

Overview: LNP was formulated using a total lipid concentration of 77mM comprising several ionizable lipids/DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). The double lipid stock (ionizable/cholesterol, DSPC/PEG-DMG) was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a 77mM total lipid concentration. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration, reaching 1.5mg/ml in 20mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 6x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12KHEK293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

A: in vivo firefly luciferase expression at the injection site in IM administration. 1ug of total mRNA was injected into mice by intramuscular (I.M.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at the injection site at 4 hours. The ROI was calculated using the IVIS system.

B: ex vivo firefly luciferase expression in IM administration. 1ug of total mRNA was injected into mice by intramuscular (I.M.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system. For ex vivo, organs are extracted and imaged immediately after in vivo imaging.

Example 31: for rapid microfluidic mixing, LNP with a total lipid concentration of 77mM was assembled with mRNA at a concentration of 1.5mg/ml to demonstrate LNP delivery efficiency in vivo. (in vivo-13)

Overview: LNP was formulated using a total lipid concentration of 77mM comprising several ionizable lipids/DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). The double lipid stock (ionizable/cholesterol, DSPC/PEG-DMG) was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a 77mM total lipid concentration. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration, reaching 1.5mg/ml in 15mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against sucrose buffer pH 7.5 for 6x1 hour. Frozen LNP was maintained at-80C for in vivo injection. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

A: in vivo firefly luciferase expression at the injection site in IM administration. 1ug of total mRNA was injected into mice by intramuscular (I.M.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system.

B: ex vivo firefly luciferase expression in IM administration. 1ug of total mRNA was injected into mice by intramuscular (I.M.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system. For ex vivo, organs are extracted and imaged immediately after in vivo imaging.

Example 32: in rapid microfluidic mixing, lipid concentrations of about 77mM and mRNA of 1.5mg/ml showed LNP delivery efficiency in vitro at different sodium acetate concentrations. In the absence of any buffer, direct protonation of the ionizable lipid prior to mixing with mRNA in water results in improved in vitro delivery efficiency (TRANS-54 v.1)

Overview: BOD C2/C4 PipZ (C24 PipZ) LNP was formulated using a total lipid concentration of 77mM with DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). The double lipid stock (ionizable/cholesterol, DSPC/PEG-DMG) was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a 77mM total lipid concentration. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 1.5mg/ml in 15-50mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. In addition, mRNA was diluted in water and the lipid mixture was protonated at different levels of 200% -25% to be mixed later under the same conditions as described above. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and discharged into 48. Mu.l of 1XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1XDPBS pH 7.4. LNP was then dialyzed against 1x DPBS pH 7.4 for 6x1 hour. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 3.9x10 ^-5 mg/ml to 4.88x10 ^-3 50ul of standard spots in the mg/ml range were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in 1X DPBS pH 7.4 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) in Zetasizer Nano ZS (Malvern Panalytical) with particle RI of 1.45 and absorption of 0.001 in 1X PBS at 25 ℃, viscosity of 0.88cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

Example 33: for rapid microfluidic mixing, increasing lipid concentration from about 10mM to about 77mM and mRNA from 0.2mg/ml to 1.5mg/ml showed LNP delivery efficiency at different sodium acetate concentrations in vitro. In the absence of any buffer, directly protonating the ionizable lipid prior to mixing with mRNA in water results in improved delivery efficiency in vivo IM and IV injection. (TRANS-54 v.2/in vivo-12 v.1)

Overview: BOD C2/C4 PipZ (C24 PipZ) LNP was formulated using a total lipid concentration of 77mM with DSPC/cholesterol/PEG-DMG (47:13:37:2 mol%). The double lipid stock (ionizable/cholesterol, DSPC/PEG-DMG) was dissolved in ethanol until a clear solution was observed. The four lipids were combined to obtain a 77mM total lipid concentration and diluted to about 10mM. Codon optimized firefly luciferase (Fluc) sequences were cloned into mRNA plasmids (optimized 3 'and 5' utrs and containing 101polyA tails) for co-transcriptional capping, transcribed in vitro using N1 methyl pseudouridine modified nucleosides and cellulose purified to remove dsRNA. Purified mRNA was ethanol precipitated, washed and resuspended in nuclease free water to a concentration of 5.5 mg/mL. The NP ratio was kept constant at 4, fluc mRNA stock was diluted in serial dilutions from higher to lower concentration solutions, reaching 1.5mg/ml in 15-50mM sodium acetate buffer pH 4, then mixed in Spark NanoAssmblr (Precision NanoSystems), which allowed for higher reproducibility in formulations using microfluidic mixing techniques. In addition, mRNA was diluted in water and the lipid mixture was protonated at different levels of 200% -25% to be mixed later under the same conditions as described above. Mu.l of the lipid mixture and 32. Mu.l of the mRNA solution were mixed on setting 3 and drained into 48. Mu.l of 1 XDPBS pH 7.4. The LNP formed was then diluted into an additional 96. Mu.l of 1 XDPBS pH 7.4. LNP was then dialyzed against sucrose buffer pH7.5 for 6x1 hour. LNP was kept frozen at-80C for in vivo injection. LNP was then diluted so that 32ul contained 25-200ng and 12k hek293 cells were transfected at the same dose but made at different concentrations in a microfluidic mixer.

Firefly luciferase assay of mrna delivery efficiency. After 24 hours transfection, the transfected cells were conditioned to room temperature 30 minutes prior to firefly luciferase assay. Quantilum recombinant luciferase standard curves were prepared in 5-fold serial dilutions in 10% EMEM. 50ul of standard spots ranging from 3.9X10-5 mg/ml to 4.88X10-3 mg/ml were included in the microwell plates as positive enzyme activity controls (data not shown) to maintain linearity of 107 RLU/mg/ml. ONE-Glo substrates previously conditioned to room temperature for at least 4 hours were added to each untransfected, transfected and Quantilum well at a 1:1 ratio. Assay plates were incubated in the dark for 3 minutes and immediately introduced into a station 5 cell imaging multimode reader (Biotek) to read fluorescence.

B: ribogreen assay of mRNA encapsulation efficiency. 1 XTE buffer and Triton buffer (2% v/v in 1 XTE buffer) were added in duplicate to the black microwell plates of each LNP. LNP was diluted to 4ng/ul in 1 XDPBS pH 7.4 and added to each TE/Triton well at a 1:1 ratio. Two standard curves were included in the Ribogreen assay, one containing mRNA and 1X TE buffer and the other containing mRNA and Triton buffer. Each of these standard curves was used to calculate mRNA concentrations in the respective TE buffer or Triton buffer. This method of using two standard curves more accurately calculates encapsulation efficiency and mRNA concentration than a single standard curve. After the diluted LNP was added to the plate, standards were included in the microplate. Microplates were incubated at 37 ℃ for 10 minutes to extract LNP with Triton. Ribogreen reagent was diluted 1:100 in 1 XTE buffer and added to each well at a 1:1 ratio. Microplates were immediately introduced into a Cystation 5 cell imaging multimode reader (Biotek) to read fluorescence (Ex 485/Em 528).

C: LNP-sized dynamic light scattering (red dots are PDI to the right of the y-axis). The dialyzed LNP was diluted to 6.25ng/ul in sucrose buffer pH 7.5 and transferred into a quartz cuvette (ZEN 2112) to measure size by Dynamic Light Scattering (DLS) at 25 ℃ in 1X PBS with particle RI of 1.45 and absorption of 0.001, in Zetasizer Nano ZS (Malvern Panalytical), viscosity of 1.1cP and RI of 1.335. Measurements were made using 173 ° backscatter detection angles that were previously repeated twice to equilibrate to 25 ℃ for 30 seconds, each run 5 times and for 10 seconds, with no delay between measurements. Each measurement had a fixed position of 4.65 in a quartz cuvette with automatic decay selection. The data was analyzed using a generic model with normal parsing.

D: in vivo firefly luciferase expression in IM administration of LNP mixed at 1.5 mg/ml. 1ug of total mRNA was injected into mice by intramuscular (I.M.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system.

E in vivo firefly luciferase expression in IV administration of LNP in a 1.5mg/ml mix. 1ug of total mRNA was injected into mice by intravascular (I.V.) injection. Luciferin was administered intraperitoneally 4 hours after injection, and luciferase expression was monitored by live animal imaging at 4 hours. The ROI was calculated using the IVIS system.

For all patents, applications, or other references cited herein, such as non-patent documents and reference sequence information, it is to be understood that they are incorporated by reference in their entirety for all purposes and for the stated perspectives. In the event of any conflict between a document incorporated by reference and the present application, the present application will control. All information related to the reference gene sequences disclosed in the present application, such as GeneID or accession numbers (often referred to NCBI accession numbers), including, for example, genomic loci (genomic loci), genomic sequences, functional annotations, allelic variants and reference mrnas (including, for example, exon boundaries or response elements) and protein sequences (such as conserved domain structures) as well as chemical references (e.g., pubhem compounds, pubhem substances or pubhem biological entities, including annotations therein, such as structures and assays, etc.), are hereby incorporated by reference in their entirety.

Headings used in this application are for convenience only and do not affect the interpretation of the application.

The preferred features of each aspect provided by the application are in principle applicable to all other aspects of the application and are exemplified (without limitation) by the dependent claims and also encompass combinations and permutations of individual features (e.g., elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the application, including working examples. For example, the specific experimental parameters exemplified in the working examples may be adapted for use in the claimed application one by one without departing from the application. For example, for the disclosed materials, each is specifically contemplated and described herein, although specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed. Thus, if a class of elements A, B and C and a class of elements D, E and F are disclosed and an embodiment of a combination of elements a-D is disclosed, each is individually and collectively contemplated even if each is not recited individually. Thus, in this embodiment, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F is specifically contemplated and should be considered as being from A, B and C; D. e and F; and the disclosure of example combinations a-D. Likewise, any subset or combination of these components is specifically contemplated and disclosed. Thus, for example, a subset of A-E, B-F and C-E specifically encompasses and should be considered from A, B and C; D. e and F; and the disclosure of example combinations a-D. This concept applies to all aspects of the present application including the elements of the subject compositions and the method steps of making or using the compositions.

As one of ordinary skill in the art will recognize in light of the teachings of this specification, the foregoing aspects of the invention can be claimed in any combination or permutation so long as they are novel and non-obvious relative to the prior art—thus, when elements are described in one or more references known to those of ordinary skill in the art, they can be excluded from the claimed invention by a negative condition or disclaimer of the feature or combination of features.

Claims (59)

1. A method for preparing a lipid nanoparticle comprising a nucleic acid ("naLNP"), the method comprising the steps of:

a. providing a nucleic acid solution comprising at least one nucleic acid at a concentration of nucleic acids;

b. providing a lipid solution comprising at least one lipid at a lipid concentration; and

c. combining a portion of the nucleic acid solution and a portion of the lipid solution to produce a mixed solution comprising a mixed nitrogen-phosphate ratio and a lipid: nucleic acid ratio; and

d. optionally adjusting the pH in the final mixed solution to a physiological pH to obtain a pH-adjusted mixed solution; and

e. obtaining the naLNP from the mixed solution; and is also provided with

Wherein the naLNP has greater potency than a reference lipid nanoparticle ("refLNP"), wherein the refLNP comprises the at least one lipid and the at least one nucleic acid and is prepared by a reference LNP manufacturing method.

2. The method of claim 1, wherein the portion of nucleic acid solution and the portion of lipid solution are combined in step (c) in a volume ratio selected from the group consisting of: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, and 7:1.

3. The method of claim 1, wherein the naLNP has an average diameter in the range of about 40 to about 150 nm.

4. The method of claim 1, wherein the naLNP has an average diameter in the range of about 50 to about 100 nm.

5. The method of claim 1, wherein the naLNP has a nucleic acid encapsulation efficiency of from about 40% to about 100%.

6. The method of claim 1, wherein the naLNP has a nucleic acid encapsulation efficiency of about 50% to about 85%.

7. The method of claim 1, wherein the naLNP has a nucleic acid encapsulation efficiency of about 60% to about 85%.

8. The method of claim 1, wherein the naLNP has a nucleic acid encapsulation efficiency of about 68% to about 83%.

9. The method of claim 1, wherein the naLNP has a lower nucleic acid encapsulation efficiency than the refLNP.

10. The method of claim 1, wherein the at least one nucleic acid is DNA or RNA.

11. The method of claim 1, wherein the at least one nucleic acid is RNA.

12. The method of claim 1, wherein the at least one nucleic acid is mRNA.

13. The method of claim 1, wherein the at least one nucleic acid is an mRNA encoding at least one open reading frame.

14. The method of claim 1, wherein the at least one nucleic acid is an mRNA encoding at least one open reading frame encoding an immunogen.

15. The method of claim 1, wherein the nucleic acid solution comprises a buffer.

16. The method of claim 1, wherein an acid is added to the lipid solution to protonate the ionizable lipid.

17. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.21 to about 3mg/ml.

18. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.23 to about 3mg/ml.

19. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.25 to about 3mg/ml.

20. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.28 to about 3mg/ml.

21. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.29 to about 3mg/ml.

22. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.30 to about 3mg/ml.

23. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.40 to about 3mg/ml.

24. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.50 to about 3mg/ml.

25. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.60 to about 3mg/ml.

26. The method of claim 1, wherein the nucleic acid concentration is at least or about 0.70 to about 3mg/ml.

27. The method of claim 1, wherein the nucleic acid concentration is at least or about 1 to about 3mg/ml.

28. The method of claim 1, wherein the lipid solution comprises an organic solvent selected from the group consisting of: methanol, ethanol, acetone, benzene and toluene.

29. The method of claim 1, wherein the at least one lipid in the lipid solution is selected from the group consisting of: MC3, KC2, DLin, DODMA, DODAP, formula I, formula II, formula III, formula IV, and combinations thereof.

30. The method of claim 1, wherein the at least one lipid in the lipid solution is an ionizable cationic lipid having a pKa.

31. The method of claim 1, wherein the mixed solution has a pH of about 0 to about 2 pH units below the pKa of the lipids in the refLNP.

32. The method of claim 1, wherein the mixed solution has a pH of about 0.5 to about 1.5 pH units below the pKa of the lipids in the refLNP.

33. The method of claim 1, wherein the mixed solution has a pH of about 0.75 to about 1.25 pH units below the pKa of the lipids in the refLNP.

34. The method of claim 1, wherein the lipid concentration is at least or about 1mM to about 200mM.

35. The method of claim 1, wherein the lipid concentration is at least or about 10mM to about 150mM.

36. The method of claim 1, wherein the lipid concentration is at least or about 50mM to about 100mM.

37. The method of claim 1, wherein the mixed liquid nitrogen-phosphate ratio is at least or about 2 to at least or about 10.

38. The method of claim 1, wherein the mixed solution lipid to nucleic acid weight ratio is at least or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 15:1, 17:1, 18:1, 20:1, 25:1, 30:1, 35:1, 40:1, or 50:1.

39. The method of claim 1, wherein the refLNP is prepared using a reference nucleic acid concentration of less than 0.21 mg/ml.

40. The method of claim 1, wherein the refLNP is prepared using a reference lipid concentration of less than 10.5 mM.

41. The method of claim 1, wherein the refLNP is prepared using a reference nucleic acid concentration of less than 0.21mg/ml and a reference lipid concentration of less than 10.5 mM.

42. The method of claim 1, wherein the potency is about 1.5 times higher than the refLNP.

43. The method of claim 1, wherein the potency is about 2 times higher than the refLNP.

44. The method of claim 1, wherein the potency is about 3 times higher than the refLNP.

45. The method of claim 1, wherein the potency is about 4 times higher than the refLNP.

46. The method of claim 1, wherein the potency is at least or about 5 times higher than the refLNP.

47. The method of claim 1, wherein the potency is at least or about 6 times higher than the refLNP.

48. The method of claim 1, wherein the potency is at least or about 7 times higher than the refLNP.

49. The method of claim 1, wherein the potency is at least or about 8-fold higher than the refLNP.

50. The method of claim 1, wherein the potency is at least or about 9 times higher than the refLNP.

51. The method of claim 1, wherein the potency is at least or about 10 times higher than the refLNP.

52. The method of claim 1, wherein the potency is at least or about 11 times higher than the refLNP.

53. The method of claim 1, wherein the potency is at least or about 12 times higher than the refLNP.

54. The method of claim 1, wherein the potency is at least or about 13 times higher than the refLNP.

55. The method of claim 1, wherein the potency is at least or about 14 times higher than the refLNP.

56. The method of claim 1, wherein the potency is at least or about 15 times higher than the refLNP.

57. The method of claim 1, wherein the potency is at least or about 20 times higher than the refLNP.

58. The method of claim 1, wherein the potency is at least or about 25 times higher than the refLNP.

59. The method of claim 1, wherein the potency is at least or about 50 times higher than the refLNP.