LYOPHILIZED NANOPARTICLE COMPOSITIONS AND METHODS OF USE

THEREOF

Technical Field

The present disclosure provides novel methods and compositions for producing nanoparticles to deliver one or more therapeutic and/or prophylactics to and/or produce polypeptides in mammalian cells or organs.

Background

Delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids including mRNA is a medical challenge. In particular, the delivery of nucleic acids to cells is made difficult by the relative instability, low cell permeability of such molecules, storage, and shipment. Thus, there exists a need to develop compounds, compositions, and methods for improved stability and storage of therapeutic and/or prophylactics molecules into cells or organs.

Summary of the Invention

The present disclosure provides methods and formulations for making, storing, reconstituting, and using lyophilized lipid nanoparticles, including methods to deliver one or more therapeutic and/or prophylactics to and/or produce polypeptides in mammalian cells or organs. The present disclosure details lyophilization and reconstitution of lipid nanoparticles for 3-component lipid nanoparticles.

In one aspect, the present disclosure provides a method of making a nanoparticle composition, comprising the following steps: (a) freezing a solution comprising a lipid nanoparticle (LNP) composition and a nucleic acid at a temperature that is 2°C to 10°C below a eutectic point of the solution to produce a frozen solution, wherein the LNP composition comprises a cationic or ionizable lipid-containing component, a steroidal or structural lipid-containing component, and a Stabilizing lipid- containing component; (b) placing the frozen solution from step (a) under vacuum to produce a sample; (c) warming the sample from step (b) to a temperature from 20°C to 35°C; and (d) placing the warmed sample from (c) under vacuum.

In some embodiments, the method further comprises, before step (a), bringing a temperature of the solution to a range from 0°C to 10°C. In some embodiments, step (a) comprises: freezing the solution at a temperature in a range of -50°C to -40°C; and warming the frozen solution to a temperature in a range of -30°C to -20°C. In some embodiments, step (a) comprises freezing the solution at the temperature in the range of -50°C to -40°C for at least 6 hours, at least 7 hours, or at least 8 hours, or for about 4 to about 10 hours, about 5 to about 8 hours, or about 6 to about 7 hours. In some embodiments, step (b) comprises placing the frozen solution from step (a) under vacuum for at least 24 hours, at least 48 hours, or at least 72 hours. In some embodiments, step (d) comprises placing the warmed sample under vacuum for at least 4 hours, at least 6 hours, or at least 8 hours, or for about 4 to about 10 hours, about 5 to about 8 hours, or about 6 to about 7 hours.

In some embodiments, the method further comprises dissolving the sample from step (d) in water or an aqueous solution to produce a reconstituted sample. In some embodiments, the reconstituted sample is stable at 4°C for at least 1 day, at least 2 days, at least 3 days, or at least 4 days.

In some embodiments, the components in the LNP composition have the following relative mole percentages: 5 to 60 mole % of a steroidal or structural lipid- containing component; 0.5 to 20 mole % of a Stabilizing lipid-containing component; and 30 to 70 mole % of a cationic or ionizable lipid-containing component. In some embodiments, the components in the LNP composition have the following relative mole percentages: 20 to 50 mole % of a steroidal or structural lipid-containing component; 0.8 to 10 mole % of a Stabilizing lipid-containing component; and 40 to 62 mole % of a cationic or ionizable lipid-containing component. In some embodiments, the components in the LNP composition have the following relative mole percentages: 25 to 46 mole % of a steroidal or structural lipid-containing component; 1 to 7 mole % of a Stabilizing lipid-containing component; and 44 to 58 mole % of a cationic or ionizable lipid-containing component. In some embodiments, the components in the LNP composition have the following relative mole percentages: 35 to 44 mole % of a steroidal or structural lipid-containing component; 1 .2 to 5 mole % of a Stabilizing lipid- containing component; and 48 to 57 mole % of a cationic or ionizable lipid-containing component. In some embodiments, the components in the LNP composition have the following relative mole percentages: 37 to 43 mole % of a steroidal or structural lipid- containing component; 0.5 to 5, 1 to 4, or 1 .4 to 3 mole % of a Stabilizing lipid- containing component; and 50 to 56 mole % of a cationic or ionizable lipid-containing component.

In some embodiments, the components in the LNP composition have the following relative mole percentages: 54-57% of a cationic or ionizable lipid-containing component, 40-44% of a steroidal or structural lipid-containing component, and 1 -3% of a Stabilizing lipid-containing component. In some embodiments, the LNP composition comprises SM-102, cholesterol, and DMG-PEG2000. In some embodiments, the components in the LNP composition have the following relative mole percentages: 54- 57% of SM-102, 40-44% of cholesterol, and 1 -3% of DMG-PEG2000.

In some embodiments, the components in the LNP composition have the following relative mole percentages: 55.9% of a cationic or ionizable lipid-containing component, 42.4% of a steroidal or structural lipid-containing component, and 1 .7% of a Stabilizing lipid-containing component. In some embodiments, the LNP composition comprises SM-102, cholesterol, and DMG-PEG2000. In some embodiments, the components in the LNP composition have the following relative mole percentages: 55.9% of SM-102, 42.4% of cholesterol, and 1 .7% of DMG-PEG2000.

In some embodiments, the solution in step (a) is produced by mixing a first stock solution comprising the LNP composition and a second stock solution comprising the nucleic acid to produce a mixture; and dialyzing the mixture in a dialysis buffer with a lyoprotectant.

In some embodiments, a total lipid concentration in the first stock solution is from 8 to 30 mM, 10 to 25 mM, 12 to 20 mM, 14 to 18 mM,12 mM to 13 mM, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 mM. In some embodiments, a total lipid concentration in the first stock solution is from 10.5 mM to 1 1.5 mM.

In some embodiments, the second stock solution has a pH from 3.5 to 4.5. In some embodiments, the second stock solution has a pH from 4.5 to 5.5. In some embodiments, the second stock solution has a pH from 5.5 to 6.5. In some embodiments, the second stock solution has a pH from 6.5 to 7.5. In some embodiments, the second stock solution comprises sodium citrate. In some embodiments, the second stock solution comprises sodium acetate.

In some embodiments, the solution in (a) comprises a lyoprotectant. In some embodiments, the lyoprotectant is sucrose. In some embodiments, the lyoprotectant is trehalose. In some embodiments, the lyoprotectant is mannose or mannitol. In some embodiments, the lyoprotectant is a combination of trehalose and sucrose. In some embodiments, the lyoprotectant is a combination of mannitol and sucrose. In some embodiments, the lyoprotectant is a combination of two lyoprotectants (e.g., mannose, mannitol, sucrose, trehalose, glucose) at a ratio of 3:1 to 1 :3. In some embodiments, the lyoprotectant has a volume/volume concentration from 1% to 50%, 2% to 45%, 3 to 40%, 4 to 35%, 5 to 30%, 6 to 25%, 6 to 20%, 6 to 15%, 6% to 10%, 7% to 9%, 5% to 16%, from 6% to 14%, or from 7% to 12%, e.g., 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16%. In some aspects, the solution in (a) comprises a lyoprotective buffer or agent such as histidine and/or glycine.

In some embodiments, the nucleic acid is mRNA.

In some aspects, the present disclosure provides novel compositions and methods involving LNPs formed from a mixture of three components.

In another aspect, the present disclosure provides a prelyophilization, lyophilized, or reconstituted three-component LNP composition wherein the three components in the prelyophilization, lyophilized, or reconstituted composition are:

1 ) a steroidal or structural lipid-containing component;

2) a Stabilizing lipid-containing component; and

3) a cationic or ionizable lipid-containing component. Thus, the three-component LNP composition of the present disclosure does not contain a phospholipid-containing component.

In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:

1 ) 5 to 60 mole % of a steroidal or structural lipid-containing component;

2) 0.5 to 20 mole % of a Stabilizing lipid-containing component; and

3) 30 to 70 mole % of a cationic or ionizable lipid-containing component.

In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:

1 ) 20 to 50 mole % of a steroidal or structural lipid-containing component;

2) 0.8 to 10 mole % of a Stabilizing lipid-containing component; and

3) 40 to 62 mole % of a cationic or ionizable lipid-containing component.

In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:

1 ) 25 to 46 mole % of a steroidal or structural lipid-containing component;

2) 1 to 7 mole % of a Stabilizing lipid-containing component; and

3) 44 to 58 mole % of a cationic or ionizable lipid-containing component.

In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:

1 ) 35 to 44 mole % of a steroidal or structural lipid-containing component;

2) 1 .2 to 5 mole % of a Stabilizing lipid-containing component; and

3) 48 to 57 mole % of a cationic or ionizable lipid-containing component. In one aspect, the cationic or ionizable lipid-containing component may comprise

MC3, ALC-0315, ALC-0159, SM-102, 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), or a cationic and/or ionizable lipid disclosed in WO2017049245A2 (Benenato), which is incorporated herein by reference. In one aspect, the cationic or ionizable lipid-containing component may comprise compounds of Formula (IA) : or a salt or isomer thereof, wherein m is 0-9; n is 0-9; o is 0-12; p is 0-12;

Ri is a linear C1-12 alkyl;

R2 is H or a linear C1-12 alkyl;

R3 is a linear C1-12 alkyl;

R4 is H or linear C1-12 alkyl; and

Mi and M2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

In certain aspects, compounds of Formula IA may include, for example, the following compounds:

(lAc).

In another aspect, the present disclosure provides compounds of Formula (IB):

TB or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9;

0 is selected from 0-12; p is selected from 0-12;

R is the side chain of an independently selected amino acid;

R1 is a linear C1-12 alkyl;

R2 is H or linear C1-12 alkyl;

R3 is a linear C1-12 alkyl; F is H or linear C1-12 alkyl;

Rs is the side chain of an independently selected amino acid;

Xi is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;

X2 is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;

X ₃ is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-; and

X ₄ is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-.

In some aspects, R or Rs comprises the side chain of a Serine (S), Threonine (T),

Cysteine (C), Selenocysteine (U), Glycine (G), Alanine (A), Isoleucine (I), Leucine (L),

Methionine (M), or Valine (V). In some aspects, the carbonyl group in Formula IB is bonded to the amino terminus of the amino acid. In some aspects, the carbonyl group in Formula IB is bonded to the carboxy terminus of the amino acid.

In certain aspects, compounds of Formula IB may include, for example, the following compounds:

or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-5;

Ri is a linear C1-12 alkyl;

R2 is H or a linear C1-12 alkyl; R3 is a linear C1-12 alkyl;

R4 is H or a linear C1-12 alkyl;

Rs is H or CH3;

Mi and M2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, -

OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H; and

X is selected from -CH2-, -O-, -S-, or -P(O)(OR)O-.

In certain aspects, compounds of Formula IC may include, for example, the following compounds:

In one aspect, the cationic or ionizable lipid-containing component may comprise compounds of Formula (HA): or a salt or isomer thereof, wherein m is selected from 0-5; n is selected from 0-12; o is selected from 0-12; q is selected from 1 -3;

Ri is a linear C1-12 alkyl;

R2 is H or linear C1-12 alkyl;

R3 is a linear C1-12 alkyl;

R4 is H or a linear C1-12 alkyl; and

X is selected from C(R)2, N(R), or O, wherein R is independently selected from a methyl and H.

In certain aspects, compounds of Formula IIA may include, for example, the following compound.

(IIAb).

In another aspect, the present disclosure provides compounds of Formula (I IB) : or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-6;

Ri is a linear C1-12 alkyl;

R2 is H or a linear C1-12 alkyl; Rs is a linear C1-12 alkyl;

R4 is H or a linear C1-12 alkyl;

Rs is a linear C1-4 alkyl alcohol;

Re is a linear C1-4 alkyl alcohol; Mi and M2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

In certain aspects, compounds of Formula I IB may include, for example

(IIBa). In another aspect, the present disclosure provides compounds of Formula (IIC): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; 0 is selected from 0-12; p is selected from 0-12; q is selected from 2-6; Ri is a linear C1-12 alkyl;

R2 is H or linear C1-12 alkyl;

R3 is a linear C1-12 alkyl;

R4 is H or a linear C1-12 alkyl;

Rs is a linear C1-4 alkyl alcohol;

Re is a linear C1-4 alkyl alcohol;

Mi and M2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

In certain aspects, compounds of Formula IIC may include, for example

In another aspect, the present disclosure provides compounds of Formula (I ID): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 1 -7;

0 is selected from 0-12; p is selected from 0-12; Ri is a linear C1-12 alkyl;

R2 is H or a linear C1-12 alkyl;

R3 is a linear C1-12 alkyl;

R4 is H or a linear C1-12 alkyl; and Mi and M2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

In certain aspects, compounds of Formula I ID may include, for example In another aspect, the present disclosure provides compounds of Formula (HE): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; 0 is selected from 0-12; p is selected from 0-12; q is selected from 2-6;

R1 is a linear C1-12 alkyl; R2 is H or linear C1-12 alkyl;

R3 is a linear C1-12 alkyl;

R4 is H or a linear C1-12 alkyl; and

Mi and M2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H. In another aspect, the present disclosure provides compounds of Formula (I IF) : or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;

Ri is a linear C1-12 alkyl;

R2 is H or linear C1-12 alkyl; R3 is a linear C1-12 alkyl;

R4 is H or a linear C1-12 alkyl; and

Mi and M2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H. In certain aspects, compounds of Formula I IF may include, for example

In another aspect, the present disclosure provides a method of delivering a therapeutic and/or prophylactic (e.g., an mRNA) to a cell (e.g., a mammalian cell) by administering a three-component LNP composition containing the steroidal or structural lipid-containing component, the Stabilizing lipid-containing component, and the cationic or ionizable lipid-containing component, to deliver the therapeutic and/or prophylactic to a subject (e.g., a mammal, such as a human), in which administering involves contacting the cell with the three-component LNP composition composition such that the therapeutic and/or prophylactic is delivered to the cell.

In another aspect, the present disclosure provides a method of producing a polypeptide of interest in a cell (e.g., a mammalian cell) by contacting the cell with the three-component LNP composition and an mRNA encoding the polypeptide of interest, whereby the mRNA is capable of being translated in the cell to produce the polypeptide.

Brief Description of the Drawings

Fig. 1 A shows the in vitro expression of 3-component LNPs before and after lyophilization compared to 4-component controls (lanes 1 and 7) using 20 mM Tris-HCI, pH 7.4, 8% sucrose. The tested nanoparticle formulations are produced according to methods in Example 1 .

Fig. 1 B is the quantification of the expression in the Western Blog data in Fig. 1 A.

Fig. 2. The in vitro expression of 3-component LNPs before and after lyophilization compared to 4-component controls (lanesl and 7) using 20 mM Tris-HCI, pH 7.4, 8% trehalose.

Fig. 3 shows a Western blot and data of in vitro expression of protein from mRNA encapsulated in the three-component LNP composition of the present disclosure and four-component LNP (comparator) composition (“RL007”). The top image is raw data and the bottom graph is processed data by Imaged.

Fig. 4 is the ELISA results of animal study. Briefly, plates were coated with SARS-CoV-2 delta S1 (from Sino Biological) and SARS-CoV-2 delta RBD (from eEnzyme) proteins, respectively. Then the sera from immunized mice were added to the plates coated with delta S1 and delta RBD. The results showed the amount of antibody that can bind to antigen.for mRNA encapsulated in the three-component LNP composition of the present disclosure (right) and four-component LNP (comparator) (left) composition in mice.

Fig. 5 shows encapsulation data for 3-component and 4-component LNPs. One through four (1 -4) are the encapsulation results for 3-component LNPs prepared at pH 4.0 with different concentrations of lipids and different Stabilizing lipids. Five (5) is the encapsulation result for the control 4-component LNP (“RL007”). Six (6) and seven (7) are LNPs prepared at pH 6.0. Six (6) is the control 4-component LNP (RL007) and seven (7) is the 3-component LNP that was also used in the in vivo study (Fig. 2).

Fig. 6 shows four images of 4-component LNPs and 3-component LNPs after reconstitution taken using transmission electron microscopy. The four images on the left are of 4-component LNPs and the four images on the right are of 3-component LNPs. Two of the four images of both groups show the LNPs at a 100 nm length scale and two of the four images of both groups show the LNPs at a 50 nm length scale. The arrows point to blebs formed on the surfaces of the LNPs.

Fig. 7 shows a Western blot and normalization data of in vitro expression of protein from mRNA encapsulated in the three-component LNP composition of the present disclosure (mLNP) suspended in solutions comprising different formulations (reconstitution media, dialysis buffers and lyoprotectants). The expression of protein from mRNA encapsulated in the mLNP in each formulation was shown in a graph normalized against actin both before and after lyophilization. Fig. 8 shows the strength of immune response mounted against the Delta S1 protein from mRNA encapsulated in the three-component LNP composition of the present disclosure (mLNP) and a 4-component LNP 14 days after immunization. Three different compositions of mLNPs tested were: SM-102 (59.8%), Cholesterol (38.5%), DMG-PEG2000 (1.7%) (M1 and M2, before and after lyophilization, respectively); SM- 102 (54.6%), Cholesterol (42%), DMG-PEG2000 (3.4%) (M3 and M4, before and after lyophilization, respectively); and a 4 component LNP (L1 and L2, before and after lyophilization, respectively).

Fig. 9 shows the strength of immune response mounted against the Delta S1 protein from mRNA encapsulated in the three-component LNP composition of the present disclosure (mLNP) and 4-component LNP 35 days after immunization.

Fig. 10 shows the strength of immune response mounted against the RBD protein from mRNA encapsulated in the three-component LNP composition of the present disclosure (mLNP) and 4 component LNP 35 days after immunization.

Detailed Description

The present disclosure provides methods and compositions for producing nanoparticles with improved stability and/or function (e.g., capability of expressing nucleic acid payload in the nanoparticle in a cell, tissue, organ, or subject at a higher level). In some aspects, the present disclosure provides a method of making a nanoparticle by lyophilizing a solution comprising a LNP composition and a nucleic acid. The lyophilized sample may be reconstituted in water or an aqueous solution for administering to a subject to deliver the payload. In some embodiments, the method comprises freezing a solution comprising a LNP and a nucleic acid at a temperature several degree Celsius below the eutectic point of the solution, and applying vacuum to the frozen solution to remove water to produce a sample. The sample may then be placed under vacuum at a warmer temperature (e.g., a temperature around or above room temperature, e.g., 25°C to 35°C, such as 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35°C) to produce a lyophilized sample. The lyophilized sample produced according to the present disclosure may be reconstituted very easily in a small amount of water or an aqueous solution. The nanoparticle composition produced by the method may be capable of expressing the payload nucleic acid (e.g., mRNA) at a higher level (e.g., at least 2-fold) compared to the same nanoparticle composition before lyophilization. After reconstitution, the nanoparticle composition produced by the method may remain stable.

In one aspect, the present disclosure includes a method comprising forming a pre-lyophilization formulation comprising a nucleic acid and a three-component LNP composition wherein the three components are:

1 ) a steroidal or structural lipid-containing component;

2) a Stabilizing lipid-containing component; and

3) a cationic or ionizable lipid-containing component.

For example, the three components in the following relative mole percentages:

1 ) 5 to 60 mole % of a steroidal or structural lipid-containing component;

2) 0.5 to 20 mole % of a Stabilizing lipid-containing component; and

3) 30 to 70 mole % of a cationic or ionizable lipid-containing component. In one aspect, the pre-lyophilization formulation is subjected to dialysis at 2 to 10°C, e.g., 4°C for 10-20 hours, e.g., 18 hours. The dialysis solution includes a buffer and a lyoprotectant.

In one aspect, the present disclosure includes a method comprising: 1 ) freezing a solution comprising a LNP and a nucleic acid at -50°C to -40°C; 2) warming to -30°C to - 20°C over 0.5 to 1 hour; 3) applying vacuum (e.g., 100-250 mbar) for 60-84 hours; 4) warming to 25 to 35°C over 6 hours. For step 3, vacuum may be applied until the amount of liquid in the sample is less than 2%, less than 1 %, or less than 0.5%.

In one aspect, the present disclosure includes a method comprising: 1 ) cooling to 4 to 10°C; 2) freezing a solution comprising a LNP and a nucleic acid at -50°C to -40°C over 1 to 2 hours; 3) applying vacuum (e.g., 100-250 mbar) for 60-84 hours; 4) warming to 25 to 35°C over 6 hours; 5) applying vacuum at 25 to 35°C for 10-16 hours. Vacuum may be applied until the amount of liquid in the sample is less than 2%, less than 1 %, or less than 0.5%.

In one aspect, the present disclosure includes a method comprising: 1 ) freezing a solution comprising a LNP and a nucleic acid at -50°C to -40°C; 2) warming to -30°C to - 20°C over 0.5 to 1 hour; 3) cooling to -50°C to -40°C; 4) warming to -30°C to -20°C; 5) cooling to -50°C to -40°C; 6) applying vacuum (e.g., 100-250 mbar) for 60-84 hours; 7) warming to 25 to 35°C over 6 hours; 8) applying vacuum at 25 to 35°C for 10-16 hours. Vacuum may be applied until the amount of liquid in the sample is less than 2%, less than 1 %, or less than 0.5%.

In one aspect, the present disclosure includes reconstituting with pure water or 1 - 500 mM NaCI in pure water. In one aspect, the present disclosure includes reconstituting the lyophilized solution with the original volume of solution that was lyophilized.

In one aspect, the present disclosure includes reconstituting the lyophilized material with a buffer. In some aspects, the buffer may be PBS or Tris-HCl. In some aspects, a buffer may have a concentration of 20-50 mM and a pH from 6.5 to 7.4. In some aspects, the reconstitution buffer may be the same as the dialysis buffer. In some aspects, the reconstitution may be different from the dialysis buffer. In some aspects, if lyophilization is performed with low molarity buffer, e.g., sodium citrate, then present disclosure includes reconstitution with PBS or Tris-HCl.

It is noted that at each stage, there is a chance for the LNPs to be rendered less stable, e.g., size growth, increased impurities, and/or loss of encapsulation efficiency. It is surprisingly discovered that lipid nanoparticles containing nucleic acid are rendered more stable throughout its life cycle with the use of the method of the present disclosure. “Stability,” “stabilized,” and “stable” in the context of the present disclosure refers to the resistance of LNPs to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given manufacturing, preparation, transportation, storage and/or in-use conditions, e.g., when stress is applied such as shear force, freeze/thaw stress, etc.

The “stabilized” formulations of the disclosure preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the purity (e.g., chromatographic purity) of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and/or in-use conditions. The “stabilized” formulations of the disclosure also preferably has an increase of about 20%, 10%, 5%, 1%, 0.5% or less of a starting, standard, or reference LNP mean size under given manufacturing, preparation, transportation, storage and/or in-use conditions.

For example, the formulation has an increase in LNP mean size of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at 4° C or lower for at least one month. For example, the formulation has an increase in LNP mean size of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at -20° C or lower for at least six months (e.g., at least one year, two years, or three years). For example, the formulation has an increase in LNP mean size of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about -80° C or lower for at least six months (e.g., at least one year, two years, or three years). For example, the formulation has an increase in LNP mean size of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after up to 30 freeze/thaw cycles.

For example, the formulation has an increase in LNP mean size of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after a purification process as compared to that prior to purification. For example, the purification process includes filtration. For example, the formulation has an increase in LNP mean size of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after lyophilization as compared to that prior to lyophilization.

The “stabilized” formulations of the disclosure preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the LNP size distribution of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and/or in- use conditions. The “stabilized” formulations of the disclosure preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the encapsulation efficiency of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and/or in- use conditions.

For example, the encapsulation efficiency is substantially the same after storage at about 4° C or lower (e.g., about -20° C or lower or about -80° C or lower) for at least one month (e.g., for at least six months, one year, two years, or three years). For example, the encapsulation efficiency may decrease for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about 4° C or lower for at least one month. For example, the encapsulation efficiency may decrease for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about -20° C or lower for at least six months (e.g., at least one year, two years, or three years). For example, the encapsulation efficiency may decrease for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about -80° C or lower for at least six months (e.g., at least one year, two years, or three years). For example, the encapsulation efficiency is substantially the same after up to 30 freeze/thaw cycles.

For example, the encapsulation efficiency is substantially the same after a purification process as compared to that prior to purification. For example, the purification process includes filtration. For example, the encapsulation efficiency is substantially the same after lyophilization as compared to that prior to lyophilization.

The “stabilized” formulations of the disclosure also preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the biological activity of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and/or in-use conditions.

For example, the formulation has little or no immunogenicity (e.g., inducement of an innate immune response). For example, the immunogenicity (e.g., inducement of an innate immune response) is substantially the same after storage at about 4° C or lower (e.g., about -20° C or lower or about -80° C or lower) for at least one month (e.g., for at least six months, one year, two years, or three years). For example, the immunogenicity (e.g., inducement of an innate immune response) may increase for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about 4° C or lower for at least one month. For example, immunogenicity (e.g., inducement of an innate immune response) may increase for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about -20° C or lower for at least six months (e.g., at least one year, two years, or three years). For example, the immunogenicity (e.g., inducement of an innate immune response) may increase for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about -80° C or lower for at least six months (e.g., at least one year, two years, or three years).

For example, the immunogenicity (e.g., inducement of an innate immune response) is substantially the same after up to 30 freeze/thaw cycles. For example, the formulation has a lower immunogenicity (e.g., inducement of an innate immune response) as compared to a corresponding formulation which is not made according to the method of the present disclosure. For example, the therapeutic index of therapeutic or prophylactic agent-loaded LNP formulation is substantially the same after storage at about 4° C or lower (e.g., about -20° C or lower or about -80° C or lower) for at least one month (e.g., for at least six months, one year, two years, or three years). For example, the therapeutic index may decrease for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about 4° C or lower for at least one month. For example, the therapeutic index may decrease for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about -20° C or lower for at least six months (e.g., at least one year, two years, or three years). For example, the therapeutic index may decrease for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about -80° C or lower for at least six months (e.g., at least one year, two years, or three years).

For example, the therapeutic index is substantially the same after up to 30 freeze/thaw cycles.

For example, the formulation comprising a therapeutic or prophylactic agent has an increased therapeutic index as compared to a corresponding formulation which is not made according to the method of the present disclosure.

The “stabilized” formulations of the disclosure also preferably has an increase of about 20%, 10%, 5%, 1%, 0.5% or less of a starting, standard, or reference amount of impurities under given manufacturing, preparation, transportation, storage and/or in-use conditions.

The “stabilized” formulations of the disclosure also preferably has an increase of about 20%, 10%, 5%, 1%, 0.5% or less of a starting, standard, or reference amount of sub-visible particles under given manufacturing, preparation, transportation, storage, and/or in-use conditions.

The zeta potential (“ZP”) is the electrostatic potential surrounding the LNP. In general, a near-neutral zeta potential is desirable. Anionic LNPs may be electrostatically repelled from negatively charged plasma membranes, and cationic LNPs can be cytotoxic.

The polydispersity index (“PDI”) is a measure of the LNP size distribution. Homogeneous, uniformly sized samples have small PDIs, and samples with heterogeneous size distributions have large PDIs. For example, PDI can be measured via dynamic light scattering.

The purity, LNP mean size, encapsulation efficiency, biological activity, immunogenicity, therapeutic index, amount of impurities can be determined using any art- recognized method. For example, the LNP mean size can be measured dynamic light scattering (DLS). For example, the concentration of a component of the formulation can be determined using routine methods such as UV-Vis spectrophotometry and high pressure liquid chromatography (HPLC). For example, amount of sub-visible particles can be determined by micro-flow imaging (MFI).

In certain embodiments, the present formulations are stabilized at temperatures ranging from about 2 to 8° C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In one embodiment, the formulation is stabilized for at least 2 months at 2 to 8° C.

In certain embodiments, the present formulations are stabilized at a temperature of about 4° C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months. In one embodiment, the formulation is stabilized for at least 2 months at about 4° C.

In certain embodiments, the present formulations are stabilized at temperatures of about -20° C for at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In one embodiment, the formulation is stabilized for at least 6-12 months at -20° C. In one embodiment, the formulation is stabilized for at least 24-36 months at -20° C.

In a particular embodiment, a formulation of the disclosure is stabilized at a temperature ranging between about -20° C and 4° C at a nucleic acid concentration (e.g., an mRNA concentration) of up to 2 mg/mL for at least 2 weeks, for at least 4 weeks, for at least 8 weeks, for at least 12 weeks, or for at least 16 weeks.

In a particular embodiment, a formulation of the disclosure is stabilized at a temperature ranging between about -20° C and 4° C at a nucleic acid concentration (e.g., an mRNA concentration) of up to 1 mg/mL for at least 2 weeks, for at least 4 weeks, for at least 8 weeks, for at least 12 weeks, or for at least 16 weeks.

In some aspects, the disclosure relates to novel three-component LNP composition compositions. The disclosure also provides methods of delivering a therapeutic and/or prophylactic to a mammalian cell, specifically delivering a therapeutic and/or prophylactic to a mammalian organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof. For example, a method of producing a polypeptide of interest in a cell involves contacting a three-component LNP composition comprising an mRNA with a mammalian cell, whereby the mRNA may be translated to produce the polypeptide of interest. A method of delivering a therapeutic and/or prophylactic to a mammalian cell or organ may involve administration of a three-component LNP composition including the therapeutic and/or prophylactic to a subject, in which the administration involves contacting the cell or organ with the three-component LNP composition, whereby the therapeutic and/or prophylactic is delivered to the cell or organ.

As used herein, the term “alkyl” or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation “C1-14 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1 -14 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups. As used herein, the term “alkenyl” or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. The notation “C2-14 alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carboncarbon double bonds. For example, Cis alkenyl may include one or more double bonds. A C alkenyl group including two double bonds may be a linoleyl group. Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.

As used herein, the term “alkynyl” or “alkynyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted. The notation “C2-14 alkynyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds. For example, Cis alkynyl may include one or more carbon-carbon triple bonds. Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified. As used herein, the terms “approximately” and “about,” as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” may refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

As used herein, the term “compound,” is meant to include all isomers and isotopes of the structure depicted. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. Further, a compound, salt, or complex of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.

As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and a nanoparticle are made to share a physical connection. Methods of contacting cells with external entities both in vivo and ex vivo are well known in the biological arts. For example, contacting a nanoparticle composition and a mammalian cell disposed within a mammal may be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve varied amounts of nanoparticle compositions. Moreover, more than one mammalian cell may be contacted by a nanoparticle composition. As used herein, the term “delivering” means providing an entity to a destination. For example, delivering a therapeutic and/or prophylactic to a subject may involve administering a nanoparticle composition including the therapeutic and/or prophylactic to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

As used herein, “encapsulation efficiency” refers to the amount of a therapeutic and/or prophylactic that becomes part of a nanoparticle composition, relative to the initial total amount of therapeutic and/or prophylactic used in the preparation of a nanoparticle composition. For example, if 97 mg of therapeutic and/or prophylactic are encapsulated in a nanoparticle composition out of a total 100 mg of therapeutic and/or prophylactic initially provided to the composition, the encapsulation efficiency may be given as 97%. As used herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide or protein and/or post-translational modification of a polypeptide or protein.

As used herein, the term “isomer” means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. Compounds may include one or more chiral centers and/or double bonds and may thus exist as stereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis/trans isomers). The present disclosure encompasses any and all isomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known.

As used herein, a “lipid component” is that component of a nanoparticle composition that includes one or more lipids. For example, the lipid component may include one or more cationic/ionizable, Stabilizing, or steroidal/structural lipid.

As used herein, a “linker” is a moiety connecting two moieties, for example, the connection between two nucleosides of a cap species. A linker may include one or more groups including but not limited to phosphate groups (e.g., phosphates, boranophosphates, thiophosphates, selenophosphates, and phosphonates), alkyl groups, amidates, or glycerols. For example, two nucleosides of a cap analog may be linked at their 5' positions by a triphosphate group or by a chain including two phosphate moieties and a boranophosphate moiety.

As used herein, “methods of administration” may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. A method of administration may be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body.

As used herein, “modified” means non-natural. For example, an RNA may be a modified RNA. That is, an RNA may include one or more nucleobases, nucleosides, nucleotides, or linkers that are non-naturally occurring. A “modified” species may also be referred to herein as an “altered” species. Species may be modified or altered chemically, structurally, or functionally. For example, a modified nucleobase species may include one or more substitutions that are not naturally occurring.

As used herein, the “N:P ratio” is the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a lipid to phosphate groups in an RNA, e.g., in a nanoparticle composition including a lipid component and an RNA.

As used herein, a “nanoparticle composition” is a composition comprising one or more lipids. Nanoparticle compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or less.

As used herein, “naturally occurring” means existing in nature without artificial aid.

As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.

As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.

The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending, complexing, or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin D, vitamin E (alphatocopherol), vitamin C, vitamin K, xylitol, and other species disclosed herein.

In the present specification, the structural formula of the compound represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like, it being understood that not all isomers may have the same level of activity. In addition, a crystal polymorphism may be present for the compounds represented by the formula. It is noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.

Compositions may also include salts of one or more compounds. Salts may be pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety to its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17 ^th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et aL, Journal of Pharmaceutical Science, 66, 1 -19 (1977), each of which is incorporated herein by reference in its entirety.

As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell. In certain aspects, the three-component LNP of the present disclosure is free of phospholipids, i.e., does not have the phospholipid component used in the traditional four-component LNP compositions.

As used herein, the term “polypeptide” or “polypeptide of interest” refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically.

As used herein, an “RNA” refers to a ribonucleic acid that may be naturally or non-naturally occurring. For example, an RNA may include modified and/or non- naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and/or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, an RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. RNAs may be selected from the non-liming group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, and mixtures thereof.

As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

The term “therapeutic agent” or “prophylactic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. Therapeutic agents are also referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

As used herein, the term “eutectic point” refers to a temperature of a solid-liquid composition of a mixture of two or more components that has the lowest possible complete melting temperature. At the eutectic point, further lowering the temperature of the eutectic mixture does not change the composition of the solid or liquid phase. As a result, a mixture having a composition at the eutectic point may not be further purified by conventional crystallization. The eutectic point temperature and composition can be calculated from enthalpy and entropy of fusion of each components, e.g., according to the method described in Brunet L et al., Thermodynamic calculation of n-component eutectic mixtures, International Journal of Modern Physics C, Volume 15, Issue 05, pp. 675-687 (2004), which is incorporated by reference herein in its entirety.

Method of Producing Nanoparticles

In some embodiments, the method for producing a nanoparticle composition comprises freezing a solution comprising a lipid nanoparticle (LNP) composition and a nucleic acid at a temperature that is below the eutectic point of the solution to produce a frozen solution.

The temperature below the eutectic point of the solution may be 1 °C to 10°C, 1 °C to 5°C, 5°C to 10°C, 1 °C to 3°C, 2°C to 4°C, 3°C to 5°C, 4°C to 6°C, 5°C to 7°C, 6°C to 8°C, 7°C to 9°C, or 8°C to 10°C below the eutectic point of the solution. For example, the temperature below the eutectic point of the solution may be about 1 °C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C below the eutectic point of the solution. For example, the freezing temperature may be -60°C to -50°C, -50°C to -40°C, -40°C to -30°C, -30°C to -20°C, -20°C to -10°C, or -10°C to 0°C.

In some embodiments, the freezing may comprise freezing the solution at a lower temperature and warming the frozen solution to a higher (but still freezing) temperature. For example, the freezing may comprise freezing the solution at a temperature in the range of -60°C to -50°C, -50°C to -40°C, or -40°C to -30°C and warming the frozen solution to a temperature in the range of -30°C to -20°C, -20°C to -10°C, or -10°C to 0°C. In one example, the freezing may comprise freezing the solution a temperature in the range of -50°C to -40°C (e.g., -45°C) and warming the frozen solution to a temperature in the range of -30°C to -20°C (e.g., -25°C). The solution may be frozen (e.g., at the lower temperature) for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, or at least 10 hours.

The frozen solution may be placed under vacuum to produce a sample with less water. In some cases, the solution may be placed under vacuum after frozen. In certain cases, the solution may be placed under vacuum while being frozen. In cases where the freezing comprises freezing the solution at lower temperature and warming the frozen solution to a warmer (but still freezing) temperature, the vacuum may be applied when the frozen solution is at the warmer temperature. In some example, the vacuum may be applied for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, or at least 72 hours. Vacuum may be applied until the amount of liquid in the sample is less than 2%, less than 1%, or less than 0.5%.

The sample from vacuum may be warmed to a temperature in the range of 20°C to 40°C, 20°C to 30°C, 25°C to 35°C, or 30°C to 40°C. The warmed sample may be placed under vacuum to produce a lyophilized sample, e.g., for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, or at least 10 hours.

In some embodiments, the method may further comprise reconstituting the lyophilized sample in water or an aqueous solution (e.g., a buffer solution) to produce a reconstituted sample. The reconstituted sample may have improved stability than the solution before lyophilization. For example, the reconstituted sample may remain stable for at least 1 day, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 days. The reconstituted sample may be used to administer to a subject for expressing the payload nucleic acid in the subject. In some examples, the reconstituted sample may be capable of expressing the payload nucleic acid (e.g., mRNA) at least 10%, at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher compared to a reference sample comprising the same components at the same concentrations but produced without lyophilization,

The LNP composition may be any LNP composition described in the disclosure or known in the art. In general, the LNP composition may comprise a cationic or ionizable lipid-containing component, a steroidal or structural lipid-containing component, and a Stabilizing lipid-containing component. The LNP composition may further comprise a phospholipid. For example, the LNP composition may be any 3- component LNP compositions disclosed herein or other LNP compositions (e.g., the 4- component LNP compositions). Examples of the LNP compositions also include those described in U.S. Provisional Application Nos. 63/287,647, 63/287,657, and 63/328,367, each of which is incorporated by reference herein in its entirety.

In some embodiments, the solution subject to freezing may be produced mixing a first stock solution comprising the LNP composition and a second stock solution comprising the nucleic acid to produce a mixture; and dialyzing the mixture in a dialysis buffer with a lyoprotectant.

In some examples, the total lipid concentration in the first stock solution may be from 8 to 30 mM, 10 to 25 mM, 12 to 20 mM, 14 to 18 mM,12 mM to 13 mM, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 mM, 10 mM to 1 1 mM, from 10.5 mM to 11 .5 mM, from 11 mM to 12 mM, from 1 1 .5 mM, to 12.5 mM, from 12 mM to 13 mM, from 12.5 mM to 13.5 mM, from 13 mM to 14 mM, from

13.5 mM to 14.5 mM, or from 14 mM to 15mM. In some examples, the total lipid concentration in the first stock solution may be about 10.0 mM, about 10.1 mM, about 10.2 mM, about 10.3 mM, about 10.4 mM, about 10.5 mM, about 10.6 mM, about 10.7 mM, about 10.8 mM, about 10.9 mM, about 11.0 mM, about 11.1 mM, about 11.2 mM, about 1 1 .3 mM, about 11 .4 mM, about 11 .5 mM, about 11 .6 mM, about 11 .7 mM, about 1 1 .8 mM, about 1 1 .9 mM, about 12.0 mM, about 12.1 mM, about 12.2 mM, about 12.3 mM, about 12.4 mM, about 12.5 mM, about 12.6 mM, about 12.7 mM, about 12.8 mM, about 12.9 mM, about 13.0 mM, about 13.1 mM, about 13.2 mM, about 13.3 mM, about 13.4 mM, about 13.5 mM, about 13.6 mM, about 13.7 mM, about 13.8 mM, about 13.9 mM, or about 14.0 mM.

The second stock solution may have a pH from 3 to 4, from 3.5 to 4.5, from 4 to 5, from 4.5 to 5.5, from 5 to 6, from 5.5 to 6.5, or from 6 to 7. For example, the second stock solution may have a pH of about 3, about 3.1 , about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0. The second stock solution may comprise a buffer. In some examples, the nucleic acid solution may comprise sodium citrate (e.g., from 20 mM to 30 mM, e.g., 25mM). In some examples, the second stock solution may comprise sodium acetate (e.g., from 20 mM to 30 mM, e.g., 25mM). The dialysis buffer may comprise a lyoprotectant such as those described herein, e.g., sucrose or trehalose. The dialysis buffer may comprise a lyoprotectant at a concentration from 1% to 50%, 2% to 45%, 3 to 40%, 4 to 35%, 5 to 30%, 6 to 25%, 6 to 20%, 6 to 15%, 6% to 10%, or 7% to 9%, 1 % to 15%, from 5% to 12%, from 6% to 10%, or from 7% to 9%. For example, the concentration of the lyoprotectant may be about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11 %, about 12%, about 13%, about 14%, or about 15%.

The dialysis buffer may be a buffer such as those described herein, e.g., a Tris- HCI buffer.

The solution processed by the method herein may comprise a lyoprotectant such as those described herein, e.g., sucrose or trehalose. The solution may comprise a lyoprotectant at a concentration from 1% to 50%, 2% to 45%, 3 to 40%, 4 to 35%, 5 to 30%, 6 to 25%, 6 to 20%, 6 to 15%, 6% to 10%, 7% to 9%, 1 % to 15%, from 5% to 12%, from 6% to 10%, or from 7% to 9%. For example, the concentration of the lyoprotectant may be about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 1 1%, about 12%, about 13%, about 14%, or about 15%.

Three-component Lipid Nanoparticle Compositions

The disclosure includes three-component LNP compositions containing:

1 ) a steroidal or structural lipid-containing component;

2) a stabilizing lipid-containing component (such as a PEGylated lipid); and

3) a cationic or ionizable lipid-containing component. In some embodiments, the largest dimension of a nanoparticle composition is 1 pm or shorter (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter), e.g., when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and/or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels.

Cationic/lonizable Lipid-containing Component

A three component LNP composition of the present disclosure may include one or more cationic and/or ionizable lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH) including, but not limited to, MC3, ALC-0315, ALC- 0159, SM-102, DOTAP, or a cationic and/or ionizable lipid disclosed in WO201 7049245 A2 (Benenato), lipids of Formulae IA, IB, IC, IIA, IIB, IIC, HD, HE, HF, including lAa-IAc, IBa-lbe, ICa-ICc, IIAa-HAb, IIBa, HCa, HDa, HEa-HEb, and HFa-IIFb, and any combination thereof.

Stabilizing Lipid Component

A three-component LNP composition of the present disclosure may include one or more PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

Steroidal/Structural Lipid-containing Component. The 3-component formulation may also comprise of alternatives to PEGylated lipids, such as polysarcosine, polysaccharides, etc.

A three component LNP composition of the present disclosure may include one or more structural lipids. Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

A three component LNP composition of the present disclosure is free of helper lipids, such as phospholipids. For example, the three component LNP composition of the present disclosure is free of 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1 ,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 - palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1 -oleoyl-2-cholesterylhemisuccinoyl-sn- glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn- glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1 ,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), and sphingomyelin.

Adjuvants

In some embodiments, the three-component LNP may be combined in a composition with one or more adjuvants, e.g., Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(l:C), aluminum hydroxide, Pam3CSK4, saponin extracts (e.g. Quil-A®), and Lipid A.

Therapeutic Agents

The three-component LNP composition may include one or more therapeutic and/or prophylactics. The disclosure features methods of delivering a therapeutic and/or prophylactic to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof comprising administering to a mammal and/or contacting a mammalian cell with a nanoparticle composition including a therapeutic and/or prophylactic. Therapeutic and/or prophylactics include biologically active substances and are alternately referred to as “active agents.” A therapeutic and/or prophylactic may be a substance that, once delivered to a cell or organ, brings about a desirable change in the cell, organ, or other bodily tissue or system. Such species may be useful in the treatment of one or more diseases, disorders, or conditions. In some embodiments, a therapeutic and/or prophylactic is a small molecule drug useful in the treatment of a particular disease, disorder, or condition. Examples of drugs useful in the nanoparticle compositions include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylative agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetolol), antihypertensive agents (e.g., clonidine and hydralazine), anti-depressants (e.g., imipramine, amitriptyline, and doxepim), anti-conversants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorphenirimine, and promethazine), antibiotic/antibacterial agents (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics, and imaging agents.

Polynucleotides and Nucleic Acids In some embodiments, a therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide,” in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. In some embodiments, a therapeutic and/or prophylactic is an RNA. RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is an mRNA.

In certain embodiments, a therapeutic and/or prophylactic is an mRNA. An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

In other embodiments, a therapeutic and/or prophylactic is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

In some embodiments, a therapeutic and/or prophylactic is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.

Nucleic acids and polynucleotides useful in the disclosure typically include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5'-terminus of the first region (e.g., a 5'-UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'-UTR), at least one 5'-cap region, and a 3’-stabilizing region. In some embodiments, a nucleic acid or polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5'- UTR). In some cases, polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, a polynucleotide or nucleic acid (e.g., an mRNA) may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and/or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside). For example, the 3'-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-O-methyl nucleoside and/or the coding region, 5'-UTR, 3'-UTR, or cap region may include an alternative nucleoside such as a 5-substituted undine (e.g., 5- methoxyuridine), a 1 -substituted pseudouridine (e.g., 1 -methyl-pseudouridine or 1 -ethyl- pseudouridine), and/or a 5-substituted cytidine (e.g., 5-methyl-cytidine).

Formulations

The three-component LNP composition may include, for example, the three components in the following relative mole percentages:

1 ) 5 to 60 mole % of a steroidal or structural lipid-containing component;

2) 0.5 to 20 mole % of a stabilizing lipid-containing component; and

3) 30 to 70 mole % of a cationic or ionizable lipid-containing component.

In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:

1 ) 20 to 50 mole % of a steroidal or structural lipid-containing component;

2) 0.8 to 10 mole % of a stabilizing lipid-containing component; and

3) 40 to 62 mole % of a cationic or ionizable lipid-containing component.

In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:

1 ) 25 to 46 mole % of a steroidal or structural lipid-containing component;

2) 1 to 7 mole % of a Stabilizing lipid-containing component; and

3) 44 to 58 mole % of a cationic or ionizable lipid-containing component.

In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:

1 ) 35 to 44 mole % of a steroidal or structural lipid-containing component;

2) 1 .2 to 5 mole % of a Stabilizing lipid-containing component; and 3) 48 to 57 mole % of a cationic or ionizable lipid-containing component.

In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:

1 ) 37 to 43 mole % of a steroidal or structural lipid-containing component;

2) 0.5 to 5, 1 to 4, or 1 .4 to 3 mole % of a Stabilizing lipid-containing component; and

3) 50 to 56 mole % of a cationic or ionizable lipid-containing component.

Any numerical value within the recited ranges and any combination of ranges and specific numerical values within the claimed ranges are contemplated and supported by the foregoing disclosures, i.e., 5 to 60 mole % of a steroidal or structural lipid-containing component includes any numerical value and range within the range of 5 to 60, e.g., 5, 5.01 , 5.02, ...59.97, 59.98, 59.99, 60, 5-10, 5-20, 10-30, 15-25, etc. Similarly, 0.5 to 20 mole % of a Stabilizing lipid-containing component includes any numerical value and range within the range of 0.5 to 20, e.g., 0.5, 0.501 , 0.502, ...19.97, 19.98, 19.99, 20, 0.5-10, 0.52-15, 1 -12, 5-13, etc. Similarly, 30 to 70 mole % of a cationic or ionizable lipid-containing component includes any numerical value and range within the range of 30 to 70, e.g., 30, 30.01 , 30.02, ...69.97, 69.98, 69.99, 70, 30.5-68, 35-51 , 40-52, 45-63, etc.

The amount of a therapeutic and/or prophylactic in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic and/or prophylactic. For example, the amount of an RNA useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic and/or prophylactic and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic in a nanoparticle composition may be from about 5:1 to about 60:1 , such as 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 , 11 :1 , 12:1 , 13:1 , 14:1 , 15:1 , 16:1 , 17:1 , 18:1 , 19:1 , 20:1 , 25:1 , 30:1 , 35:1 , 40:1 , 45:1 , 50:1 , and 60:1 . For example, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic may be from about 10:1 to about 40:1 . In certain embodiments, the wt/wt ratio is about 20:1 . The amount of a therapeutic and/or prophylactic in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

In some embodiments, a nanoparticle composition includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA. In general, a lower N:P ratio is preferred. The one or more RNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 30:1 , such as 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 , 12:1 , 14:1 , 16:1 , 18:1 , 20:1 , 22:1 , 24:1 , 26:1 , 28:1 , or 30:1 . In certain embodiments, the N:P ratio may be from about 2:1 to about 8:1 . In other embodiments, the N:P ratio is from about 5:1 to about 8:1 . For example, the N:P ratio may be about 5.0:1 , about 5.5:1 , about 5.67:1 , about 6.0:1 , about 6.5:1 , or about 7.0:1 . For example, the N:P ratio may be about 5.67:1 .

Pharmaceutical Compositions Nanoparticle compositions may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more nanoparticle compositions. For example, a pharmaceutical composition may include one or more nanoparticle compositions including one or more different therapeutic and/or prophylactics. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21 ^st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a nanoparticle composition. An excipient or accessory ingredient may be incompatible with a component of a nanoparticle composition if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.

In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a nanoparticle composition. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Relative amounts of the one or more nanoparticle compositions, the one or more pharmaceutically acceptable excipients, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt/wt) of one or more nanoparticle compositions.

In certain embodiments, the LNP is lyoprotected with 8% v/v sucrose. However, any lyoprotectant will suffice (e.g. trehalose). The concentration of the lyoprotectant can be from 4%-32% v/v.

In certain embodiments, the nanoparticle compositions and/or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and/or shipment (e.g., being stored at a temperature of 4° C or lower, such as a temperature between about -150° C and about 0° C or between about -80° C and about -20° C (e.g., about -5° C, -10° C, -15° C, -20° C, -25° C, -30° C, -40° C, -50° C, -60° C, -70° C, -80° C, -90° C, -130° C or -150° C). In certain embodiments, the disclosure also relates to a method of increasing stability of the three-component LNP compositions and/or pharmaceutical compositions by storing the nanoparticle compositions and/or pharmaceutical compositions at a temperature of 4° C or lower, such as a temperature between about -150° C and about 0° C or between about -80° C and about -20° C., e.g., about -5° C, -10° C, -15° C, -20° C, -25° C, -30° C, -40° C, -50° C, -60° C, -70° C, -80° C, -90° C, -130° C or -150° C). For example, the three-component LNP compositions and/or pharmaceutical compositions disclosed herein are stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months, e.g., at a temperature of 4° C. or lower (e.g., between about 4° C and -20° C). In one embodiment, the formulation is stabilized for at least 4 weeks at about 4° C. In certain embodiments, the pharmaceutical composition of the disclosure comprises a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, an acetate (e.g., sodium acetate), an citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the carrier may be at a concentration of 1 -100 mM (e.g., including but not limited to any numerical value or range within the range of 1 -100mM such as 1 , 2, 3, 4, ...97, 98, 99, 100, 10-90 mM, 20- 80 mM, 30-70 mM and so on).

LNP buffer exchange may be performed by dialysis, tangential flow filtration, or any other method that effectively removes and replaces buffer.

In certain embodiments, the pharmaceutical composition of the disclosure has a pH value between about 4 and 8 (e.g., 4, 4.1 , 4.2, ... 6.8 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, or between 4 and 7 or between 5 and 6.5). For example, a pharmaceutical composition of the disclosure comprises a nanoparticle composition disclosed herein, Tris, saline and sucrose, and has a pH of about 7-8, which is suitable for storage and/or shipment at, for example, about -20° C. For example, a pharmaceutical composition of the disclosure comprises a nanoparticle composition disclosed herein and PBS and has a pH of about 7-7.8, suitable for storage and/or shipment at, for example, about 4° C or lower.

In certain embodiments, the pharmaceutical composition of the disclosure contain the therapeutic or prophylactic agent at a ratio of 0.05 to 25 mg/ml, 0.1 to 20 mg/ml, 0.2 to 18 mg/ml, 0.5 to 15 mg/ml, 0.7 to 12 mg/ml, 0.9 to 10 mg/ml, 1 to 8 mg/ml, 1 .5 to 6 mg/ml, 2 to 5 mg/ml, 2.5 to 4 mg/ml, 0.5 to 3.0 mg/ml, 0.2 to 4.0 mg/ml, 0.4 to 2.0 mg/ml, and any numerical value or range within the range of 0.05 to 25 mg/ml.

Nanoparticle compositions and/or pharmaceutical compositions including one or more nanoparticle compositions may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of a therapeutic and/or prophylactic to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system. Although the descriptions provided herein of nanoparticle compositions and pharmaceutical compositions including nanoparticle compositions are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats.

A pharmaceutical composition including the three-component LNP compositions may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and/or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., nanoparticle composition). The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Methods of Producing Polypeptides in Cells

The present disclosure provides methods of producing a polypeptide of interest in a mammalian cell. Methods of producing polypeptides involve contacting a cell with a nanoparticle composition including an mRNA encoding the polypeptide of interest. Upon contacting the cell with the nanoparticle composition, the mRNA may be taken up and translated in the cell to produce the polypeptide of interest.

In general, the step of contacting a mammalian cell with a nanoparticle composition including an mRNA encoding a polypeptide of interest may be performed in vivo, ex vivo, in culture, or in vitro. The amount of nanoparticle composition contacted with a cell, and/or the amount of mRNA therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the nanoparticle composition and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors. In general, an effective amount of the nanoparticle composition will allow for efficient polypeptide production in the cell. Metrics for efficiency may include polypeptide translation (indicated by polypeptide expression), level of mRNA degradation, and immune response indicators.

The step of contacting a nanoparticle composition including an mRNA with a cell may involve or cause transfection. Transfection may allow for the translation of the mRNA within the cell.

Methods of Delivering Therapeutic Agents to Cells and Organs

The present disclosure provides methods of delivering a therapeutic and/or prophylactic to a mammalian cell or organ. Delivery of a therapeutic and/or prophylactic to a cell involves administering a nanoparticle composition including the therapeutic and/or prophylactic to a subject, where administration of the composition involves contacting the cell with the composition. For example, a protein, cytotoxic agent, radioactive ion, chemotherapeutic agent, or nucleic acid (such as an RNA, e.g., mRNA) may be delivered to a cell or organ. In the instance that a therapeutic and/or prophylactic is an mRNA, upon contacting a cell with the nanoparticle composition, a translatable mRNA may be translated in the cell to produce a polypeptide of interest. However, mRNAs that are substantially not translatable may also be delivered to cells. Substantially non-translatable mRNAs may be useful as vaccines and/or may sequester translational components of a cell to reduce expression of other species in the cell.

In some embodiments, a nanoparticle composition may target a particular type or class of cells (e.g., cells of a particular organ or system thereof). For example, a nanoparticle composition including a therapeutic and/or prophylactic of interest may be specifically delivered to a mammalian liver, kidney, spleen, femur, or lung. Specific delivery to a particular class of cells, an organ, or a system or group thereof implies that a higher proportion of nanoparticle compositions including a therapeutic and/or prophylactic are delivered to the destination (e.g., tissue) of interest relative to other destinations, e.g., upon administration of a nanoparticle composition to a mammal. In some embodiments, specific delivery may result in a greater than 2 fold, 5 fold, 10 fold, 15 fold, or 20 fold increase in the amount of therapeutic and/or prophylactic per 1 g of tissue of the targeted destination (e.g., tissue of interest, such as a liver) as compared to another destination (e.g., the spleen). In some embodiments, the tissue of interest is selected from the group consisting of a liver, kidney, a lung, a spleen, a femur, an ocular tissue (e.g., via intraocular, subretinal, or intravitreal injection), vascular endothelium in vessels (e.g., intra-coronary or intra-femoral) or kidney, and tumor tissue (e.g., via intratumoral injection).

As another example of targeted or specific delivery, an mRNA that encodes a protein-binding partner (e.g., an antibody or functional fragment thereof, a scaffold protein, or a peptide) or a receptor on a cell surface may be included in a nanoparticle composition. An mRNA may additionally or instead be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties. Alternatively, other therapeutic and/or prophylactics or elements (e.g., lipids or ligands) of a nanoparticle composition may be selected based on their affinity for particular receptors (e.g., low density lipoprotein receptors) such that a nanoparticle composition may more readily interact with a target cell population including the receptors. For example, ligands may include, but are not limited to, members of a specific binding pair, antibodies, monoclonal antibodies, Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding reagents including mono- or bi-specific antibodies such as disulfide stabilized Fv fragments, scFv tandems, diabodies, tribodies, or tetrabodies; and aptamers, receptors, and fusion proteins.

In some embodiments, a ligand may be a surface-bound antibody, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site. In one embodiment, multiple antibodies are expressed on the surface of a cell, and each antibody can have a different specificity for a desired target. Such approaches can increase the avidity and specificity of targeting interactions.

In certain embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 10 mg/kg, from about 0.001 mg/kg to about 10 mg/kg, from about 0.005 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.05 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about 0.001 mg/kg to about 5 mg/kg, from about 0.005 mg/kg to about 5 mg/kg, from about 0.01 mg/kg to about 5 mg/kg, from about 0.05 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 0.0001 mg/kg to about 2.5 mg/kg, from about 0.001 mg/kg to about 2.5 mg/kg, from about 0.005 mg/kg to about 2.5 mg/kg, from about 0.01 mg/kg to about 2.5 mg/kg, from about 0.05 mg/kg to about 2.5 mg/kg, from about 0.1 mg/kg to about 2.5 mg/kg, from about 1 mg/kg to about 2.5 mg/kg, from about 2 mg/kg to about 2.5 mg/kg, from about 0.0001 mg/kg to about 1 mg/kg, from about 0.001 mg/kg to about 1 mg/kg, from about 0.005 mg/kg to about 1 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, from about 0.05 mg/kg to about 1 mg/kg, from about 0.1 mg/kg to about 1 mg/kg, from about 0.0001 mg/kg to about 0.25 mg/kg, from about 0.001 mg/kg to about 0.25 mg/kg, from about 0.005 mg/kg to about 0.25 mg/kg, from about 0.01 mg/kg to about 0.25 mg/kg, from about 0.05 mg/kg to about 0.25 mg/kg, or from about 0.1 mg/kg to about 0.25 mg/kg of a therapeutic and/or prophylactic (e.g., an mRNA) in a given dose, where a dose of 1 mg/kg (mpk) provides 1 mg of a therapeutic and/or prophylactic per 1 kg of subject body weight. In some embodiments, a dose of about 0.001 mg/kg to about 10 mg/kg of a therapeutic and/or prophylactic (e.g., mRNA) of a nanoparticle composition may be administered. In other embodiments, a dose of about 0.005 mg/kg to about 2.5 mg/kg of a therapeutic and/or prophylactic may be administered. In certain embodiments, a dose of about 0.1 mg/kg to about 1 mg/kg may be administered. In other embodiments, a dose of about 0.05 mg/kg to about 0.25 mg/kg may be administered. A dose may be administered one or more times per day, in the same or a different amount, to obtain a desired level of mRNA expression and/or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered, for example, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In some embodiments, a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition.

Nanoparticle compositions including one or more therapeutic and/or prophylactics may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. For example, one or more nanoparticle compositions including one or more different therapeutic and/or prophylactics may be administered in combination. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of compositions, or imaging, diagnostic, or prophylactic compositions thereof in combination with agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.

It will further be appreciated that therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than those utilized individually. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, a composition useful for treating cancer may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., control of any adverse effects, such as infusion related reactions).

EXAMPLES

Example 1

This example describes exemplary lyophilization protocol and reconstitution of lipid nanoparticles for 3-component lipid nanoparticles.

The first sets of exemplary nanoparticle compositions include 3-component lipid nanoparticles, where there are 3 lipids in the LNP (25 mM SM-102 in ethanol, 19 mM cholesterol in ethanol, and 0.75 mM DMG-PEG2000). Equal volumes of each lipid and of a blank ethanol were combined to give lipid moles ratios as follows: SM-102 (55.9%), cholesterol (42.4%), and DMG-PEG2000 (1 .7%) to give a total lipid concentration of 1 1 .2 mM. In one example, the 0.13 mg/g mRNA in 25 mM sodium acetate pH 5.0 was prepared. In a second example, the 0.13 mg/g mRNA in 25 mM sodium citrate pH 4.0 was prepared. In a third example, the 0.13 mg/g mRNA in 25 mM sodium citrate pH 5.0 was prepared. In a fourth example, the 0.13 mg/g mRNA in 25 mM sodium citrate pH 6.0 was prepared. Each of these formulations were dialyzed into 20 mM Tris-HCI with 8% sucrose or 8% trehalose, respectively. The second sets of exemplary nanoparticle compositions include 3-component lipid nanoparticles, where there are 3 lipids comprising the LNP (25 mM SM-102 in ethanol, 19 mM cholesterol in ethanol, and 0.75 mM DMG-PEG2000). Equal volumes of each lipid were combined and diluted with 0.57 vol% an aliquot with pure ethanol to give mole ratios as follows: SM-102 (55.9%), cholesterol (42.4%), and DMG-PEG2000 (1.7%) with a total lipid concentration of 12.5 mM. The LNP was formulated with 0.13 mg/g mRNA in 25 mM sodium acetate. This formulation was dialyzed into a sample with either 8% sucrose or 8% trehalose, respectively.

A 4-component control was formulated, where there are 4 lipids comprising the LNP (25 mM SM-102 in ethanol, 19 mM cholesterol in ethanol, 5 mM DSPC in ethanol, and 0.75 mM DMG-PEG2000). Equal volumes of each lipid were combined to give lipid moles ratios as follows: SM-102 (50%), cholesterol (38.5%), DSPC (10.0%), and DMG- PEG2000 (1 .5%) to give a total lipid concentration of 12.5 mM.

The LNP was formulated with a mRNA aqueous solution to lipid ethanolic solution ratio of 3:1 using microfludics to give unimodal peaks. The sample was then dialyzed using 10 kDa MWCO cassettes at 4 °C against 20 mM Tris-HCI, with either 8% sucrose or 8% trehalose to give the final LNP. The sample was concentrated to an mRNA concentration over 0.2 mg/mL by UV and sterilization filtration performed. The encapsulation was found to be comparable to current 4-component systems as shown in Fig. 5.

The samples were lyophilized on a SP Scientific Lyobeta. Small aliquots 0.5 mL) of each LNP formulation were micropipetted into glass vials and lyophilized. The samples were cooled to 4°C and held at temperature for 30 min. The samples were then cooled to -45°C and held for 8h. The samples were warmed to -25°C and vacuum (100 mbar) was applied for 72 hr. The samples were then warmed to 30°C over 6 hr and held under vacuum (100 mbar) for 8 hr.

The lyophilized samples were reconstituted with ultrapure water 0.5 mL. All samples reconstituted very quickly and showed no solids within less than 5 seconds of reconstitution by visual inspection and confirmed by size measurements by dynamic light scattering (DLS). The samples were kept at 4°C on an orbital shaker until in vitro analysis four days later. Also, no precipitate was observed after 1 week by visual inspection.

In vitro expression was determined and is shown in the western blot of Figs. 1 A- 1 B and 2. Figs. 1 A-1 B show the in vitro data for the LNPs dialyzed with 20 mM Tris-HCI, pH 7.4, 8% sucrose. Fig. 2 shows the in vitro data for the LNPs dialyzed with 20 mM Tris- HCI, pH 7.4, 8% trehalose. In Figs. 1 A-1 B, lanes 1 -6 correspond to the in vitro data for the reconstituted lyophilized LNPs and lanes 7-12 correspond to the in vitro data for the same samples before they were lyophilized. Lanes 1 and 7: formulations produced from 4-component LNP I mRNA stock solution with sodium acetate pH 5; lanes 2 and 8: formulations produced from LNP stock solution with 12.5 mM total lipid / mRNA stock solution with sodium acetate pH 5; lanes 3 and 9: formulations produced from LNP stock solution with 1 1.2 mM total lipid / mRNA stock solution with sodium acetate pH 5; lanes 4 and 10: formulations produced from LNP stock solution with 11 .2 mM total lipid / mRNA stock solution with sodium citrate pH 4: lanes 5 and 11 : formulations produced from LNP stock solution with 1 1.2 mM total lipid / mRNA stock with solution sodium citrate pH 5; lanes 6 and 12: formulations produced from LNP stock solution with 11 .2 mM total lipid I mRNA stock solution with sodium citrate pH 6.

Fig. 2 shows the in vitro data for the LNPs dialyzed with 20 mM Tris-HCI, pH 7.4, 8% trehalose. In Fig. 2, lanes 1 -6 correspond to the in vitro data for the reconstituted lyophilized LNPs and lanes 7-12 correspond to the in vitro data for the same samples before they were lyophilized. Lanes 1 and 7: formulations produced from 4-component LNP / mRNA stock solution with sodium acetate pH 5; lanes 2 and 8: formulations produced from LNP stock solution with 12.5 mM total lipid / mRNA stock solution with sodium acetate pH 5; lanes 3 and 9: formulations produced from LNP stock solution with 11 .2 mM total lipid / mRNA stock solution with sodium acetate pH 5; lanes 4 and 10: formulations produced from LNP stock solution with 1 1 .2 mM total lipid / mRNA stock solution with sodium citrate pH 4: lanes 5 and 11 : formulations produced from LNP stock solution with 11 .2 mM total lipid I mRNA stock with solution sodium citrate pH 5; lanes 6 and 12: formulations produced from LNP stock solution with 1 1.2 mM total lipid / mRNA stock solution with sodium citrate pH 6.

Surprisingly, in Figs. 1 A-1 B, the in vitro data after lyophilization was superior to the in vitro data of the same samples before lyophilization. This indicates that the method of the present disclosure and using a lyoprotectant such as sucrose improved stability of the 3-component LNP performance.

EXAMPLE 2

A three-component LNP composition of the present disclosure was made containing 25 mM SM-102 in ethanol, 19 mM cholesterol in ethanol, and 0.75 mM DMG- PEG2000. Equal volumes of each lipid and of a blank ethanol were combined to give lipid mole ratios as follows: SM-102 (55.9%), cholesterol (42.4%), and DMG-PEG2000 (1.7%). The 0.13 mg/g mRNA in 25 mM sodium acetate pH 6.0 was prepared.

The LNP was formulated with a mRNA aqueous solution to lipid ethanolic solution ratio of 3:1 using microfludics to give unimodal peaks. The sample was then dialyzed using 10 kDa MWCO cassettes at 4 °C against 20 mM Tris-HCI, 8% sucrose to produce the final three-component LNP composition. The sample was concentrated to an mRNA concentration over 0.2 mg/mL by UV and filter-sterilization was performed. Surprisingly, the encapsulation was found to be comparable to current 4-component LNP systems.

A control (four-component LNP) composition was formulated using SM-102 (50%), cholesterol (38.5%), DSPC (10%), and DMG-PEG2000 (1.5%) in ethanol mixed by microfluidics with mRNA (0.13 mg/mL) in 25 mM sodium acetate pH 6.0 and dialyzed into 20 mM Tris-HCI pH 7.4, 8% sucrose (“RL-007”). The LNPs were filter-sterilized and concentrated to give an mRNA concentration over 0.2 mg/mL by UV.

In vitro expression was determined and is shown in the western blot of Fig. 3, channels 6 (4-component control) and 7 (novel 3-component LNP) and in Fig. 3, lower the last (right side) two bars.

In vivo expression was determined from IM injection in mice. The expression after the first dose is shown in Fig. 4. In Fig. 4, the novel 3-component LNP composition was compared to a 4-component LNP control. The expression after one dose was very similar to the 4-component (DSPC-containing) control.

The encapsulation of both the 3-component LNP and the 4-component LNP control were excellent (Fig. 5, green).

EXAMPLE 3 Further formulations were made and tested as summarized in the Table 1 and characterized in Figs. 3-5.

TABLE 1 In another aspect of this invention, the same lipids were prepared in relation to the same 4-component control except, the formulation was performed with mRNA (0.13 mg/mL) in 50 mM sodium citrate, pH 4.0. The results are shown in Fig. 3, upper channel 3 and lower 3 ^rd bar with the control in channel 5 and bar 5. The encapsulation, determined by ribogreen assay, of the 3-component and 4-component systems were very similar.

In another aspect, the same components were prepared for LNP, but the total concentration of the lipids in ethanol solution was adjusted (see Table), see Fig. 3, channel 1 and bar 1. At the elevated concentration the in vitro data showed worse performance relative to the control used in the previous example. However, contrary to the in vitro data, the in vivo data show that the 3-component LNP of the present disclosure performed very well, which was very surprising.

In another aspect of this invention, a 3-component LNP was prepared using SM- 102, cholesterol, and DSPE-PEG2000. The in vitro data were gathered, see Fig. 3 channel 4 and bar 4. In vitro expression was observed but it was less than the 3- component LNP using DMG-PEG2000 with same concentration and buffer conditions. The encapsulation was similar to the control (Fig. 5).

In another aspect of this invention, the previous 3-component LNP was prepared using lower concentreations of the lipids in ethanol, (Fig. 3, channel 2 and bar 2). The expression was the lowest of all of the attempted combinations of the three-component LNPs. The encapsulation was similar to the control (Fig. 5, bar 2).

EXAMPLE 4

Images of 4-component LNPs (LNP) and 3-component LNPs (mLNP) of the disclosure were taken after reconstitution using transmission electron microscopy (TEM). As shown in Fig. 6, the 4-component LNPs possess blebs that are larger and more distinct than the mLNPs. Therefore, mLNPs retain their spherical shapes after reconstitution better than LNPs. EXAMPLE 5

Several different lyoprotectant formulations were tested to determine the effect of the lyoprotectant on reconstututed mLNPs comprising ionizable lipids of the disclosure.

First, two different lyoprotectants, sucrose and trehalose were studied at three different concentrations (8, 12, and 16% w/v). Combinations containing 1 :1 ratios of the two lyoprotectants were also tested. mLNPs were dialyzed into 20mM Tris-HCI, pH 7.4 with either sucrose and/or trehalose at the above concentrations. The dialyzed products were lyophilized and reconstituted and then characterized using Malvern zetasizer for size, PDI, and ZP. As shown in Table 2 both sucrose and trehalose effectively protected the mLNP with similar results.

TABLE 2 Second, the formulation of the reconstitution media containing a lyoprotectant were tested using a reconsitution formulation containing 8% sucrose, mLNPs encapsulating mRNA and water, at pH 7.4. Seven different formulations were tested as shown in Table 3. TABLE 3

The mLNP contained in the reconstutution media formulations described above were lyophilized and reconstituted and then characterized using Malvern Zetasizer for size, PDI, and ZP. The samples were formulated and dialyzed under conditions shown under the “Dialysis Buffer” column in Table 3. After lyophilization, the product was reconstituted under conditions shown under the “Reconstitution Media” column in Table

The mLNP in the reconstitution media formulations described in Table 3 above were characterized before and after reconstitution. As shown in Table 4, the quality of the mLNP after lyophilization varied based on the reconstitution media chosen. The reconstitution medium comprising 147mM NaCI resulted in a PDI value of the reconstituted mLNP of less than 0.1 , thereby demonstrating very high structural integrity based on uniform size.

TABLE 4

The reconstutution media formulations containing mLNPs described in Tables 3 and 4 above were also studied in T-cells over time. 293-T cells were transfected with mLNPs contained in reconstitution media according to groups 1 -7 of Tables 3 and 4 before and after lyophilization. Expression of the mRNA encapsulated by the mLNP was observed after 24 hours. As shown in Fig. 7, Group 6 demonstrated higher in vitro expression compared to the other groups. Group 3 and 4 show the lowest in vitro expression. This result indicate that in vitro expression does not necessarily correlate to characterization data, however, the choice of reconstitution media influences the characteristics of the reconstituted mLNP.

EXAMPLE 6

Reconstitution media formulations were also tested in vivo. The four mLNP compositions tested are shown in Table 5. The DMG-PEG2000 lipid component comprised 1 .7 to 2.5 to 3.4% of the mLNP compositions tested. TABLE 5

Table 6 shows characterization data of each mLNP formulation and a control 4 component LNP formulation collected using a Malvern Zetasizer (characterizing size, PDI, and ZP) and a ribogreen assay (characterizing encapsulation efficiency). The effect of the different reconstitution buffers on particle size, PDI, zeta potential and encapsulation efficiency are shown under the “after” column of each tested characteristic. From top to bottom for each sample, each mLNP or LNP was reconstituted in different dialysis buffers comprising lyoprotectants (A)-(C): 8% w/v sucrose (A), 8%w/v sucrose+4%w/v mannitol (B), and 8% w/v sucrose + 4%w/v trehalose (C). Each formulation of the reconstitution media also contained 100mM NaCI (aq). Each mLNP and LNP was formulated using methods known in the art, then dialyzed into one of the three dialysis buffer formulations. After dialysis, the LNP and mLNP compositions were lyophilized. After lyophilization, each LNP and mLNP was reconstituted with the same reconstitution buffer, which was 100mM NaCI. The mLNPs and LNPs were characterized before and after lyophilization.

TABLE 6

The size and PDI were all acceptable before and after lyophilization under the tested conditions. The encapsulation efficiency varied based on the lyoprotectant contained in each reconstitution media formulation. Therefore, the choice of lyoprotectant is important to protect the integrity of the LNP or mLNP. Additionally, increasing the PEG concentration from 1 .7 to 3.4% appeared to decrease the encapsulation efficiency after lyophilization specifically when the mLNP was reconstituted using a formulation containing 8% sucrose+4% mannitol. The other tested reconstitution media formulations did not show any trends in mLNP characterization data based on mLNP composition or reconstitution media formulation.

EXAMPLE 7

The study described in Example 6 showed that PEG concentration can affect mLNP encapsulation efficiency in certain reconstruction media formulations. This study tested the effect of cholesterol on mLNP compositions. The concentration of cholesterol in each tested mLNP composition varied between 38.5 and 50 mole% as shown in

Table 7 below.

TABLE 7:

After formulation of each tested mLNP at the lipid molar concentrations described in Table 7, the mLNPs and a control 4 component LNP were dialyzed into a medium comprising 20mM Tris-HCI, pH 7.4, and 8% sucrose. Then the mLNP and LNP compositions were lyophilized and reconstituted with water. The characterization data were collected using a Malvern Zetasizer and encapsulation efficiency was determined by ribogreen assay. Table 8 shows the particle size, PDI, zeta potential and encapsulation efficiency of each mLNP and LNP composition characterized before and after lyophilization. TABLE 8

Groups 3 and 4 maintained disparity of particle integrity before and after lyophilization. Group 3 showed significantly lower PDI compared to the other tested groups before and after lyophilization. EXAMPLE 8

The effect of DMG-PEG2000 ratio of mLNP compositions was studied in a mouse model using Delta S1 and RBD mRNA payloads. Table 9 shows two different mLNP lipid compositions and one 4 component LNP tested in vivo. Five mice were used in each Group and the mLNP and LNP formulations before and after lyophilization were administered by intramuscular injection (i.m.). The dose for each mouse was 5 ug. PBS was used as a negative control. M1 and M2 were Group 2 and Group 3. M3 and M4 were Group 4 and Group 5. L1 and L2 were Group 6 and Group 7.

TABLE 9

After formulation of each tested mLNP and LNP at the lipid molar concentrations described in Table 9, the mLNPs and LNPs were dialyzed into a medium comprising 20mM Tris-HCI, pH 7.4, 8% sucrose. Half of the sample in Group 2, Group 4 and Group 6 were lyophilized and reconstituted in water to produce Group 3, Group 5, and Group 7 respectively. 100 uL of each Group was injected into the mice, blood from each mouse was sampled 14 days after immunization, a second immunization was administered by the same way after 21 days from the first immunization, and a second bleeding was performed 35 days after the first immunization. IgG titer data was collected after the first timepoint (Fig. 8) and after the second timepoint (Figs. 9 and 10) to determine the strength of induced immune response. IgG titer against Delta S1 was shown in Fig. 8 and Fig. 9 and IgG titer against Delta RBD was tested in Fig. 10. Before lyophilization, the mLNP with an increased SM-102 content (M1 ) and the 4 component LNP (L1 ) produced equivalent immune response in mice models. Comparing mLNP with an increased SM-102 content (M1 ) and the 4 component LNP (L1 ), M1 (with a PEG content of 3.4%) produced less immune response compared to the 4 component LNP. Additionally, mLNP with increasing SM-102 content (M1 ) produced equivalent immune response before and after lyophilization. The 4 component LNP formulation showed less immune response after lyophilization. At both timepoints, the 1 ,7mol% DMG-PEG2000 mLNP formulation performed better than the 3.4 mol% DMG-PEG2000 mLNP for both S1 and RBD.

Before and after lyophilization, mLNP with increasing SM-102 content (M1 ) produced equivalent immune response, while LNP showed less immune response after lyophilization

In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein.

It is to be understood that while the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and alterations are within the scope of the following claims.